Professional Documents
Culture Documents
10.1007@978 3 662 53270 6
10.1007@978 3 662 53270 6
Penetration Enhancers
Drug Penetration
Into/Through the Skin
Methodology and
General Considerations
Nina Dragicevic
Howard I. Maibach
Editors
123
Percutaneous Penetration Enhancers
Drug Penetration Into/Through the Skin
Nina Dragicevic Howard I. Maibach
Editors
Percutaneous
Penetration Enhancers
Drug Penetration
Into/Through the Skin
Methodology and General
Considerations
Editors
Nina Dragicevic Howard I. Maibach
Apoteka "Beograd" San Francisco
Belgrade California
Serbia USA
The main function of skin is the protection of the body from the external
environment by preventing loss of water and the ingress of exogenous sub-
stances. This implies that the skin acts as a barrier for the diffusion of sub-
stances into the underlying tissue. Despite this role, the skin has become
recognized as an important drug delivery route which can be reached directly.
It is an ideal site for the application of drugs for achieving local (topical) and
systemic (transdermal) drug effects. Local or topical drug delivery assumes
treating various skin diseases, while transdermal delivery aims to achieve
systemically active drug levels in order to treat systemic diseases. Drugs have
been applied to the skin to achieve also regional drug delivery which involves
drug application to the skin to treat or alleviate disease symptoms in deep tis-
sues beneath the skin (such as in musculature, etc.). Topical and transdermal
drug delivery offer a number of advantages compared to other conventional
routes, and hence they are of great interest to pharmaceutical research, which
explains the increasing interest in skin as a site of drug application.
However, skin represents a formidable barrier for percutaneous drug
absorption, being of crucial importance for achieving topical and transdermal
effects of drugs. Significant efforts have been devoted to developing strate-
gies to overcome the impermeability of intact human skin. There are many
ways for circumventing the stratum corneum, which provides the main bar-
rier to drug penetration. These methods can be divided into chemical and
physical penetration enhancement methods, i.e., percutaneous penetration
enhancers(as well as the skin structure are described in the other volumes in
this book series).
The aim of this book is to provide to readers working in academia and
industry, including young researchers, an up-to-date comprehensive work
describing all the important topics required to understand the principles of
enhancing transdermal and dermal drug delivery. We have divided this book
into five volumes.
Volume 1 begins with a description of the skin, as understanding of its
structure, function, and especially its penetration pathways is fundamental to
understanding how topical and transdermal dosage forms work and how dif-
ferent methods may be employed and work to enhance percutaneous drug
penetration as well as how they work. The first parts devoted to skin and the
stratum corneum, representing its uppermost layer being responsible for its
protection, discuss their structure, the importance of the lipid organization in
the stratum corneum, the different penetration pathways through the skin
v
vi Preface
others from Springer, for their dedicated work which was necessary to achieve
such a high standard of publication. We highly appreciate readers comments,
suggestions, and criticisms to improve the next edition of this book.
ix
x Contents
Index 405
Contributors
xiii
xiv Contributors
Here, (x,t) is the chemical potential of the sub- Here, MW is the solute molecular weight, and n
stance, T is the temperature, and k is Boltzmans and Dm0 are constants, the characteristics of the
constant. For the homogeneous case with membrane at a specific temperature. Other theo-
j ( x,t ) = kT ln(c ( x,t ) and a constant diffusivity, ries derived from polymer research have also
Eq.1.4 simplifies to the standard diffusion equa- been applied successfully to the field of describ-
tion as stated in Eq.1.3. ing diffusivities in the skin domain. For deeper
The general case equation that describes the insight, the interested reader is kindly referred to
diffusion-partition problem with the chemical
Hansen etal. (2013).
( )
potential defined as j ( x,t ) = kT ln ( c ( x,t ) / K ( x ) , Where the diffusivity D denotes the speed
and the position- dependent partition coefficient of a diffusant through a membrane and is
K is given by Eq.1.5 (Anissimov and Roberts inversely related to the weight of the solute, the
2004): partition coefficient K accounts for jumps in
concentrations at the interface of two adjacent
d
d c ( x,t ) d D ( x ) d x c ( x,t ) skin layers and is often related to a measure of
= solute lipophilicity, most commonly the loga-
dt d x D x c x,t d ln K x (1.5)
( ) ( ) ( ) rithmic octanolwater partition coefficient. K
dx is a thermodynamic parameter reflecting the
Besides studies with variable partition and/or relative affinity of a solute for a certain phase
diffusion coefficients inside a certain skin layer over another phase. Hence, the partition coef-
(Anissimov and Roberts 2004), it is more ficient between layer m2 and layer m1 can be
common (especially for more complex multilayer defined as Eq. 1.8
models) to assign specific diffusion coefficients
Cmeq2
to the different layers and specific partition K m2 / m1 = (1.8)
coefficients to the interfaces of two adjacent lay- Cmeq1
ers. In this chapter, the presented solutions of the
diffusion equation and determination of distinc- with Cmeq2 and Cmeq1 being the respective solute
tive parameters almost exclusively depend on concentrations at the interface of the two adjacent
predicted, fitted, or experimentally determined phases at equilibrium. In general, the partition
diffusivities and partition coefficients. Therefore, coefficient is concentration-dependent, but if the
at least a basic understanding of the relationship solubilities in the two phases are relatively low
between molecular properties and the aforemen- (which holds true for many cases), this effect can
tioned parameters is essential when it comes to be neglected, and the partition coefficient can be
the preparation, setup, and evaluation of transder- defined by using the saturation concentrations
mal skin transport. S m1 and S m2 in the different layers (Anissimov
For spherical particles in a continuous fluid, etal. 2013), with Eq. 1.9
the diffusion coefficient can be calculated by
S m2
using the well-known StokesEinstein relation K m2 / m1 = (1.9)
(Eq.1.6), with viscosity of the solvent and par- S m1
ticle radius r:
A typical strategy is to establish a relationship
kT
D= (1.6) between an easy-to-observe parameter that
6ph r mimics the membrane properties and the
For diffusion in polymers, it could be found parameter of interest. A prominent example is
empirically that for a small particle the following the ocanol/water partition coefficient Ko/w that
relationship holds (Eq.1.7) (Anderson and could be related to the stratum corneum lipid
Raykar 1989): phase/water partition coefficient Klip,with the
help of a linear free-energy relationship
Dm = Dm0 MW - n (1.7)
(Eq.1.10) (Anderson etal. 1988)
6 D. Selzer et al.
a b
c d
Fig. 1.1 Theoretical change of concentration in the donor o utgoing flux (d) for infinite (solid line), semi-infinite
compartment over time (a), change of mass inside the (dashed line), and finite dosing (dotted line). Simulations
stratum corneum (b), accumulated mass inside the accep- were performed using the DSkin software
tor compartment (c), and change of stratum corneum
The chosen values correspond to a model com- corresponds to a plateau of the flux-over-time
pound of approximately 300Da with a logKO/W profile (Fig.1.1d). In contrast, the finite dose sce-
of 2in an aqueous vehicle, and model parameters nario will reach a theoretical mass plateau in the
were estimated by DSkin. The concentration- acceptor compartment if all substance has trav-
over-time profile depicted in Fig.1.1a shows the eled through the membrane. Obviously, the flux
characteristic depletion of the donor for the finite reaches a maximum and subsequently decreases
dose case and less pronounced for the semi-infi- with time. For the simulation of the semi-infinite
nite case. The barrier mass-over-time curve for dose case shown in Fig.1.1d, the applied volume
an infinite dose setup does reach a plateau as per area was set slightly higher than the finite
soon as the steady state is reached (Fig.1.1b). As dose threshold defined by the OECD.This shows
opposed to this, the mass of the finite dose case clearly that the assumption of an infinite dose (no
decreases after reaching a m aximum (Fig.1.1b). significant depletion) does not automatically hold
In case of the infinite dose setup, the accumulated by applying fixed volume-based rules. This is
mass inside the acceptor compartment reaches the crucial when applying the mathematical concepts
typical straight steady-state line (Fig.1.1c). This presented in the next subsections, since c hoosing
8 D. Selzer et al.
Steady-state
meation is low, the system might behave like an
Mass
infinite dose case due to the fact that significant
donor depletion only occurs at very long experi-
mental time periods in relation to the permeated
solute amount in the acceptor compartment.
Investigation of infinite and finite dose experi-
ments typically differ concerning their parame-
ters of interest and application of mathematical
Time
concepts (e.g., analytical solutions of the diffu-
sion equation are always tailored to certain
boundary and initial conditions). In the next two Fig. 1.2 Accumlated mass over time in the acceptor com-
partment of an infinite dose experiment (solid line) and
sections, analytical solutions of the diffusion
linear part of the steady-state phase (dashed line). The
equation and mathematical concepts for the most intersection of the linearized steady-state phase and time
typical experimental settings are presented. For axis denotes the lag time
an exhaustive compilation of solutions regarding
the diffusion equation for various boundary and
initial conditions, the reader is kindly referred to (see Fig.1.2). From a mathematical point of
the excellent book The Mathematics of Diffusion view, a few assumptions must be made to derive
by J.Crank (Crank 1975). an analytical solution for the diffusion equation
by defining initial and boundary conditions. In
this case, we assume a constant and steady con-
1.2.1 D
ealing withInfinite Dose centration in the donor compartment, perfect sink
Skin Permeation conditions (zero concentration in the acceptor at
all times), and that no compound of interest is
Analysis of infinite dose invitro skin permeation located inside the barrier at time t = 0 .
is typically done by measuring the cumulative By incorporating these rules for a homogeneous
amount of substance inside the acceptor compart- membrane, the absorption curve can be described
ment over time. For short times, the amount by an analytical solution of Ficks second law of
increases exponentially until reaching a steady diffusion (Scheuplein 1967; Crank 1975), with
line (the steady state) with constant flux JSS
( -1) Dn 2p 2 t
n
1 2
m ( t ) = A K l C0 K l t - - 2
6 p
n2
exp -
l 2
(1.11)
n =1
Here, A denotes the area of application, K is steadystate), the solution simplifies to the linear
the partition coefficient between donor and bar- part of the steady state (Fig.1.2), with
rier, C0 is the concentration of applied formula-
DK l2
tion in the donor which is assumed not to change m (t ) = A C0 t - (1.12)
significantly during experimental time periods, D
l 6D
is the macroscopic diffusion coefficient, l is the
macroscopic thickness of the barrier, and t is the From Eq.1.12, we can examine important
time after application. It is obvious that with t parameters when it comes to analysis of infinite
leaning toward infinity (and hence reaching the dose experiments. The first parameter is the
1 Basic Mathematics inSkin Absorption 9
so-
called apparent permeability coefficient, kp, intrinsic permeability of a solute in a certain
which is often given in units of cm/h and is medium, making it an ideal parameter to com-
defined as pare permeability of different solutes. Obviously,
this is the case, as long as the vehicle does not
DK
kp = (1.13) affect the transport kinetics in the barrier (Zhang
l etal. 2011).
It is independent of the area of application and In contrast to epidermal kp which is
initial concentration, and hence a direct parame- optimally correlated to the molecular weight
ter for the strength of permeation for a compound and lipophilicity of a compound (Fiserova-
through a certain barrier from a specific vehicle Bergerova etal. 1990; Potts and Guy 1992;
under infinite dose and perfect sink conditions. McKone and Howd 1992), Magnusson etal.
Mathematically, it denotes a normalization of (2004) could show that molecular weight is the
steady-state flux JSS with main determinant when it comes to predicting
solute maximum flux.
J SS
kp = (1.14) A further parameter to characterize infinite
C0 dose absorption is the so-called lag time tlag
given by
Such a parameter might heavily depend on exper-
l2
imental conditions. Hence, this has to be kept in tlag = (1.16)
mind when comparing parameters (interlabora- 6D
tory and intralaboratory). Although kp may be a It is a measure that relates to the time it takes for
useful and popular parameter when it comes to a compound to travel through the barrier and
examination of permeation experiments, it can be establish a steady state. A word of caution is nec-
sometimes misleading when comparing the per- essary concerning the meaning of tlag. tlag does not
meation of several compounds (Michaels etal. directly represent the time when the steady state
1975; Anissimov etal. 2013). The apparent per- is achieved (see Fig.1.2); however, it can be
meability coefficient kp describes an intrinsic approximated by multiplying tlag with 2.7 (Crank
property of a solute to permeate across a specific showed that steady state is achieved when
medium (e.g., the skin) which is independent of Dt
= 0.45 approximately (Crank 1975)).
the dose but influenced by the applied vehicle. l2
Therefore, comparisons are only possible From a practical point of view, determination
between compounds which are applied in identi- of permeability coefficient and lag time can gen-
cal vehicles. In 2006, Sloan etal. introduced a erally be accomplished in two fashions:
change of paradigm when it comes to explaining
experimental data (Sloan etal. 2006). They sug- 1. The easiest approach utilizes the linearization
gested to use the more expressive parameter Jmax of the steady state (see Fig.1.2) which inter-
which denotes the maximum possible flux of a sects the time axis at tlag. This can be accom-
solute through a barrier for comparing permea- plished graphically by manual interpolation of
bility (Eq.1.15). By using Jmax, it is possible to the last data points that contribute to the steady
overcome the limitations addressed before state or mathematically more soundly by a lin-
ear fit (Schfer-Korting etal. 2008). It is
Sm
J max = kp S v = D (1.15) important to mention that the occurrence of
l physically untenable negative lag times often
Here, Sv is the saturated permeant concentration indicates experimental problems such as
in the vehicle and Sm is the solubility of the solute donor depletion (Barbero and Frasch 2009) or
within the barrier. In other words, by removing insufficient sink conditions (Anissimov and
the influence of the partition coefficient between Roberts 1999). The permeability coefficient
the skin and vehicle, Jmax should be independent can be determined by dividing the slope of the
of the vehicle applied. Thus, it describes an linear part by the initial concentration C0 and
10 D. Selzer et al.
application area A or simply by using the two discarding data often yields wrong results
steady-state data points with and can produce a high variability, depend-
ing on which points are chosen for evalua-
mt1 - mt2
kp = (1.17) tion. This is an obvious but often neglected
AC0 ( t1 - t2 ) problem in processing experimental data.
2. A more sophisticated approach is the use of
If the slope of the curve decreases after reaching the mathematical representation of the entire
steady state, a general strategy is to employ curve (Eq. 1.11). In 2009, Henning etal. per-
the steepest part of the curve for evaluation formed an in-depth analysis of problems that
(Buist etal. 2010). The clear advantage is the arise in data evaluation of infinite dose perme-
simplicity in calculations, but several prob- ation experiments and showed the superiority
lems arise using this approach that makes it of this procedure over the manual approach
extremely prone to errors from an experi- (Henning etal. 2009). A huge advantage is the
mental and evaluation point of view: ability to deliver sound results even if only a
(a) This approach cannot be applied if the partial representation of the steady state is
steady state was not clearly reached at the available. Often, the macroscopic thickness of
end of the experiment. the swollen skin is not exactly known. By
(b) It is often difficult to identify which data defining partition parameter P1 and diffusion
points contribute to the steady state and parameter P2 with
which do not. Often, a frequent sampling
P1 = K l (1.18)
and stepwise addition of data points from
tend (last data point) toward t0 (first data D (1.19)
P2 =
point) and analyzing the linear interpola- l2
tions can overcome this problem.
(c) Using only a limited amount of data points and reformulating Eq.1.11 yields (Dez-Sales
generally ignores information. Carelessly etal. 1991; Kubota etal. 1993)
( -1)
n
1 2
m ( t ) = A P1 C0 P2 t - - 2 exp ( - P2 n 2p 2 t ) (1.20)
6 p n =1 n 2
This reduces the number of unknown parameters. reach a steady state, but builds a mass plateau
Hence, P1 and P2 can be easily fitted to experi- inside the acceptor compartment at late time points
mental data using a nonlinear least squares (see Fig.1.1c). Finite dose experiments also show
approach. Permeability and lag time can be a characteristic depletion of donor concentration
subsequently computed by (Fig.1.1a) and increase in flux until reaching the
so-called peak flux Jpeak at time tpeak (Fig.1.3).
kp = P1 P2 (1.21)
As opposed to the infinite dose case, deduc-
1 (1.22) tion and application of an analytical solution
tlag =
6 P2 for the description of absorption curves require
a more sound mathematical foundation. Based
1.2.2 D
ealing withFinite Dose Skin on the theory of heat flow by Carslaw and
Permeation Jaeger, a description of the flux of the com-
pound leaving the barrier at time t is given by
In contrast to the infinite dose exposure scenario, a Eq.1.23 (Carslaw and Jaeger 1959; Cooper and
typical finite dose absorption profile does not Berner 1985):
D a i2 a i2 Dt
J (t ) = 2 A M b
l 2 i =i cos a i ( b + b 2 + a i2 )
exp - 2 ,
l
(1.23)
1 Basic Mathematics inSkin Absorption 11
with
l
b =K , (1.24)
hv
Flux
a i tan a i = b (1.25)
Here, M denotes the applied mass, and hv is
Time
the height of the formulation in the donor com-
partment. The remaining parameters keep their
Fig. 1.3 Sketch of outgoing flux across the barrier meaning as introduced above.
acceptor interface over time of a finite dose experiment
(solid line). The maximum of the curve denotes the peak Integrating Eq.1.23 yields the accumulated
flux Jmax at time to peak flux tmax mass per area (Kasting 2001) with
M (t )
1 a i2 Dt
= 1 - 2b exp - 2 (1.26)
M i =i cos a i ( b + b 2 + a i2 ) l
In comparison to Eq.1.11 for the infinite dose worked for the infinite dose case (Eqs.1.18 and
case, solving Eq.1.26 requires a more skillful 1.19) can be applied.
evaluation, since Eq.1.25 must be solved for Equation 1.26 can be fitted to experimental
arbitrary values of i. A basic strategy to solve data by a nonlinear least squares approach and
thetranscendal equation is to use logistic yields values for , diffusivity D, and macro-
regressionto tabulated values of the first roots scopic path length l.
(see Fig.1.4).To find further roots a subsequent Important parameters for evaluation of finite
linear extrapolation from previous roots dose permeation experiments are the peak flux
( root n = 2 root n -1 - root n - 2 ) followed by a Jpeak and time to peak flux tpeak.
refinement step (root-finding with, e.g., Newtons A general strategy to easily find Jpeak and tpeak
method) (Kasting 2001) is key. To reduce the is finding the root of the first derivative of J(t) and
number of unknowns, the same approach that hence solving the following equation for tpeak:
D a i2 a i2 Dtpeak a i2 D
2 M b
l 2 i =i cos a i ( b + b 2 + a i2 )
exp -
l2
2 = 0 (1.27)
l
A reliable solution for the root-finding prob- If the thickness of the applied formulation is
lem is, for example, Brents method (Brent 1973). reasonably small in comparison to the macro-
For small doses ( b < ~ 0.1 ), Jpeak and tpeak can scopic diffusion path length, it can be neglected,
be calculated by the following simple equations and Eq.1.29 simplifies to the familiar
(Kasting 2001; Scheuplein and Ross 1974): expression:
D l2
J peak = 1.85M , (1.28) tpeak = (1.30)
l2 6D
In contrast to fitting solutions of the diffusion
tpeak =
(l 2
- hv2 )
(1.29) equation to experimental data, some researchers
6D use the steepest linear part of the absorption curve
12 D. Selzer et al.
a b
Finite dose
Fig. 1.5 Theoretical change of concentration in the barrier over time for the infinite dose (a), semi-infinite dose (b), and
finite dose (c) cases. Simulations were performed using the DSkin software
line in the concentration over space kinetics is over time and space by using the appropriate ini-
typically not reached, and a significant drop can tial and boundary conditions together with
be recognized over time (Fig.1.5c). Eq. 1.3. For the infinite dose case, assuming a
As for permeation experiments, analytical homogeneous membrane, one can calculate the
solutions for the diffusion equation can be concentration inside the barrier at point x and
obtained to describe the change of concentration time t (Crank 1975; Hansen etal. 2008), with
x 2 1 np x Dn 2p 2 t
c ( x,t ) = K C0 1 - - sin exp - (1.32)
l p n =1 n l l 2
As for the permeation case (Eq.1.11), K denotes deplete over time (typical infinite dose assump-
the barrier/vehicle partition coefficient, C0 the tion), the receptor compartment is kept at a theo-
initial concentration in the vehicle, D the appar- retical zero concentration (perfect sink
ent diffusivity, and l the macroscopic thickness of conditions), and no solute is present inside the
the barrier membrane with 0 x l . The equa- barrier at t = 0 . Obviously, only the passive dif-
tion can only be applied if the donor does not fusion process is modeled. Hence, permeation
14 D. Selzer et al.
enhancement, binding phenomena, and convec- Obviously, information about diffusivity can
tion effects are not part of the model. only be obtained before the steady state is
Eq.1.32 can be of great benefit to estimate the reached. Hence, in order to obtain kinetic param-
values for partition coefficient (K) and diffusion eters, fitting Eq.1.32 to experimental data should
D
coefficient ( 2 ) and extrapolate the skin- focus on short incubation times.
l
concentration depth profile for later time points If experimental data that account for the depth
(Naegel etal. 2008) or even other exposure sce- profile at a certain time cannot be obtained
narios (Selzer etal. 2013a, b; Naegel etal. 2011). immediately at the end of an experiment (e.g.,
For late times, the skin-concentration depth due to a long tape-stripping procedure), the
profile will reach steady state, and Eq.1.32 sim- obtained values have to be adjusted according to
plifies to the time delay (Reddy etal. 2000b), and the
skin-concentration depth profile can be described
x
css ( x ) = K C0 1 - (1.33) with
l
8 ( 2n + 1) p x - ( 2n + 1)2 p 2 t
c ( x,t ) = K C0 cos exp d
(1.34)
n=0 ( 2 n + 1) p 2 l
24 t
lag
1 4
1 ( 2n + 1)2 p 2 Dt
AUC = K C0 - 2
2 p
n = 0 ( 2n + 1)
2
exp -
l 2
(1.35)
In 1998, the Food and Drug Administration For the steady state, the amount of solute in
released a draft guidance on dermatopharmacoki- the membrane can be easily calculated from a
netics for the evaluation of topically applied com- simplified formulation of Eq.1.35:
pounds (Shah etal. 1998). Besides the maximum
K C0
amount in the outermost skin layer (stratum cor- M ss = A l (1.36)
neum) and the time to reach the maximum amount, 2
the AUC was defined as a parameter of interest. For the finite dose case, an analytical solution for
Hence, Eq.1.35 can be useful to predict the AUC the diffusion equation can be obtained with and
as a function of time after obtaining K and 2
D (as defined earlier in Eqs.1.25 and 1.24)
from parameter fits (Herkenne etal. 2007). l (Kasting 2001):
b cos (a n x / h ) - a n sin (a n x / h ) a n2 Dt
c ( x,t ) = 2 K C0 exp - 2 (1.37)
n =1 b + b 2 + a n2 l
1 Basic Mathematics inSkin Absorption 15
As for the infinite dose case, the same limitations For the two-compartment model, Eqs.1.39 and
hold (e.g., perfect sink conditions and no solute 1.40 hold, respectively:
in the barrier at t = 0 ).
dC2
V2 = k1C1 - k-1C2 + k-2 C3 - k2 C2 ,
dt (1.39)
1.3.2 Skin Compartmental
Approaches dC3
V3 = k2 C2 - k-2 C3 + k-3C4 - k3C3
dt (1.40)
Compartmental or pharmacokinetic models (PK)
are used since the early beginnings of mathemati- The separation of the skin in a lipophilic part
cal description to study the fate of a substance (the stratum corneum) and hydrophilic part (via-
that was applied in the systemic circulation and ble epidermis) is common practice to mimic the
are also part of the history of describing transder- heterogeneity of the skin (McCarley and Bunge
mal drug absorption. They treat the body as a 2001; Seta etal. 1992). The underlying ODEs
series of well-stirred compartments. The basic can be solved either analytically (for the most
idea is the elimination of space dependence of the part, only for easy equations) or numerically.
partial differential diffusion equation (Eq.1.2) to Numerical solvers for nonstiff equations are, for
obtain a series of ordinary differential equations example, Eulers method or the famous fourth-
(ODEs) that describe the change of amount of order RungeKutta method (widely known as
solute in different compartments over time. RK4). For stiff equations, the backward differen-
For the family of PK models, a set of first- tiation formula (BDF) is a well-known algo-
order rate constant can be assigned to denote the rithm. Commercial and free mathematical
transfer of a compound from one compartment to software package usually provide various possi-
another. Figure1.6 shows two examples of skin bilities for the convenient solving of these kinds
compartment models: a one-comparment skin of equations.
model and a two-comparment skin model. In comparison to solutions of the diffusion
The corresponding first-order ODE of the equation, using compartmental models can have
one-compartment model can be expressed by some advantages.
Eq.1.38:
A. Obviously, the solution of a set of ODEs can
dC2
V2 = k1C1 - k-1C2 + k-2 C3 - k2 C2 (1.38) often be derived with little hassle in
dt comparison to complex multicompartmental
e.g. Vehicle e.g. Stratum corneum e.g. Viable epidermis e.g. Systemic circulation
Fig. 1.6 Exemplary sketch of a one-compartment (a) and C denotes the average concentration in the compartment,
two-compartment (b) skin model. Here, the number denotes assuming simplified well-stirred conditions, and V is the
the number of compartments used to describe the skin bar- volume of the compartment that is accessible for the solute
rier, rather than the number of overall compartments. distribution
16 D. Selzer et al.
Microscopic SC model
1.4.2 Numerical Diffusion Models
Fig. 1.7 Simplified depiction of a macroscopic diffusion
Numerical methods for solving the diffusion model with effective diffusivities and partition coefficients
equation for various initial and boundary condi- (a), and a microscopic representation of the brick-and-
mortar structure (Elias 1983) of the stratum corneum (SC)
tions are important techniques and allow a more with diffusivities and partition parameters for the lipid and
flexible construction of advanced skin models the corneocyte phase (b). Figures are not drawn to scale
(e.g., incorporation of binding effects (George
2005)), iontophoretic transdermal delivery
(Wearley etal. 1990), repeated applications the interplay among input parameters. The basic
(Kubota etal. 2002), controlled release vehicles idea consists of the discretization of time and
(Kurnik and Potts (1997), and invitro lateral space domain (gridding) and a numerical approx-
transport (Selzer etal. 2013a, b)). In comparison imation of the partial differential diffusion equa-
with classical PK skin compartment models, it is tion (e.g., for the most trivial form, see Eq.1.2).
typically much more convenient to relate the nec- The discretization allows the construction of
essary input parameters (for the simple diffusion coarse-grained macroscopic and fine-grained
problem, only diffusivities and partition coeffi- microscopic models (a basic example is depicted
cients are needed) to physicochemical parame- in Fig.1.7).
ters of the diffusant (Hansen etal. 2013). These Macroscopic models do neglect the heteroge-
models can be used for descriptive and predictive neity of different skin layers and use effective
tasks as well as for the theoretical investigation of kinetic parameters of a diffusant for each layer to
18 D. Selzer et al.
describe the transport process. Usually, these with a system of constant space step size x and
kinds of models are only needed for solving the time step size t. Obviously, the above-mentioned
underlying diffusion equation for the one- equation can be easily generalized to problems in
dimensional case and hence treat the skin as a 2D and 3D space. Reformulation of Eq.1.42
series of multilayered slabs (Fig.1.7a). yields
Microscopic models allow a heterogeneous
Cit +1 = MCit-1 + (1 - 2 M ) Cit + MCit+1 (1.43)
representation of the stratum corneum (Hansen
etal. 2009) (lipid phase and protein phase, see
Fig.1.7b) and hence yield a more in-depth analy- with
sis of the underlying transport processes. In this
t
case, the absorption kinetics do not only rely on M =D (1.44)
( x)
2
the definition of diffusion parameters but also on
the definition of spatial geometry. Due to the high
spatial resolution, these models typically require Because of the fact that every point of a certain
much higher computational costs in comparison layer only depends on points from the past,
to coarse-grained macroscopic models. By math- there is no functional relationship between dif-
ematical homogenization, it is possible to reduce ferent points of one time layer (it is a so-called
a microscopic model to a macroscopic one with uncoupled problem). This leads to the problem
preservation of model properties (e.g., anisotro- of numerical instabilities that depend on the
pic diffusivity in the stratum corneum) (Muha value of M (Eq.1.44) and hence on the choice of
etal. 2010; Selzer etal. 2013b). t for a given spatial resolution x. It can be
From a numerical perspective, several tech- shown that this explicit method is only stable for
niques exist to approximate the solution of the values of M 0.5 (for the one-dimensional
diffusion equation. For the one-dimensional case, case). A violation of this recommendation can
probably the most prominent method is the finite produce noise which may result (empirically, it
differences approach. Assuming a constant dif- always does for the diffusion equation) in nons-
fusivity, Eq.1.2 can be approximated by moothable rounding errors. For small diffusion
coefficients (e.g., in the stratum corneum), this
Cit +1 Cit Cit1 2Cit + Cit+1
=D (1.42) restriction can lead to extremely small time
t ( x )
2
This equation yields a system of linear equa- can solve tridiagonal systems of equations).
tions (tridiagonal matrix) that can be efficiently Using this implicit method, it is possible to
solved by, for example, the Thomas algorithm choose larger time steps, which decrease the
(an optimized form of Gaussian elimination that number of computational steps in spite of the
1 Basic Mathematics inSkin Absorption 19
fact that the solving of the system of linear data can be used for the purpose of establishing
equations is computationally more complex
IVIVC.
compared to the explicit method. Various works The topic of IVIVC is especially relevant for
on modeling transdermal transport that are based estimating bioavailability (BA) and for bioequiv-
on finite differences exist (Kurnik and Potts alence (BE) testing of topically applied dosage
1997; Kubota etal. 2002; George 2005; Nitsche forms. For systemically acting drugs, BE is gen-
and Frasch 2011). erally demonstrated based on a pharmacokinetic
Finite differences are not the only mesh-based study comparing the plasma levels of test and ref-
technique to find approximate solutions to partial erence product, following the idea that the
differential equations, as the diffusion equations. pharmacokinetics are correlated to the pharmaco-
The method of finite elements that is widely used logical effect. Similarly, it is possible for
in the field of mechanical engineering and finite transdermal patches which act systemically to
volume approaches that can efficiently handle demonstrate BE based on plasma levels or
unstructured girds and is often applied in the field excretion data. In particular, a transdermal patch
of computational fluid dynamics are known to provides an infinite source and controls the inva-
successfully model transdermal transport sion rate into the blood similar to an i.v. infusion.
(Barbero and Frasch 2005; Heisig etal. 1996; Consequently, in this case, the transdermal
Naegel etal. 2008, 2011; Selzer etal. 2013b). For absorption rates which are measured invitro can
excellent overviews about numerical models, the be correlated with the plasma concentrations
interested reader is kindly referred to references (Chien etal. 1989; Franz etal. 2009).
(Frasch and Barbero 2013; Naegel etal. 2013). Finite dosing is of course more relevant to the
real exposure to chemicals or actives, and conse-
quently also for demonstrating IVIVC (Bronaugh
1.5 I n VitroIn Vivo Correlation and Maibach 1985). At the same time, accurately
(IVIVC) detecting the low systemically absorbed and
excreted amounts of solutes which are applied to
In vitroin vivo correlations (IVIVC) address the the skin as a finite dose requires sensitive and
question of the significance of invitro data for selective analysis in complex biological media
predicting the situation in the living being. In (blood, urine). Data which were obtained during
vitro data are often compared to invivo data from the 1960s1980s therefore often report radioac-
studies obtained under different experimental cir- tivity labeling as a technique to obtain pharmaco-
cumstances, and exact information on relevant kinetic data, for example, form excretion to the
experimental conditions is frequently lacking urine in human volunteers (Feldmann and
(ECETOC 1993). Factors which are known to Maibach 1969; Bartek etal. 1972; Feldmann and
influence transdermal absorption include among Maibach 1974; Wester and Maibach 1976).
others the exposure conditions (such as dose, Indeed, if skin penetration is the rate-limiting
vehicle, humidity, temperature, and exposure step, the rate at which the compound appears in
duration), the skin type, and anatomical site of the urine will represent the rate at which it pene-
application (for more information, the interested trates the skin. For solutes which are also fecally
reader is referred to Chapter 10 in this book). excreted, a correction for the proportion of uri-
Therefore, only data which were obtained using nary to fecal excretion is required. Results are
harmonized protocols should be compared. The calculated as percent of the applied doses which
type of data obtained in invitro and invivo stud- are excreted over time and corrected for applica-
ies is usually quite different mostly due to experi- tion area and incomplete urinary excretion; divi-
mental, physiological, and ethical constraints. sion by time gives the absorption rate (Feldmann
Several different endpoints can be used to relate and Maibach 1969, 1974).
invitro to invivo skin absorption data. This para- Likewise, if one assumes that the steady-state
graph should give an overview on which kind of flux through skin invitro (Jss [ g/cm2/h]) is
20 D. Selzer et al.
equivalent to the rate of input to the systemic cir- collected substantial evidence that invitro skin
culation invivo, then invitro percutaneous absorption testing may be used as a surrogate
absorption data can be used, for example, during method for invivo BA or BE testing (Franz etal.
formulation development for formulation optimi- 2009). In particular, two types of measurements
zation based on known clinical data: are being discussed for this purpose. First, these
are the already mentioned tape-stripping DPK
J ss A = Css Cl (1.46)
measurements which can be performed invivo as
Here, A [cm2]=area of skin, Css [ well as invitro (for more details, the interested
g/l]=steady-state blood concentration, and Cl reader is referred to references (Herkenne etal.
[l/h]=systemic clearance. With Css and Cl being 2008; Russell and Guy 2009)). Second, these are
known, for example, from i.v. application, a tar- also permeability measurements which are per-
get flux can be calculated which needs to be formed invitro.
achieved with a developed dosage form to obtain In fact, Franz etal. reported that the invitro
plasma levels accordingly (Franz etal. 2009). method was able to discriminate between differ-
Cnubben etal. further used linear systems ent vehicles, a finding which was also supported
dynamics (Opdam 1991; Vaughan and Dennis by differences in clinical efficacy, while this dif-
1978; Fisher etal. 1985) to calculate an invivo ference was not picked up by the vasoconstric-
percutaneous absorption rate ( g cm2h1) based tion assay (Franz etal. 2009). Importantly, Franz
on the plasma concentrationtime profile of sol- etal. as well as others demonstrated excellent
utes after dermal application and after a reference IVIVIC, provided that the study protocols
i.v. administration (Cnubben etal. 2002). From between invitro and invivo studies were harmo-
this, they derived invivo permeability coeffi- nized (Franz etal. 2009; Lehman etal. 2011).
cients and lag times and compared these to values The latter aspect is certainly very important to
obtained invitro. ensure comparability between invitro and invivo
Difficulties arise for topically (i.e., on or in the results. Treffel and Gabard as well as Chatelain
skin) or regionally (i.e., in nearby tissues, such as etal., later in a follow-up study, noticed that dif-
the joints, muscle, or cartilage) acting drugs for ferences between several sun protection products
which the plasma concentration is neither quanti- which were obvious in tape-stripping experi-
fiable nor relevant for their local activity. For ments as well as sun protection factor (SPF) mea-
those drugs, measuring the drug concentration at surements in human volunteers as early as 30min
the site of action, or dermatopharmacokinetics after application could invitro not be detected
(DPK), will be more relevant. Unfortunately, a until later time points, that is, 6h (Treffel and
draft guidance document recommending tape- Gabard 1996; Chatelain etal. 2003). In vivo and
stripping for demonstrating BE of topically act- invitro protocols differed in terms of the applied
ing drugs was withdrawn in 2002, due to issues dose (2mg/cm2 vs. 3mg/cm2). The differences
arising from poorly defined experimental stan- may be also explained by artifacts from nonphys-
dardization. Currently, tape-stripping is only rec- iological swelling of the skin inside the Franz dif-
ommended by the FDA for BE testing for certain fusion cell obscuring early effects.
classes of drugs, such as for antifungals that tar- Comparing the skin penetration of the nonste-
get the stratum corneum itself (Narkar 2010). roidal anti-inflammatory drug flufenamic acid
This results in the unfortunate situation that the invitro and invivo by tape-stripping, Wagner
invivo skin blanching or vasoconstriction assay etal. observed that the invivo levels were consis-
(only for corticosteroids) is currently the only tently lower than the invitro levels which they
FDA-recommended assay for demonstrating BE found inside the stratum corneum after different
of generics which does not rely on clinical stud- incubation times (Wagner etal. 2000). The rea-
ies (with known problems such as poor sensitiv- sons for these differences can be manifold, such
ity, requirement for high number of test persons, as interindividual differences, regional variances,
and consequently high costs). Franz etal. have different pressures applied during tape-stripping
1 Basic Mathematics inSkin Absorption 21
to name but a few. Nonetheless, correlating the From a practical point of view, the infinite
amounts found after different incubation times, sum has to be truncated at some point. Some
they demonstrated an excellent IVIVC (Wagner researchers only use <10 summations, for exam-
etal. 2000). To exclude interindividual differ- ple, for the infinite dose solution presented in
ences in a follow-up study, Wagner etal. designed Sect.1.3.1, since the subsequent terms do not sig-
a special workflow, where they used abdominal nificantly contribute to the overall result (Frasch
skin from the same exact individual before and and Barbero 2008). To achieve maximal accu-
after undergoing abdominal reduction surgery racy, a more precise way to truncate the sum is to
(Wagner etal. 2002). Unfortunately, the results incorporate a relative threshold, like the so-called
obtained from tape-stripping were not usable due machine epsilon of the computer a relative error
to the disinfectant wipe which was necessary for due to rounding in floating point arithmetic
the surgery and which significantly changed the (Hansen etal. 2008). The machine epsilon 0 is
drug levels inside the upper skin layers. However, defined as the smallest number, such that
they could show excellent agreement of the drug 1 + e 0 > 1 holds for a certain machine.
levels inside the deeper skin layers (Wagner etal. Nowadays, implementing more complex
2002). This collection of reports shows that functions and fitting to experimental data can be
obtaining a good IVIVC is possible. However, achieved easily using commercial or free data
the parameters which should be correlated need analysis and statistical software packages or free
to be carefully selected, and the conditions under scripting languages. For example, the authors
which these parameters are obtained are most used the popular scripting language Python2 in
critical. combination with the SciPy package (scientific
python) (Jones etal. 2001) to compute various
equations presented for the finite dose case.
1.6 Tips and Tricks These kinds of packages provide predefined
functionalities for root-finding, fitting, and
This section supplies information about common plotting.
difficulties when computing analytical solutions
of the diffusion equation and fitting these solu-
tions to experimental data. It should work as a 1.6.2 Fitting ofExperimental Data
guide when conducting mathematical analysis of
experimental data. As reported here, fitting of experimental data to
analytical solutions of the diffusion equation is
an excellent way of analysis. To gain confidence,
1.6.1 I nfinite Sums inAnalytical experiments are typically conducted in a recur-
Solutions rent manner. For data evaluation, researchers can
fit the objective function to either each individual
Many analytical solutions presented in this chap- dataset (of every single experiment) or a combi-
ter include an infinite sum of form nation of experimental data (e.g., mean accumu-
lated mass over time).
F ( x ) = a H i ( x ) + b, (1.47) It is strongly recommended to report the eval-
i uation of single experiments (OECD 2004b), and
with Hi(x) going to zero for i going to infinity. the authors support the approach of individual
These are typical examples of solutions of the evaluation of experiments and averaging of indi-
diffusion equation for various scenarios. Being vidual determined parameters. Despite this fact,
able to deal with this kind of equations is a key many researchers prefer to handle arithmetic
requirement for a sound evaluation of skin trans- means of combined experiments (Schfer-
port experiments. Hence, we will give a few hints
for the solid handling. Python: http://www.python.org
2
22 D. Selzer et al.
Korting etal. 2008). Fitting is typically done by such mathematical tools is a fundamental
applying a linear or nonlinear least-squares understanding of the underlying physical and
approach. The basic idea is to minimize the sum biological mechanistics but also of experi-
of squared residual errors (e.g., minimization of mental and model constraints. These were
the so-called weighted root mean square function highlighted in this chapter.
displayed in Eq.1.48):
1 n
RMS = wi ( diexp - dicalc )
n i =1
(1.48) References
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(1998) AAPS/FDA Workshop report: bioequivalence
Occlusive Versus Nonocclusive
Application inTransdermal Drug 2
Delivery
GamalM.El Maghraby
view on the occlusive versus nonocclusive appli- with the concentration of water increasing as we
cation and how it can alter the transdermal drug move from the skin surface to deep strata. At the
delivery. SC, the water concentration is less than 15%, but
increases deeper into the skin where it reaches a
five times higher value at the basal skin layers
2.2 Permeability Barrier (Warner etal. 1988). This creates a transdermal
ofHuman Skin hydration gradient which is characteristic for
normal skin (Cevc and Blume 1992).
The advancement in research methods resulted in
clear and detailed identification of skin structure
and its barrier function (Scheuplein and Blank 2.3 Enhancing Effect
1971; Elias 1981; Orland 1983; Barry 1983). The ofOcclusion onTransdermal
studies were extended to cover the barrier func- Delivery
tion of the skin in the healthy and diseased state.
This has been reviewed and documented Skin occlusion can inhibit the continuous water
(Bouwstra and Ponec 2006). It is now accepted loss from skin surface. This can result in over-
that the stratum corneum (SC) is the main barrier hydration of the SC with subsequent reduction
for transdermal drug delivery. Two pathways of in the barrier nature of the SC (Bucks etal.
drug permeation have been identified as the tran- 1991). The penetration-enhancing effect of
sepidermal and the transappendageal pathways water can be explained on the basis that water
(Scheuplein 1965). The former is considered as forms hydration shells around the head groups
the main route with the latter playing only minor of the SC lipids. This leads to the disruption of
role in transdermal drug delivery (Scheuplein the closely packed hydrocarbon chains result-
1967). In the transepidermal pathway, drugs pass ing in a loose organization of SC lipids. In
through the intact SC.This major pathway con- addition, excess water can extend the hydro-
tains two routes: the intercellular route, which is philic domains of the lamellar structure of the
a continuous but tortuous way through the inter- SC lipids providing room for drug permeation
cellular lipids, and the transcellular route through (Barry 1987). However, occlusion does not
the keratinocytes and across the intercellular lip- increase percutaneous absorption of all types
ids. Passing through these routes, drugs will face of drugs. For example, occlusion increased the
many defense lines which hinder their progress permeation of citropten (lipophilic compound)
into and through the skin. These lines of defense 1.6 times, but the transdermal flux of caffeine
are shown in Fig.2.1, which considered both the (amphiphilic compound) was the same under
intercellular and the transcellular pathways occlusive or open application (Treffel etal.
(Barry 1987). The diagram represents the keratin 1992). In a more recent study, the effect of
in the cells which contains hydrogen-bonding occlusion was shown to depend on the vehicle.
sites that can sequester drugs providing the first In this study, the authors evaluated the penetra-
difficulty in their penetration through the trans- tion of a mixture of paraben ester preservatives
cellular pathway. The second defense line is the from a commercially available test ointment
closely packed hydrocarbon chains of the inter- and two commonly employed solvent vehicles
cellular lipid lamellae. This line provides the (acetone and ethanol), together with the effect
main barrier in both the intercellular and trans- of occlusion on the rate of delivery of the pre-
cellular routes. In addition to this, the SC con- servatives from these systems. Occlusion was
tains minimum amount of water which is achieved by the placement of a piece of high-
provided by the hydrophilic domains in the dia- density polyethylene on the application site
gram (Fig.2.1; Barry 1987). immediately after dosing. There was a signifi-
The integrity of human skin is also linked to cant difference in the epidermal flux of para-
its water content which varies across skin strata bens provided by the different vehicles applied
2 Occlusive Versus Nonocclusive Application inTransdermal Drug Delivery 29
Fig. 2.1Simplified
diagram showing a model
Keratin in cells with
for the stratum corneum
hydrogen bonding sites
with various defense lines
against penetration of
xenobiotics (Modified
from Barry 1987)
Closely packed
hydrocarbon chains
hydrophilic domain
under occlusion. Whereas increased flux was 2.4 elivery Systems Utilizing
D
observed from acetone and ethanol used as theMethod ofApplication
vehicles, a decreased flux was seen upon the
occlusive application of the ointment formula- 2.4.1 TransfersomesTM
tion (Cross and Roberts 2000). These results
can be explained on the basis that occlusion While most of the application studies employ
increased the contact time between the skin mainly occlusive application with no attention
and the volatile solvents (ethanol and acetone) paid to the possible effect of the application
which possess the penetration-enhancing abil- method, Cevc and Blume (1992) highlighted the
ity. In contrast, open application of these sol- importance of the application method on the
vents will lead to their rapid evaporation and transdermal delivery of drugs from the highly
hence shorter contact time with the skin and deformable lipid vesicles, TransfersomesTM. They
lower penetration-enhancing effect. Open indicated that these vesicles can penetrate into
application of these solvents will thus provide and through intact skin, carrying therapeutic
a less damaging effect on the skin compared to amounts of drugs if applied under nonocclusive
the occlusive application of the same solvents. conditions. The ability of such carriers to pene-
30 G.M. El Maghraby
trate intact skin was attributed to their xerophobia 2.4.2 Supersaturable Transdermal
(tendency to avoid dry surrounding), which Delivery Systems
causes the vesicles to resist dehydration at the
skin surface by moving into the skin along with Supersaturation is the process of creation of a
the local transdermal hydration gradient (Cevc transient, metastable, supersaturated state in
and Blume 1992; Cevc etal. 1995). Vesicles which the thermodynamic activity of the drug is
deformation ability was considered as another increased above unity. This can increase the
essential property which made the transdermal driving force for transdermal drug delivery.
hydration gradient to provide sufficient driving Alternative methods have been adopted to
force for vesicle penetration through the skin. obtain drug supersaturation leading to increased
The vesicles were thus given the name ultrade- chemical potential and enhanced transdermal
formable vesicles (El Maghraby etal. 2000, drug delivery. The first method was based on
2001a, b; Cevc etal. 2002). Based on this hypoth- mixing of cosolvents for a certain drug, with the
esis, ultradeformable vesicles need to be applied rapid addition of a nonsolvent thereby creating a
under nonocclusive conditions, as occlusion is solution in which the drug concentration exceeds
believed to abolish the driving force for the skin its equilibrium solubility (Megrab etal. 1995).
penetration of vesicles (Cevc and Blume 1992). In the second method, supersaturation can be
The same group applied the local anesthetic lido- achieved by the uptake of water diffusing pas-
caine entrapped in TransfersomesTM under occlu- sively out of the skin into the formulation, in
sion using a watertight wrapping for 25min over which case water can act as the nonsolvent. This
the applied formulation (Planas etal. 1992). The can represent a special case of the first method
results showed the superiority of TransfersomesTM and can gain the benefit of occlusive application
compared to traditional formulations. in which the formulation can mix with skin
The above hypothesis was not accepted easily secretion. This process was used to explain the
by researchers in the field of dermal and transder- enhanced pharmacodynamic effect of buprano-
mal drug delivery. To test this hypothesis, it was lol after dermal administration using micro-
important to test occlusive versus nonocclusive emulsion (ME) (Kemken etal. 1992). Taking
application of liposomes. On doing so, it was this process into consideration, a self-micro-
important to maintain the hydration gradient even emulsifying drug delivery system (SMEDDS)
in the invitro experiments. To verify this, El of ethyl oleate, polyoxyethylene 20 sorbitan
Maghraby and coworkers developed an invitro monooleate (Tween80) and sorbitan monolau-
experiment which preserves the transepidermal rate (Span20), was developed and used to
hydration gradient (El Maghraby etal. 1999). This enhance the transdermal delivery of indometha-
was achieved by the developed open hydration cin compared to the corresponding ME formula-
protocol in which the epidermal membrane was tion containing increasing concentration of
hydrated from viable epidermal side with the stra- water (El Maghraby 2010). The results indi-
tum corneal surface being left open to the atmo- cated the superiority of SMEDDS compared to
sphere, to mimic the invivo conditions. This the tested ME as indicated from the recorded
protocol was employed to study the transdermal transdermal drug flux values which revealed
delivery of estradiol from ultradeformable lipo- greater transdermal drug delivery from
somes after their occlusive and nonocclusive SMEDDS compared to that obtained from
application (El Maghraby etal. 2001b). Occlusion ME.This superiority was attributed to possible
resulted in a significant reduction of the transder- supersaturation of the drug after dilution of the
mal flux of the drug which confirms the impor- SMEDDS with the hydroalcoholic receptor
tance of the application protocol for the transdermal which will diffuse from the receptor upward
drug delivery from ultradeformable vesicles. through the skin, acting as nonsolvent. The
2 Occlusive Versus Nonocclusive Application inTransdermal Drug Delivery 31
Solubility (mg/ml)
pared by diluting saturated drug solution in s
Fig. 2.3Transdermal 16
delivery of progesterone
after occlusive and open Occluisve application
application of microemul- 14
Open application
sion. EFME is ethanol-
free microemulsion, EME 12
is ethanol-containing
microemulsion, ETW is
10
Flux ( g cm-2h-1)
40% ethanol in water, and
control is 14% propylene
glycol in water (This 8
figure was produced using
data published by El
Maghraby 2012) 6
0
EFME EME ETW Control
El Maghraby GM, Williams AC, Barry BW (2001b) Skin Planas ME, Gonzalez P, Rodriguez L, Sanchez S, Cevc G
hydration and possible shunt route penetration in con- (1992) Noninvasive percutaneous induction of topical
trolled skin delivery of estradiol from ultradeformable analgesia by a new type of drug carrier, and prolonga-
and standard liposomes invitro. JPharm Pharmacol tion of local pain insensitivity by anesthetic liposomes.
53:13111322 Aneth Analg 75:615621
Elias PM (1981) Epidermal lipids; membranes and kerati- Scheuplein RJ (1965) Mechanisms of percutaneous
nization. Int JDermatol 20:19 absorption. I.Routes of penetration and the influence
Kemken J, Ziegler A, Muller BW (1992) Influence of of solubility. JInvest Dermatol 45:334346
supersaturation on the pharmacodynamic effect of Scheuplein RJ (1967) Mechanisms of percutaneous
bupranolol after dermal administration using micro- absorption. II.Transient diffusion and the relative
emulsion as vehicle. Pharm Res 9:554558 importance of various routes of skin penetration.
Leichtnam ML, Rolland H, Wthrich P, Guy RH (2007) JInvest Dermatol 48:7988
Impact of antinucleants on transdermal delivery of tes- Scheuplein RJ, Blank IH (1971) Permeability of the skin.
tosterone from a spray. JPharm Sci 96:8492 Physiol Rev 51:702747
Megrab NA, Williams AC, Barry BW (1995) Oestradiol Treffel P, Muret P, Muret-DAniello P, Coumes-Marquet S,
permeation across human skin and silastic mem- Agache P (1992) Effect of occlusion on invitro percuta-
branes: effects of propylene glycol and supersatura- neous absorption of two compounds with different phys-
tion. JControl Rel 36:277294 icochemical properties. Skin Pharmacol 5:108113
Moser K, Kriwet K, Kalia YN, Guy RH (2001) Enhanced Warner RR, Myers MC, Taylor DA (1988) Electron
skin permeation of a lipophilic drug using supersatu- probe analysis of human skin: determination of the
rated formulations. JControl Rel 73:245253 water concentration profile. JInvest Dermatol
Orland GF (1983) Structure of the skin. In: Goldsmith LA 90:218224
(ed) Biochemistry and physiology of the skin, vol 1. Zhai H, Maibach HI (2002) Occlusion vs. skin barrier
Oxford University Press, NewYork, pp363 function. Skin Res Technol 8:16
Finite andInfinite Dosing
3
WingManLau andKengWooiNg
formulation large enough to preclude any sig- p ermeant passing through a unit area of mem-
nificant reduction in the test compound or any brane at time t (Crank 1975):
other component in the donor phase during the
dc
course of the experiment (Franz etal. 1993). In J = -D (3.1)
these cases, a significant reduction usually d x
occurs when the permeant concentration in the where J is the flux of the permeant (mass per
donor phase declines by more than 10% of the cm2), D is the diffusion coefficient and c/x is
initial value (Kielhorn etal. 2006). the concentration gradient. To determine how dif-
Under finite dose conditions, permeant con- fusion affects the permeant concentration with
centration in the formulation changes during time, Ficks second law of diffusion (Eq.3.2),
the experiment, due to penetration of the per- which is derived from Ficks first law of diffusion
meant into and permeation through the skin and the differential mass balance, can be
barrier, or due to evaporation. Generally, the employed:
experimental conditions should mimic as
dc d 2c
closely as possible the invivo situation in order =D 2 (3.2)
to provide suitable data. The amount and con- dt dx
centration of permeant formulation to be This equation assumes that the permeant does not
applied to the skin surface, as well as the dura- bind, nor is it metabolised. It is also assumed that
tion and procedure of the protocols, depend on the diffusion coefficient does not vary with the
the study aims. For example, a small amount of position or composition of the permeant, and that
permeant formulation may be applied in order the barrier properties of the skin remain constant
to mimic invivo application of an ointment. over time (Crank 1975).
According to the Organisation for Economic
Co-operation and Development (OECD), the
application of 10l cm2 of a liquid or 15mg 3.2.1 Permeation Kinetics
cm2 of a solid formulation should be used for
finite dose studies (OECD 2004a). For infinite 3.2.1.1 Infinite-Dose Permeation
dose, amounts of >100l cm2 or >10mg cm2 A typical infinite dosing regimen will produce a
are required. permeation profile of cumulative amount perme-
ated across a unit area of membrane versus time,
as shown in Fig.3.1. Initially, when a permeant is
3.2 Skin Absorption Kinetics applied to the skin, molecules penetrate into and
diffuse through the stratum corneum. Depending
Kinetically, the absorption of a substance into or on the permeants physical and chemical proper-
through the skin is regarded as a passive diffusion ties (e.g. lipophilicity, hydrogen bond acceptor or
process. It is also assumed that the skin is a pseu- donor potential), it may penetrate and diffuse
dohomogeneous membrane providing a single rapidly or may bind to stratum corneum compo-
transport route. Thus, both infinite dose and finite nents. A lag phase is seen where the amount of
dose experiments can be described by Ficks laws permeant in the receptor compartment increases
of diffusion. exponentially due to binding and the increasing
Typically, the parameters describing the concentration of permeant in the stratum cor-
absorption of a substance through the skin barrier neum. After a sufficient time (again dependent on
(such as the permeability coefficient and lag the nature of the permeant, typically shorter for
time) are obtained by evaluating the time- lipophilic non-bound molecules, longer for
dependency of its cumulative permeated amount. hydrophilic molecules), binding sites become
An important parameter is the flux, J, which is fully occupied (or at steady-state equilibrium),
proportional to the concentration gradient accord- and a steady-state concentration gradient of the
ing to Ficks first law of diffusion (Eq.3.1). J can permeant develops across the membrane. Under
be estimated from the cumulative amount of these conditions, the flux profile becomes essen-
3 Finite andInfinite Dosing 37
L t1
Time, t
tially constant, and the curve approaches a athematical model in examining skin perme-
m
straight line. The linear portion of the graph rep- ation data:
resents the steady-state flux (Jss) and can be
DK m Cv
determined by simple linear regression of the lin- J ss = (3.5)
ear portion of the graph (Crank 1975; Scheuplein h
and Blank 1971). The lag time can be obtained by extrapolating
Typically, the steady-state flux is assessed the steady-state (linear) portion of the permeation
from an invitro experiment under infinite dose graph to the intercept on the horizontal axis.
conditions, with perfect sink conditions Crank (1975) showed that the lag time, L, can be
where the receptor compartment is at zero determined mathematically by
concentration throughout. Jss can also be esti-
h2
mated by L= (3.6)
6D
DC0
J ss = (3.3) It has been estimated that the time required for
h most permeants to achieve steady-state flux is
where C0 is the concentration in the outermost about 2.7 times the lag time (Barry 1983).
first layer of the skin and h is the membrane Permeant transport across the skin is some-
thickness. Practically, it is very difficult to mea- times described in terms of the permeability
sure C0. However, the concentration of the per- coefficient (Kp), essentially a measure for the
meant in the donor vehicle, Cv, is easily obtainable speed of transport across a membrane (often as
or is usually known. Since C0 and Cv are related cm/h):
to the partition coefficient between donor and
K m D J ss
membrane (Km), where Kp = = (3.7)
h Cv
C0 = K m Cv (3.4)
then substitution of Eq.3.4 into Eq.3.3 gives Typically, the steady-state flux Jss and the perme-
Eq. 3.5, which is the most widely applied ability coefficient Kp are obtained from an invitro
38 W.M. Lau and K.W. Ng
infinite dose experiment. As described above, Jss but this may lead to dilution of the penetrant
is obtained from the gradient of the linear portion below the detection limit. For static diffusion
of the permeation profile, and therefore, if the cells, it is commonly regarded that sink condi-
concentration of the permeant in the applied tions are maintained when the receptor fluid does
vehicle (Cv) is known, then Kp can be determined. not contain more than 10% of the saturated con-
Kp is often used to characterise the skin perme- centration of the penetrant (Ng etal. 2010; Skelly
ation of permeants, as calculations for other etal. 1987). The most widely used receptor fluid
parameters such as D and Km can be problematic is isotonic buffered saline, pH7.4. However, for
as the membrane thickness (h), or the diffusional highly lipophilic compounds, the solubility in the
path length, is often unknown. receptor fluid may become the rate-determining
Estimates of steady-state flux and permeabil- step in skin absorption and may have a significant
ity coefficients should only be derived from data effect on the total flux measured. When solubility
points beyond 2.7 times the lag time when is a concern, the receptor phase can include lipo-
(pseudo-) steady-state conditions are established philic solvents which do not affect the skin bar-
(Kielhorn etal. 2006); using data before steady rier or a solubilising agent (e.g. 50% ethanol, 6%
state is established leads to false estimates of per- polyethylene glycol, 20% oleyl ether or 5%
meability coefficients (Shah etal. 1994). It has bovine serum albumin) (Skelly etal. 1987;
been recommended that infinite dose permeation OECD 2004a).
experiments should last for at least 24h (OECD In reality, even under infinite dose conditions,
2004a). However, an increase in exposure time depletion of the donor phase, non-sink receptor
may alter the integrity of the skin barrier conditions and deterioration of the skin can occur
(Kleszczyski and Fischer 2012). Typically, with time. These factors may result in inaccura-
experiments are performed for 2448h (Boonen cies in steady-state flux and lag time estimations
etal. 2012; Karadzovska etal. 2012; Fasano etal. (Moody 2000). Therefore, particular caution has
2012; Brain etal. 2005; Walters etal. 1997), to be exercised in experiments where prolonged
although shorter durations have also been incubation times are necessary.
reported (Chen etal. 2011). It has been suggested
that 72h or even longer may be needed in some 3.2.1.2 Finite-Dose Permeation
cases for an infinite dose to establish steady-state In vitro percutaneous absorption studies often
flux, especially for permanents that have very utilise an infinite dose regimen to define a perme-
low fluxes or present difficulties for detection ants properties, that is, steady-state flux, perme-
(Franz etal. 1993; Howes etal. 1996). ability coefficient and lag time. However, a major
Although Eqs.3.5, 3.6 and 3.7 are commonly limitation of infinite dosing is its failure to mimic
used to evaluate infinite dose experiments, it is the application of topical drug formulations in
assumed that the skin membrane acts as a homo- common clinical situations, where a relatively
geneous membrane and that permeation through small amount (i.e. a finite dose) of the formula-
the stratum corneum is the rate-limiting factor for tion is used. Under these circumstances, steady-
transdermal transport. This assumption is usually state permeation seldom occurs and, therefore,
valid for most permeants, but for highly viscous the permeability coefficient cannot be deter-
vehicles, highly lipophilic or highly hydrophilic mined. Most invivo experiments are based on
molecules, partitioning behaviour may become finite dosing, although in some cases, infinite
the limiting factor (Cross etal. 2001). Also, the dose conditions may result when finite doses are
equations assume perfect sink conditions applied repeatedly.
throughout the experiment in order to ensure In contrast to the infinite-dose permeation
drug permeation is not affected by solubility in profile, finite-dose application may result in a
the receptor phase. For invitro studies, flow- pseudo steady-state condition, where the
through diffusion cells maintain sink conditions amount permeated may be transiently linear but
by constant replenishment of the receptor fluid, then reaches a plateau, beyond which the amount
3 Finite andInfinite Dosing 39
Time, t
Jmax
commonly reported parameters in finite dosing.
These can be represented by (Scheuplein and
Ross 1974; Crank 1975; Kasting 2001)
1.85 DC0d
J max = (3.8)
h2
h2 d 2
Tmax = (3.9)
6D
Here, D is the apparent diffusion coefficient, C0 is
Tmax the concentration of the permeant in the first
Time, t
layer of the stratum corneum, h is the thickness of
the stratum corneum and is the thickness of the
Fig. 3.3 Estimation of maximum flux (Jmax) from a graph finite dose layer on the skin surface. For a finite
of amount penetrated per unit time (assuming constant dose, since is considerably smaller in compari-
permeation area) versus time. In this case, the vertical axis
represents instantaneous flux. The time taken to reach
son with h, can be neglected, leading to
maximum flux is referred to as Tmax
h2
Tmax = (3.10)
6D
permeated remains constant due to donor deple- Thus, it is possible to estimate the apparent diffu-
tion (Fig.3.2). sion coefficient (D), if the values of Jmax and Tmax
Alternatively, by plotting the amount pene- are known. In addition to this mathematical model,
trated between the time points (i.e. instantaneous Jmax and Tmax are sometimes obtained graphically
flux) against time (Figs.3.3 and 3.4), a peak is from experimental data (Figs.3.2 and 3.3) (van de
observed which corresponds to the maximum Sandt etal. 2004; Wilkinson etal. 2006).
flux before appreciable donor depletion (Franz Due to the nature of finite dosing, experimen-
1983; Kasting 2001). The maximum flux (Jmax) tal problems can ensue, since applying a small
40 W.M. Lau and K.W. Ng
1400 400
Lop. 10 mg/cm2
1000 PG 40 mg/cm2
PG Flux (g/cm2/hr)
800
200
600
400
100
200
0 0
0 4 8 12 16 20 24
Time (hours)
Fig. 3.4 Exemplar data showing instantaneous flux of (2004). Figure copyright (2012) reprinted with permission
finite doses of loperamide and propylene glycol (PG) from Elsevier)
through human skin over time (Data are from Trottet etal.
finite dose formulation evenly across a mem- homogenous distribution (Kasting and Miller
brane invitro can be difficult. Since the 2006; Southwell and Barry 1984). The volatile
bioavailability of drugs applied to the skin in solvent evaporates, leaving a thin film of the drug
finite doses depends on the amount of drug on the skin surface. However, the volatile solvent
applied per unit area (Wester and Maibach 1976; may extract some lipids from the stratum cor-
Franz etal. 1993), a homogenous drug distribu- neum, affecting the integrity of the skin mem-
tion over the whole donor area is needed to mini- brane. Another method used for uniform dose
mise variation within and between experiments. distribution is by massaging the formulation onto
Applying semisolid formulations homoge- the skin surface, but this mechanical stress may
nously poses more complications than liquid dos- influence the absorption rate in some cases
ing. Semisolids are usually applied manually, (Amidouche etal. 1994; Lademann etal. 2007,
using a mechanical device such as a glass rod 2009). To date, a very limited number of publica-
(Hadgraft etal. 2003; Franz etal. 1993; Brain tions have investigated drug formulation distribu-
etal. 1995; Gupta etal. 1999) or spatula (Dreher tion over the incubation area (Lademann etal.
etal. 2002; Trottet etal. 2004; Youenang Piemi 2008); homogenous distribution is generally
etal. 1998) to help distribute the formulation assumed in many reports.
evenly on the skin surface. In this case, it is The degree of occlusion of diffusion cells dur-
important that the application device is also anal- ing experiments can affect skin hydration and,
ysed in order to determine the actual dose applied consequently, drug permeation (Akhter and
to the skin. This is commonly done by extraction Barry 1985; Zhai and Maibach 2001; Sarpotdar
(Gupta etal. 1999) of the remaining drug from and Zatz 1986). Some researchers recommend
the application device, or by weight difference that the skin be exposed to ambient conditions to
(Walters etal. 1997; Brain etal. 1995; Vallet etal. simulate the in-use conditions of topically applied
2007). Sometimes, the permeant is dissolved in a formulations (Wilkinson and Williams 2002;
volatile medium to allow easy application and Grgoire etal. 2009). However, evaporation of
3 Finite andInfinite Dosing 41
the vehicle and/or permeant can occur in non- should include permeant remaining in the donor
occluded experiments which can impact signifi- phase, that within the skin and all equipment in
cantly permeant absorption (Frasch etal. 2011). contact with the test substance (e.g. the donor and
Moreover, the skin may become dry, and its bar- receptor chambers of the diffusion cell). In some
rier function can thus be affected (Selzer etal. cases, the recommended total recovery may not
2013). Occlusion is also used during finite dosing be achieved, for example, due to the limits of
to avoid drying of the skin surface due to applica- analytical detection or difficulties with simple
tion of small amounts of formulation, or to pre- extraction methods. Degradation of the permeant
vent evaporation, which could lead to a change in could also aggravate the accuracy of total recov-
permeant concentration (van de Sandt etal. 2004; ery. For example, skin esterase has been shown to
Koyama etal. 1994). However, care is required to degrade permeants in situ (Lau etal. 2010, 2012).
avoid damage to skin integrity caused by exces- Chemical degradation could also have a signifi-
sive stratum corneum hydration (Zhai and cant impact on permeant stability (Kubota etal.
Maibach 2002). 1995; Mller etal. 2003). The degradation prod-
For finite dose studies, exposure times usually ucts are likely to have lower permeation rates
reflect in-use scenarios to allow quantification than the test compound. Thus, failure to detect
of permeant absorption over a realistic period of degradation products using suitable analytical
time that relates to potential human exposure methods may confound experimental results.
(OECD 2004a). Exposure times of up to 48h are
commonly used (Hadgraft etal. 2003; Gupta
etal. 1999; Walters etal. 1998), but may be 3.2.2 Penetration Kinetics
shorter to mimic exposure to rinse-off products
(Brain etal. 1995, 2005; Okuda etal. 2011), or in 3.2.2.1 Infinite-Dose Penetration
studies on occupational exposure to hazardous Although skin permeation data allow the trans-
materials (Howes etal. 1996; Moody etal. 2007). port parameters across skin to be determined,
In such cases, data collection commonly contin- constructing a drug concentration/depth profile
ues for at least 24h. On the other hand, if the can be valuable. The tape-stripping technique is
drug formulation is for a once-a-week product, most commonly used to assess the change in per-
for example, the experiment design should mimic meant concentration throughout the stratum cor-
the application exposure period and thus should neum (Lau etal. 2010; Thomas and Heard 2007;
be performed over 7 days (Williams 2003). Weerheim and Ponec 2001; Pellett etal. 1997;
Due to the limited amount of test substance Mohammed etal. 2012; Stinchcomb etal. 1999;
used in finite dose studies, it is important that a Dreher etal. 1998). This is a method, whereby
mass balance is performed on completion of the the stratum corneum is progressively removed by
experiment to ensure the total amount of applied adhesive tape and the drug within each tape is
permeant could be recovered. The OECD guide- then extracted and determined to calculate the
lines state that the mean recovery of the permeant diffusivity and solubility of the drug within the
and metabolites should be in the range of stratum corneum.
10010%, and reason must be sought if this is Under infinite dosing, it is possible to predict
not met (OECD 2004a, b). However, under cer- the concentration of the penetrant (c(x, t)) as a
tain circumstances, the test of a volatile substance function of position and time within the stratum
that had to be trapped in a filter, recovery can be corneum, using the appropriate solution of Ficks
broadened to 10020% (OECD 2004a; second law of diffusion (Anissimov etal. 2012;
European Commission 2006). The mass balance Moser etal. 2001; Selzer etal. 2013):
x 2 1 np x - Dn 2p 2 t (3.11)
c( x , t ) = KC0 1 - - sin exp
h p n =1 n h h2
42 W.M. Lau and K.W. Ng
In Eq.3.11, K is the partition coefficient between diffusion. In addition, the formulation should not
the stratum corneum and the formulation vehicle, alter the membrane integrity or act as a carrier for
x is the vertical position within the stratum cor- the test permeant. It is also assumed that the
neum (where 0xh), t is the time at which the experiment is maintained under sink conditions,
permeant concentration is to be determined and and the stratum corneum is the rate-limiting
D/h2 gives the characteristic diffusion parameter. barrier.
If the concentration of the permeant in the stra-
tum corneum is obtained experimentally, the 3.2.2.2 Finite-Dose Penetration
experimental concentration profiles can be fitted Similarly, with finite dosing, it is possible to
into Eq.3.11 to determine K and D/h2 (Pirot etal. predict permeant concentration at a given posi-
1997). Again, it is assumed that the skin is a tion inside the stratum corneum and at a given
homogenous membrane and that the permeant time, using Eq.3.12 (Kasting 2001; Selzer
transports across the stratum corneum by passive etal. 2013):
b cos (a n x / h ) a n sin (a n x / h ) a n 2 Dt
c( x ,t ) = 2 KCv 0 exp 2 (3.12)
n =1 b + b 2 + a n2 h
where Cv0 is the initial concentration of the per- attenuation of tretinoin induced skin irritation by 2-
meant in the donor compartment, =K(h/hv), hv cyclodextrin complexation. Int JPharm 111:111116
Anissimov YG, Jepps OG, Dancik Y, Roberts MS (2012)
being the theoretical height of the volume of Mathematical and pharmacokinetic modelling of epi-
donor formulation and n is related to such that dermal and dermal transport processes. Adv Drug
ntan n=. However, very few studies have Deliv Rev 65(2):169190
made use of this mathematical model. Barry B (1983) Dermatological formulations: percutane-
ous absorption. Marcel Dekker, NewYork
Boonen J, Malysheva SV, Taevernier L, Diana di Mavungu
Conclusion J, De Saeger S, De Spiegeleer B (2012) Human skin
Finite and infinite dose regimens have differ- penetration of selected model mycotoxins. Toxicology
ent applications in transdermal delivery. Each 301:2132
Brain KR, Walters KA, James VJ, Dressler WE, Howes
approach presents its own advantages and D, Kelling CK, Moloney SJ, Gettings SD (1995)
challenges. Mathematical models allow us to Percutaneous penetration of dimethylnitrosamine
predict, characterise and compare the skin through human skin invitro: application from cos-
absorption kinetics relating to finite or infinite metic vehicles. Food Chem Toxicol 33:315322
Brain KR, Walters KA, Green DM, Brain S, Loretz LJ,
dosing. In this respect, they are an invaluable Sharma RK, Dressler WE (2005) Percutaneous pene-
tool for transdermal delivery. However, in tration of diethanolamine through human skin invitro:
applying these models, it is important to application from cosmetic vehicles. Food Chem
appreciate the underlying assumptions and Toxicol 43:681690
Chen M, Liu X, Fahr A (2011) Skin penetration and deposi-
limitations of the models. tion of carboxyfluorescein and temoporfin from differ-
ent lipid vesicular systems: invitro study with finite and
infinite dosage application. Int JPharm 408:223234
Crank J(1975) The mathematics of diffusion. Oxford
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Effect of finite doses of propylene glycol on
Non-formulation Parameters
That Affect Penetrant-Skin-Vehicle 4
Interactions andPercutaneous
Absorption
JeffreyE.Grice, HamidR.Moghimi,
ElizabethRyan, QianZhang, IshaHaridass,
YousufMohammed, andMichaelS.Roberts
Surface: Hair:
Insect repellents Depilatory agents
Sunscreens Conditioners
Antimicrobial agents Colors Stratum corneum:
Moisturizers
Keratolytics
Sebaceous gland:
Anti acne products
Dermis
sweet gland
Dermis:
Anti-inflammatory agents
Antihistamines
Anaesthetics
Sweat glands:
Antiperspirants
Hypodermis
Systemic delivery:
Nerves Nitro glycerin
Blood vessels
Lymph vessels Nicotine
Fig. 4.1 Schematic illustration of skin (showing epidermis, dermis, hypodermis and skin appendages) and underlying
muscles and joints, showing targets of drugs and cosmetics applied to skin for local, systemic or regional action
epithelial permeability in biological systems A limitation of Eq.4.3 is that it really only reflects
where low solute concentrations are used. This the effect of diffusivity on skin transport, noting
approach defines transport in terms of a perme- the diffusivity is a function of solute size, as
ability coefficient; fluxvehicle concentration defined by free volume and other theories
(Kuswahyuning and Roberts 2014) and is lim- (Kasting etal. 1987). It does not reflect the con-
ited by the variability in permeability coeffi- tribution of solubility of solutes to the flux. The
cient between vehicles, in accordance with the theoretical importance of solubility is evident
variation in solute solubility in the different when it is assumed that the SC is the main barrier
vehicles (Roberts 2013). Care should be applied for solute penetration and when the saturated sol-
when using such relationships, as most vehicles ute flux in expressed terms of its underlying
do in fact affect skin permeability, they may determinants for skin penetration: diffusivity in
affect dermal blood flow, and solutes could be the SC(Dsc), the path length for transport across
in solution in a supersaturated form, i.e. above the SC(hsc) and the solubility in the SC(Ssc).
the usual solubility of the solute in the vehicle Hence, incorporating these into Eq.4.1 and
and therefore be associated with higher fluxes. expanding by recognising that the SC-water par-
The use of saturated or maximum flux has the tition coefficient (Ksc,v) is given by (Ssc/Sv) yields:
advantage of directly showing the maximum
dose deliverable over a period of time
(Magnusson etal. 2004; Sloan etal. 2006; Q D
J sskin
, sat = = sc Ssc
Milewski and Stinchcomb 2012). Further, if A T hsc (4.5)
neither the formulation nor the solute affects D
the membrane, it should be an invariant in the = sc K sc , v Sv = k pskin Sv
hsc
thermodynamic description of the penetration
process, whereas the permeability coefficient
depends on the formulation used (Zhang etal. Thus, it is evident from Eq.4.5 that the solubility
2009; Scheuplein 2013). However, a changed of the solute in the SC, Ssc, can be expressed in
maximum flux after formulation application terms of a solubility of the solute in the vehicle
may suggest an alteration of the skin bioproper- and a partition coefficient Ksc,v between the SC
ties by the components of the vehicle, either and vehicle (i.e. Ssc = K sc , v Sv ) and, in turn, the
during solute partitioning or the diffusion permeability coefficient is the product of this par-
process. tition coefficient multiplied by Dsc/hsc (i.e.
Relevant to the first approach, we showed that k p = K sc , v Dsc / hsc ). We have previously applied
the main determinant for maximal (or more accu-
Ssc = K sc , v Sv and the relationships between
rately, saturated) solute flux from aqueous vehi-
logKsc and log P (Roberts etal. 1996), the depen-
cles is the solute size (molecular weight or
dence of solubility of the solutes in water on the
molecular volume) with an r2 of 0.688 (n=278,
octanol-water partition coefficient and the solute
p<0.001) (Magnusson etal. 2004):
melting point above 25C (Yalkowsky and
log J s ,sat = log J max = - 4.52 - 0.0141MW (4.4) Valvani 1980) to yield (Roberts etal. 2002):
Thus, the overall saturated flux is given by a solubility of different solutes. The most recent
combination of Eqs.4.3 and 4.4. A difficulty in relationship, recognising most of the previous
this area is the accuracy of the predictions for studies, suggests the slope for the linear relation-
both SC-water partition coefficients and in water ship for a large data set of lipophilic solutes is
4 Non-formulation Parameters Affecting Skin Permeation 51
Fig. 4.3 Relationships between maximum flux (Jmax, tions to excised human epidermal membranes. (a) log
estimated from a 10% solution), stratum corneum solu- Jmax vs log P; (b) log Ssc vs log P; (c) linear relationship
bility (Ssc) and log P (experimentally determined) for a between log Jmax and log Ssc (Data adapted from (Zhang
series of similar-sized phenols applied in aqueous solu- etal. 2009). The curves are for illustrative purposes only
of these solutes by increasing their solubility in Whilst this model probably is currently limited to
the intercellular lipids of the SC, but again aqueous solutions, it may be applied to other
showing that diffusivity was not dependent on the vehicles if the concentration of the solute in the
lipophilicity of the solute. vehicle can be expressed as a fractional solubility
The second approach is to examine skin perme- in a simple or complex vehicle, Raoults Law is
ation in terms of the skin permeability coefficient approximately obeyed and the vehicle has either
(kp), which is defined by two main parameters: dif- not affected the skin or its extent of skin perme-
fusion and partition coefficients (Eq.4.5). ation enhancement is known. We have pointed
Traditionally, these have been represented by a out that when the fractional solubilities for the
combination of organic water partition coeffi- vehicle f v ( = Cv / Sv ) and skin f s ( = Cs / S s ) can
cients (presented by log P) and size (represented be assumed to be equal, the skin flux is given by
by molecular weight MW or molecular volume Eq. 4.1, i.e. J s = J s ,sat f v (Roberts 2013).
MV) (Lien etal. 1971). The most comprehensive Despite much effort in developing quantitative
analysis of kp in terms of solute size and lipophilic- structureactivity relationship (QSAR) models,
ity was presented by Potts and Guy (Potts and Guy the predictability and quality of these models for
1992), as shown in Eq4.7: risk assessment purposes can be further improved.
A key issue is the large set of historical experi-
log k p = - 6.3 + 0.71 log P
mental data, in which variability is likely to have
- 0.0061 MW
arisen from the use of different experimental
( n = 93; r 2 = 0.67 )
(4.7) approaches and human skin donors. In an attempt
to cover as many formulations and conditions as
Many others have since attempted to improve possible, Samaras etal. (Samaras etal. 2012)
estimation of kp (Lian etal. 2008; Chen etal. examined the invitro skin fluxes from the
2010; Moss etal. 2012). In a recent work, EDETOX database in their regression analyses in
Milewski and Stinchcomb (Milewski and terms of not only the molecular descriptors of the
Stinchcomb 2012) have arrived at a predictive penetrants and the vehicle, but also the range of
model for saturated skin fluxes by combining the various experimental and vehicle conditions
Potts Guy (Eq.4.7) with that from the Yalkowsky used including finite/infinite dosing, skin
group (Yalkowsky and Valvani 1980) for water pre-hydration, occlusion and the skin thickness.
solubility to yield Eq.4.8 for estimation of Js,sat They concluded that the donor concentration,
from log P, MW and MP (n=208, r2=0.81). solute lipophilicity, solute size and solute polar-
log J s ,sat = 4.6 - 0.219 log P - 0.0086 MW - 0.0102 ( MP - 25 ) ( n = 208, r 2 = 0.81) (4.8)
4 Non-formulation Parameters Affecting Skin Permeation 53
ity, as well as the vehicle melting and boiling The pilosebaceous unit comprises four differ-
points were prominent factors influencing skin ent areas for topical application: the sebaceous
flux. gland, the bulb region, the hair matrix cells and
In reality, the SC is a heterogeneous mem- the hair follicle infundibulum. Sebum excreted
brane consisting of several layers of corneocytes by the sebaceous gland is composed of short-
with the space filled with lipids. Some hydro- chain fatty acids, resulting in a microenviron-
philic and polar solutes penetrate through the ment that is rich is neutral, non-polar lipids
SC faster than would be anticipated based on (Meidan etal. 2005). The lower infundibulum
lipophilicity alone (Nitsche etal. 2006; Wang provides decreased barrier properties and is sur-
etal. 2007; Mitragotri etal. 2011). Consequently, rounded by a dense network of blood capillaries.
the porous pathway model was introduced to The infundibulum increases the possible surface
address this challenge by describing skin per- area for targeted delivery and is an area of addi-
meability to hydrophilic solutes (Peck etal. tional absorption. The immediate region sur-
1994; Lai and Roberts 1999; Tang etal. 2001; rounding the infundibulum is rich in
Tezel etal. 2002; Mitragotri 2003). In this antigen-presenting cells, whereas the lower bulge
model, hydrophilic solutes are assumed to dif- region is surrounded by stem cells (Patzelt and
fuse through pores in the skin, where pore size Lademann 2013), providing target sites for pen-
distribution, skin porosity and tortuosity are etrated molecules (Knorr etal. 2009).
considered. The brick-and-mortar model has Work by a number of groups has found that
also been adapted and expressed in different the follicular route predominates for hydrophilic
forms (Wang etal. 2006, 2007; Chen etal. 2010, molecules (Essa etal. 2002; Ogiso etal. 2002;
2013). This mathematic model aims to include Mitragotri 2003) that cannot permeate the epider-
permeation through the corneocytes (i.e. trans- mal pathway easily. A study by Frum etal. (2007)
cellular pathway) and intercellular lipids (inter- quantified the influence of the drug lipophilicity
cellular pathway), where the geometrical and on follicular penetration by using a range of
compositional parameters of the SC have been drugs with similar molecular weights. A para-
included. bolic profile shows a maximal follicular contribu-
tion occurs at log P around 1. The two most
lipophilic drugs, estradiol and corticosterone, had
4.5 Physicochemical Properties minimal entry into the follicular pores, whereas a
That Favour Appendageal 34% contribution to total skin flux was found for
Permeation hydrophilic adenosine (log P=1.05). For man-
nitol, which is also hydrophilic (log P=2.47)
As an alternative to the stratum corneum inter- there was a 54% follicular contribution associ-
and intracellular permeation routes, the bulk ated with its flux through human skin, though its
pathway or shunt pathway by appendages molecular size is slightly smaller than the above
such as hair follicles also acts as a permeation penetrants (Essa etal. 2002).
route through the skin (Fig.4.1). This pathway The invivo follicular penetration of caffeine
has previously been considered as insignificant was also systemically examined using pharmaco-
as appendageal structures only make up 0.11% kinetic modelling (Liu etal. 2011). There was a
(forearm to forehead) of the total skin surface 10 times faster absorption from hair follicles, and
area (Otberg etal. 2004). However, in recent a 10-min delay for transport through the stratum
years, hair follicles and their associated piloseba- corneum, with the follicular route contributing
ceous structures acting as a permeation pathway 2535% to the overall skin absorption. The
and/or reservoirs have been increasingly explored invivo absorption of caffeine was fast and high
and received more attention, especially for compared to the invitro absorption, and this was
nanoparticle delivery (Lademann etal. 2008; considered to be mainly due to a fast resorption
Patzelt and Lademann 2013). by invivo blood flow, the physiological differ-
54 J.E. Grice et al.
Fig. 4.4 Above: Penetration depths in m of different sizes Below: The hair surface structure, showing the layers of
of PLGA particles in relation to the target sites within ter- overlapping cells (cuticula pili) (Reproduced with permis-
minal hair follicles (THF) and vellus hair follicles (VHF). sion from Patzelt etal. 2011 and Lademann etal. 2011)
ence in hair follicles of the investigated skin and without massage, whereas, massage drives the
a contraction of hair follicles of the excised skin. particles but not the dye deep into the follicle
Besides polar molecules, the transfollicular (Lademann etal. 2007).
pathway is also an important route for nanopar- Particles, which may be solid or vesicular,
ticles, which may be targeted to deep follicular with therapeutic substances bound to the surface
regions and function as drug delivery vehicles or encapsulated, may be ideal follicular delivery
(Lademann etal. 2007). Topically applied poly- agents, because of their long residence time
meric particles were shown to penetrate porcine within the follicle and their targetable penetration
skin with massage via the follicular pathway in a depth (Lademann etal. 2008). In addition, Vogt
size-dependent manner (Patzelt etal. 2011), with etal. found that antigen-presenting cells sur-
643-nm particles penetrating deepest into the fol- rounding the infundibulum are able to internalise
licle, to a maximum depth of approximately 40-nm particles that have permeated through the
1100m (see Fig.4.4). follicular epithelium (Vogt etal. 2006), thus pro-
Lademann has suggested that the optimal size viding a promising mechanism for vaccination
of particles for deep penetration is governed by via the follicular route.
the size of the cuticula on the hair shafts Targeted follicular delivery can be achieved
(Lademann etal. 2011) (see Fig.4.4), which may by manipulating the formulation (i.e. particle-/
act as geared pumps to drive the particles into vesicle-based dosage forms and using sebum
the follicles during massage (Lademann etal. miscible ingredients) or modifying the target
2009). The effect of massage is demonstrated in molecule (optimising physicochemical proper-
Fig.4.5, where both particles and dye molecules ties such as size, lipophilicity, solubility parame-
remain in the superficial regions of the follicle ters and charge) (Trommer and Neubert 2006).
4 Non-formulation Parameters Affecting Skin Permeation 55
a b
c d
Fig. 4.5 Superposition of transmission and fluorescent and (b) dye in non-particle form with massage. (c)
images, demonstrating the invitro penetration of the dye- Particle form and (d) non-particle form without massage
containing formulation into the hair follicles of porcine (Reproduced with permission from Lademann etal. 2007)
skin after application of massage. (a) Dye in particle form
56 J.E. Grice et al.
An example reported by Grice etal. (2010) shows They observed that, in general, the concentration
that formulations that contained ethanol could gradients for highly protein bound drugs for
enhance the early minoxidil uptake into the hair deeper tissue penetration tended to be shallow,
follicles. As minoxidil is six times more soluble whereas the poorly bound drugs showed an expo-
in ethanol as it is in water, this was likely due to nential concentration gradient. They applied a
the partitioning of ethanol (plus minoxidil) physiological pharmacokinetic analysis to this
through the lipid rich compartments. Some other data and concluded that highly plasma protein
methods like fractional laser and massage could bound drugs, in addition to normal diffusive
also selectively enhance permeant targeting to transport, may be transported in deeper tissues
follicles (Lademann etal. 2007; Lee etal. 2013). via the blood and lymphatic circulation as well as
The importance and required molecular and via interstitial convection (Fig.4.6) and that these
particular properties for an optimum follicular transport routes may dominate in some cases.
delivery are yet to be fully investigated. Such vascular transport can increase deep tissue
concentrations beyond those resulting from
extravascular diffusion alone, and decrease the
4.6 Physicochemical Properties drugs lag time into deep dermis, subcutaneous
That Favour Direct and muscle tissue.
Subcutaneous andDeeper
Tissue Permeation
4.7 Skin Hydration
There are now a large number of studies showing andPercutaneous
that deep tissue penetration can occur after topi- Absorption
cal application to specific areas of the body.
Various human biopsy (Rabinowitz etal. 1982; Transdermal delivery can also be controlled by
Grundmann-Kollmann etal. 2002; Anissimov the extent of skin hydration, which mainly refers
and Roberts 2011) or cutaneous microdialysis to the extent of hydration in the SC.A major
(Cross etal. 1998; Benfeldt etal. 1999; Brunner controller of hydration is natural moisturising
etal. 2005) studies have been used to show direct factor (NMF). The original bricks and mortar
and deep tissue penetration of solutes after topi- description of the SC has been refined substan-
cal application. tially and it is now described as being a continu-
Rat wound deposition studies have been used to ous poly-proteinaceous structure, with the bricks
show that the main determinant for the penetration being likened to NMF-containing keratin
of solutes into deeper tissues after topical applica- sponges. NMF functions as a plasticiser of the
tion is the size of the solute (Singh and Roberts stratum corneum under basal conditions (Laden
1996; Cross and Roberts 1999; Kretsos etal. 1967; Jokura etal. 1995). Following the addition
2008). The cutaneous vascular appears to be impli- of water or under conditions of high humidity,
cated in the penetration of solutes in studies con- the NMF-bound water assists in the swelling of
ducted in rats, pigs and man (McNeill etal. 1992; the corneocytes. Swelling is at its greatest in
Monteiro-Riviere etal. 1993; Cross etal. 1999). regions where the NMF concentration is at its
Dancik etal. (2012) combined experimental highest (Bouwstra etal. 2003). It has been shown
percutaneous permeation studies with the avail- that when the skin is highly hydrated, water is
able microdialysis literature on the penetration of predominantly located either in the intercellular
topically compounds into subcutaneous tissue regions in separate domains or trapped in cor-
and muscle. The solutes studied were of similar neocytes (Roberts etal. 2008). In addition, swol-
size and included diclofenac, ibuprofen, flucon- len corneocytes and separation of lipid bilayers
azole, lidocaine, nicotine, propranolol, ethanol, in the intercellular space to form cisternae and
5-fluorouracil, methyl salicylate, salicylic acid, networks of spherical particulates are found in
salicylic compounds and trolamine salicylate. porcine skin after a 410-h hydration period
4 Non-formulation Parameters Affecting Skin Permeation 57
Fig. 4.6Physiological
pharmacokinetic model for
topical drug transport
processes in deeper skin.
(a) Blood vessels,
lymphatic vessels and
interstitial convection in
dermis. (b) Schematic of
physiological
pharmacokinetic model
used to analyse human
cutaneous microdialysis
data at two depths, the
concentrationtime profile
in the superficial
microdialysis probe being
used as an input to predict
the deeper microdialysis
probe concentrationtime
profile (Reproduced with
permission from (Dancik
etal. 2012))
(Tan etal. 2010). Figure4.7 shows high magnifi- known, just as disturbed skin barrier function has
cation cryo- scanning electron microscopy been correlated with low SC hydration (Proksch
images of human SC in various hydration states, etal. 2008).
showing clearly the swollen corneocytes and In the presence of an intact barrier, increasing
water pools present at high levels of hydration the water content of the skin can have substantial
(Bouwstra etal. 2003). effects on the transdermal delivery of penetrants
The hydration state of the stratum corneum is by altering skin physiology. In clinical practice,
mainly influenced by environmental humidity, occlusion of the skin, by which the hydration of
temperature, transepidermal water loss (TEWL) the SC can be greatly increased, is widely used to
rate, keratinisation rate and the type of moisturis- enhance the penetration of topically applied
ers (Vergnanini etal. 2010). The inverse relation- drugs (Hafeez and Maibach 2013). A number of
ship between TEWL and SC hydration is well factors could contribute to the increased permea-
58 J.E. Grice et al.
a b
c d
Fig. 4.7High magnification cryo-scanning electron black asterisks) indicating the presence of water. In the
microscopy images of SC hydrated to various levels. upper and lower part, the appearance of the SC is similar to
Arrows indicate undulations or interdigitations of cells. that of dry skin (white asterisks). (d) A high magnification
Arrowheads indicate the cell boundaries. (a) Dry SC char- of a fully hydrated SC sheet. The keratin network is clearly
acterised by low contrast images. The cells are approxi- depicted. This network is surrounded by the cornified enve-
mately 360nm in thickness. The spaces between the lope. Between the cells frequently large and very small
corneocytes are mostly air-filled, possibly caused by drying water pools are observed (see white arrows). If no water
of the SC required for low hydration levels. Arrows indicate pools are present a close cell to cell contact is observed. The
undulations or interdigitations of cells. Arrowheads indi- cell ends are round and fewer undulations are observed
cate the cell boundaries. T, tissue-freezing medium. (b) SC compared to dry SC or SC hydrated to approximately 20%
hydrated to 17% wt/wt reveals a low contrast image, simi- wt/wt. A difference in the degree of swelling between the
lar to that observed for dry skin. This indicates the absence various cells is noticed. The cells that are less swollen are
of water pools. (c) SC hydrated to 70% wt/wt reveals in the indicated by short arrows (Reproduced with permission
central part slightly swollen cells with a higher contrast (see from Bouwstra etal. 2003)
bility. Firstly, it is likely to be due to an increase water in the stratum corneum, also illustrated by
in the solubility of the permeant in the SC.This cryo- electron microscopy (Fig.4.7) could disor-
increases the partitioning of the solute from the der the structures of lipids and proteins
vehicle into the membrane. Another possibility is (Vyumvuhore etal. 2013), thereby altering the
that the increased hydration could lead to swell- permeation rate of topically applied solutes.
ing of the structure and rearrangement of the lip- Changes in skin hydration may affect skin per-
ids. Recent work using Raman spectroscopy meation by several orders of magnitude as previ-
demonstrated that increased amounts of unbound ously described (Roberts 1991; Roberts and
4 Non-formulation Parameters Affecting Skin Permeation 59
% Dose absorbed
rin, fusidic acid, methylethylketone, various cor- 40
ticosteroids, etc. (Roberts and Walker 1993).
30
Interestingly, current findings suggest that
there are no quantitatively defined relationships 20
penetration enhancer (Wohlrab 1984, 1990; found to be higher in infants, as were the pH
Beastall etal. 1986). Glycerin based creams, very readings. No TEWL, capacitance or pH varia-
familiar to most consumers, also have humectant tions were observed in infants according to sex or
properties. Humectants with a small molecular age.
size (e.g. urea and glycerin) are assumed to pen- As the skin ages, certain structural changes
etrate into the stratum corneum, mimicking the occur, so that the aged SC is considerably drier
role of natural moisturising factors to retain than that of a young adult, it has a reduced lipid
moisture (Caussin etal. 2007). However, the content and frequently has a less extensive micro-
mode of action is still poorly understood (Loden circulation. The epidermis becomes thinner and
2008). the keratinocytes become less adjacent to one
another. The dermis also becomes relatively atro-
phic, acellular and avascular. Collagen, elastin
4.8 ge-Related Skin Barrier
A and glycosaminoglycan are also altered. Exactly
Performance how the penetration barrier of the skin changes
with age remains unclear; however, it should be
Epidermal development, including SC structure, assumed that aged skin provides a more variable
is complete in utero by 34 weeks. Pre-term babies environment for the diffusion of topically applied
have elevated levels of both TEWL and transcu- drugs, as discussed below (Byl 1995).
taneous heat transfer, which as expected, are Waller and Maibach reviewed the literature on
largely dependent on how premature the baby is. the effect of aging on blood flow, pH, thickness
Ultra-low birth weight infants from the ages of and ultrasound echogenicity (Waller and Maibach
2325 weeks were shown to need at least 4 weeks 2005). They concluded that the data which
to develop a fully functional SC, whereas babies described age-related changes in skin structure
of 3032 weeks had barrier functions comparable and function were often conflicting and difficult
to adults (Kalia etal. 1998; Fluhr etal. 2004). to interpret. The cutaneous blood flow results
It has been widely reported that premature suggested that there may be a trend towards
infant skin is more susceptible to penetration than decreased cutaneous perfusion in older individu-
fully formed infant skin. A study by Anderson als, especially in photo-aged areas. The skin pH
etal. showed that when hexachlorophene was data were more convincing, as it appears that skin
used as a disinfectant on the skin of pre-term pH is constant throughout adulthood to approxi-
infants, dermal absorption occurred which mately 70 years of age. After 70, the pH increased,
resulted in myelinopathy in the premature infants which was suggested to be due to stasis of blood,
(Anderson etal. 1975). Another study by Barker especially in the lower limbs. The change in
etal. examined the permeation of sodium salicy- thickness of the skin layers seemed to have been
late through exvivo newborn infant skin. It was influenced more by site on the body than by age.
found that absorption of the compound was 100 The epidermis thickens with age on certain sites
1000 times higher in infants who had a gestation such as the volar and dorsal upper arm, but
period of 30 weeks or less, compared to full term remains constant in areas such as the buttock,
infants (Barker etal. 1987). dorsal forearm and shoulder. Similar variations
Giusti etal. measured the TEWL, capacitance were noted for dermal thickness.
and pH of the volar forearm and buttocks of 70 TEWL has been used as an indication of the
infants aged 824 months and compared these to effect of aging on barrier function. Hillebrand
values in adult skin (Giusti etal. 2001). As and Wickett noted considerable variability in
expected, no differences in TEWL were found TEWL with age in Chinese female women living
between the infants and adults at either site, con- in Beijing, with a quadratic regression showing
firming that the infant skin had a fully developed little change between 10 and 70 years (Hillebrand
barrier function. Capacitance values, which and Wickett 2008). They point out that an alter-
quantify the amount of water in the SC, were native explanation for these findings is that the
4 Non-formulation Parameters Affecting Skin Permeation 61
performance in elderly; the effects of which, Maibach 1967), pesticides (Maibach etal. 1971)
however, depend on the physicochemical proper- and other chemicals (Rougier etal. 1987) have
ties of the permeant. However, there are also a been reported, with scrotal, forehead and wrist
number of confounding environmental and skin generally more permeable than skin on the
genetic factors at play and these may dominate trunk and periphery. Rougier etal. showed a good
over any underlying apparent relationship correlation between benzoic acid permeation
between skin permeation and aging. A combina- (measured as 14C-benzoic acid excretion and
tion of factors, including reduced lipid and water TEWL) across seven different body sites in
content of the SC, may be responsible for the per- humans (Rougier etal. 1988). This work also
meation behaviour of certain types of molecules showed the forehead to be the site of greatest per-
through aging skin. meability for water loss (scrotal skin was not
examined).
In addition to mapping, attempts have been
4.9 ite-Related Skin Barrier
S made to relate the regional variations in skin
Performance permeability to skin properties. Early work by
Plewig and Marples showed significant
Site-related or regional variations in SC structure regional differences in corneocyte dimensions,
are well recognised and may be reflected in dif- with the smallest cells found in the forehead
ferences in permeability of the skin (Feldmann (Plewig and Marples 1970). The authors sug-
and Maibach 1967; Scheuplein and Blank 1971). gested that these cellular variations could
A key determinant for differences between sites explain previous observations of regional dif-
is SC thickness, which can vary from 400 to ferences in TEWL (Baker and Kligman 1967)
600 m for the plantar and palmar callus to and hydrocortisone (Feldmann and Maibach
1020m for the back, arms, legs, and abdomen 1967). Recent work from Hadgrafts laboratory
(Rushmer etal. 1966). The differences in thick- (Hadgraft and Lane 2009; Machado etal.
ness are manifested in delayed responses to 2010a) has suggested a mechanism, in which
materials applied to plantar and palmar skin, smaller corneocytes can pack more closely
compared to other sites. As seen in data for water together, resulting in a shorter intercellular
permeation quoted by Scheuplein and Blank, the path length for permeating molecules to tra-
greater thickness of plantar skin leads to longer verse the layers of corneocytes. Machado etal.
lag times, despite its having greater permeability have calculated the intercellular path length
to water than abdominal skin. The increased lag from the corneocyte surface area and the num-
time effectively masks the increased permeability ber of cell layers at six body sites, and shown
(Scheuplein and Blank 1971). Indeed, it has been an excellent correlation between the reciprocal
suggested that plantar and palmar SC should be of path length and TEWL (Machado etal.
considered separately from the thinner horny lay- 2010a). As noted previously, the forehead had
ers at other sites (Kligman 1964; Scheuplein and the smallest corneocyte surface area, the small-
Blank 1971). Results from the Roberts group est number of cell layers and the greatest
showing 48-h cumulative urinary salicylate TEWL.Earlier work by Rougier etal. (1988)
excretion following topical application of methyl examined the effect of corneocyte size on ben-
salicylate on five separate body sites for 10h zoic acid permeation as well as TEWL in
(Fig.4.10) clearly show the effect of increased humans and concluded that regional differ-
SC thickness (Roberts etal. 1982). ences in permeation could not be entirely
Various studies mapping the regional varia- explained by corneocyte size alone and that
tions in TEWL, hydration and other skin param- other factors were important, especially for
eters skin (Marrakchi and Maibach 2007; Kleesz corneocytes <600m2 or >1000m2. A re-anal-
etal. 2012; Luebberding etal. 2013) and the per- ysis of Rougier etal.s data for benzoic acid
meability of hydrocortisone (Feldmann and permeation supports this conclusion. If 1/path
4 Non-formulation Parameters Affecting Skin Permeation 63
100
50
0
plantar heel instep forearm abdomen
length is calculated and plotted against TEWL Luebberding etal. 2013) impacts on the perme-
and permeation results for four body sites, the ation of molecules with different physicochemi-
correlation for TEWL (R2=0.98) is very strong, cal properties, although decreasing or artificially
while that for benzoic acid permeation is increasing the level of sebum on the skin surface
weaker (R2=0.75). has no effect on transepidermal water loss
Noting that the forehead contains a high con- (Kligman 1963).
centration of sebaceous glands, Maibach Based on their results from permeation of
explained the relatively high hydrocortisone water and salicylic acid across abdominal and leg
permeation at this site by suggesting that a sub- skin, Elias etal. have suggested that the regional
stantial amount of permeation was probably variations in skin permeability arise mainly from
occurring through the follicles rather than the differences in the lipid content in the epidermal
SC (Feldmann and Maibach 1967; Rougier etal. layers (Elias and Brown 1978). The permeation
1987). This may be considered unlikely, since it of each solute was found to correlate inversely
had been accepted that hair follicles made up with the percentage lipid content in each piece of
less that 0.1% of the surface area of the skin skin but did not relate to the number of cell layers
(Schaefer and Redelmeier 1996). However, the or the SC thickness. Given the relatively low per-
Lademann group has identified significant meation of chemicals at these two sites compared
regional variations in follicle size and distribu- to the forehead (Wester etal. 1984), their similar-
tion in seven body sites (Otberg etal. 2004). The ity in SC thickness (Ya-Xian etal. 1999) and the
volume and surface area of the follicular infun- likelihood of a similar contribution by follicles
dibula per square centimetre of skin was great- (Otberg etal. 2004) Elias and Brown may have
est in the forehead and least in the forearm, with been able to reveal a dependence on SC lipids in
the overall pattern of follicular distribution the absence of other strong contributing
being similar to that seen in variations in TEWL differences.
(Rougier etal. 1988) and chemical permeation Laser Doppler techniques allow the mea-
(Feldmann and Maibach 1967; Maibach etal. surement of blood flow to surface tissues.
1971; Scheuplein and Blank 1971; Rougier Johnson etal. found regional differences in
etal. 1987, 1988). These correlations indicate basal skin blood flow across the forearm
that the follicular route may contribute signifi- (Johnson etal. 1984), while in kinetic studies
cantly to chemical permeation and water trans- with topical application of the vasodilator ben-
port and that variations in follicular size and zyl nicotinate, variations in cutaneous blood
distribution may contribute to regional varia- flow, temperature and redness were seen across
tions in skin permeability. It is not clear whether the forehead, forearm and calf, both basally
the significantly greater sebum content on the and in response to the drug (Jacobi etal. 2006).
forehead skin surface (Rougier etal. 1987; The forehead had a higher blood flow and
64 J.E. Grice et al.
greater and more rapid responses to the vasodi- As well, the site of application should be specified
lator. These results suggest that differences in on the product if the site dependency in absorp-
cutaneous blood flow may also account for tion affects the treatment or safety.
regional differences in skin permeability
invivo.
Figure4.11 attempts to summarise the state of 4.10 G
ender-Related Skin Barrier
knowledge on regional variations in skin perme- Performance
ation and some of the factors that may contribute
to this. As far as the skin structure is concerned, studies
In conclusion, the influence of the site of appli- by Fluhr etal. in 33 pre-menopausal women, 21
cation on percutaneous permeation of topical for- post-menopausal women on no hormone replace-
mulations or any material which comes into ment and 25 male subjects found that the skin of
contact with the skin is substantial and depends pre-menopausal women contained significantly
on different variables as discussed above. This smaller corneocytes compared to the other two
implies that for optimised delivery, dermatologi- groups (Fluhr etal. 2004). The authors hypothe-
cal formulations should be designed in a way that sised that sex hormones may have an effect on
the formulation, and not the skin, controls the rate corneocyte area, since sex hormone levels are
of delivery if it is designed for different skin areas. normally elevated in pre-/ post-menopausal
Low High
Fig. 4.11 Regional differences in responses and proper- and the number of cell layers (Machado etal. 2010a); (d)
ties of human skin based on rank order. (a) TEWL cutaneous blood flow determined by Laser Doppler flow-
(Rougier etal. 2005); (b) follicle density, expressed as metry (Jacobi etal. 2006); (e) skin permeation of hydro-
infundibular surface area mm2 per cm2 of skin (Otberg cortisone (Feldman and Maibach 1967) and parathion
etal. 2004); (c) minimum intercellular path length through (Maibach etal. 1971), both measured as total urinary
the stratum corneum, based on corneocyte surface area excretion of the 14C-compound over five days
4 Non-formulation Parameters Affecting Skin Permeation 65
women compared to the other two groups. No change in barrier performance is, therefore,
differences in corneocyte size were observed by anticipated. This is important because the appli-
Machado etal. when male and female subjects cation of therapeutic agents is sometimes accom-
aged 2060 years were compared (Machado panied by the application of cosmetics. As a
etal. 2010a). result, impaired skin barrier properties that may
Prather etal. carried out a study in which the occur in cosmetically treated skin can increase
differences in permeation of nicotine from a the risk of dermatotoxicity. In this section, we
Nicoderm transdermal delivery system (Alza will examine the influence of certain actives
Corp.) based on gender was examined (Prather found in cosmetic products on skin barrier
etal. 1993). Following a 24-h application of the function.
transdermal nicotine system, the levels in nico-
tine plasma concentration (Cmax) and area under
the curve (AUC) did not vary significantly 4.11.1 Insect Repellents
between women and normal-sized men.
A recent review by Machado etal. supports Insect repellent creams, lotions and sprays are
the above observations, and suggests that skin widely used products, having been available to
barrier function does not seem to vary with gen- the public for more than 50 years. N,N-diethyl-
der (Machado etal. 2010b). Reed etal. also con- m-toluamide (DEET), a hydrophobic com-
cluded that there were no gender-related pound, is the most common ingredient of insect
differences in barrier integrity and barrier recov- repellents with more than 200 million people
ery and that any conflicting results may be due to using products containing various concentra-
changes in the skins barrier function related to tions of this active (10% or higher) worldwide
the menstrual cycle (Reed etal. 1995). (Debboun etal. 2006). The absorption of DEET
To conclude, the above-mentioned studies into the circulatory system and its relative toxic-
might reveal that among different biological fac- ity are topics that have been of interest. However,
tors, gender seems to have lowest effect on percu- it is equally interesting to note that DEET is also
taneous absorption of drugs. Accordingly, as a considered to be a permeation enhancer, shown
rule of thumb, and until proven otherwise, all to improve the dermal and transdermal delivery
laboratories use mixtures of male and female of a variety of drugs through hairless mouse
skin samples for their investigations indiscrimi- skin (Windheuser etal. 1982).
nately and there is no gender-specified product in Kondo etal. (1988) reported no increase of
the market. absorption of nifedipine in the presence of DEET
while Ogiso etal. (2002) observed a slight increase
in indomethacin flux. However, it has been
4.11 Barrier Performance reported that mixtures of DEET and DES (diethyl
inCosmetically Treated Skin sebacate, a lipophilic vehicle with enhancer prop-
erties used in medications and cosmetics such as
Application of products on the skin for cosmetic shaving lotions) resulted in a remarkable increase
purposes is widespread. Some products, such as in nifedipine penetration (Kondo etal. 1988). This
moisturising creams, insect repellents and sun- finding might indicate that DEET is not an effec-
screen creams are used by everyone irrespective tive permeation enhancer when employed alone. It
of age and gender, while depilatory creams, would be interesting to discover if the application
deodorants, antiperspirants, and anti-aging prod- of a cosmetic cream containing DES before or
ucts are more age and gender specific. Cosmetic after the application of insect repellent would
products contain ingredients that can alter both increase the penetration of the actives contained in
the structure and function of the skin and a the aforementioned cream.
66 J.E. Grice et al.
4.11.2 Chemical Depilatories tural changes in the SC were observed and the defi-
nition of cell borders was disrupted, presenting a
A plethora of products and services is available homogenised pattern (Lee etal. 2008). Secondly,
on the market for hair removal, ranging from the volume of intercellular spaces and gaps
high-tech laser removal to the more conventional increased (Shiozuka etal. 2010). Consequently, the
systems, such as shaving and depilatory formula- barrier function of the SC is compromised and
tions, that are the subject of the present review. resistance to transdermal delivery is greatly
The two main components of these formulations reduced. However, no studies have been conducted
are calcium thioglycolate and potassium thiogly- to date exploring the effects of chronic application
colate. Keratin filaments contained in hair strands of calcium or potassium thioglycolate. Therefore,
are strengthened by intercellular disulphide one would not be able to conclude if continued use
bridges formed between two cysteine residues. of depilatory creams induces any long-term struc-
Calcium and potassium thioglycolates reduce tural or permeability changes in the skin. Some
these intercellular bonds thus compromising the other hair removal methods also can affect skin
structural integrity of hair strands. As a result, the permeation of compounds. It has been shown that
hair can be easily removed by scraping or rub- shaved skin enhances the permeation of aluminium
bing (Goddard and Michaelis 1934). salts (Anane etal. 1997).
The absence of the hair shaft is influential in
promoting cutaneous absorption through the
transfollicular route. Hair removal induces the 4.11.3 Peeling Agents
hair follicle and greatly enhances the low-
resistance character of this pathway (Han etal. The anti-ageing industry, worth billions of dol-
2004). Corneocytes also contain keratin fila- lars, has an overwhelming number of products
ments, which constitute a major structural pro- and treatments promising to turn back the clock.
tein, located within the cornified cells and in the One example is chemical peeling, which
cell envelope (Madison 2003). Therefore, depila- involves treating the skin with low concentra-
tory agents will also be able to reduce the disul- tions of phenol or -hydroxy acids (AHAs),
phide bridges contained in corneocytes, such as glycolic or salicylic acid to remove the
weakening their structure. Lee etal. found that superficial layer of the skin, the SC and to stim-
the transdermal penetration of both hydrophilic ulate its renewal. These acids are known to
and lipophilic compounds through rat skin pre- cause irritation to the skin by producing one of
treated with a depilatory agent was significantly the following reactions: keratolysis or keratoco-
enhanced and that this enhancement persisted for agulation (Brown etal. 1961). Keratolysis leads
24h after application (Lee etal. 2008). This sug- to the necrosis of the SC and its separation from
gests that the structural changes induced by the the viable epidermis whereas keratocoagulation
depilatory agent may be restored later. involves the irreversible phenomenon of dena-
The mechanism of thioglycolate-induced per- turation and coagulation of keratin (Rothman
meation enhancement is not well understood. It has 1943). Consequently, the skin barrier function is
been reported that the structural changes induced significantly altered after this procedure. AHAs
by thioglycolate-based depilatory creams enhanced reduce the skin barrier integrity by reducing
drug delivery through both the transcellular and corneocyte cohesion and their influence extends
intercellular SC pathways (Lee etal. 2008). to the newly forming layers of the SC, resulting
Shiozuka etal. also reported changes in SC struc- in the formation of more supple superficial lay-
ture after treatment by a depilatory agent (Shiozuka ers (Van Scott and Yu 1984). In this direction,
etal. 2010). Whereas the normal SC is character- Song etal. reported that the increase in TEWL
ised by tight packing of clearly defined cells, treat- observed invivo after glycolic acid treatment
ment with a depilatory agent produced some highly returned to pre-treatments values after one day
noticeable morphological changes. Firstly, struc- (Song etal. 2004).
4 Non-formulation Parameters Affecting Skin Permeation 67
continued to increase with increasing temperature, meate the burn eschar, the underlying tissues are
with a sharp transition in barrier properties occur- permeable to such a macromolecule (Wang etal.
ring between 70 and 80C (Behl etal. 1981). The 2009). Consequently, there has been interest in
effects at lower temperatures were attributed to the techniques to enhance burn eschar permeability to
increased hydration associated with scalding, while therapeutic substances. For example, Zadeh and
at higher temperatures, the increase in permeability Moghimi showed that the permeability of human
was attributed to morphological and/or chemical third-degree burn eschar was very sensitive to the
alterations in the SC (Behl etal. 1981). level of hydration and that prolonged exposure to
The effects of higher temperatures have been water might open the compact structure of eschar
studied recently by Park etal. using full-thickness and increase drug permeation through this barrier
skin, epidermis and SC samples from human and (Zadeh etal. 2008). The Moghimi group has also
porcine cadavers heated to temperatures ranging achieved success in the use of chemical penetration
from 100 to 315C.They found that skin perme- enhancers with burn eschar. While outside the
ability was increased by a few fold after heating scope of this review, the work showing that water,
to 100150C, by one to two orders of magni- glycerin, hexane:ethanol and ethyl acetate:ethanol
tude after heating to 150250C and by three (Manafi etal. 2008) as well as terpenes such as
orders of magnitude after heating above limonene, geraniol, 1,8-cineole and -pinene oxide
300C.These permeability changes were respec- (Moghimi etal. 2009) were able to increase perme-
tively attributed to the disordering of SC lipid ation of silversulfadiazine significantly through
structure for temperatures lower than 150C, dis- human third-degree burn eschar should be noted.
ruption of SC keratin network structure at 150
250C and finally decomposition and
vaporisation of keratin to create micron-scale 4.13 P
revention ofPercutaneous
holes in the SC above 300C (Park etal. 2008). Permeation: APossibility
The experiments mentioned above were per- andaNecessity
formed on burnt skin separated from the body,
whereas in the invivo situation, barrier function Intact skin is a very effective barrier and inhibits
is more fully reflective of the range of skin permeation of most compounds; therefore, skin
responses caused by the burn. In vivo experi- permeation enhancement has been the subject of
ments also allow the dynamic effects of the burn much research over last few decades. In spite of
to be studied. Flynn etal. followed the progress this, skin penetration retardation is also a very
of mice burned with an 80C metal surface and crucial issue, not only to prevent permeation of
showed that the permeability to methanol and toxic chemicals, but also due to the fact that some
butanol gradually increased after the burn, reach- materials (e.g. polyethylene glycol) that are
ing a maximum on the tenth day, then decreasing employed in dermatological formulations for
until termination of the studies at 14 days (Flynn another purposes (e.g. as solvents) can reduce
etal. 1982). The Moghimi group has investigated percutaneous absorption of drugs (Moghimi and
barrier function in humans, showing increased Shakerinejad 1998).
permeability of fully hydrated full-thickness Human skin is exposed to a number of irri-
(1500 m) third-degree burn eschar to silver tants and toxic material in daily life, some of
sulphadiazine (Zadeh etal. 2008, 2010). which have the ability to permeate the skin bar-
Despite the evidence of reduced barrier function rier in toxic amounts and cause local or systemic
in burned skin, it has been shown that some drugs toxicities, particularly if the barrier is damaged
cannot permeate the burn eschar in therapeutic (OFlaherty 2000). Organophosphates, such as
amounts to overcome infection and drug delivery parathion and malathion that are used as insecti-
to and through burn eschar is still a challenge cides, have caused toxicity and deaths after
(Moghimi and Manafi 2009). Wang etal. also absorption through the skin (Aaron 2001). The
showed that while fetuin-A (40kDa) cannot per- nerve agent VX (an organophosphorus ester)
4 Non-formulation Parameters Affecting Skin Permeation 69
shows a percutaneous LD50 of 10mg for a 70kg retardants may be explained by their actions to
person (Somani and Romano 2000). Another increase the organisation of SC lipids (Kaushik
example is hexane that can produce a peripheral etal. 2008, 2010; Kaushik and Michniak-Kohn
neurotoxicity through the skin (OFlaherty 2000). 2010). Stabilisation of lipid lamellae in the more
Different techniques have been developed over ordered orthorhombic phase has been associated
time to overcome such transdermal toxicities, as with enhanced SC barrier properties (Pilgram
discussed below. etal. 1999).
Protective clothing and gloves can provide
some degree of protection and are considered to Conclusion
be essential protective items. However, care is The interaction of a penetrant with the skin
necessary, as some gloves cannot prevent pene- barrier determines its rate of percutaneous
tration of low molecular weight chemicals absorption and is influenced by the physico-
(Wigger-Alberti and Elsner 1998; Moghimi etal. chemical properties of the molecule or particle
2010). Another class of protectants is barrier and the properties of the barrier itself. There is
creams, which contain ingredients that are sup- considerable variability in the skin barrier, due
posed to trap or transform unwanted material or to factors such as the degree of hydration, age,
prevent/delay their contact with the skin by form- gender, anatomical site, abnormalities due to
ing a protective barrier at the skin surface (Berndt disease or injury and prior treatment with vari-
etal. 2000). Similar to other creams, barrier ous skin treatments. While most research has
creams contain different materials such as surfac- concentrated on finding ways to increase per-
tants (that can act as penetration enhancers) and cutaneous absorption, retardation may be nec-
oily phases (that can increase skin hydration), so essary in some situations, for example, to
that under some circumstances, barrier creams prevent permeation of toxic chemicals.
have the potential to increase the permeation of
drugs. For example, it has been shown that the Acknowledgements Thanks go to the National Health
percutaneous absorption of trimethylbenzene & Medical Research Council of Australia for financial
was increased through skin treated with barrier support. The authors also thank Navin Chandrasekaran
creams (Korinth etal. 2003). Therefore, in prac- (Therapeutics Research Centre, School of Medicine, the
University of Queensland) for help in drawing figures
tice, barrier creams are recommended only for and Dr Amy Holmes, from (Therapeutics Research
low-grade irritants and should be not used as pri- Centre, School of Pharmacy and Medical Sciences,
mary protection against high-risk substances. University of South Australia), for assistance in editing
This area has been extensively reviewed the chapter.
(Machado etal. 2010b).
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The Influence ofEmollients
onDermal andTransdermal Drug 5
Delivery
V.R.Leite-Silva, JeffreyE.Grice,
YousufMohammed, HamidR.Moghimi,
andMichaelS.Roberts
Table 5.1 The nature, uses, and various definitions of emollients over time
Source Date Definition
Oxford English Dictionary 1643 Substance that has the power of softening or relaxing the living
animal textures
Edwards (1940) 1940 Since oils, fats and glycerin when applied to the skin tend to
soften the epidermis they are termed emollients
An adhesive coat is produced which prevents the irritation of
drying, and the access of bacterial, chemical and mechanical
irritants
Blank (1957) 1957 Any material that tends to prevent or alleviate the symptoms and
signs of dry skin
Idson (1982) 1982 Emollients substances lubricating and/or occluding the skin
with water-insoluble material (Moisturizers substances actively
increasing the water content of the skin)
Wilkinson and Moore (1982) 1982 Emolliency is only associated with imparting smoothness and
general sense of well-being to the skin, as determined by touch
Wehr and Krochmal (1987) 1987 Emollients systems that smooth the roughened surface of the
SC, but do not occlude the skin. No effect on TEWL after
application
Loden (1992) 1992 Similar action to moisturizers
Dederen etal. (2012) 2012 Emollients oily ingredients used for skin care formulations
recorded in 1643. In general, emollients are lipid press over the years, leading to the terms often
in nature and are ingredients in a product, which being used interchangeably, although the sensory
when applied to the skin deposit a lipid film that responses are different.
can also replenish any lost skin lipids. The resul- With a history of several thousands of years, the
tant effect is improved skin lubrication, a smoother nature, uses, and various definitions of emollients
skin surface, a soothing effect as it protects the have changed with time (Table5.1). Many of the
exposed viable epidermis, and hydration of the earliest emollients were derived from animal fats.
stratum corneum (by moisturizing the skin Marks refers to Egyptians and ancient Greeks
through reduced transepidermal water loss). using oils and pleasant smelling fatty concoctions
Overall, the skin treated with an emollient is on the skin, the use of wool fat by the Greeks in
described as being soft and supple, whereas that about 700 BC, the processing of lanolin from
treated with a humectant (a substance that attracts sheeps wool by a Greek physician in a Materia
water to the skin (Idson 1992), improving its Medica in 60AD, and the patenting of petrolatum
hydration) has the sensory feel of softness due to (also known as petroleum jelly, white soft paraffin,
moisturization of the stratum corneum, but with- and Vaseline) in 1872. As he points out, lanolin is a
out the sensorial suppleness feel associated with complex emollient in being a two-phase liquid and
the oily film. A moisturizer usually refers to a wax system consisting of multiple complex sterols,
product, and it may contain an emollient and/or a fatty alcohols, and fatty acids, dependent on the
humectant and/or water to provide direct hydra- nature of the sheep sourced, its manufacturing, and
tion of the stratum corneum. In practice, however, its storage (Marks 2001). Interestingly, goose
the term emollients has been interchangeable grease and even human fat have been used as emol-
with moisturizers and lubricants, and being lients, and emolliency has also been referred to in
used as bases, vehicles, or to make vanishing the soothing of the throat (Coxe 1825). A prevail-
creams, revitalizing creams, or regenerating ing view is that lipids (fats, waxes, and oils) are
milk (Nola etal. 2003). Regrettably, the nature seen as the essential components of emollients
and actions of an emollient, a humectant, and a and that the total lipid content in an emollient for-
moisturizer often have become blurred in the mulation is usually 2040% (Marks 2001).
5 The Influence ofEmollients onDermal andTransdermal Drug Delivery 79
Fig. 5.1 The effect of various products on the stratum cor- occlusion, for example, with a plastic covering; (f).
neum, ranked in order of increasing lubrication of the stra- Semipolar emollient; (g). Hydrocarbon emollient. The fig-
tum corneum surface after several hours of application ure also shows (i) the effect of products on stratum corneum
(except B) for: (a). Control; (b). Humectant, early times hydration (the darker the box, the higher the skin hydration)
only; (c). Humectant, later times; (d). Partial mechanical and (ii) the transepidermal water loss (TEWL), with the
occlusion with a breathable membrane; (e). Mechanical number of arrows indicating the magnitude of the TEWL
Other definitions of emollients have included sepidermal water loss (TEWL) promotes skin
the preparations themselves (Ray and Blank hydration. The application of a semipolar emol-
1940; Harry 1941), and ointments designed for lient like vegetable oil is likely to reduce TEWL
deeper skin penetration (Wild 1911). Confusion to promote skin hydration to a lesser effect than
has arisen, in part, from the early emphasis on the the more occlusive hydrocarbon emollient.
emollient lipid film interactions with skin lipids We now describe the emollients used in prac-
and scales and the more mechanistic approach tice, their potential effects on percutaneous
advocated by Blank, in 1957, which emphasized absorption, and some practical examples of prod-
the skin hydration associated with emolliency. ucts containing emollients. In recognizing that the
He defined an emollient as any externally stratum corneum is the main barrier for both der-
applied material that tends to prevent or counter- mal and transdermal absorption, we have focused
act the symptoms and signs of dryness of the this chapter on the effects of emolliency on skin
skin (Blank 1957). An occlusive dressing is also function and the skin penetration of the active.
often used to increase skin moisturization.
Figure5.1 summarizes our current view of the
effects of humectants, semipermeable or imper- 5.2 Current Emollients, Their
vious occlusive films, semipolar emollients, and Modes ofAction, andTheir
hydrocarbon emollients on stratum corneum Use inPractice
(SC) roughness, hydration, and transepidermal
water loss (TEWL). It is evident from this figure 5.2.1 Sebum
that emollients differ in their actions on normal
skin, compared with other skin treatments such Sebum is the natural emollient of skin. It is pro-
as application of humectants and occlusive dress- duced from the sebaceous glands adjacent to the
ings. As shown in Fig.5.1, emollients affect both hair follicle and consists predominantly of squa-
the transepidermal water loss and the roughness lene, wax esters, triglycerides, cholesterol esters,
of skin surface through the oily film that they cre- and possibly free cholesterol (Stewart 1992). The
ate. The lipid surface film on the stratum cor- sebum provides a pliable film on the skin surface
neum and its resulting lubrication of the surface that lubricates the skin, inhibits percutaneous
gives a feel of suppleness. The reduction in tran- absorption of unwanted substances, and impairs
80 V.R. Leite-Silva et al.
TEWL, leading to increased skin hydration (e.g., PEG-150 distearate, PEG/PPG-8/3 laurate)
(Stoughton 1959). As well as providing lubrica- and are used not only for skin but also for hair
tion and hydration of the skin, the sebum can also (e.g., PEG-7 glyceryl cocoate, PPG-3 benzyl
provide immunological and antimicrobial protec- ether myristate). Others may feel dry to the touch
tion through its surfactant proteins and peptides, (e.g., oleyl alcohol, C1215 alkyl benzoate,
especially when their expression in human skin is phenethyl benzoate, cyclomethicone, and isono-
upregulated (Mo etal. 2007). In addition, the nyl isononanoate). In general, the lipophilicities
sebum can also act as a buffer, impairing adverse of the emollients in (Table5.2) are such that those
irritation from acidic or basic compounds. containing hydrogen bonding groups, such as
Regular washing of the skin, however, can ethers, esters, vegetable oils, and lanolin are more
remove the sebum and result in greater skin polar than those without these groups, for exam-
roughness and reduce stratum corneum hydra- ple, hydrocarbons. Todays emollients are used to
tion. Low sebum levels have been regarded as a meet many different functional needs and to sup-
contributing factor in the development of dry skin port multiple claims, and hence, a formulator
(Clarys and Barel 1995). Emollients are widely has to select appropriate emollients to meet not
used to provide the desired lubrication and skin only the consumer and regulatory needs but also
hydration that is normally supported by the to cater for whether the final product is designed
sebum. In addition, a number of common skin for a cosmetic or a therapeutic application.
care ingredients, including mineral oil, white pet-
rolatum, and isopropyl myristate, have been
shown to enhance sebocyte counts, and hence, 5.2.3 E
ffect ofEmollients onSkin
potentially, sebum production, in a hairless Lubrication
mouse model (Lesnik etal. 1992).
Sebum has been shown to contribute to stra- The choice of an emollient is often based on the
tum corneum hydration by a glycerol-dependent tactile properties of these substances on the skin
mechanism. Based on the identification of glyc- surface and is often of higher importance in cos-
erol as the putative product of triglyceride metology than in the formulation of topical thera-
hydrolysis in sebaceous glands, Fluhr etal (2003) peutic drugs, where sensory properties are not
treated asebia mice, showing profound sebaceous necessarily the main priority (Dederen etal.
gland hypoplasia, with glycerol, and were able to 2012). A plethora of imaginative terms may be
restore normal stratum corneum hydration. Urea, used to describe these subjective properties.
another commonly used humectant, had no Words such as tacky, oily, dry-velvety, or
effect. waxy are readily understood, whereas other
more esoteric terms such as scroopy (the textile
chemists description of the rough, soft-draggy
5.2.2 Emollient Classes feel of raw silk) are less obvious (Goldemberg
andProperties and De La Rosa 1971). All these terms are used
in an attempt to describe the sensory responses
Members of the different classes of emollients caused by the lubricating actions of emollients on
have different physicochemical properties that the skin. A special issue of the Journal of
result in a range of functional and sensorial Investigative Dermatology was devoted to the
effects when left as a lipid film after being applied effects of emollients on the mechanical proper-
to the skin in a cosmetic or dermatological prod- ties of the skin in 1977, with articles on the visco-
uct. Traditionally, emollients have been regarded elastic (Christensen etal. 1977) and frictional
as having a number of common properties: (i) fat (Highley etal. 1977) properties of human skin, as
solubility, (ii) the ability to soften the skin, and well as measurement of skin hydration (Campbell
(iii) an oily feel. However, they can differ quite etal. 1977), among others.
markedly in their physicochemical properties. To the formulator, the tactile sensory proper-
Some emollients are partially soluble in water ties of the neat oils are the first consideration in
5 The Influence ofEmollients onDermal andTransdermal Drug Delivery 81
Table 5.3 Spreading values for selected ester constituent chain lengths can alter the skin-surface
emollients
spreading characteristics of ester emollients. For
High >850mm2/10min For example, example, isopropyl myristate and palmitate, with
spreading isopropyl short-chain alcohol components and the shortest
values myristate and
isopropyl acid chain lengths (C14 and C16, respectively) in
palmitate this table, have the highest spreading values. The
Medium 501850mm2/10min For example, C16 palmitate is greasier than the C14 myristate,
spreading ethylhexyl but the spreading values are similar. Ethylhexyl
values stearate and
stearate and decyl oleate, with longer chain com-
decyl oleate
ponents, have medium spreading values, whereas
Low 0500mm2/10min For example,
spreading C1215 alkyl the longer chain alcohol (C12C15), alkyl benzo-
values benzoate ates, are low spreading esters.
Many attempts have been made to achieve a
measure of sensory softness of the skin. In 2013,
choosing an emollient for a cosmetic product Nakatani suggested that conventional methods for
(Goldemberg and De La Rosa 1971; Zeidler measuring the mechanical properties of the skin,
1992). The key property of the emollient that this such as the elongation in response to suction, elas-
is reflecting is its ability to lubricate and reduce tic responses to ballistic impact, and rheological
any friction between the skin surface and its envi- responses to torsional stress, were restricted to
ronment (skin with skin, clothing with skin, etc.), measuring the properties of the whole skin and
as this reduces possible discomfort, irritation, and were unable to look at different skin layers sepa-
pain (Dederen etal. 2012). The lubrication inten- rately. They developed a novel piezoelectric tac-
sity of the emollient on the skin can be partly tile sensor system that could simultaneously
explained by the properties of the emollient itself; measure the mechanical properties of the whole
the residual film thickness, by dynamic spread- skin and its superficial layer. Such a technique has
ability and the viscosity. However, the skin is not obvious advantages to the cosmetic industry, but
a rigid, inert surface, and emollients can directly can also be applied clinically to the quantitative
or indirectly modify its mechanical properties. evaluation of skin disorders such as atopic derma-
This must also contribute to the overall sensory titis (Nakatani etal. 2013).
response (Dederen etal. 2012). This important
property of emollients is defined by their ability to
disperse more or less quickly on the skin surface 5.2.4 E
ffect ofEmollients onSkin
by forming a film. This can be assessed quantita- TEWL andSkin Hydration
tively using a parameter known as the spreading
value. A common technique for determining the The residual lipid film on the stratum corneum
spreading values has been described by Zeidler surface after the application of products
(1985). Spreading values, in units of mm2/10min, containing emollients will limit the evaporation
are determined by applying 20l of an emollient of water from the skin surface and therefore
to the center of an ashless, medium-to-fast filter cause an increase in skin hydration. Accordingly,
paper at 25C and measuring the area covered by emollients have been described as indirect skin
the applied material in 10min. Examples of moisturizers (Dederen etal. 2012). In general,
spreading values for some of the most widely the presence of hydrogen bonds in emollients
used group of emollient for skin lubrication, the also facilitates the transport of water through the
esters, are shown in Table5.3. Esters are useful to lipid films, so that for lipid films of similar thick-
formulators because of their versatility and the ness and viscosity, the semipolar emollients will
unique properties they can give to the final prod- be more permeable to water than the hydrocarbon
uct, influenced by the chemical properties, includ- emollients, resulting in a lower occlusive state
ing chain length, of their constituent fatty acids than that induced by the hydrocarbon emollients.
and alcohols. As can be seen, changes in the However, these findings can differ significantly,
5 The Influence ofEmollients onDermal andTransdermal Drug Delivery 83
depending on the nature of the emollient. Patzelt 2001). On the other hand, some moisturizers do
etal (2012) recently showed that vegetable oils act by penetrating the intercellular lipid regions.
(except Jojoba oil) led to a similar occlusion of A novel mechanism known as internal occlu-
the human skin surface invivo as paraffin oil, but sion (Wiechers etal. 2009) has been described,
the semisolid, petrolatum, was the most effective where moisturizers such as isostearyl isostearate
occlusive. The occlusive effects of an emollient and isopropyl isostearate cause improved skin
then result in a reduced transepidermal water loss hydration and barrier function by stabilizing the
(TEWL) and, in turn, an increase in the hydration SC lipid organization in the more tightly packed
of the stratum corneum relative to normal mois- orthorhombic phase (Caussin etal. 2007).
ture conditions. By occluding the skin and pro- A different approach to the emollient treatment
viding an additional barrier to water loss, skin of skin diseases such as atopic dermatitis, relying
hydration can be increased by up to 50% (Hafeez on the use of emollient treatments containing
and Maibach 2013a). This increase in hydration ceramide-dominant physiological mixtures of the
as an effect of occlusion has also been seen with three key lipids, cholesterol, free fatty acids, and
physical occlusives like wound dressings and ceramides at the appropriate physiological pH,
bandages (Voegeli etal. 2009, 2011). Increasing has been pioneered by Elias (Chamlin etal. 2002;
the thickness of the lipid film and/or increasing Elias 2010). The mechanism leading to skin bar-
the viscosity of the lipid film will reduce the rier enhancement is believed to involve more than
TEWL and increase stratum corneum hydration, a simple augmentation of intercellular lipid popu-
so that a wax will have a low TEWL and higher lations and structure. On passing through the SC,
SC hydration than an oil. the applied lipids migrate to the nucleated cell
The presence of water in a formulation can add regions, to be taken up by keratinocytes and then
to the moisturizing properties of that formulation trafficked to lamellar bodies, where they are
on the skin, but generally for only a short time. mixed with endogenous epidermal lipids. The
Indeed, the moisturizing effect of topically augmented lipid mixture is then secreted into the
applied water is often lost after 1020min of SC intercellular spaces (Mao-Qiang etal. 1995;
application (Batt and Fairhurst 1986; Paepe and Chamlin etal. 2002) to enhance skin barrier func-
Rogiers 2009). Blank showed that the main effect tion and normalize skin hydration. According to
associated with skin moisturization was an Elias, the effectiveness of any such treatment
increase in its softness and pliability (Blank depends primarily on understanding the mecha-
1952). The moisturizing effect of water can be nism responsible for a particular skin barrier
prolonged when an emollient is present in the defect, in order to judge whether lipid replace-
moisturizing formulation. The presence of a ment is appropriate for that condition (Chamlin
humectant, such as glycerol, in the aqueous phase, etal. 2002). An alternative approach to address
as well as the emollient will increase the moistur- imbalance in the SC proteolytic cascade leading
ization of the skin. Indeed, Batt etal. showed that to dry skin is to use serine protease inhibitors to
the enhanced moisturizing effect of glycerol by treat mild-to-severe barrier abnormalities (Voegeli
different emollients and oils was present even 12 etal. 2009; Rawlings and Voegeli 2013).
h after application (Batt etal. 1988).
Nonphysiological occlusive moisturizers such
as petrolatum remain on the skin surface without 5.2.5 Emollient Substantivity
being incorporated into the deeper skin layers.
While they may provide some benefit by improv- This is defined here as a measure of the retention
ing skin hydration, they are not effective in of an emollient in and persistence of its effect on
directly treating the disordered lipid states in such the skin after exposure to water, perspiration, and
diseases. For example, petrolatum treatment had resistance to being rubbed off. Another definition
no effect on the abnormal lipid organization asso- of substantivity is: the property of continuing
ciated with barrier defects in patients with atopic therapeutic action despite removal of the vehicle,
dermatitis or laminar ichthyosis (Pilgram etal. such as the action of certain shampoos (Mosby
84 V.R. Leite-Silva et al.
Fig. 5.2 Relative performance indices (RPI, as %) for a Substantivity (using a Sebumeter, with petrolatum as a posi-
range of emollient ingredients shown as individual bars. tive control and untreated skin as the negative control). For
(a) Moisturization (by skin hydration using a Corneometer, each property, ingredients were classified as: good-perform-
with glycerin as a positive control and untreated skin as a ing ingredients, RPI 70%; medium-performing ingredi-
negative control); (b) Elasticity (using a Dermal Torque ents, RPI between 30 and 70%; low-performing ingredients,
Meter, with water applied 30min under occlusion as a posi- RPI 30%. The 30 and 70% cutoffs are shown as dotted
tive control and untreated skin as a negative control) and (c) lines (Adapted from Wiechers etal. 1997)
5 The Influence ofEmollients onDermal andTransdermal Drug Delivery 85
c
86 V.R. Leite-Silva et al.
s olubility in the formulation, that is, at saturation, generally deliver its active for a longer period of
providing the formulation does not affect the time. On the other hand, with a semipolar emol-
skin. Accordingly, the maximal flux for smaller lient, the product will be more readily washed off
actives is greater than that for larger ones, and by water. Silicone emollients appear to offer both
those with the lowest melting point (Magnusson persistence in their retention in the skin and a
etal. 2004) and highest flux for a series of actives high solubility, and thus substantivity, for actives
of similar size will occur at a lipophilicity similar dissolved in them (Sene 2003).
to that of the skin lipids (a log P of about 3)
(Zhang etal. 2009). There are two general prin-
ciples defining the ability of an emollient to dis- 5.3.2 H
ow Do theEmollients Differ
solve an active: Like dissolves like and Oils intheir Ability toAffect
aint all oils! In other words, lipid-soluble andEnter theSkin (Size
actives generally dissolve better in emollients andSolubility Parameter
than polar actives, AND not all emollients have Determined) andtoPromote
the same properties. In general, actives dissolve Skin Penetration ofActives?
better in semipolar emollients (e.g., esters) than
in nonpolar, for example, hydrocarbon emol- The primary action by which an emollient pro-
lients. Actives can dissolve in both liquid and motes skin penetration is by hydration of the
waxy emollients after heating, but will be released stratum corneum. When the skin barrier is nor-
more slowly from the waxy than the liquid emol- mal, increasing the water content by occlusion
lient. However, increasing the viscosity of a for- can lead to enhanced penetration of some, but
mulation can sometimes result in an enhanced not all compounds (Hafeez and Maibach 2013a).
skin penetration as a result of the formulation Some possible mechanisms include increased
excipients on evaporation, leaving behind a semi- solubility of the compound in the SC, increased
solid barrier that may impede TEWL, promote partitioning from the vehicle into the hydrated
skin hydration, and in turn skin penetration flux membrane, and structural alterations due to the
(Cross etal. 2001a). swelling of corneocytes and a rearrangement of
These findings have the following implica- the intercellular lipid domains (Bjorklund etal.
tions for how the solubility of an active in an 2010; Hafeez and Maibach 2013b). Occlusion is
emollient may affect its percutaneous absorption. a well-recognized strategy for enhancing skin
Firsty, the thermodynamic activity for two equal penetration (Roberts etal. 2008). The recent
concentrations of an active in two emollients will reviews by Hafeez and Maibach examined litera-
be higher in the hydrocarbon than in the semipo- ture data on the effects of occlusion on the pen-
lar emollient in accordance with their different etration of compounds of varying lipophilicities
relative fractional solubilities, resulting in a invivo (Hafeez and Maibach 2013b) and invitro
higher flux of the active through the skin (Hafeez and Maibach 2013a). They concluded
(Wiechers etal. 2012; Roberts 2013). Hence, an that occlusion tends to enhance the penetration
active formulated with a hydrocarbon emollient of lipophilic compounds more than hydrophilic
will usually have a faster rate of skin penetration compounds, which would be expected, given the
than the one formulated in a semipolar emollient. relatively lipophilic nature of the intercellular
Accordingly, the skin flux for the sunscreen oxy- domains into which the compound must parti-
benzone for petrolatum was found to be greater tion. However, there appears to be a fall-off in
than that for an oil-in-water (o/w) emulsion penetration for very lipophilic compounds,
(Kurul and Hekimoglu 2001). However, there which would also be expected as the hydrated
can be a downside. As the active in the hydrocar- conditions under occlusion increase the water
bon emollient has a limited solubility, it will also content in the intercellular regions. Additionally,
exhaust much more quickly than in the semipolar the penetration of highly lipophilic compounds
emollient, that is, the semipolar emollient will may be further limited, as they will not readily
5 The Influence ofEmollients onDermal andTransdermal Drug Delivery 87
partition from the stratum corneum to an increas- the ester emollients in Table5.2, the liquid di-
ingly hydrated viable epidermis. These findings isopropyl adipate (MW 230Da) is most likely to
show an interesting parallel to those of Zhang, enter the skin, whereas the solid, glycol distea-
who showed a parabolic relationship between rate (MW 595Da) is the least likely to enter.
maximum flux and lipophilicity for a series of Zhang etal (2013) showed that the ester emol-
phenols penetrating human skin invitro. Zhang lient isopropyl myristate (MW 270Da) rapidly
saw the greatest penetration at an octanolwater enters the skin and could change its properties,
partition coefficient of about 3, with a reduced whereas the hydrocarbon emollient liquid paraf-
flux at higher values, and concluded that the fin appears not to enter the skin. Thus, isopropyl
relationship was driven by the solubility of the myristate is an emollient that enhanced the skin
compound in the stratum corneum (Zhang etal. penetration of phenols, whereas mineral oil did
2009). In both of these examples, stratum cor- not. The main effect of isopropyl myristate was
neum solubility can be seen to be largely depen- to carry the phenols into skin lipids, increasing
dent on the relative lipophilicities of the their overall solubility and maximum flux.
penetrating compound and the intercellular lipid However, isopropyl myristate also appeared to
domain. act as a reservoir, retarding the penetration for the
As we have seen, a large part of the strategy of more lipophilic phenols.
using emollients to moisturize and soften the skin Limited information is available about the
is concerned with replacing, replenishing, and extent to which emollients can modify the skin
reorganizing the population of intercellular lip- reservoir effect. It seems likely that, in a similar
ids. At the same time, a restored lipid domain way to occlusion, emollients may promote the
may strengthen the skin barrier and most likely release of actives from the horny layer (Roberts
lead to a reduced permeability to applied chemi- etal. 2004). However, there may be some waxy
cals. Results from infrared spectroscopy on emollients which have a very slow release rate
human stratum corneum suggested that increased of actives. Therefore, such emollients, if
lipid organization occurred as a result of increased retained in the horny layer, could potentially
hydrogen bonding (Kaushik and Michniak-Kohn cause an enhanced reservoir effect. A more
2010). On the other hand, petrolatum (Ghadially desirable effect is to have an enhanced substan-
etal. 1992) and nonphysiological lipophilic tivity as a consequence of the emollients sub-
moisturizers (Caussin etal. 2007) were shown to stantive properties, as shown for silicone esters
become localized in separate intercellular (Sene 2003).
domains, with little effect on lamellar organiza-
tion or barrier function.
Conversely, the application of emollients to 5.3.3 W
hat Do Other Ingredients
the skin may have the effect of reducing barrier inaMoisturizing Formulation
function and enhancing the penetration of Do toEnhance or Inhibit
applied compounds. This may occur as a result theEffects ofEmollients
of a direct effect on intercellular lipids, where onSkin Penetration?
they become disrupted or fluidized (Kaushik
and Michniak-Kohn 2010). Certain silicone Formulations, especially emulsions, may contain
polymers, although functioning as effective a wide range of ingredients with many different
moisturizers, were shown by electron micros- functions, including: preservants, coloring mate-
copy to disrupt lipid bilayers, leading to reduced rials, fragrances, thickeners, surfactants,
barrier function (Menon and Ghadially 1997). humectants, emollients, buffers to control the pH,
In the same way as active solutes will pene- chelating agents, and others. The balance between
trate the skin according to their size, melting them is very important for the stability and final
point, and lipophilicity, similar considerations function of the formulation. For example, if a fra-
determine how emollients enter the skin. Thus, of grance causes skin irritation, it may also change
88 V.R. Leite-Silva et al.
skin penetration of actives. As they are major d isordering of the intercellular lipid structure.
ingredients in many topical formulations, the Nonionic surfactants tend to cause less irritation
effects of humectants and surfactants on the skin and barrier damage, with polysorbates being
barrier may be significant. accorded GRAS (generally regarded as safe) sta-
Humectants such as glycerol, propylene gly- tus by the US Food and Drug Administration
col, or sorbitol are used to accentuate moisturiza- (Predmore and Li 2011). They act to fluidize lip-
tion, but can also accentuate emollient effects on ids and bind to keratin filaments (Nokhodchi
skin lipids by inhibiting the SC lipid phase transi- etal. 2003). As expected, alterations in SC bar-
tion. Indeed, in a 1990 study done in a dry atmo- rier properties by surfactants may lead to
sphere, Froebe et.al. (1990) showed that glycerol enhanced permeation of topically applied mate-
acts as a skin moisturizer by inhibiting the lipid rials. Most studies have examined the effects of
phase transition from liquid to solid crystal, anionic and nonionic surfactants, with the more
rather than by acting primarily as a humectant. disruptive anionic materials such as sodium lau-
More recently, invitro studies using mixtures of ryl sulfate and sodium dodecyl sulfate causing
glycerol and the powerful skin irritant and pene- the greatest enhancement (Williams and Barry
tration enhancer, sodium dodecyl sulfate (SDS), 2004). Nonionic surfactants like ethers and poly-
showed that glycerol was able to attenuate the sorbates (e.g., Tween 80) cause more modest
effects of SDS on the skin barrier by reducing the degrees of enhancement (Som etal. 2012).
ability of SDS to penetrate into the SC (Ghosh Nonionic surfactants have also been incorpo-
and Blankschtein 2007). rated into W/O emulsions which are compatible
Surfactants are used widely in topical formu- with the lipophilic sebum environment in hair
lations, usually to solubilize more lipophilic follicles, in order to target the follicular route of
actives. As the name suggests, they interact at skin penetration by hydrophilic solutes (Wu
membrane interfaces and, in particular, are capa- etal. 2001).
ble of modifying the structure and properties of Figure5.3 shows maximum fluxes for a series
the skin (Williams and Barry 2004). Anionic and of phenols of similar molecular weight applied to
cationic surfactants in particular may cause irri- human epidermal membranes from a range of
tation and skin damage by strong binding and simple vehicles, plotted against log P (Zhang
denaturing of skin surface proteins with swelling etal. 2013). It is clear that the maximum fluxes
and disruption of the corneocytes, as well as for water and the occlusive emollient mineral oil
(MO) are similar across this range of log P, as 5.4 Practical Aspects
expected for inert solvents that do not
permanently alter the properties of the skin (Barry Table5.4 shows five formulations containing dif-
etal. 1985; Cross etal. 2001b). In contrast, the ferent emollients and their likely effect on the skin
ester emollient IPM led to increased maximum properties and on the skin penetration of an active.
fluxes, particularly for the more polar phenols, The first impression that a consumer has in using
which was due to penetration of IPM into deeper a product is the sensorial feel. Formulation 1 feels
layers of the stratum corneum (Zhang etal. 2013), light and soft, because it lacks oils but is capable
where it has been suggested that it integrates into of lowering TEWL sufficiently to increase hydra-
the stratum corneum lipid matrix and disrupts the tion and potentially promote skin penetration of
organization of the lipid lamellae (Brinkmann and an active. The second formulation contains ester
Muller-Goymann 2005). Figure5.3 also illus- emollients to give a smooth and soft feel. The
trates similar increases in maximum flux seen smoothness derives from the lubrication of the
with mixtures of the humectant propylene glycol skin surface by the ester emollients lipid film. As
and water, most likely due to increased solubility this film has a greater effect on TEWL and skin
of the phenols in stratum corneum lipids follow- hydration than formulation 1, it may promote skin
ing propylene glycol absorption (Zhang etal. penetration more. On the other hand, the lipid
2011). Such a mechanism was also used to explain active is probably more soluble in formulation 2,
the enhanced penetration of minoxidil into human and this may inhibit skin penetration of the active.
skin from vehicles containing propylene glycol Formulation 3 contains mineral oil as an emol-
(Grice etal. 2010). lient, and it will feel oily and very soft, as this is
Table 5.4 Examples of some formulations containing different emollients and their effects on the skin and the likely
skin penetration of an active
Raw materials 1 (control) 2 (ester) 3 (mineral oil) 4 (animal fat) 5 (IPM)
Ceteareth-20 2.0% 2.0% 2.0% 2.0% 2.0%
Cetearyl alcohol 3.0% 3.0% 3.0% 3.0% 3.0%
Triethanolamine pH5.56.5 pH5.56.5 pH5.56.5 pH5.56.5 pH5.56.5
Carbomer 0.15% 0.15% 0.15% 0.15% 0.15%
Caprylic/capric triglyceride 5%
(CCT)
Lanolin 5%
Mineral oil 5%
Isopropyl myristate 5%
Glycerin 4.0% 4.0% 4.0% 4.0% 4.0%
Phenoxyethanol and 0.8% 0.8% 0.8% 0.8% 0.8%
parabens (methyl, ethyl,
and propyl)
Water Qsp 100% Qsp 100% Qsp 100% Qsp 100% Qsp 100%
Effects on skin
Sensorial feel Light and soft Smooth and Oily and soft Greasy and Smooth and
soft heavy soft
Reduction in TEWL
Skin hydration
Change in solubility of
nonpolar active
Potential effect on skin /
penetration
90 V.R. Leite-Silva et al.
the most occlusive formulation of all described 2001). Other ingredients, such as the humectant,
formulations and therefore will provide the great- propylene glycol (>15%), and ingredients used
est inhibition of TEWL and the greatest skin to stabilize emollients in products, such as sur-
hydration. An active is also likely to have poorer factants (e.g., oleic acid), can also cause irritation
solubility in formulation 3. Accordingly, this for- (Marks 2001).
mulation is likely to provide better skin penetra-
tion of the active. Formulation 4 feels greasy, Conclusion
because it contains animal fat such as lanolin, and This chapter has attempted to clarify the defi-
heavy because of the lanolin waxes. This formula- nition of an emollient, show the range of
tion is expected to be occlusive and to promote the emollients available in the market, and explore
solubility of the active. However, lanolin also con- how these emollients are likely to affect skin
tains cholesterol, cholesterol derivatives, and free penetration. The chapter concludes with an
fatty acids, which may act as skin penetration examination of some emollient- containing
enhancers. The overall effect is likely to be an products and their effects on the skin and on
enhancement of skin penetration. Formulation 5 the likely skin penetration of an active.
is very similar to formulation 2. However, it con- The key property of an emollient is that it
tains the ester emollient, isopropyl myristate, a softens the skin through occlusion and resul-
well-known skin penetration enhancer. tant moisturization as well as by forming a
Accordingly, formulation 5 should provide greater lipid film which smooths the skin by lubrica-
skin penetration of the active than formulation 2. tion. Moisturization of the skin is well known
There are other practical considerations in the to be the main means by which skin penetra-
use of emollients. They can provide a number of tion enhancement can be achieved (Roberts
functions, including the relief of potential dis- etal. 2008). However, emollients can also
comfort and irritation caused by solvents, promo- affect the solubility of an active and penetra-
tion of penetration, particle coating, stabilization tion enhancers, and thus their effects on the
of suspensions, brightness control for makeup, skin penetration of an active may be uncertain.
among others (Dederen etal. 2012). Another The final section discusses some potential out-
practical consideration is emollient stability. comes for different formulations containing
Ester emollient stability may be affected at low or emollients.
high pH, due to the possibility of hydrolysis or It also needs to be recognized that all cos-
saponification, respectively. While formulations metic and pharmaceutical products contain
are generally prepared at neutral or slightly acidic different ingredients, each of which can
pH, care must be taken to ensure that these condi- impinge on the sensorial feel and skin penetra-
tions will be maintained in a product over time. tion properties of a product. One concern is
There are some raw materials that can promote the potential skin irritation caused by these
stability of emollient esters at extreme pH. ingredients. Of the emollients, lanolin and iso-
Additionally, it should be recognized that if the propyl myristate are the most likely to be asso-
finished products are to come into contact with ciated with skin irritation. The most inert
the mouth (e.g., in lipstick), emollients and other emollient is probably mineral oil and, as it is
ingredients must not have an unpleasant taste. In very hydrophobic, it may also promote skin
summary, each product has unique physicochem- penetration by inducing a high thermody-
ical properties that can promote different interac- namic activity of the active as a consequence
tions and reactions with the skin surface. of the actives low solubility in the mineral oil.
Another consideration is the potential irri- Treatments designed to improve dry skin or
tancy of the formulations. As a general principle, treat skin diseases, such as lipid replenish-
in order to minimize skin irritancy, the sensitiz- ment, may also enhance the skin barrier func-
ing lanolin alcohols should be used in formula- tion and reduce its permeability to topical
tions in concentrations less than 3% (Marks chemicals. On the other hand, emollients may
5 The Influence ofEmollients onDermal andTransdermal Drug Delivery 91
enhance the barriers permeability by mecha- microelectrode method. JInvest Dermatol 69(3):
nisms such as increased hydration following 290295
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occlusion, disruption of the intercellular lipid Bouwstra JA (2007) Interaction of lipophilic moistur-
organization, or increasing the solubility of a izers on stratum corneum lipid domains invitro and
chemical or active ingredient in the stratum invivo. Skin Pharmacol Physiol 20(4):175186
corneum. Formulation strategies designed to Chamlin SL, Kao J, Frieden IJ, Sheu MY, Fowler AJ,
Fluhr JW, Williams ML, Elias PM (2002) Ceramide-
enhance skin penetration can be devised by dominant barrier repair lipids alleviate childhood
the judicious choice of emollient ingredients; atopic dermatitis: changes in barrier function provide
however, it is important to stress that each skin a sensitive indicator of disease activity. JAm Acad
characteristic varies widely between individu- Dermatol 47(2):198208
Christensen MS, Hargens CW 3rd, Nacht S, Gans EH
als. In addition, factors such as safety, cost, (1977) Viscoelastic properties of intact human skin:
sensory properties, sustainability, and market- instrumentation, hydration effects, and the contribu-
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Acknowledgments This chapter is dedicated to Johann Coxe JR (1825) The American dispensatory. Carey &
Wiechers who was to write this chapter and brought us all Lea, Philadelphia
together. It was his fervent wish that we looked at the Cross SE, Jiang R, Benson HA, Roberts MS (2001a) Can
impact of practical formulations on skin penetration. We increasing the viscosity of formulations be used to
thank the NHMRC of Australia for its support of our reduce the human skin penetration of the sunscreen
work. We are also grateful to Ricardo Azzini, Fabricio oxybenzone? JInvest Dermatol 117(1):147150
Sousa, and Joaosinho Di Domenico for their valuable Cross SE, Pugh WJ, Hadgraft J, Roberts MS (2001b)
guidance on the construction of Table5.2 and their gen- Probing the effect of vehicles on topical delivery:
eral comments. understanding the basic relationship between solvent
and solute penetration using silicone membranes.
Pharm Res 18(7):9991005
Dederen JC, Chavan B, Rawlings AV (2012) Emollients
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The Effects ofVehicle Mixtures
onTransdermal Absorption: 6
Thermodynamics, Mechanisms,
Assessment, andPrediction
JasonT.Chittenden andJimE.Riviere
or ternary mixtures (Baynes and Riviere 2004; experimental apparatuses gives insight into the
Baynes etal. 2001; El Maghraby etal. 2009; Van strengths and limitations of available data.
der Merwe and Riviere 2005b; Monti etal. 1995; Finally, a thorough review of the available litera-
Rosado etal. 2003). Enhancement of diazepam ture on predicting mixture effects on permeabil-
permeability in rat skin from a 50:50 mixture of ity shows the promise and failings of work to
propylene glycol and water was found to vary date.
with the concentration of added surfactants
(Shokri etal. 2001). One study explored over
4000 binary vehicles to develop rules for predict- 6.2 Thermodynamics
ing synergistic permeability enhancements ofMixtures
(Karande etal. 2006). An extensive study of up to
quaternary combinations of ten enhancers on The key properties solubility, partition coeffi-
skin permeability found synergistic enhance- cient, and diffusivity that drive transdermal
ment, with the best results on average for ternary absorption can be related back to a single prop-
combinations (Arora etal. 2010). Despite such a erty of a penetrant, the chemical potential, which
large body of work, a comprehensive is a function of the composition of the multicom-
understanding of the effect of complex vehicle ponent system also including solvents and
mixtures is still elusive. enhancers. The chemical potential measures the
A review (Moss etal. 2002) of quantitative differential change in Gibbs energy of a system
structure to permeability relationships (QSPRs) with respect to one of its constituents. In practical
for percutaneous permeation prior to 2002 terms, the chemical potential is the definitive
showed only one example where a vehicle effect property which is equated in various equilibria
was accounted for, and it only provided an indi- (e.g., reaction, phase, adsorption). For compo-
cator variable to adapt between two vehicles. nent i in a solution, one can write the chemical
Most of the prior work on skin absorption is done potential i as
with few compounds, aqueous or single vehicles,
and in nonstandardized conditions. Building G
mi = = RT ln g i xi + mi ,
models to predict vehicle mixture effects requires N i T , P , N j i
an extensive dataset with dosing applied in vari-
ous vehicle compositions. Single penetrant or where R is the ideal gas constant, T and P are
enhancer studies, even if combined, are not suit- temperature and pressure, xi is the mole fraction
able to study synergistic effects. Studies should of component i, Nj are the numbers of molecules
be specifically designed for the purpose. Thus, it of each component j, mi is the chemical potential
is only recently that predictive models that of pure component i at a reference state ( T , P ),
include vehicle effects, and especially mixture and i is the activity coefficient. Rearranging the
effects, have been developed. equation leads to an expression of the change in
The goal of this chapter is to exhibit some of the partial Gibbs energy of mixing (Gi) as a
the most recent works in the modeling and pre- function of concentration:
diction of skin permeability from complex vehi-
cle mixtures. The chemical thermodynamics that DG i = mi - mi o = RT Inxi + RT In g i
underlie many of the interactions are briefly = DGi ID + DGi EX ,
reviewed to highlight the mixture effects that can
affect some key properties driving permeability. where GiID and GiEX are the ideal and
The structure of the skin is discussed in the con- excess Gibbs mixing energies for component i.
text of potential mechanisms for interaction with The activity coefficient of component i can then
vehicle constituents. An overview of some of the be taken as:
6 The Effects ofVehicle Mixtures onTransdermal Absorption 97
Fig. 6.1Activity
coefficient of salicylic 100.0
acid in water and ethanol
mixture, by one 50.0
parameter (Wilson
model with parameters
from Matsuda etal.
2010) 10.0
1.0
0.5
c hemistry (SPARC) system has been used to pre- vary by an order of magnitude with the concen-
dict solubility (N=707, RMS=0.487), activity tration of the solute and that the Maxwellian dif-
coefficient (N=2647, RMS=.272), and distribu- fusivity D12 as a function of concentration of
tion coefficient (N=698, RMS=0.44) (Hilal solute was log-linear, being described by:
etal. 2004), where N is the number of compounds
D12 = ( D12 ) (D )
x1 x2
in the dataset and RMS is the root mean square 12 , where D12 and D12 are the
error in the prediction in log units. Ingram used infinite dilution diffusivities. The implication is
COSMO-RS in the prediction of partition and that with an activity coefficient model for a sol-
distribution coefficients of 19 ionizable drugs vent/solvent system, and a prediction of dilute
over a range of pH and with various counterions diffusivity, that diffusivity can be predicted over
(Ingram etal. 2011). the entire range of concentrations.
6.2.3 Diffusion
6.3 Potential Mechanisms
Chemical potential plays a role in diffusion as ofInteraction
well. The generalized Maxwell-Stefan equation
relates the diffusional flux of components of a Thermodynamics provides a mechanism for
mixture to the driving force for diffusion (Taylor understanding how components in a mixture can
and Kooijman 1991): interact to modulate key properties related to
transdermal penetration. How these properties
n x J -x J
di =
i j j i
interact, however, at different levels of the pene-
j =1 c D tration process is the key driver of the rate and
t ij
n -1
extent of absorption. As the penetrant moves
x ln g i
di = i mi = d ij + xi x j from the dosing vehicle (if there is one), into
RT
j =1 x j solution at the skin surface, then into the stratum
n -1
corneum and dermis, it encounters different
= Gij x j
j =1
environments at each level. The vehicle can then
have synergistic effects by acting in multiple lev-
In a binary system (or perhaps one with only one els. In addition, mechanisms that change the
diffusing solute), the equation simplifies to: composition of the solvent and/or absorption
environment can have dynamic effects on the
J1 = -ct D12 Gx1 = -ct Dx1
chemical properties related to penetration and
ln g 1 absorption.
D = D12 G = D12 1 + x1
x1
The diffusion coefficient usually estimated from 6.3.1 Skin Surface
experimental data is the Fickian diffusion coeffi-
cient D, which is the product of the Maxwell dif- Several mechanisms at the skins surface affect the
fusion coefficient D12 and the thermodynamic dissolution, diffusion, and penetration of drug from
factor . Note that the mixture molar density ct is the dosing vehicle into the stratum corneum.
taken to be constant here. The thermodynamic Transport of the penetrant from the dosing vehicle
factor can be computed from any activity coeffi- to the surface of the skin may be limited by the
cient model. solubility of the penetrant in the dosing vehicle or
The activity-adjusted diffusion coefficient D12 the rate of diffusion across a boundary layer. If the
has been computed from observed Fickian diffu- penetrant is dosed as a suspension, then dissolution
sion coefficients D for several binary systems may also become a rate-limiting factor. In the case
(Vignes 1966). The results showed that D could of a neutral solute (penetrant) in a pure solvent, the
100 J.T. Chittenden and J.E. Riviere
thermodynamics of the system are relatively If volatile solvents are used, evaporation can
simple, even if not ideal (in the sense of ideal cause an increase in solute concentration
thermodynamic behavior). For binary or higher astheeffective volume of solvent decreases
mixtures of solvents, however, the combined (Stinchcomb etal. 1999). This disappearance of
effects on the solute properties such as solubility solvent volume can also reduce the depth of the
and diffusion coefficient are not necessarily simple boundary layer, which also increases flux. When
combinations of the properties in the pure solvent a solvent mixture is used, the evaporation of one
systems, and as such may exhibit synergistic of the solvents results in a change in the solvent
effects. composition over time (except in the case of an
Consider a boundary layer of the dosing vehi- azeotrope), which can drastically change the
cle sandwiched between the skin surface and physical properties of solubility, diffusivity, and
some bulk fluid or dosing apparatus (e.g., a drug partition coefficient. As the solute concentrates
eluting patch). The flux of penetrant through the due to solvent evaporation, it may begin to pre-
boundary layer at steady state is determined by cipitate. If the solvent evaporates completely
C before the solute is fully absorbed into the stra-
Ficks law as: J = - D , where J is flux of
z tum corneum, rapid intial flux will give way to
penetrant, C is concentration of penetrant, D is slower flux and crystallization of penetrant on
the diffusion coefficient, z is the distance through the skin surface, with the end result of incom-
the boundary layer. Assuming a constant gradient plete absorption (Oliveira etal. 2012b). If, how-
J = - D ( Ch - C0 ) / h , where Ch is the bulk ever, the solvent evaporates more slowly than
concentration at the top of the layer and C0 is the the absorption rate, the effect will be to increase
penetrant concentration at the skin surface (note, the overall absorption rate. One other possibility
not in SC membrane itself). If the bulk concentra- is that the solvent does not evaporate com-
tion of penetrant is near saturation, then the mix- pletely, which is more likely in a solvent mix-
ture effects that increase solubility can increase ture where one of the components is not
flux as long as diffusion to the skin surface is the volatile.
rate-limiting step. In the case of incomplete evaporation, the
One way to increase solubility is to add sur- impact on absorption is more complex. Up to
factants, which can aid in the solvation of hydro- the point of precipitation, the absorption rate
phobic molecules. Solvation imposes increased may be increased, presuming no other
order around the solute molecule, however, and effectson solubility or diffusion coefficient.
reduces rate of diffusion, as the solute and sur- Supersaturation of lipophilic solutes in the
rounding surfactant molecules have an increased donor well has been shown to increase their
tendency to move as a unit. In essence, the penetration by increasing thermodynamic
effective radius of the solute increases. This activity while not affecting the stratum cor-
implies a trade-off between increased solubility neum barrier function (Moser etal. 2001).
increasing flux and reduced diffusivity reducing When the evaporation stops, Ch will be at (or
flux. When surfactant concentrations are above possibly above) the saturation concentration
the critical micelle concentration (CMC), phase Csat and should remain at the saturation con-
separation into micelles will occur, further com- centration, until all the precipitated drug redis-
plicating the system. Micelles diffuse much solves. If redissolution is slow, the net effect
more slowly than the single solute molecule, but may be an initial increase in flux followed by
due to their size, can deliver multiple solute lowering of flux to the dissolution rate, which
molecules instantaneously when they disassem- may be overall slower than if the system were
ble at the stratum corneum interface. occluded and evaporation could not occur.
Nonetheless, with the introduction of micelles, These same effects may occur if, instead of
penetration is generally reduced (Wiechers evaporation, loss of solvent is through its pen-
1989). etration into the skin.
6 The Effects ofVehicle Mixtures onTransdermal Absorption 101
lipid domains of the stratum corneum (Walker corneum increased for some compounds, possibly
and Smith 1996). At high concentrations, ethanol due to increased exposure to a protein domain that
has been shown to extract lipids, while at lower is normally shielded by the lipid domain (Surber
concentrations it disrupts the lipid head groups etal. 1990). Depending upon the desorption rate,
resulting in disruption of the crystal structure and this could result in an extended time of absorption
increased permeability (Krill etal. 1992; Suhonen or even reduced absorption, if desorption is so slow
etal. 1999). Fatty acids (Aungst 1989), various that the penetrant is eliminated by desquamation.
ceramides (Vvrov etal. 2003), a variety of For penetrants with local action or toxicity, modula-
other compounds including ethers and alcohols tion of this accumulation may be crucial.
(Ibrahim and Li 2010), and numerous terpenes Hydration of the stratum corneum results in
(Williams and Barry 1991) have been shown to the inclusion of bulk water that may pool in cor-
increase permeation by fluidization of the lipid neocytes or within the intercellular lipid matrix
lamellae. Acidic pH may also contribute to fluidi- (Pieper etal. 2003). Inclusion of water in the stra-
zation (Kitagawa etal. 1995). tum corneum, as a function of relative humidity
Modulation of the partition coefficient can (RH), was found to sharply increase the diffusiv-
occur by modification of the mixture properties ity of water in the membrane at values over 80%
on the skin surface or through changes induced RH (Wiechers 1989). These results may indicate
in the stratum corneum lipid structure. The parti- the formation of a hydrophilic pathway, when the
tion coefficient between the stratum corneum stratum corneum is in a highly hydrated state.
lipids and the vehicle at the skin surface is
C v x g
Psc = sc = sc sc sc , which is a function of the
C0 v0 x0g 0 6.3.3 Epidermis andDermis
mixture composition through the activity coeffi-
cients. So, vehicle components that raise sc or As the penetrant moves through the epidermis, it
lower 0 of the penetrant increase the partitioning must cross cell membranes and diffuse through
into the stratum corneum. A study of the effect cells (Monteiro-Riviere 2010). Alternatively, it is
of vehicle mixtures of SLS, water, ethanol, and possible that small polar molecules could follow
propylene glycol demonstrated that partitioning an aqueous paracellular pathway through tight
into the stratum corneum was determined by the junctions to the basal lamina (Brandner 2009;
relative solubility of the solute in the vehicle and ONeill and Garrod 2011). By the time the pene-
stratum corneum lipids (Van der Merwe and trant arrives in the epidermis, it is likely to be sepa-
Riviere 2005b). Experiments in model mem- rated from the hydrophilic penetration enhancers
branes and skin support a model of vehicle inter- and other vehicle components. Remaining compo-
actions where high solvent uptake promotes nents can interact in a few interesting ways to
drug partitioning by enabling the solute to exist enhance absorption rate. Increasing the partition-
within the solvent fraction/solvent-rich areas ing into the cell membranes or the fluidity of the
inside the membrane or skin by increasing solu- membranes will increase diffusion of the pene-
bility in the membrane (Oliveira etal. 2012a). trant. Metabolizing enzymes in the epidermis may
Conversely, lowering the solubility in the vehicle reduce overall bioavailability of the penetrant
has been found to also contribute to greater par- (Stinchcomb 2003; Storm etal. 1990), though a
titioning into the stratum corneum (Hilton etal. co-penetrant that inhibits or downregulates expres-
1994). sion of the enzymes will diminish that effect.
Another potential interaction in the stratum cor- In the dermis, the vasculature acts as the final
neum is binding of the drug to proteins in the kera- barrier between the skin and the systemic avail-
tinocytes (Frasch etal. 2011), which may cause a ability of the penetrant. Systemic uptake is a
depot effect of the drug. That is, penetrant may function of the perfusion rate and the partitioning
move rapidly into the stratum corneum but accumu- between the tissue and plasma. Penetration
late there rather than enter the epidermis. For exam- enhancers that increase blood flow would create a
ple, partition coefficient in delipidized stratum stronger sink condition for diffusion, increasing
6 The Effects ofVehicle Mixtures onTransdermal Absorption 103
permeation rate (Riviere and Williams 1992; trant (and perhaps some of the vehicle constitu-
Wiechers 1989). Co-penetrants that affect protein ents) partition into and diffuse through the
binding of the penetrant in the extracellular membrane to the receptor well. In the Franz-type
spaces of the dermis or blood plasma will affect static cells, the receptor well is a closed system
the equilibration into the blood and the eventual which is periodically sampled, while in the flow-
venous uptake of the penetrant. Finally, like through cell, fresh perfusate is continually intro-
many tissues in the body, the skin expresses sev- duced and collected (Bronaugh and Stewart
eral drug transporter systems. The P-glycoprotein 1985; Franz 1975). In both cases, it is common
(P-gp) transporter system is one of the most to include a binding agent such as albumin in the
important in presenting barriers to drug uptake by perfusate to create a sink condition on the recep-
tissues. In the dermis, P-gp is concentrated at the tor side. If the boundary layer in the donor and
vasculature and presents a barrier to drug uptake receptor wells is kept small, by stirring or turbu-
from the systemic circulation. For penetrants, lence, or if diffusion is rapid, then the rate-limit-
P-gp may provide a boost to penetration rate that ing barrier to permeation will be diffusion
co-penetrants could inhibit (Skazik etal. 2011). through the membrane.
The use of different membranes such as silas-
tic and porcine skin allows the combination of
6.4 Experimental Assessment the obtained data to factor out the contributions
ofInteractions from the membrane and account for effects in the
donor and receptor wells, such as solubility,
The interaction of vehicle constituents to modify evaporation, binding, and if the penetrant parti-
the transport properties of a penetrant can be tioning/binding to silicone membrane is negligi-
assessed in several ways. In vivo experiments ble or otherwise accounted for diffusivity.
offer the most direct assessment of effects via
endpoints such as Cmax (maximum plasma con-
centration) and Tmax (time of Cmax), which indi- 6.4.2 I solated Perfused Porcine
cate rate of exposure, and AUC (area under the SkinFlap
concentration profile) which indicates extent of
exposure. But, invivo experiments can be expen- A deficiency with invitro systems is the inabil-
sive, time consuming, unethical (in the case of ity to assess effects due to perfusion or metabo-
toxicants), and may not provide much informa- lism which only manifest in viable, integrated
tion on the mechanism of the interaction. In vitro tissues. The isolated perfused porcine skin flap
and exvivo experiments provide a way to isolate (IPPSF) addresses these deficiencies by provid-
mechanisms of interaction as well as increase the ing an exvivo system for percutaneous penetra-
throughput of experiments. In trying to identify tion studies. The IPPSF is surgically prepared in
molecular properties of vehicle constituents that situ on weanling swine and excised after closure
contribute to changes in penetrant transport prop- of the incisions. The flap is cannulated and per-
erties, collection of data over several penetrants fused with a modified Krebs-Ringer solution
and vehicle mixture combinations is required and containing glucose, antimicrobial agents, and
only feasible in a high-throughput system. serum albumin to maintain viability. The perfus-
ate effluent can be collected or sampled for
analysis to determine the absorptive flux of pen-
6.4.1 Diffusion Cells etrants placed on the surface of the flap. The
integral, viable nature of the IPPSF allows the
Diffusion cells, whether static or flow-through, investigation of effects on permeation and parti-
presents a conceptually simple system for tioning due to changes in perfusion, metabo-
determining skin permeation rate. A donor well lism, inflammation, and cytokine signaling
above the fixated membrane sample is dosed (Riviere etal. 1986; Riviere and Monteiro-
with a penetrant and vehicle mixture. The pene- Riviere 1991).
104 J.T. Chittenden and J.E. Riviere
sion or partition coefficients, to chemical tional problem with this approach is that it relies
descriptors of the permeant (and possibly on saturation conditions in the donor well; so, it
vehicle) such as molecular weight, Ko:w, dipolar- may be of limited use in practical application.
ity, etc. An early relationship that could be Another approach to the incorporation of
considered a QSPR related partition coefficient activity coefficients is through linear free
in skin to solubility parameters, which in turn is energy relations (LFER) (Karadzovska etal.
a method for computing activity coefficients 2013a). These are models that relate a property
(Barton 1975; Sloan etal. 1986). Sloan reasoned of interest, which has its basis in Gibbs free
that for small molecules of a similar size, diffu- energy, to a linear function of chemical descrip-
sivity would be approximately constant, and tors. LFERs have been used to relate various
therefore the permeability rate constant kp would molecular properties to overall permeability
depend only on partition coefficient. Using a coefficients. The use of LFER equations for
relationship for the activity coefficient of the sol- predicting Kp has arisen from their use in pre-
ute i in solution n: dicting various other physicochemical proper-
ties such as log partition coefficient (Abraham
( )
2
ln g i ( ) = d i - d v ( n ) Vifv2( n ) / RT
v n
and Martins 2004). Extrapolation across exper-
imental systems should not be expected.
where V is the molar volume, i and v(n) are the Potts and Guy found that a simple model (6.1)
solubility parameters of the solute i and vehicle that accounts for lipid partitioning and molecular
n, v(n) is the volume fraction of solvent n size is sufficient to describe stratum corneum
(which approaches unity for dilute solutions), R permeability, and they discount competing ideas
is the gas contant, and T is temperature. that aqueous pathways play a significant role in
Substituting into the definition of partition the absorption kinetics in SC (Potts and Guy
coefficient: 1992).
ln Pi
v ( 2 ,1) sat
= ln
Xi (
v 2 ) sat
Xi ( )
= ln
g iv (1) sat ( d ) + f log ( K
log ( k p ) = log D
0
oct )
v 1 sat
g iv ( 2) sat - b MW
0
D - hypothetical diffusivity of MW =
for vehicle n and skin s, gives:
0 compound
Vi
(
= d i - d v ( n )
- (d i - d s ) ) d - diffusion path length
2
s , v ( n ) sat 2
log Pi
2.3RT K oct - octonal : water partition
The correlation of the predicted partition coeffi- coefficient
cients to skin permeability coefficient worked MW - Molecular weight (6.1)
well for theophylline in various vehicles, includ-
ing dimethylformamide, propylene glycol, eth- In (6.1), molecular weight is a surrogate for
ylene glycol, and formamide. However, it failed molecular size (volume) and Koct is a surrogate
to predict much higher fluxes from isopropyl for SC lipid partitioning. This first widely
myristate and octanol vehicles, which have simi- accepted QSAR for predicting skin permeability
lar solubility parameters to skin. This might have coefficient showed a strong correlation
been due to an interaction between the skin and ( R 2 = 0.67 ) for the 93 compounds in the Flynn
vehicle that changed the diffusivity or chemical dataset (Flynn 1990).
nature of the skin, resulting in a permanent Abraham and Martins performed a metastudy
change of barrier function lipid extraction, for involving experiments on human skin for 119
instance. So, this approach does not provide a compounds. They note that ionization of the
mechanism for predicting changes in barrier compounds does not necessarily have a deleteri-
function due to vehicle application. An addi- ous effect on absorption, especially in the case of
106 J.T. Chittenden and J.E. Riviere
hydrogen-basic molecules. They fit a model of system rather than separated out as features of
the form: interest (Moss etal. 2002). Hostynek and Magee
(1997) provide a simple model for accounting for
sp = c + eE + sS + aA+bB + vV
the vehicle effect, but the approach lacks general
sp - log ( k p ) or log ( k sc ) applicability, because it uses an indicator variable
E - excess molar volume that ignores the relative concentrations of the
vehicle and compound. In addition, the relation-
S - dipolarity / polarizability
ship is not extensible to new vehicle effects.
A - hydrogen bond acidity Rosado etal. (2003) examined the effect of
B - hydrogen bond basicity pretreating skin samples with various vehicles
V - McGowan characteristic volume before applying permeants in a diffusion cell.
Flux and retention enhancement ratios were cal-
The final fitted model for kp shows it is decreas- culated for four compounds over 13 vehicles.
ing for all terms except characteristic volume: While most of the studied vehicles enhanced par-
titioning into SC, a few modulated diffusion
log ( k p ) = 5.426 ( 0.101) 0.106 ( 0.129 ) coefficients of the permeants.
E .473 ( 0.095 ) S To separate and model the effect of complex
0.473 ( 0.148 ) A 3.000 ( 0.152 ) vehicle mixtures on the permeability of a particu-
B + 2.296 ( 0.137 )V lar compound, Qiao etal. (1996) proposed a
2
mechanistically defined chemical mixture
N = 119, R = 0.832, SD = 0.461, F = 112 (MDCM) experimental design. The MDCM is
defined as containing a penetrant, solvent(s), and
The positive term for volume seems nonintuitive, selectively a surfactant, a reducing agent, and/or
but is due to negative correlation between molec- a vasodilator. The effects of 16 MDCMs on para-
ular volume and hydrophilicity (Geinoz etal. thion absorption in IPPSF were evaluated in a full
2002). 24 factorial design, with the MDCMs com-
Hostynek and Magee proposed a model posed of a solvent (acetone or, full name,
involving molar refractivity (MR), number of dimethyl sulfoxide (DMSO)), surfactant (sodium
hydrogen bond acceptors (HBA), and number of lauryl sulfate (SLS)), metabolism modifier (stan-
hydrogen bond donors (HBD). This model also nous chloride), a vasoactive compound (methyl
accounts for effects of lipophilicity and molecu- nicotinic acid (MNA)), and 20 % water.
lar size (Hostynek and Magee 1997). One addi- Assessment of absorption and residue levels indi-
tional feature of their model is a factor for dosing cated not only effects due to single vehicles, but
vehicle (VEH): also interaction effects. A similar approach was
also used to study benzidine absorption in IPPSF
log ( k p ) = i + aVEH +bMR + cHBA+ dHBD (Baynes etal. 1996).
Williams etal. (1996) used the MDCM data of
Accounting for the vehicle effects on the perme- Qiao etal. (1996) to predict and compare trans-
ability constant is an important aspect in trans- dermal flux and skin residues using a multicom-
dermal absorption modeling. Because most partment dermato-pharmacokinetic model for
exposure to, or dosing of, compounds will occur parathion that compared quite well to the
in the presence of solubilizers and/or permeabil- observed flux in IPPSF.In this model, each of the
ity enhancers, the accurate prediction of the components of the MDCM is modeled, and their
uptake of the compound must necessarily include concentrations in the various compartments are
some factor to account for the presence of the allowed to affect the rate parameters in mecha-
dosing vehicle. Yet, in most of the literature up to nistically chosen, nearby compartments, accord-
the turn of the 21st century, these important ing to a modified Hill equation. That is, for
aspects were integrated into the experimental instance, DMSO in the SC increases the
6 The Effects ofVehicle Mixtures onTransdermal Absorption 107
p ermeability of parathion, but does not affect its treatments with four to five replicates per treat-
transfer to the depot compartment. Parameter ment. A validation dataset with five compounds
values for vehicle transit were directly gathered (also included triazine) in various chemical mix-
from previous studies, while those for parathion tures added 56 additional treatments. Finite doses
were adjusted from a previous study. While this were applied which resulted in dynamic flux pro-
model is certainly mechanistic and thorough, it files; so, permeability constant (kp) was assessed
requires many parameters for each penetrant, and from the regressed slope of the cumulative flux
many more for the interactions, making estima- profiles.
tion and validations of such a large model imprac- Among the 16 LFER relationships reviewed
tical for prospective applications. by Geinoz etal. (2004), the training set of 288
Extending the experimental approach of the treatments was fit well by Abrahams model,
MDCMs, Riviere and Brooks have found signifi- which was used as a baseline fit for investigating
cant improvements in LFER fits, using the potential mixture factors. Potential MFs were
Abraham and Martins models to describe perme- identified by trends in residual values against MF
ability in porcine skin flow-through (PSFT) cells values. Three MFs identified as having potential
by accounting for mixture effects with a mixture to reduce the residual variance (based on R2 of
factor (MF) (Riviere and Brooks 2005). The MF the trend) were refractive index, polarizability,
approach adds a new term to the LFER that is a and log(1/HC).Inclusion of these MFs in the
mass fraction weighted mixture property of the LFER improved the correlation as shown in
penetrant-vehicle mixture. The basic form of the Table6.1. Predictions on the validation set data
mixture factor (MF) is as follows: showed that the MF approach extended well to
the new treatment combinations as well.
MF = mi pi
chemcial = i
It is worth noting that the descriptors used in
mi - mass fraction of chemicali the MFs have a link back to chemical thermody-
namics that make their effectiveness less than
pi - property of chhemicali surprising. Consider that Abrahams LFER
These experiments examined 20 chemical prop- accounts for diffusivity and partitioning compo-
erties to assess the influence of their MF on nents of kp and that the primary role of the MF is
permeability, including descriptors of molecular to account for a change in partitioning due to the
size, volume, boiling point, hydrogen bonding vehicle. The partition coefficient is a function of
properties, polarizability, refractivity, melting the activity coefficients of the solute in the vehi-
point (MP), and more. Twelve chemically diverse cle and skin, but the LFER is optimized for an
penetrants and 24 vehicle mixtures (comprised of aqueous vehicle. Correcting a partition coeffi-
water, ethanol, propylene glycol, sodium lauryl cient for the vehicle composition, then, would
sulfate, and methyl nicotinate) were studied in require multiplying by the ratio of activity coef-
PSFT in a full factorial design resulting in 288 ficients in vehicle and water. Henrys law con-
Table 6.1 Coefficient of determination (R2), of predicted vs. observed log kp and regression residuals vs. mixture factor
(MF) value
Original dataset (288 treatments) Validation dataset (56 treatments)
Predicted vs. Predicted vs.
Mixture factor observed log kp Residuals vs. MF observed log kp Residuals vs. MF
None 0.58 0 0.62 0
Refractive index 0.80 0.53 0.78 0.41
Polarizability 0.76 0.43 0.69 0.17
log(1/HC) 0.77 0.45 0.75 0.34
Table modified from Riviere and Brooks (2005)
HC Henrys law constant, log Kp log of permeability coefficient, MF mixture factor
108 J.T. Chittenden and J.E. Riviere
stant is the activity coefficient of an infinitely d ifferent experimental systems to extrapolate sin-
dilute solute; so, it has direct bearing on the com- gle-compound LFER models across different dos-
putation. Polarizability relates to the attractive ing vehicles, although the specific chemical
forces between molecules in solution, which property that best describes the vehicle effect has
have direct bearing on activity. Refractive index yet to be identified. An additional study in PSFTs
itself is influenced by polarizability with a posi- using a different set of compounds (caffeine, cor-
tive correlation. The MFs, however, are an imper- tisone, diclofenac, mannitol, salicylic acid, and
fect measure of the required activity coefficient testosterone) was recently reported where the
ratio due to the incomplete information provided optimal MF was 1/MP for these drugs
by these descriptors and the potentially inade- (Karadzovska etal. 2013b). This study also
quate linear mass fraction mixing rule used. allowed the incorporation of datasets from differ-
Nonetheless, the success with this approach high- ent invitro models (infinite versus finite dosing,
lights the promise and necessity of incorporating saturation level, vehicle) to be accomplished by
the vehicle mixture properties when predicting incorporating indicator variables and a MF of 1/
skin permeability. MP.
A further study used the same data from PSFT A QSAR model developed with the already
cells (Riviere and Brooks 2005) and additional published data (Riviere and Brooks 2005) again
data for ten penetrants in five vehicle mixtures in highlighted the importance of vehicle effects by
IPPSF to investigate the effect of adding mixture finding a significant correlation of kp with the dif-
factors to the LFERs of Potts and Guy, Hostyneck ference between melting and boiling point of the
and Magee, and Abraham and Martins (Riviere vehicle (Ghafourian etal. 2010a). Stepwise
and Brooks 2007). The mixture factor approach regression with descriptors for the penetrants
was able to improve the kp prediction in each of including connectivity indices, quantum molecu-
the LFERs tested in PSFT.The results from the lar properties, and group counts, along with vehi-
Abraham and Martins based model are shown in cle mixture properties comprising melting point,
Fig.6.2. Note the vertical banding in (a) due to boiling point, solubility, vapor pressure, and
the penetrant predictions being unadjusted for Henrys law constant, resulted in a best fit model
mixture effects, as well as the large variation in which included octanol/water partition coeffi-
observed logkp values. In many cases, the vari- cient, ninth order molecular connectivity index,
ance within a penetrant, due to the formulation and difference in boiling and melting point of the
effects, is larger than the variance between pene- solvent system.
trants. Inclusion of topical polar surface area
log k p = 0.909 0.610 log K o:w + 2.62 c p ,9
(TPSA) as a MF allows the predictions to vary
within a penetrant, removing the banding effect, 0.00917 ( BPs MPs )
(6.2)
and reducing the prediction error. Application of
MFs to predict area under the flux curve (AUC) In the resulting model, Eq. (6.2), Ko:w is the octanol-
in IPPSF also improved the prediction over the water partition coefficient, p,9 is the ninth order
unmodified LFER models. molecular connectivity index, and BPs and MPs are
Influential MFs in PSFT were number of the boiling and melting points of the solvent. The
hydrogen bond acceptors, refractive index, and negative effect of partition coefficient was surpris-
TPSA.TPSA provided the best fit among all of ing, and it was hypothesized that this was due to the
the investigated LFERs. For IPPSF, octanol:water overall lipophilic nature of the dataset. This moti-
partition coefficient and inverse water solubility vated the author to compare the chemical space of
were important MFs for the Potts and Guy LFER, the dataset with the datasets of Flynn (1990) and
while log water solubility and ovality (a measure Wilschut etal. (1995). The comparison was con-
of deviation from spherical shape) were important ducted using principal component analysis, where
in the other two LFERs investigated. The results linear combinations of the descriptors are used to
showed that the MF approach works in two describe the variability of the response variable.
6 The Effects ofVehicle Mixtures onTransdermal Absorption 109
Fig. 6.2 Predicted vs. observed of penetrant absorption topical polar surface area (Figure from (Riviere and
in PSFT cells, with the Abraham and Martins LFER Brooks 2007), used with permission)
(Abraham and Martins 2004). (a) With no MF. (b) MF is
Plotting the first two principal components against With the data for the four additional chemicals
each other (Fig.6.3) is enlightening as it demon- expanding the dataset to 384 treatments and
strates that the chemical space of the dataset is increasing the diversity of the chemical space, a
quite constrained when compared to the chemical QSAR study was again performed using stepwise
space of the Flynn and Wilschut datasets. selection across a wide array of chemical
110 J.T. Chittenden and J.E. Riviere
Fig. 6.3The chemical diversity of the combined datasets in PCA analysis (Figure modified from (Ghafourian etal.
(Riviere solid circles, Flynn (1990) and Wilschut etal. 2010a), reprinted with permission. *PC1 and PC2 are the
(1995) empty circles) using all of the chemical descriptors first and second principal components of the chemical space)
sodium lauryl sulfate, propylene glycol, and eth- absorption. The IPPSF includes dermis, vascula-
anol (Riviere and Brooks 2011). A dataset with ture, active enzymes, and is a live tissue all of
seven compounds in mixtures of 14 vehicle com- which can contribute to not only differences in bar-
ponents (89 treatments) in IPPSF was used for rier function, but also to tissue partitioning and
validation. QSPR models based on the LFERs of depot effects. In addition, the MF approach appears
Potts and Guy, and Abraham and Martins, were to confound effects in the solvent with effects in the
estimated separately for the two experimental barrier, such that for the same vehicle mixture dif-
systems. A stepwise selection process was ferent MFs are required. It is suggested that using
applied to the mixture factor components added the estimation from PSFT to fix some portion of
to the LFER models, resulting in the final QSPRs the model for IPPSF may help to isolate effects that
(6.4) and (6.5). are particular to the exvivo IPPSF system.
A QSPR for the absorption of cosmetic and
log Y = i + mMF + a a 2H + bb 2H
dermatological formulations in human skin was
+ sp 2H + eR2 + vVx (6.4) developed from a model for predicting cumula-
tive absorption (Bunge etal. 1995; Bunge and
log Y = i + mMF + a log K o:w + bMW (6.5)
Cleek 1995; Cleek and Bunge 1993), adjusting
In Eqs. (6.4) and (6.5), is a placeholder for it to account for ionization of the penetrant in
either kp (in the case of PSFT) or AUC (in the the vehicle as well as distribution to aqueous
case of IPPSF); MF is a placeholder for a specific and organic phases of an emulsion (Grgoire
etal. 2009). The adjustment for ionization
mixture factor; a H
2
is hydrogen bond donor affects the prediction of partitioning into skin
acidity; b H
2
is hydrogen bond acceptor basic- and is accomplished by replacing the logPo:w
term in the Potts and Guy LFER with the log
ity; 2H is dipolarity/polarizability; R2 is excess
distribution coefficient of the penetrant in the
molar refractivity; Vx is McGowan volume; Ko:w
octanol-water system at the pH of the vehicle
is octanol:water partition coefficient; MW is
(logDpH). The effective area of diffusion was
molecular weight; and m, a, b, s, e, v, and i are
corrected for the aqueous fraction, which is
estimated parameters. In both the PSFT and
assumed to be the phase contributing the major-
IPPSF datasets, the MFs that contributed most to
ity of the flux. The model assumes steady-state
improving the model, as evaluated by correlation
diffusion, ignores the effect of penetration
coefficients and cross-validation metrics, were
enhancers, approximates all vehicles as emul-
the number of hydrogen bond acceptors (HBA)
sions, and considers only absorption from the
and the inverse of melting point (1/MP). However,
aqueous phase of the vehicle.
different MFs were needed in each model system
The dataset comprised cumulative amount of
to achieve the best fit. The QSPR based on the
absorbed measurements from human skin in 101
model of Abraham and Martins could be simpli-
individual studies, with a total of 36 compounds
fied by removing a parameter, but it was a differ-
in various vehicles and experimental settings.
ent parameter in each of the model systems:
The estimated parameter of the model is a coef-
hydrogen bond donor basicity in PSFT and polar-
ficient to control the distribution of the penetrant
izability in IPPSF.The addition of a MF to a
between the phases of the emulsion. The result-
LFER model has little impact on the estimated
ing QSPR predicted 91% of the data within 0.2
parameters (Table6.2) for the chemical descrip-
tors, which indicates the independence of the MF to 5-fold ( R 2 = 0.7115 ). This model is unique in
from the descriptors. the literature in incorporating biphasic vehicle
The results would indicate that different barrier and ionization interactions, but more research
functions are being evaluated between the two sys- needs to be done to extend it to ionic or metabo-
tems, but they could also indicate a fundamental lized compounds, monophasic vehicles, and to
difference between predicting rate and extent of account for penetration-enhancing excipients.
112 J.T. Chittenden and J.E. Riviere
Table 6.2 Compilation of QSPR results from Riviere and Brooks (2011), showing best fits with and without mixture
factor (MF)
R2 System Equation MF m i a b s e v
0.53 PSFT (6.4) 2.55 1.45 0.01 0.27 0.55 1.39
0.73 PSFT (6.4) HBA 1.21 4.27 1.44 0.28 0.55 1.38
0.60 IPPSF (6.4) 1.61 2.00 0.39 0.04 0.38 1.54
0.69 IPPSF (6.4) 1/MP 35.63 1.81 2.00 0.35 0.47 1.61
0.33 PSFT (6.5) 1.13 0.10 0.0058
0.55 PSFT (6.5) HBA 1.23 2.89 0.10 0.0058
HBA hydrogen bond acceptors, MP melting point, PSFT porcine skin flow-through cell, IPPSF isolated perfused por-
cine skin flap, m, i, a, b, s, e, v, which are parameters in Eqs.6.4 or 6.5, as indicated by the equation column (NB
Parameters and carry different meanings in the two equations)
The effects of vehicle properties and experi- and melting point ( BP - MP ( mix ) ), showing
mental conditions have recently been studied in a the strong contribution of vehicle effect on per-
large database of 536 flux data points from excised meability. Each of the factors for experimental
human skin (Samaras etal. 2012). The QSARs condition was then added to the regression
that were developed account for different dosing equation, whereby skin thickness and exposure
conditions, vehicles, membrane thickness, and type were found to be statistically significant
sample pretreatment. Vehicle properties were variables. With the RT models, inclusion of
computed by weighted averaging. For melting BP - MP ( mix ) had the lowest mean absolute
and boiling points, solute effects of boiling point error (MAE) on the full dataset. These results
elevation and freezing point depression were fac- clearly indicate the importance of accounting
tored in. Models were estimated by stepwise for the vehicle mixture.
regression as well as regression tree. Regression It has also been demonstrated that results from
tree models split the problem space according to one model system can extend to predictions in
covariate regions (leaves) and fit simpler models another model system. The MCF array has been
(often just the mean of the data) in each leaf. The used to predict absorption in IPPSF in a study
RT models were used to investigate the effects of that tested the effect of two surfactants (SLS and
experimental conditions, but only one condition at linear alkylbenzene sulfate (LAS)) on penetra-
a time. That is, no model included all of the exper- tion of six compounds (Riviere etal. 2010).
imental condition effects, possibly because the Penetrant partition coefficient from each vehicle
response space would become too fractured, into MCFs coated with PA, PDMS, and CW was
resulting in subsets too small to estimate parame- determined by mass balance. The partition coef-
ters for even simple models. ficients were then used in a QSPR (6.6) to predict
Neither stepwise regression nor regression log AUC ( R 2 = 0.718 ) .
tree models selected variables for skin thick-
log AUC = I + a ( log K PDMS ) + b ( log K PA )
ness, infinite vs. finite dosing, prehydration of
the membrane, or occlusion. The failure to auto- +c ( log K CW )
(6.6)
matically select for what were found to be oth-
erwise statistically significant effects may be The rank ordering of absorption, penetration, and
due to the large number of descriptors (375) peak flux across the three mixtures in IPPSF was
used in the automated procedure. The most sig- consistent: water>SLS>LAS, except for sima-
nificant variable selected in both models was zine, where SLS>water>LAS.Remarkably, rank
donor concentration, with other descriptors for ordering of logKMCF followed the same pattern
molecular size, hydrophilicity, lipophilicity, and (water>SLS>LAS) except for simazine in all of
polarizability. The regression equation also the MCFs, and propazine in just the CW MCF,
selected the difference between vehicle boiling where the ordering was water>LAS>SLS.Seeing
6 The Effects ofVehicle Mixtures onTransdermal Absorption 113
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Barton AFM (1975) Solubility parameters. Chem Rev
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Mechanistic Studies ofPermeation
Enhancers 7
S.KevinLi andWilliamI.Higuchi
etal. 1992; Yoneto etal. 1995; Warner etal. 2003; transport across skin, to determine the intrinsic
He etal. 2004). First, if mechanistic insight is to be potencies of the enhancers, and to establish a
collected directly, a symmetric and equilibrium quantitative structureenhancement relationship
configuration (with respect to the enhancer) should between the enhancers and permeation enhance-
be used. In the symmetric configuration, the ment. This chapter is a review of our studies
enhancer is present at equal concentrations in both employing this strategy and the experimental
the donor and receiver chambers of a side-by-side approaches.
diffusion cell and in equilibrium with the mem-
brane. Under these conditions, the complications
arising from enhancer concentration (or activity) 7.2 Methods
gradients across the membrane (Liu etal. 1991,
1992; Chantasart and Li 2010) can be avoided. 7.2.1 Animal Model
These enhancer gradients would otherwise lead to
a situation in which the local permeation enhance- Experiments were conducted with freshly sepa-
ment varies with the position within the membrane rated hairless mouse skin (HMS) obtained from
and make mechanistic data analysis difficult. With the abdomen region and freed from adhering fat
the symmetric configuration, the permeability and other visceral debris. HMS was selected as
coefficients obtained for the permeants can be the model for human skin for the following rea-
used directly to determine the effectiveness of an sons. HMS has relatively constant lipid content
enhancer in enhancing transdermal transport (Yoneto etal. 1998), and a large body of HMS
(enhancer potency). Second, good enhancers are data is available in the literature allowing the
usually lipophilic and relatively water-insoluble. direct comparisons of our results with those in
Because they are water-insoluble, well absorbed, previous studies. HMS stratum corneum (SC)
and need to be solubilized for effective presenta- lipid composition (Grubauer etal. 1989) is also
tion, they cannot be systematically investigated similar to that of human skin (Lampe etal. 1983).
conveniently for structureenhancement activity. In certain cases, such as for experiments requiring
Establishing equilibrium between the enhancer long-term skin stability in aqueous solution, HMS
and the membrane is therefore necessary in obtain- is not a good model of human skin (Lambert etal.
ing mechanistic insights into the action of perme- 1989). However, in the investigation of chemical
ation enhancers and for establishing a permeation enhancers for the lipoidal pathway
structureenhancement relationship. Third, model and where relatively short experimental times are
analyses to separate the effects of permeation involved, HMS can be an adequate quantitative
enhancement on transport across the lipoidal and model for human skin (Kim etal. 1992; Li etal.
pore transport pathways using model permeants of 1997). For example, Chantasart etal. (2007) com-
different polarity have been used. The effects of pared the effects of permeation enhancers on the
the enhancers upon the intercellular lipoidal and permeability coefficients of the lipoidal pathways
pore pathway transport have been delineated in of HMS and human epidermal membrane (HEM)
order to understand the mechanism of action of the under the symmetric and equilibrium configura-
enhancers. Last, in the assessment of skin perme- tion. The quantitative structureenhancement
ation enhancement, changes in the chemical relationships of the enhancers in HMS and HEM
potential (activity) of the permeant in the enhancer were also compared. The results suggest that for
solution with respect to that in the buffer solution the assessment of skin permeation across the lip-
(the control) are corrected for, so the effects of per- oidal pathway and for the mechanistic studies of
meant activity alteration upon transport in the the effects of skin permeation enhancers upon this
presence of the enhancers are taken into account. pathway, HMS can be a reliable model for human
Taking the above issues into consideration, we skin. There is no direct evidence of significant dis-
have developed a research strategy to gain mecha- crepancies between the mechanisms of action of
nistic insights into the effects of enhancers upon permeation enhancers in HMS and human skin.
7 Mechanistic Studies ofPermeation Enhancers 121
N R
N R
R R
HO
O
O
OH
N R HO
R
HO
O
O
HO
N
HO HO R
O R
n-alkyl-b-D-glucopyranosides (AG)
OH
O OH R
R OH
R
OH
R OH
R'
cis-3-alken-1-ols (CAL) Branched alkanols (bAL)
7 Mechanistic Studies ofPermeation Enhancers 123
configuration, and equilibrium between the mem- into parallel lipoidal and pore pathway compo-
brane and the enhancer solution was allowed to nents in SC via the following equation:
take place. However, in this protocol, both cham-
PSC = PL + PP (7.3)
bers of the diffusion cell were then rinsed with
PBS to remove the enhancer equilibrated in the where PL and PP are the permeability coefficients
membrane. Following the PBS rinsing regime, for the lipoidal pathway and the pore pathway
transport studies were carried out with PBS in (TEA is used as the probe permeant for estimat-
both chambers. The permeability coefficients ing the magnitude of PP), respectively, in the
obtained with PBS after pretreatment with SC.The intercellular lipid domain in SC is gener-
enhancers were then compared with those ally accepted as the lipoidal transport pathway
obtained with pretreatment with PBS only. across SC.Substituting Eq. (7.3) into Eq. (7.2)
Except for the highly lipophilic enhancers, all yields:
enhancers were tested for reversibility at enhancer
1
concentrations up to enhancement factor (E) at PT = (7.4)
1 1
E=10 (Warner etal. 2001, 2003; He etal. 2003, +
2004; Chantasart etal. 2004), and their effects PL + PP PD / E
upon permeation across SC were shown to be
essentially reversible (permeability coefficients Based on the results from previous studies, the
in PBS after enhancer pretreatment were within a use of CS as the probe permeant allows Eq. (7.4)
factor of 2 of those in PBS without enhancer to be approximated by:
pretreatment).
PT PL (7.5)
7.2.2.3 Model Description andAnalysis For other steroidal permeants, PL can be calcu-
ofExperimental Data lated by Eq. (7.4) with PD/E and PP values obtained
The permeability coefficient (P) of a probe per- from transport experiments with stripped skin
meant was calculated according to Eq. (7.1) and TEA, respectively. The equation for the lipoi-
(Warner etal. 2001): dal pathway transport enhancement factor (E) is:
1 dQ PL , X S X
P= (7.1) E= (7.6)
ACD dt PL , O SO
where A is the diffusional area of the diffusion where PL,X and PL,O are the permeability coeffi-
cell, CD is the concentration in the donor cham- cients for the lipoidal pathway when the solvent
ber, and dQ/dt is the slope of the linear region of is enhancer/PBS and PBS, respectively, and SX
the cumulative amount of permeant in receiver and SO are the permeant solubilities in enhancer/
chamber (Q) vs. time plot. PBS and in PBS, respectively. The solubility ratio
Total permeability coefficient expression for corrects for any activity coefficient differences
full-thickness skin is written as follows: between the activity coefficient in PBS and that
in the enhancer solution. Use of the solubility
1
PT = (7.2) ratio assumes that Henrys law is obeyed for the
1 1
+ permeant in both PBS and enhancer solutions
PSC PD / E (Kim etal. 1992).
where PSC is the permeability coefficient for the 7.2.2.4 Permeant Solubility
stratum corneum (SC) and PD/E is the permeabil- Determination
ity coefficient for the epidermis-dermis combina- The solubilities of the steroidal permeants in PBS
tion (D/E) and can be obtained from experiments and the enhancer solutions were determined by
of tape-stripped skin. PSC can be further divided adding excess crystals of the permeant into the
124 S.K. Li and W.I. Higuchi
enhancer solution in Pyrex culture tubes. The and dried at room temperature. After drying, the
drug suspension was shaken for 72h at 37C.The SC was kept in a freezer for later use.
culture tubes were then centrifuged for 15min at
3500rpm, and the clear supernatants were ana- 7.2.3.2 HMS SC Delipidization
lyzed for permeant concentrations with HPLC. Heptane-treated HMS SC samples were prepared
as described in the previous section. The deli-
7.2.2.5 Determination ofPartition pidized HMS SC was prepared according to the
Coefficient inBulk Organic method described previously (Yoneto etal.
Solvent/PBS Systems 1998). Briefly, dried n-heptane-treated SC sam-
Organic solvent/PBS partition coefficients were ples (about 12mg) were weighed and trans-
obtained at the aqueous enhancer concentrations ferred into 5mL CHCl3/MeOH (2:1) mixture and
corresponding to E=10 and at one tenth of the equilibrated for 48h at room temperature. The
E=10 concentration, the latter to test whether residue of SC was then rinsed several times with
Henrys law is obeyed in the two liquid phases. The fresh CHCl3/MeOH (2:1) mixture and dried
two-phase systems were maintained at 37C for under room temperature for 24h. The dried resi-
72h. Both the organic and aqueous phases were due was carefully weighed and used for the parti-
centrifuged, and aliquots were carefully withdrawn tion experiments.
from both phases and appropriately diluted for sub-
sequent analysis using HPLC or GC. 7.2.3.3 Partition Experiments
withHeptane-Treated
andDelipidized HMS SC
7.2.3 Partition Experiments Partition experiments were carried out to deter-
mine the uptake amounts of the chemical perme-
7.2.3.1 n-Heptane Treatment andSC ation enhancer and of probe permeant ES into
preparation n-heptane-treated or delipidized HMS SC.Two
Before SC preparation, HMS was rinsed with different partition experimental setups were used
heptane for 310s to remove the SC surface in our laboratory. The old setup used a Franz dif-
lipids. This rinsing protocol (the number of rinses fusion cell (Yoneto etal. 1998) and would not be
and the rinse time) was shown to remove approx- discussed here. The following is a brief descrip-
imately 20% of the SC lipids but did not disrupt tion of the other method (Chantasart etal. 2004).
the SC barrier (He etal. 2003). Similar treat- Heptane-treated SC (about 12mg) or delipidized
ments with nonpolar organic solvent were also SC sample was carefully weighed and equili-
shown to remove skin surface lipids (e.g., Abrams brated in about 20mL of enhancer solution (E=10
etal. 1993; Nicolaides 1974). SC was then pre- concentration) containing trace amounts of radio-
pared according to the method described by labeled ES (3H-ES) in a screw-capped glass vial.
Kligman and Christophers (1963) and Yoneto The vial was sealed with parafilm to prevent
etal. (1998). Briefly, the skin was placed, dermis enhancer solution evaporation and put in a water
side down, on a filter paper (quantitative filter bath with shaking at 370.1C for 12h. The 12-h
paper No. 1, Whatman) mounted on a Petri dish. incubation period was chosen because prelimi-
The Petri dish was filled with 0.2% trypsin in nary studies showed that equilibrium of enhancer
PBS solution up to the surface of SC.The Petri and 3H-ES with the SC sample took place in less
dish was covered and maintained at 37C for than 12h and that a longer incubation period
16h. When the skin membrane was placed in dis- might result in too fragile a membrane sample for
tilled water after the trypsin treatment, the dermis the partitioning experiments. After 12h, the SC
and viable epidermal layers would separate and sample was taken out from the solution by twee-
fall away from the SC.The SC was then rinsed zers and blotted by Kimwipe tissue paper. The
with distilled water several times and swabbed enhancer and ES concentrations of the solution in
with Kimwipe tissue paper to remove excess the screw-capped glass vial were checked. The
water. Then, the SC was placed on aluminum foil wet SC sample was carefully weighed in a
7 Mechanistic Studies ofPermeation Enhancers 125
snap-capped glass bottle. Then, 5mL of 100% equilibrium partition enhancement data of ES a
ethanol was added into the bottle to extract the direct correlate of the partition enhancement of
enhancer and ES from the sample for 48h at room ES in SC permeation? To address this question,
temperature with occasional gentle agitation. The the partition enhancement in SC permeation was
extracted solution was then transferred to a screw- determined in skin transport experiments using a
capped Pyrex test tube. The test tube was centri- nonsteady state transport analysis (He etal.
fuged at 3500rpm for 15min. The supernatant 2005). However, a direct comparison of the parti-
was analyzed for the enhancer by GC or HPLC tion enhancement data obtained in transport
and for ES by a scintillation counter. experiments and those data obtained in equilib-
The uptake amount of enhancer in the heptane- rium partitioning experiments of ES was not
treated SC or delipidized SC was calculated as practical due to the D/E layer being a significant
follows: barrier for ES permeation across
HMS.Furthermore, significant ES metabolism
Aextracted , i Ci
Acorrected , i = - (Wwet - Wdry ) (7.7) was observed in ES transdermal permeation.
Wdry Wdry Because of these difficulties, nonsteady state ES
transport analysis was complicated, and it was
where Aextracted, i is the amount enhancer extracted decided to employ CS as the surrogate permeant
from heptane-treated or delipidized HMS SC, for ES in the following study. The strategy here
Wdry is the dried heptane-treated or delipidized was to examine the relationship between the
SC weight, and the subscript i represents the transport partitioning enhancement of CS and the
enhancer. A correction for the enhancer in the equilibrium partitioning enhancement of ES,
aqueous compartment(s) of the SC was calcu- with the assumption that ES and CS should likely
lated according to the wet weight of SC (Wwet) behave similarly. This assumption was consid-
and the concentration of the enhancer in aqueous ered to be reasonable because previous studies
bulk phase (Ci). The partition coefficient of ES had shown similar permeability coefficient
(KES) for partitioning from the aqueous phase into enhancement effects of chemical enhancers with
n-heptane-treated SC or delipidized SC was cal- ES and CS for permeation across the lipoidal
culated as follows: pathway of HMS SC (Yoneto etal. 1995).
The skin transport model (He etal. 2005) is a
Aextracted
- (Wwet - Wdry ) Ci / Wdry S two-layer numerical transport simulation with a
K ES = X
(7.8)
C i SO least squares-fitting software Scientist
(MicroMath, Salt Lake City, UT). This model
where Aextracted is the amount of extracted 3H-ES, assumes that both SC and D/E are homogenous
Ci is the concentration of 3H-ES in aqueous and divides the SC and D/E into a sufficient num-
bulk phase, and SX and SO are the solubilities of ber of layers characterized by partition, diffusion,
ES in enhancer solution and in PBS, respec- and dimension parameters. The permeant con-
tively. The solubility ratio corrects for any activ- centration in the donor chamber was assumed
ity coefficient differences between the activity constant, which was true in all transport experi-
coefficient of ES in PBS and that in the enhancer ments carried out in the study. The receiver
solution. chamber concentration was kept at sink condi-
tions. The transport data of full-thickness HMS
7.2.3.4 Permeant Partitioning were analyzed using the model to obtain the par-
intotheTransport Rate- tition coefficient (KSC) and diffusion coefficient
Limiting Domain (DSC) of SC.The reduced parameters KSC and
andEquilibrium Permeant DSC of SC were then calculated:
Partitioning intotheStratum
DSC = DSC / L2 (7.9)
Corneum Intercellular Lipids
An important question raised in the above trans- K SC = K SC L (7.10)
port and equilibrium partition studies was: is the
126 S.K. Li and W.I. Higuchi
where L is the effective path length across ers studied. The enhancement factor profiles at
SC.These reduced parameters KSC and DSC were increasing aqueous enhancer concentrations are
defined (Okamoto etal. 1988) to avoid the diffi- essentially the same for the steroidal permeants
culty and uncertainty in assigning the L value and of different lipophilicity, suggesting the same
to minimize the number of parameters for least mechanism of permeation enhancement for these
square fitting in model analyses of the experi- steroidal permeants.
mental transport data. The enhancement of KSC
and DSC (EK, SC and ED, SC, respectively) was cal-
culated by dividing the KSC and DSC parameters 7.3.2 Effects ofAlkyl Chain Length
obtained with the enhancers at E=10 by those
with PBS control. The isoenhancement concentrations at E=10 for
more than 20 different enhancers are presented in
Fig.7.3 (Warner etal. 2003); isoenhancement con-
7.3 Results andDiscussion centration is defined as the aqueous concentrations
of enhancers to induce the same enhancement fac-
7.3.1 Isoenhancement tor and in this case E=10. These isoenhancement
Concentrations andEnhancer concentrations were interpolated from the E vs.
Effects aqueous enhancer concentration plots similar to
those in Fig.7.2. Figure7.3 shows the relationship
Figure7.2 shows a representative plot of enhance- between the E=10 enhancer concentration and the
ment factor vs. aqueous enhancer concentration carbon number of the enhancer n-alkyl group (at
for ES, CS, and HC permeation across the SC constant permeant thermodynamic activity). The
lipoidal pathway with 1-butyl-2-pyrrolidone, major conclusion deduced from the data in Fig.7.3
1-hexyl-2-pyrrolidone, and 1-octyl-2-pyrrolidone is a slope of around 0.55 found for each enhancer
as permeation enhancers (Yoneto etal. 1995). series (enhancers have the same polar head func-
Similar enhancement factor vs. aqueous enhancer tional group but different alkyl chain length) in the
concentration plots were observed for the enhanc- figure. The value of 0.55 translates into an around
AP AL API AZ AD
ES BP CS BP HC BP
1 AM AG MD MG
ES HP CS HP HC HP
15
Aqueous concentration (M)
ES OP CS OP HC OP 0.1
Enhancement factor, E
0.01
10
0.001
5 0.0001
0.00001
0 2 4 6 8 10 12 14
0.01 0.1 1 10 Carbon number
Enhancer concentration (%w/w)
Fig. 7.3 Relationships between the aqueous E=10 isoen-
Fig. 7.2 Transport enhancement factors of estradiol (ES), hancement concentrations of the enhancers and the carbon
corticosterone (CS), and hydrocortisone (HC) across the number of the enhancer alkyl chain. Each data point rep-
SC lipoidal pathway in the presence of 1-butyl-2- resents the average value without showing the standard
pyrrolidone (BP), 1-hexyl-2-pyrrolidone (HP), and deviation because the error bar generally lies within the
1-octyl-2-pyrrolidone (OP). The transport enhancement symbol in the plot. Enhancer abbreviations are provided
factors were calculated using Eq. (7.6) in Fig.7.1
7 Mechanistic Studies ofPermeation Enhancers 127
the enhancers for the steroidal permeants are n-alkanols reported in Fig.7.3 are also included
related to the enhancer lipophilicities. Together in Fig.7.6 for comparison. In Fig.7.6, it is seen
with the data analysis in Figs.7.3 and 7.4, it is that the E=10 isoenhancement concentrations of
reasonable to hypothesize that (a) permeation the cis- and trans-3-alken-1-ols are two to three
enhancement is related to the ability of the per- times higher than those of the corresponding
meation enhancer to partition into the transport n-alkanols (open squares). Based on the criterion
rate-limiting domain in SC, (b) the polar head that the E=10 isoenhancement (aqueous) con-
group assists the translocation of the enhancer to centration is a measure of the enhancer potency,
the site of action through a free energy of transfer the results in Fig.7.6 would suggest the cis- and
from the bulk aqueous phase to the transport rate- trans-3-alken-1-ols are less potent than the
limiting domain, and (c) the transport rate- n-alkanols by a factor of 2 to 3. However, when
limiting domain has a microenvironment with the E=10 isoenhancement concentrations of the
polarity similar to the polarity of bulk octanol. cis- and trans-3-alken-1-ols and of the n-alkyl
These hypotheses and the results of Fig.7.5 have enhancers are plotted against their octanol/PBS
been discussed in detail in a recent book chapter partition coefficients (Koctanol/PBS) in Fig.7.7, the
(Li and Higuchi 2015). cis- and trans-3-alken-1-ol data fall closely on
the correlation line in the figure (same correlation
line as that in Fig.7.5) when the lipophilicity of
7.3.4 E
ffects ofHydrocarbon Chain the enhancers is taken into consideration. The
CarbonCarbon Double Bond correlation between the E=10 isoenhancement
concentration and octanol/PBS partition coeffi-
The effects of substituting a single carboncar- cient here is consistent with the demonstrated
bon bond on the n-alkyl chain of an enhancer structureenhancement relationship for the
with a carboncarbon double bond have been n-alkyl enhancers in Fig.7.5.
investigated, and the E=10 isoenhancement con- In the equilibrium partition experiments of the
centrations of the cis-and trans-3-alken-1-ols cis- and trans-3-alken-1-ols, the concentrations
(closed symbols) are plotted against the carbon of the enhancers in the SC intercellular lipid
numbers of the enhancer hydrocarbon chains in
Fig. 7.6 (He etal. 2004). The data for the
AL
1
CAL
Log (aqueous concentration) (M)
0 AP AL API AZ AD TAL
Aqueous concentration (M)
AM AG MD MG
0.1
1
2 0.01
3
0.001
4
5 0.0001
0.5 1.5 2.5 3.5 4.5 5.5 2 4 6 8 10 12
Log Koctanol/PBS Carbon number
Fig. 7.5 Correlation between the aqueous E=10 isoen- Fig. 7.6 Relationships between the aqueous E=10 isoen-
hancement concentration of the enhancer and its octanol/ hancement concentrations of the enhancers and the carbon
PBS partition coefficient (Koctanol/PBS). The slope of the line number of the enhancer alkyl chain. Each data point rep-
is 1. Each data point represents the average value with- resents the average value without showing the standard
out showing the standard deviation because the error bar deviation because the error bar generally lies within the
generally lies within the symbol in the plot. Enhancer symbol in the plot. Enhancer abbreviations are provided
abbreviations are provided in Fig.7.1 in Fig.7.1
7 Mechanistic Studies ofPermeation Enhancers 129
domain under the isoenhancement E=10 potent than enhancers with saturated hydrocar-
conditions are essentially constant (Fig.7.8). The bon chains based on molecular modeling of skin
substitution of a single carboncarbon bond with permeation and previous experimental results
a carboncarbon double bond on the alkyl chain (Golden etal. 1987; Aungst 1989; Brain and
here has little effect upon the enhancer potency Walters 1993; Tenjarla etal. 1999).
based on the concentrations of the enhancers in
the SC intercellular lipid domain. This is some-
what surprising because enhancers with unsatu- 7.3.5 E
ffects ofBranched Alkyl
rated hydrocarbon chains are expected to be more Chain
0
AD AM AG MD 4-alkanols, and 5-nonanol) were also investigated
MG CAL TAL
1 as another group of skin permeation enhancers to
provide insights into the mechanism of action of
2 both n-alkyl and branched chain enhancers
3
(Chantasart etal. 2004). The 2-alkanols,
3-alkanols, and 4-alkanols are 2-hexanol,
4 2-
heptanol, 2-octanol, 2-nonanol; 3-hexanol,
3-heptanol, 3-octanol, 3-nonanol; and 4-heptanol,
5 4-octanol, and 4-nonanol, respectively. In
0.5 1.5 2.5 3.5 4.5 5.5
Fig. 7.9, the isoenhancement concentrations at
Log Koctanol/PBS
E=10 of the branched alkanols (closed symbols)
Fig. 7.7 Correlation between the aqueous E=10 isoen- are plotted against their Koctanol/PBS values. Again,
hancement concentration of the enhancer and its octanol/ the data of the n-alkyl enhancers (including the
PBS partition coefficient (Koctanol/PBS). Each data point rep- n-alkanols) are included in Fig.7.9, and the
resents the average value without showing the standard
deviation because the error bar generally lies within the
straight line shown in the figure is a best fit line
symbol in the plot. The slope of the line is 1. Enhancer based on the data for more than 20 n-alkyl
abbreviations are provided in Fig.7.1 enhancers in Fig.7.5. Different from the random
deviations for the n-alkyl enhancers (crosses), the
AP AL API AZ AD
10 2-alkanols
AM AG CAL TAL
3-alkanols
(micromole/mg membrane)
1
Membrane concentration
4-alkanols
1 5-nonanol
1.5
n-alkyl enhancers
0.1 2
2.5
0.01
3
0.001 3.5
2 4 6 8 10 12 14 1 1.5 2 2.5 3 3.5 4
Carbon number Log Koctanol/PBS
Fig. 7.8 Relationship between the enhancer concentra- Fig. 7.9 Correlation between the aqueous E=10 isoen-
tions in the intercellular lipid domain of the SC membrane hancement concentration of the enhancer and its octanol/
at the E=10 isoenhancement conditions and the carbon PBS partition coefficient (Koctanol/PBS). Each data point rep-
number of the enhancer alkyl chain. Each data point rep- resents the average value without showing the standard
resents the average value. Enhancer abbreviations are pro- deviation because the error bar generally lies within the
vided in Fig.7.1 symbol in the plot. The slope of the line is 1
130 S.K. Li and W.I. Higuchi
n-alkyl enhancers
than those of the n-alkyl enhancers (crosses in the
Membrane concentration
1
figure). This result is consistent with the relatively
lower intrinsic potency of the branched chain
0.1 alkanols suggested with the data in Fig.7.9.
Despite the observed deviation of the
0.01 branched chain alkanols from the n-alkyl chain
enhancers, it should be noted that the correlation
between the logarithm of the enhancer partition
0.001
0.5 1.5 2.5 3.5 4.5 5.5 coefficient from the aqueous phase to the SC
Log Koctanol/PBS intercellular lipid phase (log KSC lipid/PBS) and log
Koctanol/PBS continues to hold for the branched
Fig. 7.10 Relationship between the enhancer concentra- chain alkanols. The microenvironment of the
tions in the intercellular lipid domain of the SC membrane enhancer site of action remains essentially the
at the E=10 isoenhancement conditions and the octanol/
PBS partition coefficient (Koctanol/PBS) of the enhancers. same and independent of alkyl-chain branching;
Each data point represents the average value the n-alkanols, branched chain alkanols, and all
other studied enhancers fall on the same regres-
sion line (Fig.5.9 in a recent book chapter;
branched chain alkanols (closed symbols) show Li and Higuchi 2015).
modest but consistent positive deviations (in the
direction of lower potency) from the quantitative
structureenhancement correlation (the straight 7.3.6 Equilibrium Partition
line). These deviations support the view, based Enhancement ofES intoSC
on the assumption that Koctanol/PBS is a valid predic- Intercellular Lipids
tor of enhancer potency, that the branched chain
alkanols are slightly less potent than the n-alkyl In addition to the equilibrium partition experi-
enhancers. The lower potencies based on the ments of the enhancers, experiments were also
E=10 aqueous concentrations of the branched conducted with a model steroidal compound ES
chain alkanols could be attributed to decreasing (He etal. 2003, 2004; Chantasart etal. 2004).
intrinsic potency and increasing effective hydro- The goal here was to determine the enhancement
philicity of the enhancers when the hydroxyl of the partitioning of a lipophilic permeant into
group moves from the terminal end towards the the SC intercellular lipids under the isoenhance-
center of the enhancer alkyl chain. Nevertheless, ment E=10 conditions. Figure 7.11 shows the
the results of the branched chain alkanols con- plots of the partition coefficients of ES from the
tinue to support the hypothesis previously estab- aqueous phase into the SC intercellular lipid
lished for the n-alkyl enhancers that the potency domain (KES) under the E=10 conditions of more
of an enhancer based on its aqueous concentra- than 20 different enhancers vs. the Koctanol/PBS val-
tion increases with enhancer lipophilicity. ues of the enhancers. The KES values were deter-
Figure 7.10 presents the concentrations of the mined with Eq. (7.8). The dotted line represents
branched chain alkanols and n-alkyl enhancers in the KES value in PBS control. As can been seen in
the SC intercellular lipids under the isoenhance- the figure, approximately the same enhancement
ment conditions of E=10. Whereas the intrinsic of KES (four- to sevenfold) was induced under the
potencies of the n-alkyl enhancers are essentially isoenhancement conditions of E=10 for all the
the same and independent of their alkyl chain enhancers studied. The relatively constant four-
length, branching of the alkyl chain decreases to sevenfold enhancement in permeant partition-
the intrinsic potencies of the enhancers; the ing suggests that (a) the same target site in the SC
7 Mechanistic Studies ofPermeation Enhancers 131
Fig. 7.12Chemical
OH OH OH
structures of the probe
permeants Ethanol Propanol Butanol
HO H2N O
OH
O
O NH
OH O
HO
2 - Acetamidophenol 4 - Ipomeanol
HO O
OH
HO
O
OH
Kaempferol
OH
O OH
HO OH
H H
H H H H
O HO
Hydrocortisone Estradiol
and ES were also employed to examine the gen- Xiang and Anderson 1994), and when enhancers
erality of the transport enhancement results, and fluidize the SC lipids, the increase in the bilayer
essentially the same permeation enhancement free volume can have different consequences on
was observed with all three steroidal permeants transport enhancement of permeants with differ-
(e.g., Fig.7.2). However, the physiochemical ent molecular sizes (Mitragotri 2001).
properties of a permeant can influence the trans- To examine the effects of permeant molecular
port of the permeant across SC.For example, it is size upon transport enhancement, transport
general knowledge that there may be a steep- experiments were conducted using permeants of
permeant molecular size dependence in perme- different molecular sizes and lipophilicities
ation across lipid bilayers (e.g., Stein 1986; (Fig.7.12) under the E=10 enhancer conditions
7 Mechanistic Studies ofPermeation Enhancers 133
10 OP subject is required.
8
6
7.3.9 Permeation Enhancers
4 inaNonaqueous System
2
inTransdermal Drug Delivery
Enhancer
concentration Ci CD, membrane
Enhancer B
Enhancer A
Blood sink Combined SC Patch
viable
epidermis and
some dermis
Fig. 7.14Enhancer concentration profiles in SC in account for (a) enhancer- induced variation inlocal
transdermal drug delivery (see Eq.7.2): dotted line, enhancement (permeation and partition enhancement) at
Enhancer A; dashed line, Enhancer B. PD/E for Enhancers different locations within the SC and (b) enhancer-
A and B are the same, log Koctanol/PBS for Enhancer induced enhancement for the enhancer. Such enhance-
A>Enhancer B, and PSC for Enhancer A>Enhancer ment will affect the enhancer concentration in SC and
B.This analysis assumes SC is homogenous and does not lead to nonlinear profiles
p ermeability across the SC, a much more signifi- for skin permeation under the symmetric and
cant concentration drop across the SC is observed. asymmetric configurations are likely to be the
Thus, a large portion of the SC is not affected by same and supports the utility of the findings in
Enhancer B and this region of the SC becomes the symmetric transport studies for skin perme-
the rate-limiting barrier for drug transport. The ation under the asymmetric configurations in
relatively constant concentration of Enhancer A practice.
in the SC would suggest that lipophilic enhancers
are likely to be more effective in providing uni- Conclusion
form transport enhancement over the entire SC New insights into the factors influencing the
and a high overall flux enhancement of drug effectiveness of chemical permeation enhanc-
transport across SC.However, simply applying ers for the lipoidal pathway of the SC have
the most lipophilic enhancer does not guarantee been obtained. The present study supports the
success. The solubility of the enhancer and deple- view that (a) the potency of an n-alkyl
tion of the enhancer in the transdermal patch are enhancer (based on its aqueous concentration)
other factors that need to be considered. is related to the enhancer lipophilicity, this
The appropriateness of extrapolating the per- being the case because of the lipophilic nature
meation experiment findings under the s ymmetric of the enhancer site of action, which is well
and equilibrium enhancer configuration to the mimicked by liquid n-octanol; (b) the intrinsic
asymmetric situation in practice has been exam- potency of the enhancer (as represented by its
ined in a recent study (Chantasart and Li 2010). concentration at the target site of action) is
In this study, the effects of enhancers upon the relatively independent of its lipophilicity; (c)
permeation of CS across HEM under the sym- the substitution of a carboncarbon single
metric and asymmetric configurations were com- bond on the hydrocarbon chain of the enhancer
pared. The data show a correlation between with a carboncarbon double bond does not
transdermal permeation enhancement under the significantly affect its intrinsic potency; and
symmetric and asymmetric configurations. This (d) with modest effects, branching of the
suggests that the mechanisms of the enhancers n-alkyl chain of the enhancer generally
7 Mechanistic Studies ofPermeation Enhancers 135
reduces the intrinsic potency of the enhancer. Grubauer G, Feingold KR, Harris RM, Elias PM (1989)
To date, we have not encountered any enhancer Lipid content and lipid type as determinants of the epi-
dermal permeability barrier. JLipid Res 30:8996
candidates that are inconsistent with this view Hadgraft J(2001) Modulation of the barrier function of the
in our research. However, skin penetration skin. Skin Pharmacol Appl Skin Physiol 14(Suppl 1):
retarders have been reported (e.g., Hadgraft 7281
etal. 1996). This suggests that further studies Hadgraft J, Peck J, Williams DG, Pugh J, Allan G (1996)
Mechanisms of action of skin penetration enhancers/
are needed for greater generalization of the retarders: azone and analogues. Int JPharm 141:1725
present findings. Nevertheless, the present He N, Li SK, Suhonen TM, Warner KS, Higuchi WI
study has demonstrated useful concepts and (2003) Mechanistic study of alkyl azacycloheptanones
effective methodologies for mechanistic stud- as skin permeation enhancers by permeation and parti-
tion experiments with hairless mouse skin. JPharm
ies of chemical permeation enhancers. Sci 92:297310
He N, Warner KS, Chantasart D, Shaker DS, Higuchi WI,
AcknowledgmentsThe authors thank Drs. Kevin Li SK (2004) Mechanistic study of chemical skin per-
S.Warner, Ning He, Doungdaw Chantasart, and Sarah meation enhancers with different polar and lipophilic
A.Ibrahim for their contributions in the project and the functional groups. JPharm Sci 93:14151430
financial support by NIH Grants GM 043181 and GM He N, Warner KS, Higuchi WI, Li SK (2005) Model analy-
063559. sis of flux enhancement across hairless mouse skin
induced by chemical permeation enhancers. Int JPharm
297:921
Ibrahim SA, Li SK (2009a) Effects of chemical enhancers
on human epidermal membrane: structure-enhancement
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High Throughput Screening
ofTransdermal Penetration 8
Enhancers: Opportunities,
Methods, andApplications
All of the above techniques have their own one has to rely on a brute force screening
benefits and specific applications. Formulation- approach. Starting with a pool of >200 individual
based approaches have a number of unique CPEs, millions of binary and billions of higher
advantages such as design simplicity and flexi- order formulations can be designed. Screening of
bility, and ease of application over a large area these mixtures is a mammoth task.
(Prausnitz etal. 2004). The last 20 years have Screening of chemical enhancers can be per-
seen extensive research in the field of chemical formed invitro as well as invivo. In vivo experi-
enhancers, which form the core component of ments are likely to yield better results; however,
formulation-based strategies for transdermal several issues including variability, cost, and
drug delivery. More than 200 chemicals have practicality limit their applications for screening
been shown to enhance skin permeability to a large database of enhancers. Accordingly,
various drugs. These include molecules from a invitro screening based on excised tissue (human
diverse group of chemicals including fatty acids or animal) presents a more practical alternative
(Golden etal. 1987; Aungst etal. 1990; Jain (Priborsky and Muhlbachova 1990). A number of
and Panchagnula 2003), fatty esters models exist to predict invivo pharmacokinetics
(Chukwumerije etal. 1989), nonionic surfac- based on invitro data (Naito and Tsai 1981; Guy
tants (Lopez etal. 2000), anionic surfactants etal. 1982; Ogiso etal. 1989; Takayama and
(Nokhodchi etal. 2003), and terpenes (Williams Nagai 1991; Wu etal. 2000). The use of invitro
and Barry 1991; Jain etal. 2002). However, models for screening is also supported by the fact
identification of potent yet safe permeation that SC, the principle site of enhancer action,
enhancers has proved challenging. To date, shows similar behavior invivo and invitro except
only few chemicals are to be found in currently for the extent of metabolic activity (Chang
marketed transdermal products. These include etal. 1998). Majority of invitro studies on trans-
oleic acid, sorbitan monooleate, and methyl dermal drug transport have been performed using
laurate among others. Franz diffusion cells (FDCs). The throughput of
Even though individual chemical penetration this traditional setup of diffusion chamber is very
enhancers (CPEs) have found limited applica- low, not more than 1015 experiments at a time.
tions, combinations of CPEs represent a huge These permeation studies are time consuming
opportunity that has been sparsely tapped. and are resource expensive as analytical methods
Several reports have indicated that combinations such as high-pressure liquid chromatography
of CPEs offer better enhancements of transder- (HPLC) or radiolabeled drugs for liquid scintilla-
mal drug transport compared to their individual tion counting are expensive. Automated in-line
constituents (Mitragotri 2000; Thomas and flow through diffusion cells has been developed
Panchagnula 2003). However, such combinations in the last few years to increase the throughput of
do not necessarily yield safer enhancers. It should skin permeation experiments (Bosman etal.
be feasible, in principle, to use CPEs as building 1996; Cordoba-Diaz etal. 2000). Although these
blocks to construct new microstructures and methods have facilitated the experiments, the
novel formulations that offer enhancement with- throughput of these methods has not been signifi-
out irritation. However, the challenge now shifts cantly improved. Furthermore, these methods are
to screening the potency of enhancer combina- also cost prohibitive. Accordingly, standard
tions. Random mixtures of CPEs are likely to FDCs still dominate the screening of CPEs.
exhibit additive properties, that is, their potency The urgent need to increase experimental
and irritancy are likely to be averages of corre- throughput has led to the development of high
sponding properties of their individual constitu- throughput screening methods. Though in early
ents. Occurrence of truly synergistic combinations stages, these methods have already shown prom-
is likely to be rare. In the absence of capabilities ise in discovering novel formulations for trans-
to predict the occurrence of such rare mixtures, dermal drug delivery. A high throughput assay to
8 High Throughput Screening ofTransdermal Penetration Enhancers 139
Electrode
Donor
Skin
Receiver
~
Electrode
Fig. 8.1 Schematic of the INSIGHT screening apparatus. and the formulations contact the stratum corneum from the
The INSIGHT screen is made up of a donor array (top) and donor array. Conductivity measurements are made with one
a symmetric receiver array (bottom). A single screen can electrode inserted in the dermis and a second electrode
screen 100 formulations at one time. The skin is sandwiched moved sequentially in the donor wells. (a) and (b) is top and
between the donor (Teflon) and receiver (Polycarbonate) side view of the INSIGHT apparatus, respectively
Throughput screening that was recently intro- efficiency comes from two factors. First, the
duced (Karande etal. 2004). This method is INSIGHT, in its current version, can perform up
described in detail with respect to its fundamen- to 50 tests per square inch of skin compared to
tals, validation, and outcomes. about >2cm2 of skin per test in the case of Franz
diffusion cells (Fig.8.1). About 100 formulations
can be screened per INSIGHT array. Second,
8.2 verview ofINSIGHT
O INSIGHT screening uses skin impedance as a
Screening surrogate marker for skin permeability.
Skin impedance has been previously used: (i)
INSIGHT screening offers >100-fold improve- to assess the skin integrity for invitro dermal test-
ment in screening rates of transdermal formula- ing (Lawrence 1997; Fasano etal. 2002; Davies
tions (Karande etal. 2004). This improvement in etal. 2004), (ii) to evaluate the irritation potential
8 High Throughput Screening ofTransdermal Penetration Enhancers 141
of chemicals in a test known as skin integrity func- electrical impedance and solute permeability is
tion test (SIFT) (Heylings etal. 2001), and (iii) to stronger at lower frequencies. Thus, an optimal
monitor skin barrier recovery invivo following the frequency must be chosen. All experiments
application of current during iontophoresis (Turner reported in chapter were performed at a fre-
etal. 1997; Curdy etal. 2002). Since it is evident quency of 100Hz.
from the literature that skin impedance can be used INSIGHT screening is founded on the rela-
to confirm skin integrity, it is logical to hypothe- tionship between skins electrical impedance
size that alteration in skin barrier due to chemical (reciprocal of skin conductance) and solute per-
enhancers can be used as an invitro surrogate meability. There is a dearth of literature on skin
marker for permeability. Scattered literature data impedance (conductivity) and permeability rela-
support this hypothesis. A study by Yamamoto tionship, and moreover in most of the studies this
and Yamamoto (1976a, b) showed that total skin relationship was used to elucidate the mechanism
impedance reduces gradually with tape stripping of transport of hydrophilic molecules across the
and after 15 stripping skin impedance approaches skin under the influence of temperature (Peck
the impedance value of deep tissues (Yamamoto etal. 1995), hydration (Tang etal. 2002), electric
and Yamamoto 1976a, b). However, quantitative current (Sims etal. 1991; Li etal. 1998), or ultra-
relationships between skin impedance and sonic waves (Tang etal. 2001; Tezel etal. 2003).
permeability in the presence of chemical enhanc- Therefore existing data cannot be used to gener-
ers and their validity for a wide range of markers alize the relationship between skin impedance
have not been previously documented. and permeability. Accordingly, a large dataset
was first generated to assess the correlation
between skin impedance and permeability to
8.2.1 Skin ImpedanceSkin small (mannitol) and macromolecule (inulin)
Permeability Correlation hydrophilic solutes in the presence of different
chemical enhancer formulations. A set of 22
Stratum corneum is a composite of proteins and enhancer formulations, chosen from the candi-
lipids in which protein-rich corneocytes are sur- date pool was used to validate the relationship
rounded by lipid bilayers (Madison etal. 1987). between skin conductivity and skin permeability.
Approximately 70100 bilayers are stacked The candidate pool comprised of 15 single
between two corneocytes (Elias etal. 1977; Elias enhancer formulations and seven binary enhancer
1983). Because of its architecture the SC is rela- formulations. In order to establish the correlation
tively nonconductive and possesses high electri- between skin impedance and permeability for
cal impedance (Lackermeier etal. 1999). Skin wide range of chemical enhancers, formulations
impedance (AC) can be measured either by were made from different classes of chemicals
applying a constant current and measuring the including cationic surfactants (CTAB, cetyl tri-
potential across the skin or by measuring tran- methyl ammonium bromide; BDAC, benzyl
sepidermal current following the application of a dodecyl ammonium chloride), anionic surfac-
constant AC potential. Data reported in this chap- tants (NLS, N-lauorylsarcosine sodium; SLA,
ter are based on measurement of transepidermal sodium laureth sulfate; SLS, sodium lauryl sul-
current following the application of a constant fate), zwitterionic surfactants (HPS, N-hexadecyl-
potential (100mV rms). Frequency of the applied N,N-dimethyl-3-ammonio-1-propanesulfonate),
potential is also an important parameter. Due to nonionic surfactants (PEGE, polyethylene
the capacitive components of the skin, the mea- dodecyl glycol ether; S20, sorbitan monolaurate;
sured electrical impedance of the skin decreases T20, polyoxyethylene sorbitan monolaurate),
with increasing frequency (Yamamoto and fatty acids and their sodium salts (LA, lauric
Yamamoto 1976a, b). While the use of higher fre- acid; OL, oleic acid; SOS, sodium octyl sulfate;
quencies facilitates measurements due to SO, sodium oleate), fatty acid esters (TET, tetra-
decreased impedance, the correlation between caine HCl; IPM, isopropyl myristate), and others
142 A. Jain et al.
a b
4 10-4
10-3
Inulin skin permeability (cm/hr)
10-4
7 10-5
10-5
4 10-5
10-6
10 100 1 10 100
Skin impedance (k-cm2) Skin impedance (k-cm2)
Fig. 8.2 Skin impedancepermeability correlation for (a) inu- -TET:HPS (2.0 %w/v, 0.1:0.9); -MEN:T20 (2.0%w/v,
lin and (b) mannitol. Test formulations used in this study (in 0.5:0.5); -DMP:TET (2.0 %w/v, 0.4:0.6); -CTAB
parenthesis total concentration of chemical enhancer w/v, (1.0%w/v). (b) -OL (1.5%w/v); -DMP (2.0%w/v);
weight fraction used): (a) -MEN (1.5%w/v); -SO -DMP:TET (2.0%w/v, 0.4:0.6); -PEGE (1.5%w/v); -TET
(1.5%w/v); -PEGE (1.5%w/v); -OL (1.5%w/v); -S20 (2.0%w/v); -LA (1.5%w/v); -S20 (1.0%w/v); -HPS
(1.0%w/v); -DMP (1.5%w/v); -OL:MEN (1.5%w/v, (1.5%w/v); -NLS (1.0%w/v); -BDAC (1.5%w/v); -
0.4:0.6); -IPM (1.5%w/v); -TET (2.0-%w/v); -LA MEN (1.5%w/v); -DMP:HPS (1.5%w/v, 0.6:0.4); -NLS:S20
(1.5%w/v); -NLS (1.0%w/v); -SOS (2.0%w/v); - (1.0%w/v, 0.6:0.4); -DMP (1.5%w/v)
NLS:S20 (1.0%w/v, 0.6:0.4); -TET:SLS (1.0%w/v, 0.6:0.4);
10-3
a 5 10-4 b
3 10-4
10-4
10-4
8 10-5
6 10-5
2 4 6 8 10
5 6 7 8 9 10 20 30
Skin impedance (k-cm2)
Skin impedance (k-cm2)
Fig. 8.3Skin impedance-permeability correlation for skin impedance in the presence of NLS (1.5%w/v in 1:1
single enhancer. (a) Plot of skin permeability to inulin vs. EtOH:PBS). A much tighter correlation can be observed
skin impedance in the presence of DMP (1.5%w/v in 1:1 compared to Fig.8.2
EtOH:PBS); (b) Plot of skin permeability to mannitol vs.
10-3
a b
4 10-4
Mannitol skin permeability (cm/hr)
3 10-4
Inulin skin permeability (cm/hr)
10-4
2 10-4
10-4 10-5
9 10-5
8 10-5
7 10-5
6 10-5
5 10-5 10-6
10 100 1 10 100
Skin impedance (k-cm2) Skin impedance (k-cm2)
Fig. 8.4 Skin impedance permeability correlation for different enhancers are grouped in the bins of 5 k-cm2
(a) inulin and (b) mannitol. Modified plot of permeability- along the x-axis representing skin impedance. The corre-
impedance data shown in Fig.8.2. Permeability data for lation is much tighter as compared to the one in Fig.8.2
144 A. Jain et al.
100 45
1000
Formulation rank based on 4 h Incubation
40
80
FDC permeability enhancement
35
30 100
60
25
40 20
10
15
20
10
0 5 1
0 10 20 30 40 50 60 70 1 10 100 1000
INSIGHT conductivity ER at 24 h Formulation rank based on 24 h Incubation
A B
Fig. 8.5Validation of INSIGHT predictions with cate conductivity enhancement numbers, and the filled
FDC.Plot of conductivity enhancement ratios in circles indicate permeability enhancement numbers in
INSIGHT at 24h vs. conductivity and permeability FDC. (a) Plot of 24h predictions in INSIGHT vs. 4h pre-
enhancement ratios in FDC at 96h for 19 enhancer formu- dictions in INSIGHT on the potency of enhancer formula-
lations. A strong linear correlation indicates the validity of tions. A strong correlation indicates that predictions on
observations in INSIGHT when compared with those potency of formulations can be obtained at significantly
from traditional tools like FDC.The closed circles indi- lower incubation periods of 4h
8 High Throughput Screening ofTransdermal Penetration Enhancers 145
incubation are demonstrated in Fig.8.5b where from 32 chemicals chosen from a list of >250
potency rankings of 438 single and binary formu- chemical enhancers belonging to various catego-
lations randomly prepared from the enhancer ries. Random pairing of CPEs from various cate-
library based on 4-h screening are compared to gories led to 496 binary chemical enhancers
those based on 24-h screening. Rank 1 corre- pairs. For each pair, 44 distinct chemical compo-
sponds to most potent formulation in the library sitions were created with the concentration of
and rank 438 to the weakest formulation. The each chemical enhancer ranging from 02%w/v,
predictions of the potency made in 4 h were con- yielding a library of 25,000 candidate SCOPE
sistent with those made after a contact time of formulations. About 20% of this library (5,040
24h, thus indicating that the efficiency of formulations) was screened using INSIGHT, the
INSIGHT screening can be further improved. largest ever-cohesive screening study reported in
the transdermal literature. Each formulation was
tested at least four times in over 20,000 experi-
8.4 pplications ofINSIGHT
A ments (Karande etal. 2004). Using the traditional
Screening tools for formulation screening, it would have
taken over 7 years to do these many experiments.
8.4.1 D
iscovery ofRare With INSIGHT screening, the same task was
Formulations accomplished in about 2 months with screening
rate of 5001,000 experiments per day.
INSIGHT screening can be used to screen huge Binary formulations exhibited a wide range of
libraries of chemicals within a short span of time enhancements. The percent of randomly gener-
and without the fear of failure that exists with tradi- ated enhancer combinations that exhibit ER
tional tools. Many current single enhancers are also above a certain threshold decreases rapidly with
potent irritants to the skin at concentrations neces- increasing threshold (Fig.8.6a). The inset shows
sary to induce meaningful penetration enhance- a section of the main figure corresponding to high
ment. Attempts have been made to synthesize ER values. Less than 0.1 % of formulations
novel chemical enhancers such as laurocapram exhibited more than 60-fold enhancement of skin
(Azone); however, achieving sufficient potency conductivity. Discovery of such rare formula-
without irritancy has proved challenging, espe- tions by brute force experimentation is contin-
cially for macromolecules. A number of studies gent on the throughput of the experimental tool.
have shown that formulations made up of combina- INSIGHT screening opens up the possibility of
tion of chemical enhancers are more potent than its discovering such rare formulations.
individual components (Karande etal. 2004; Tezel One of the formulations discovered by INSIGHT,
etal. 2002; Mitragotri 2000). The addition of com- SLA:PP (sodium laureth sulfate:phenyl piperazine)
ponents increases the number of formulations was shown to increase the permeability of macro-
exponentially. However, the use of INSIGHT molecules such as inulin across porcine skin by
screening allows one to tackle this challenge in a 80100 fold compared to passive skin permeability
more cost-effective way compared to FDCs. In of inulin (Karande etal. 2004). SLA:PP also
addition, synergies between CPEs not only lead to increased the skin permeability of molecules such
new transdermal formulations but also potentially as methotrexate, low molecular weight heparin,
offer insight into mechanisms by which CPEs luteinizing hormone releasing hormone (LHRH),
enhance skin permeability. Prediction of synergies and oligonucleotides by 50100 fold. Animal
from the first principles is challenging. INSIGHT experiments in hairless rats also confirmed delivery
screening offers an effective tool for identifying of a synthetic analogue of LHRH, leuprolide acetate
synergies (positive or negative) between the CPEs. invivo. The amount of leuprolide acetate delivered
To identify synergistic combinations of pene- using a SCOPE formulation (SLA:PP) is signifi-
tration enhancers (SCOPE) formulations, a cantly higher than that delivered from a control
library of chemical enhancers was first generated solution and lies in the therapeutic window.
146 A. Jain et al.
35% 2.0
Percent of enhancer formulations
10%
30% Percent of enhancer formulations
Total conc.(%w/v)
1%
25%
20% 0.1%
15% 0.01%
30 40 50 60
Conductivity enhancement ratio
10%
5%
0
0% 0 1.0
0 10 20 30 40 50 60 Fraction of MP
Conductivity enhancement ratio
A B
Insight
Bulk
Discrete observable
structural parametrical
behavioral
QSAR
Relating activity to structure
Fig. 8.6 Applications of INSIGHT screening. (a) Discovery screening is used to quantify the extent of interactions
of rare enhancer formulations that is significantly potent in between the components of CPE mixtures in terms of
increasing skin permeability. Such formulations are difficult Synergy. Regions of high synergy almost always overlap
to discover using the traditional tools like FDC due to their with the regions of high potency. (c) INSIGHT screening
low experimental throughput. The success rate of discover- can be used to generate large volumes of data on the interac-
ing these potent formulations is very small (~0.1%) requir- tion of CPEs with skin. The information is used to relate
ing a tool with high experimental throughput. (b) INSIGHT chemistry of the enhancer to its potency using QSAR
8.4.2 G
eneration ofDatabase ers will provide, for the first time, a platform to
forQuantitative build further investigations of the fundamental
Understanding aspects of enhancer-skin interactions. Quantitative
descriptions of structure-activity relations
Looking beyond searching for potent combina- (QSARs) for CPEs, which have had limited suc-
tions of enhancers, the sheer volume of informa- cess in the past (Moss etal. 2002; Walker etal.
tion generated via INSIGHT screening on the 2003), may lead to better outcomes in light of the
behavior of a wide variety of penetration enhanc- availability of large volumes of data collected in a
8 High Throughput Screening ofTransdermal Penetration Enhancers 147
consistent manner. As exemplified in Fig.8.6, this Elias PM, McNutt NS etal (1977) Membrane alterations
during cornification of mammalian squamous epithe-
information should help in generating hypotheses
lia: a freeze-fracture, tracer, and thin-section study.
relating the chemistry of CPEs to their potencies. Anat Rec 189(4):577594
For working hypotheses, this knowledge can then Fasano WJ, Manning LA etal (2002) Rapid integrity
help refine our selection rules for designing next assessment of rat and human epidermal membranes
for invitro dermal regulatory testing: correlation of
generation transdermal formulations. Repeating
electrical resistance with tritiated water permeability.
the experiment-hypothesis loop over a vast but Toxicol In Vitro 16(6):731740
limited number of candidate penetration enhanc- Francoeur ML, Golden GM etal (1990) Oleic acid: its
ers will provide the missing pieces in solving a effects on stratum corneum in relation to (trans)dermal
drug delivery. Pharm Res 7(6):621627
vast multivariate problem. Also, this knowledge
Golden GM, McKie JE etal (1987) Role of stratum cor-
should significantly reduce the cost and effort of neum lipid fluidity in transdermal drug flux. JPharm
designing therapeutics for use on skin in the future. Sci 76(1):2528
Guy RH, Hadgraft Jetal (1982) A pharmacokinetic
model for percutaneous bsorption. Int JPharm 11:
119129
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Priborsky J, Muhlbachova E (1990) Evaluation of in-vitro Weaver JC, Vaughan TE etal (1999) Theory of electrical
percutaneous absorption across human skin and in ani- creation of aqueous pathways across skin transport
mal models. JPharm Pharmacol 42(7):468472 barriers. Adv Drug Deliv Rev 35(1):2139
Rosado C, Cross SE etal (2003) Effect of vehicle pretreat- Williams AC, Barry BW (1991) Terpenes and the lipid-
ment on the flux, retention, and diffusion of topically protein- partitioning theory of skin penetration
applied penetrants invitro. Pharm Res 20(9):15021507 enhancement. Pharm Res 8(1):1724
8 High Throughput Screening ofTransdermal Penetration Enhancers 149
Williams AC, Barry BW (2004) Penetration enhancers. Yamamoto T, Yamamoto Y (1976a) Dielectric constant
Adv Drug Deliv Rev 56(5):603618 and resistivity of epidermal stratum corneum. Med
Wu PC, Huang YB etal (2000) Evaluation of pharmaco- Biol Eng 14(5):494500
kinetics and pharmacodynamics of captopril from Yamamoto T, Yamamoto Y (1976b) Electrical properties
transdermal hydrophilic gels in normotensive rabbits of the epidermal stratum corneum. Med Biol Eng
and spontaneously hypertensive rats. Int JPharm 14(2):151158
209(12):8794 Yamane MA, Williams AC etal (1995) Terpene penetra-
Xing QF, Lin S etal (1998) Transdermal testosterone tion enhancers in propylene glycol/water co-solvent
delivery in castrated Yucatan minipigs: pharmacokinet- systems: effectiveness and mechanism of action.
ics and metabolism. JControl Release 52(12):8998 JPharm Pharmacol 47(12A):978989
Part II
Methods for Measuring the Percutaneous
Drug Penetration
Models, Methods,
andMeasurements inTransdermal 9
Drug Delivery
DonaldM.Cropek andPankajKarande
nated by the oral route, the next millennia of providing sustained drug release for a prolonged
health care will demand more accommodating period of time. It is painless compared to needles
delivery systems for sensitive drug classes. and therefore offers superior patient compatibil-
Injections comprise the next most commonly ity. However, skin is the first line of defense of an
used method for administering therapeutics into organism and the last barrier separating the
humans. The World Health Organization (WHO) organism from its hostile environment of viruses,
estimates that 12 billion injections are given pathogens, and toxics. Evolved to impede the
annually (Kermode 2004). Despite the common flux of exogenous molecules into the body, the
use, needle-based drug administration has several skin naturally offers a very low permeability to
limitations. Needle phobia is a significant issue in the movement of foreign molecules across it. A
adults and children alike (Nir etal. 2003) and unique hierarchical structure of lipid-rich matrix
makes drug administration stressful (Breau etal. with embedded keratinocytes in the upper strata
2001). Accidental needle sticks also add to the (15m) of skin, stratum corneum (SC), is respon-
limitations of needle use in developed and devel- sible for this barrier (Bouwstra 1997). In addition
oping countries alike (Kane etal. 1999; Miller to its role as a barrier, both physical and biologi-
and Pisani 1999). Further, hepatic metabolism cal, skin performs a complimentary role, which is
results in rapid clearance of active drug from the that of a transport regulator. Skin routinely regu-
blood plasma making repeated administration lates the flux of water molecules into and out of
inevitable. This only aggravates the problem of the body. It also permits the influx of a variety of
needle pain especially for patients requiring mul- small molecules that are fairly lipophilic (parti-
tiple administrations on a daily basis. tion coefficient, log P >1.5) and have molecular
It is thus sufficiently obvious that as we move weight (MW) less than 500Da (Bos and Meinardi
toward the next era of health care, compliant, 2000). As a result there has been a natural bias of
noninvasive, and sustained delivery will become transdermal delivery systems to cash in on thera-
the key features desirable of any drug delivery peutics that meet these requirements. Drug mol-
system. Several advances to this effect have been ecules currently administered via the transdermal
made in the last two to three decades and novel route fall within a narrow range of MW and lipo-
drug delivery systems have been brought to the philicity. They are typically characterized by
forefront (Drachman 1989; Vanbrunt 1989; high log P (>1.5) and low MW (<500Da),
Langer 1990). A large contribution to these novel thereby taking advantage of the natural selectiv-
systems appeared as modifications to the active ity of skin membrane. A large fraction of drug
drug or formulation excipients to modulate drug molecules lie outside these bounds. These are
pharmacokinetics, safety, efficacy, and mostly peptide- and protein-based drugs that will
metabolism. A more radical approach has been to become the key therapeutics in the future. The
explore newer interfaces on the body for intro- biggest challenge in transdermal drug delivery
ducing therapeutics. One such approach, trans- today is to open the skin safely and reversibly to
dermal drug delivery, makes use of human skin these high molecular weight hydrophilic drugs.
as a port of entry for systemic delivery of drug Several technological advances have been
molecules (Guy 1996; Prausnitz 1997; Barry made in the past couple of decades to overcome
2001a, b; Pillai etal. 2001; Prausnitz etal. 2004; this challenge. These advances can be broadly
Thomas and Finnin 2004). divided into two categories: (1) physical
approaches including but not limited to iontopho-
resis (Panchagnula etal. 2000; Delgado-Charro
9.1.1 Transdermal Drug Delivery and Guy 2001), sonophoresis (Mitragotri and
Kost 2004; Ogura etal. 2008), microneedles
Transdermal drug delivery (TDD) offers an (McAllister etal. 2000; Prausnitz 2004; Sivamani
advantageous mode of drug administration by etal. 2007), and electroporation (Pliquett 1999;
eliminating first-pass hepatic metabolism and Denet etal. 2004) that use some form of physical
9 Models, Methods, andMeasurements inTransdermal Drug Delivery 155
energy to modulate the SC ultrastructure, and (2) Equally important is adoption of the right plat-
chemical approaches that employ chemical for- form of models, methods, and measurement tech-
mulations to modulate skin transport barrier niques to evaluate new and traditional transdermal
(Sinha and Kaur 2000; Williams and Barry 2004). delivery strategies in light of the knowledge
Each of these methods has its individual benefits gained in the last five decades in this field of
and limitations. research. The number of original publications
with transdermal delivery in the title has well
approached 1000 with the numbers rising rapidly.
9.1.2 Scope ofReview These numbers form one metric to indicate that
new scientists continue to be attracted to this
The early 1990s of the last century brought the field. This review aims to provide a synopsis of
first transdermal patch to the market. The market the nuts and bolts in transdermal drug delivery
for patch-based therapeutics has since grown to research to a new scientist. A short introduction
$4 billion perannum worldwide, a small but sig- on skin structure and constituents is followed by
nificant proportion of the total revenues from a description of different model systems and
pharmaceuticals (Barry 2001a, b). After more methods employed in this area.
than a quarter of a century since the introduction
of the first patch, the number of marketed trans-
dermal patches has not exceeded beyond a couple 9.2 Structure ofSkin
of dozen. Another four dozen are in the develop-
mental phases. Although somewhat satisfactory Elucidation of skin structure, especially in rela-
on the face value, these numbers are misleading tion to its barrier function, has drawn countless
since a huge fraction is made up of generic repli- researchers since the early 1950s (Blank 1952;
cas of similar drugs. Only 11 independent drugs Breathnach etal. 1973; Elias and Friend 1975).
make up these 80 odd products, almost all exclu- The human skin is a sandwich of two layers: a
sively below 500Da and characteristically lipo- thin layer of epidermis stacked upon a much
philic in nature. Most efforts to push the envelope thicker substrate, the dermis. The dermis is highly
on molecular weight have shown limited success. vascularized and permeable, consisting predomi-
Only one physical method (i.e., iontophoresis) nantly of a fibrous collagen meshwork that is
has successfully entered the market share of sparsely populated with cells. It houses sweat
transdermal delivery technologies but it is being glands, sebaceous glands, hair follicles, and a net-
used to deliver a low molecular weight drug, work of capillaries supported by the connective
lidocaine (235Da). On the other hand, tissue. The dermis provides most of the bulk and
laurocapram (Azone), the most widely studied toughness of the skin. The epidermis is devoid of
chemical permeation enhancer with high expec- blood vessels, receiving all of its nutrients and
tations, has failed to gain clinical acceptance in disposing of its waste products by diffusional
transdermal delivery due to its skin irritation exchange with the dermis. It is maintained by
(Okamoto etal. 1988; Lashmar etal. 1989; Wong continuous cell division in the germinative basal
etal. 1989). The landscape of transdermal deliv- layer. Differentiating daughter cells, the keratino-
ery opportunities seems grim and one cannot cytes move outward toward the surface of skin.
help but ask, Is transdermal drug delivery During this process, there is a change in the mor-
research still important today? (Barry 2001a, b). phology and composition of keratinocytes.
The concept of transdermal drug delivery is Ultimately, the keratinocytes undergo terminal
rooted in strong scientific acumen even though differentiation, forming dead, flattened corneo-
the practical realization of it has been less fasci- cytes; 2030 layers of corneocytes embedded in a
nating than expected. A sound engineering matrix of lipid, extruded from the cells immedi-
approach coupled with fundamental understand- ately before cornification, form the
ing of a complex biological tissue is required. SC.Corneocytes continually exfoliate from the
156 D.M. Cropek and P. Karande
SC to maintain a constant thickness of this layer at Forslind 1994; Kitson etal. 1994; Engstrm etal.
~2030m (Elias and Friend 1975; Odland 1983; 1995; Menon and Elias 1997; Bouwstra etal.
Matoltsy 1986; Downing 1992). In the last 60 1998; Menon etal. 1998; Norlen 2001; Norln
years of close scrutiny of the skin structure, the 2001). A comprehensive all encompassing model
SC has received by far the most attention. And not seems to be elusive as newer observations con-
surprisingly, since this superficial layer is where tinually require modulating the physical picture of
the barrier property of skin resides. The seminal this membrane (Wertz etal. 1987; Norln etal.
work of Scheuplein etal. conclusively summa- 1998). While newer models are being proposed to
rized the locus and origin of the molecular imper- accommodate minor nuances, the coarse macro-
meability of skin and established it to be a passive scopicmicroscopic structure is well agreed upon.
rather than biologically active property The most simplistic model in this respect is still
(Scheuplein 1965, 1966, 1967, 1972, 1978; Blank the brick and mortar model of Michaels etal.;
and Scheuplein 1973). Through their studies of the term itself coined by Elias (Elias and Friend
the permeability of excised human skin invitro to 1975; Michaels etal. 1975). This model treats the
a large number of permeants, they were able to skin barrier as a simplified two-compartment sys-
show conclusively that the principal barrier to tem with discontinuous protein pockets embed-
permeation is provided by the SC.Separating the ded in a continuous, homogeneous lipid matrix.
epidermis from the underlying dermis by heat Proteins held within corneocyte lipid envelopes
stripping followed by enzymatic removal of the thus form the bricks held by the mortar of a con-
live epidermal layer, Scheuplein etal. measured tinuous lipid phase in the brick and mortar
the permeability of the residual SC and dermis model. The bricks occupy, by far, the larger vol-
independently. These measurements indicated ume of this assembly. Early solvent extraction
that the SC is at least three, and frequently as experiments indicated that lipids, especially polar
much as five, orders of magnitude less permeable lipids, play a critical role in the barrier (Matoltsy
to most substances than the dermis. Moreover, the etal. 1968; Sweeney and Downing 1970). The
permeability of the entire epidermis was found to freeze fracture studies of the SC established con-
be indistinguishable from that of the SC alone. clusively that lipids form multiple broad bilayers
This prompted Scheuplein to model the skin as a filling the corneocyte intercellular spaces
three-layer laminate of SC, epidermis, and der- (Breathnach etal. 1973). These bilayers, shown to
mis, with permeation occurring by Fickian diffu- exist throughout the SC, provide the barrier to
sion of the penetrating species through the three water permeability as determined by Elias and
layers in series. Since the dominant resistance to Squier through freeze-fracture, thin-section, and
permeation of most compounds is offered by the tracer studies (Squier 1973; Elias etal. 1977;
SC, the gradient in penetrant concentration across Madison etal. 1987; Wertz etal. 1987).
the entire skin is, for all practical purposes, local- A general observation of mammalian cells
ized within the SC. indicates that their membranes do not provide a
formidable barrier to water or water-soluble mol-
ecules. These membranes are typically composed
9.2.1 U
ltra-structure ofStratum of phosphoglycerides, sphingomyelin, and cho-
Corneum lesterol where the lipid fatty acyl chains extend to
16 through 20 carbons with a varied degree of
Several models have been proposed for the struc- unsaturation (Fettiplace and Haydon 1980).
ture of the SC.These include the classic brick Occasionally, methyl branching is also observed
and mortar model of Michaels, the domain on the interior of the fatty acyl chains. This
mosaic model of Forslind, the single gel phase methyl branching coupled with unsaturation in
model of Norlen, molecular lipid lamellae mod- the interior of the chains inhibits formation of a
els of Swartzendruber and Fenske, and membrane highly ordered membrane. The membrane is dis-
folding model of Norlen (Michaels etal. 1975; ordered or fluid with high permeability to small
Swartzendruber etal. 1989; Fenske etal. 1994; hydrophilic solutes and water. Interesting to note
9 Models, Methods, andMeasurements inTransdermal Drug Delivery 157
is the lack of any phospholipids or the usual fatty lipid. In addition, 15% of the SC lipids are made
acyl chain structures in the SC (Yardley and of cholesteryl sulfate and cholesteryl esters.
Summerly 1981; Yardley 1983; Wertz 1986). Cholesterol plays a key role in providing barrier
Instead, the SC bilayers are made of cholesterol, property of the SC.This was shown conclusively
fatty acids, and ceramides (Wertz etal. 1987; by Feingold etal. in 1990 based on their observa-
Hedberg etal. 1988). The molecular structure tion that barrier recovery was severely inhibited
and composition of these constituents play an in skin treated with an enzyme that inhibits cuta-
important role in defining the barrier properties neous cholesterol synthesis (Feingold etal.
of the bilayers and in turn of the SC membrane. 1990). Later Takahashi etal. showed that choles-
Subsequent sections provide a brief descrip- terol at high concentrations (>30% molar basis)
tion of the corneocyte proteins and the lipids of promotes lamellar structures, regarded generally
the SC. to provide superior barrier properties (Takahashi
etal. 1996). Cholesterol also increases the chain
mobility of lipids in the gel state making them
9.2.2 Lipids ofStratum Corneum more pliable and thus, potentially, more resistant
to mechanical stresses (de Kruyff etal. 1974). In
9.2.2.1 Ceramides addition, cholesterol broadens phase transition
Ceramides (16) constitute ~42% of the material regions or in some cases may entirely abolish
in the bilayers followed by cholesterol (~40% subtransitions between gel phases thereby stabi-
along with cholesteryl sulfate and cholesteryl lizing them (McMullen and McElhaney 1995;
esters) and fatty acids (~13 %) (Wertz and Takahashi etal. 1996).
Downing 1983a, b; Abraham etal. 1985; Long
etal. 1985; Wertz etal. 1985). The ceramides 9.2.2.3 Fatty Acids
include both sphingosines (ceramides 1, 2, 4, and Fatty acids make up ~13% of the SC lipids. The
5) and phytosphingosines (ceramides 3 and 6). origin of these fatty acids is not completely
Also, the amide-linked fatty acids include nonhy- understood, although it is believed that some of
droxy acids (ceramides 2 and 3), -hydroxy acids them are a result of hydrolysis of ceramides. The
(ceramides 4, 5, and 6), and -hydroxy acids composition of the mixture of fatty acids is
(ceramide 1). In addition, ester-linked fatty acids unusual in consisting predominantly of very long
(ceramide 1) are also present in the epidermal chain (2028 carbons) saturated acids, with only
ceramides. The ceramides are straight and satu- 6% of monounsaturated and 1% of diunsaturated
rated with the exception of ceramide 1. Also the acids (Wertz etal. 1987; Downing 1992).
unsaturation is placed exclusively at the polar Presence of fatty acids along with cholesterol and
end of these ceramide chains thus providing little ceramides is essential to the barrier property of
room for formation of kinks. This architecture is the SC.In addition to providing structural integ-
well poised to provide a highly ordered structure rity to the SC, free fatty acids are also responsible
to the membrane formed from these ceramides. for providing a low pH or acidic surface (Blank
In addition, there is a considerable chain length 1939; Draize 1942; Beare etal. 1958; Baden and
variation in the ceramides, i.e., from 15 to 48. Pathak 1967; Qiang etal. 1993). This may be
This provides room for interdigitation of the critical to the antimicrobial activity of the SC
hydrocarbon chains, an interaction highly favor- thereby making it a physical as well as physio-
able during bilayer formation. Also, the lipids are logical barrier (Fluhr etal. 2001).
characteristically amphiphilic in nature capable
of extensive hydrogen bonding, once again a
primo in formation of self-assembled lamellae. 9.2.3 Proteins ofStratum Corneum
9.2.2.2 Cholesterol Protein pockets, the bricks in the brick and mor-
Free cholesterol is the second most abundant tar model, form the second important compo-
lipid in the SC, amounting to 25% of extractable nent of the SC.These pockets are included in flat,
158 D.M. Cropek and P. Karande
hexagonal, and physiologically dead corneo- when all the intercellular lipids are extracted the
cytes. The SC proteins are typically composed of covalently bound hydroxyceramides can inter-
keratin. Keratins are a family of -helical poly- digitate in a zip-like manner to close the inter-
peptides ranging from 40 to 70kDa in size (Green cellular space and thus maintain the integrity of
etal. 1982; Wertz and Downing 1989). They are the SC.
relatively poor in cysteine, rich in serine and gly-
cine, and contain N-acetylserine at the amino ter-
minus (Steinert and Cantieri 1983). Keratins 9.3 Routes ofPermeation
accumulate throughout epidermal differentiation
and represent the major component of the SC as There are three major routes of permeation for
well as of epidermal appendages such as hair and passive diffusion of a molecule across the
nails (Baden etal. 1973). Earlier in epidermal SC.These include: (a) diffusion through append-
differentiation, low molecular weight keratins ages such as sweat ducts, sebaceous glands, and
predominate, whereas higher molecular weight hair follicles; (b) diffusion through the corneo-
polypeptides are found in the SC (Skerrow and cytes of the SC; and (c) diffusion through the
Hunter 1978). Individual keratin molecules lipids of the SC.Diffusion across the corneocytes
aggregate to form superhelices, the detailed and lipids of the intact SC comprises the predom-
structures of which are still under investigation inant route through which most molecules pene-
(Steinert and Cantieri 1983). They are stabilized trate. The appendageal area available for diffusion
by disulfide bridges that can be solubilized only is significantly lower, ~0.1%, but has received
by reducing agents. The keratin in the SC is prob- considerable attention as an important perme-
ably responsible for maintaining the hexagonal ation pathway for ions or large polar molecules
shapes of the corneocytes and may contribute to that have slow permeation across the SC (Barry
the toughness and flexibility of the SC (Wertz and 2001a, b).
Downing 1989).
The corneocyte envelope enclosing the
keratin filaments is made of two layers. The 9.3.1 Skin Appendages
inner portion of the envelope consists of cross
linked proteins, predominantly involucrin and at The involvement of skin appendages in transcu-
least six other soluble and membrane-associated taneous permeation has received considerable
proteins (Rice and Green 1977; Watt and
attention over six decades. Early studies by many
Green 1981; Simon and Green 1984). The outer investigators implicated skin appendages as
portion is made of ester-linked -hydroxyacyl important avenues for penetration of topically
sphingosines. These hydroxyceramide molecules applied chemicals (Mackee etal. 1945; Shelley
contain mainly 3034 carbon -hydroxyacyl and Melton 1949; Fredriksson 1961; Tregear
chains and represent 2% of the dry weight of 1961; Vankooten and Mali 1966; Wahlberg 1968;
the SC (Wertz and Downing 1989). At least Rutherford and Black 1969; Wallace and Barnett
50% of the hydroxyceramides are linked to the 1978). Using full-thickness mouse skin main-
protein envelope through the -hydroxy termi- tained as short-term organ cultures in an invitro
nus. This helps the sphingosine moieties to experimental system, Kao etal. demonstrated
interdigitate with the lipid lamellae (Wertz and that permeation of topically applied benzo[a]
Downing 1987). This may explain why unlike pyrene was higher in haired mice skin compared
other membranous structures the lipid envelope to hairless mice skin (Kao etal. 1988).
persists even after extensive extraction with Histochemical techniques, autoradiographic
methanolchloroform mixture (Swartzendruber techniques, and fluorescence microscopy have
etal. 1987; Wertz and Downing 1987). The lipid been used to visualize and quantitate appenda-
envelope hydroxyceramides anchor the corneo- geal absorption. These studies revealed that topi-
cytes to the intercellular lipids. As a result, even cally applied agents concentrated and persisted in
9 Models, Methods, andMeasurements inTransdermal Drug Delivery 159
the hair follicles and sebaceous glands (Grasso 9.3.2 Intracellular Route
and Lansdown 1972; Foreman etal. 1979;
Holland etal. 1984). Certain permeation enhancers can open up the
Of all the appendageal routes, the hair folli- dense keratin structure in corneocytes creating
cle has received the most attention as a promi- porous pathways for diffusion across them. For
nent route of permeation. It also serves as an example, decylmethyl sulfoxide interacts with
important cutaneous reservoir for topically keratin and is hypothesized to enhance permea-
applied molecules (Lademann etal. 2006). bility by opening up aqueous channels within the
Hueber etal. and Tenjarla etal. showed that the corneocyte (Cooper 1982). Dimethyl sulfoxide
penetration of corticosteroids is considerably can induce reversible changes in protein struc-
lower in hairless skin compared to haired skin tures of isolated corneocytes (Mendelsohn etal.
(Hueber etal. 1994; Tenjarla etal. 1999). 2006). Hexamethyl sulfoxide and dimethyl sulf-
Hydrocortisone permeability increased in tissue oxide convert -helical keratins in the corneocyte
engineered skin on insertion of hair follicles to -sheets (Oertel 1977). Lee etal. have demon-
(Michel etal. 1999). Permeation enhancers that strated the capability of thioglycolates in depila-
specifically target hair follicles have been inves- tory creams in disrupting intracellular keratin
tigated with great success. Of these, liposomes matrix and the protein envelope using multipho-
have been shown to deliver DNA (Li etal. ton microscopy (Lee etal. 2008). He etal. have
1993), plasmids (Domashenko etal. 2000), shown that N-trimethyl chitosan is capable of
monoclonal antibodies (Balsari etal. 1994), cal- increasing transcutaneous permeation by affect-
cein (Lieb etal. 1992), and melanin (Li and ing secondary structure of keratins within the
Hoffman 1997) to hair follicles. Lee etal. corneocytes (He etal. 2008). Azone can act on
reported that the auxiliary SC associated with the keratin fibers of the corneocytes converting
the sweat glands has a reduced barrier function their rigid -helical conformation to a flexible
(Lee etal. 2001). Following up with elegant -sheet confirmation (Xueqin etal. 2005). Lauric
immunostaining studies, Wilke etal. proposed acid enhances the permeability of verapamil by
that the active permeation barriers in sweat interacting with skin proteins (Shah etal. 1992).
ducts in the epidermis and dermis are function- Dithiothreitol enhances flux of sucrose and man-
ally and morphologically distinct (Wilke etal. nitol across the SC exclusively through interac-
2005, 2006). The innermost layer of the intra- tions with corneocyte keratin matrix (Goates and
epidermal duct is completely keratinized Knutson 1993). Oleic acid and isopropyl
(Zelickson 1961; Hashimot etal. 1965). In con- myristate increase the permeability of the cor-
trast, the dermal ducts lack the presence of cor- neocytes for polar substances after pretreatment
nified corneocytes but contain luminal tight of the skin (Eder and Mller-Goymann 1995).
junctions, which seem to be absent from the epi-
dermal duct lining (Hashimot 1971a, b). Similar
to the dermal ducts, the secretory coils of the 9.3.3 Intercellular Route
sweat glands themselves lack cornified layers
but are rich in tight junctions as evidenced by Several chemicals can alter or disrupt the organi-
the colocalization of occludin and claudin-4 zation of lipid molecules in the SC bilayers
(Hashimot 1971a, b). In light of these observa- thereby facilitating the diffusion of molecules
tions, Wilke etal. propose that dermal sweat across the SC.Barry postulated different ways in
ducts and the sweat glands could serve as poten- which permeation enhancers can modify SC lip-
tial permeation routes (Wilke etal. 2006). Only ids (Barry 1988, 1991, 2004). Enhancers can act
a few experimental studies have actually been on polar head groups of lipids and modify the
dedicated to evaluating the contribution of sweat hydrogen bonding and ionic forces between them
glands and ducts to transcutaneous permeation resulting in a disruption of the packing geometry.
(Vankoote and Mali 1966). Fluidity caused at the polar plane due to the
160 D.M. Cropek and P. Karande
d isruption of packing geometry accelerates the c ompounds or therapeutics (Rao and Misra 1994;
diffusion of solute molecules across the lipid McCullough etal. 2006; Suppasrivasuseth etal.
bilayers. An alternate consequence of disrupting 2006; Elewski 2007; Kim etal. 2008). Freshly
packing geometry of lipid head groups is the cre- excised skin from autopsies, cadaver skin, or dis-
ation of aqueous pockets that facilitate diffusion carded skin from breast reduction procedures are
of hydrophilic molecules. In addition to fluidiz- excellent sources of human skin (Bronaugh etal.
ing bilayers, enhancers that disrupt lipid head 1986; Wester and Maibach 1989; Friend 1992).
group interactions can cause extraction of lipid The primary barrier to transport of molecules
molecules, phase separation, or micelle forma- across the skin is the SC.In comparison the epi-
tion (Barry 2004). Enhancers can also insert dermis and dermis offer minimal resistance to
themselves between the hydrocarbon chains of passive diffusion of solutes. The SC is composed
the lipid bilayers and thereby disrupt the packing of lipids and terminally differentiated, fully kera-
of lipid molecules. Consequent fluidization of the tinized corneocytes. It is, therefore, intuitively
lipid bilayers facilitates the diffusion of perme- expected for exvivo skin to maintain the barrier
ants. Disruption in packing of lipid chains can in integrity of the SC for an extended period of time
turn alter the packing of polar head groups of the after harvesting when stored under appropriate
lipid molecules, thereby accelerating, to a small conditions. Some investigators have indeed veri-
extent, the diffusion of permeants. fied that skin can be frozen for up to 12 months
Karande etal. studied permeation enhancers without significant deterioration of barrier prop-
from eight different categories: anionic surfac- erties (Franz 1975; Harrison etal. 1984). Barry
tants, cationic surfactants, zwitterionic surfac- etal. found that human cadaver skin stored at
tants, fatty acids, fatty alcohols, fatty amines, fatty 18C for 466 days did not show any significant
esters, and azone-like molecules, and showed that change in permeability toward tritiated water
chemicals in all these categories could be classi- (Harrison etal. 1984). Interestingly, Barry etal.
fied, more simply, as lipid extractors or lipid fluid- also found that the skin obtained from an iceman
izers (Karande etal. 2005). Lipid extractors 5000 years old and buried in glacial ice was very
increased SC permeability by extracting lipids well preserved. Several reports have documented
from bilayers or the corneocyte envelope. Loss of the comparison between invivo and exvivo SC
lipids from the SC was monitored as a decrease in and have shown that it retains its barrier proper-
the signal intensity of methylene groups of lipid ties for several days after harvesting (Berenson
chains in Fourier transform infrared (FT-IR) spec- and Burch 1951; Galey etal. 1976). Wester etal.
troscopy. Lipid fluidizers increased SC permea- monitored glucose metabolism in skin as a mea-
bility by partitioning themselves in the bilayers sure of its viability and showed that the metabolic
and disrupting the bilayers packing structure. activity was highest during the first 18h after the
Fluidization was monitored as an increase in the skin was harvested. The metabolic activity
signal intensity of methylene groups (from hydro- showed a decrease by day 2 but stayed steady
carbon tails of the enhancer) and disappearance of until day 8 (Wester etal. 1998a).
peaks related to the ordered packing of lipids in In spite of the several advantages of using
FT-IR spectroscopy. Extent of extraction or fluidi- human skin in permeation experiments there are
zation correlated very well with the extent of skin several problems associated with its use such as
permeabilization. safety concerns, difficulty in procurement, lim-
ited supply, and regulatory considerations. Also
the permeability measurements obtained on
9.4 In Vitro Skin Models human skin samples vary greatly between indi-
viduals as well as between samples from differ-
9.4.1 Excised Human skin ent anatomical sites on the same individual
(Wester and Maibach 1992; Norlen etal. 1999;
Human skin is the obvious choice in experiments Robert Peter Chilcott 2000). Chilcott etal. have
for determining the permeability of model shown that there is a statistically significant
9 Models, Methods, andMeasurements inTransdermal Drug Delivery 161
v ariation in the skin barrier property with relation large quantities of skin can be obtained for exper-
to gender, chirality, time of the day when mea- imental purpose (Friend 1992).
surement was obtained, and to some extent the One needs to be cautious, however, in extrapo-
dietary habits of the individual (Robert Peter lating animal skin data to human skin. Several
Chilcott 2000). Akomeah measured the permea- differences exist and have been documented.
bility of caffeine, methyl paraben, and butyl para- Skin from experimental animals is different from
ben on skin samples from several donors and human skin in thickness, composition, and con-
found interdonor variabilities between 33% and stitution of the SC, and distribution and density
44% (Akomeah etal. 2007). In general, the inter- of appendages such as sweat glands and hair fol-
subject skin sample variability in skin permeation licles (Schalla and Schaefer 1982; Bronaugh
was higher than that observed within the same etal. 1983). Panchagnula etal. have documented
subject. Similar observations have been reported follicular density, SC thickness, epidermis thick-
by other investigators (Southwell etal. 1984; ness, and full skin thickness for 16 animal mod-
Langguth etal. 1986; Rochefort etal. 1986). els including human skin (Panchagnula etal.
Further, these permeability measurements show a 1997). These parameters vary significantly
non-Gaussian distribution (Williams etal. 1992; between the different species studied. For two
Cornwell and Barry 1995). model compounds used in this study, water and
7-hydroxycoumarin, lag time and permeability
varied significantly across the skin models. While
9.4.2 Excised Animal Skin both compounds have similar permeabilities
across human skin, their permeabilities across
In view of the difficulties associated with human other skin models vary drastically. The lipid con-
skin, animal skin is routinely used as a model for tent of the skin is a major determinant in its bar-
human skin in invitro experiments (Haigh and rier potential and differs between species or
Smith 1994). Mouse (Bonina etal. 1993; Roy between sites on the same animal (Elias etal.
etal. 1994; Panchagnula etal. 1997; Bhandari 1980, 1981). Hairless mouse skin which is com-
etal. 2008; Cho etal. 2008), rat (Panchagnula monly used as a model for human skin is com-
etal. 1997; Hai etal. 2008; Zhao etal. 2008), paratively fragile. While permeability of human
guinea pig (Panchagnula etal. 1997; Tipre and skin exposed to water increases only twofold in
Vavia 2003; Pabla and Zia 2007), rabbit 10 days, hydration can completely disintegrate
(Panchagnula etal. 1997; Ogiso etal. 2001; hairless mouse skin (Bond and Barry 1988a, b,
Artusi etal. 2004; Sebastiani etal. 2005; c). A 2-min treatment with acetone has negligible
Elgorashi etal. 2008), porcine (Panchagnula effect on human skin but can increase hairless
etal. 1997; Karande etal. 2004; Ben-Shabat etal. mouse skin permeability by 15-fold (Bond and
2007), monkey (Wester and Maibach 1987; Roy Barry 1988a, b, c, d). Hairless mouse skin model
and Degroot 1994; Panchagnula etal. 1997), dog overestimates the effect of permeation enhancers
(Sato etal. 1991; Panchagnula etal. 1997; on skin permeability by sevenfold (Bond and
Rohatagi etal. 1997), hamster (Coutelegros etal. Barry 1988a, b, c). In contrast, another common
1992; Panchagnula etal. 1997; Bach and Lippold model, shed snake skin, underestimates the effect
1998), fish (Watanabe etal. 1989; Masson etal. of permeation enhancers on skin permeability
2002), snake (Megrab etal. 1995; Suh and Jun when compared to human skin (Rigg and Barry
1996; Panchagnula etal. 1997), cow (Panchagnula 1990). In general, it has been observed that ani-
etal. 1997; Netzlaff etal. 2006b), frog (Dewhurst mal skin permeability is higher than human skin
and Williams 1993; Smith 1993), sheep permeability (Panchagnula etal. 1997).
(Panchagnula etal. 1997), and marmoset (Scott Of all animal skin models studied, porcine
etal. 1991) are some of the animal skin models skin, and particularly porcine ear skin, is closest
studied to represent human skin. Animal skin to human skin in terms of its biochemical compo-
offers advantages over human skin in that the age sition and histological features (Gray and Yardley
and sex of the animal can be controlled as well as 1975; Dick and Scott 1992; Wester etal. 1998b;
162 D.M. Cropek and P. Karande
Sekkat etal. 2002; Muhammad etal. 2004; Jacobi been used successfully include permeable syn-
etal. 2007). Porcine skin resembles human skin thetic membranes such as nylon mesh (Slivka
most in terms of the SC thickness (Holbrook and etal. 1993; Crooke etal. 1996) and polycarbonate
Odland 1974; Wester and Maibach 1989; Jacobi membranes (MonteiroRiviere etal. 1997; Poumay
etal. 2007), epidermis thickness (Wester and etal. 2004; Kandarova etal. 2006), collagen
Maibach 1989; Sandby-Moller etal. 2003; Jacobi (Fransson etal. 1998; Flamand etal. 2006), col-
etal. 2007), follicular structure and density lagen lattices (Bell etal. 1981), glycated collagen
(Jacobi etal. 2007), lipid composition (Gray and (Pageon and Asselineau 2005), collagen-
Yardley 1975), and the underlying vasculature glycosaminoglycan matrices (Boyce etal. 1988),
(Simon and Maibach 2000). As a result, the por- chitosan cross-linked collagen-glycosaminoglycan
cine skin has gained wide acceptance as a repre- matrices (Shahabeddin etal. 1990), fibrin
sentative model for human skin. (Holland etal. 2008), dead de-epidermized der-
mis (Regnier etal. 1998; Rehder etal. 2004), syn-
thetic scaffolds (Shakespeare 2001; Mansbridge
9.4.3 Living Skin Equivalents 2002), biodegradable scaffolds (El Ghalbzouri
etal. 2004), or combinations thereof (Slivka etal.
Skin samples obtained from different species 1993; Lee etal. 2000; Barker etal. 2004; Sobral
show varying permeability responses in presence etal. 2007). Keratinocytes receive nutrients from
of the same permeation enhancer on account of the lower surface of the culture while being
the differences in their constituents, composition, pushed upward in a process of progressive differ-
and microstructure. In addition to an interspecies entiation. In 1421 days, the topmost layer
variation, there is also a variation observed in skin achieves terminal differentiation and manifests
permeability with age and anatomical location characteristics remarkably similar to those of nor-
within the same species (Bronaugh etal. 1982; mal SC, i.e., completely cornified cells sur-
Dupuis etal. 1986; Hughes etal. 1994; Duncan rounded by a lipid intercellular matrix (Nabila
etal. 2002). Cell culture or tissue culture-based Sekkat 2001). Today, several cell culture-based
models of human skin can potentially overcome skin models are commercially available for ready
this problem by offering a more consistent skin use in skin permeation or skin toxicity studies.
representation (Roguet etal. 1998; Faller and These include TestSkin and TestSkin II by
Bracher 2002; Lotte etal. 2002). In general, Organogenesis, Canton, MA (Davis 1990; Moody
invitro cell culture models of living tissues offer etal. 1995; Elyan etal. 1996; Rodriguez etal.
several advantages such as high reproducibility, 2004; Shibayama etal. 2008), EpiDerm and
rapid assessment of permeability and metabolism EpiDermFT (Hayden etal. 2004, 2005;
of drugs, stricter control over experimental condi- Kandarova etal. 2007; Borgia etal. 2008; Schafer-
tions, well-defined end points, and potential time Korting etal. 2008) by MatTek Corp., Ashland,
and cost savings when compared to animal use. MA, EpiSkin and SkinEthic RHE (Botham
The biggest advantage of cell culture models, 2004; Schafer-Korting etal. 2006, 2008; Luu-The
however, is their amenability to high-throughput etal. 2007; Netzlaff etal. 2007) by SkinEthic
studies for drug discovery or formulation optimi- Labs., Nice, France, Vitrolife-Skin (Uchino etal.
zation studies (Audus etal. 1990). 2002; Morikawa etal. 2007) by Gunze, Kyoto,
Reconstruction of skin invitro typically starts Japan. Netzlaff etal. have reviewed the
with obtaining keratinocytes from full thickness EpiDerm, EpiSkin, and SkinEthic models
or split thickness skin by enzymatic digestion based on their morphology, lipid composition,
using trypsin (Larsen etal. 1988), dispase (Green biochemical markers, and their applicability in
etal. 1979), or thermolysin (Walzer etal. 1989). tests for evaluating phototoxicity, corrosivity, irri-
Basal keratinocytes are isolated and grown at an tancy, and transport properties (Netzaff etal.
airliquid interface on a substrate that is equiva- 2005). The architecture, homeostasis, and lipid
lent of the dermis. Dermal equivalents that have composition of these models come close to human
9 Models, Methods, andMeasurements inTransdermal Drug Delivery 163
skin (Ponec and Kempenaar 1995; Ponec etal. order permeability as human skin for caffeine
2000, 2002). Faller etal. compared the models in and -tocopherol acetate from a water in oil
their ability to secrete extracellular enzymes glu- (w/o)-emulsion, an oil in water (o/w)-emulsion, a
tamic oxaloacetic transaminase (GOT) and lactate liposomal dispersion and a hydrogel (Dreher
dehydrogenase (LDH), and interleukin-1 on etal. 2002). In addition, a multilab study verified
treatment with sodium lauryl sulfate (SLS). that the permeability ranking across EpiSkin,
EpiDermTM was the most resistant to SLS and EpiDerm, and Skin Ethic RHE models was
most reproducible (Faller and Bracher 2002). comparable to the permeation through human
In general, the reconstructed skin models have epidermis for caffeine and testosterone (Schafer-
higher permeabilities compared to excised human Korting etal. 2008).
skin (Gysler etal. 1999). Schmook etal. The biggest shortcoming of commercially
compared the permeabilities of four topical der- available skin models is their relatively weak bar-
matological compounds of varying polaritysal- rier function. Impaired desquamation (Vicanova
icylic acid, hydrocortisone, clotrimazole and etal. 1996a, b), impaired transfer of desmosomes
terbinafine, across rat, human and pig skin as (Vicanova etal. 1996a, b), and presence of unke-
well as two models of human skinGraftskin ratinized microscopic foci (Mak etal. 1991) are
LSE and Skinethic HRE (Schmook etal. cited as reasons for this poor performance.
2001). In these studies pig skin performed similar Another significant impediment to the use of
to human skin with comparable flux of solute reconstructed human skin models is their high
across both tissues. Graftskin LSE provided cost. This has limited the use of such models
an adequate barrier to salicylic acid, but clotrima- mostly to industry and out of reach of most aca-
zole flux across it was 1000-fold higher and its demic labs and small enterprises. Furthermore, all
skin concentration 50-fold higher when com- commercially available models use proprietary
pared with human skin. Skinethic HRE was chemically defined media and sources for cells
approximately sevenfold more permeable com- that can put additional constraints on the flexibil-
pared to human skin for salicylic acid and 900- ity of using such models. All three leading mod-
fold more permeable to clotrimazole. In a similar els, Epiderm, EpiSkin, and SkinEthic RHE
study Marty etal. reported that trinitroglycerol are based on the epidermis raised on a minimal
and estradiol were about 20-fold more permeable dermal equivalent such as collagen gel scaffold
across Skinethic HRE compared to split- encapsulating fibroblasts. In contrast, Nakamura
thickness human skin (Marty etal. 1997). In etal. report that full-thickness models based on
cutaneous bioavailability studies on topical for- organ cultures of skin explants match the invivo
mulations, vehicle effects were observed to be situation more closely (Nakamura etal. 1990).
vastly different in EpiDerm and EpiSkin mod-
els compared to exvivo human skin (Dreher etal.
2002). Although the reconstructed human skin 9.4.4 Polymers
models underperform significantly in reproduc-
ing the barrier properties of exvivo human skin Model membrane systems can provide tremen-
they can still be used to rank order the permeabil- dous insight into mechanistic details of solute dif-
ities of solutes based on their permeabilities. fusion and thermodynamics of solutemembrane
Such a rank order has been shown to match the and solventmembrane interactions (Corrigan
order obtained on exvivo human skin for several etal. 1980; Flynn 1985; Beastall etal. 1986; Haigh
different molecules. Lotte etal. have shown that and Smith 1994). Diffusion of a solute molecule
the skin absorption and permeability of lauric across a membrane is governed by physical factors
acid, mannitol, and caffeine follow the same rank such as molecule size and shape, pore size, pore
order as they would on exvivo human skin (Lotte distribution, path length and tortuosity, and chemi-
etal. 2002). Dreher etal. found that the EpiSkin cal factors such as hydrogen bonding, hydropho-
and EpiDerm models showed the same rank bic interactions, and electrostatic interactions. The
164 D.M. Cropek and P. Karande
contribution from each of these factors can obtained between diffusion of a wide range of
potentially be decoupled by a systematic study compounds across the IPM membrane and
with model membranes. Synthetic membranes and excised skin. Transport resistance across the
polymers such as silicone (Hou and Flynn 1997; model membrane, however, was 1000-fold lower
Cross etal. 2001), cellulose acetate (Barry and as compared to excised skin (Hadgraft and Ridout
Eleini 1976; Barry and Brace 1977; Farinha etal. 1987). A three-component mixture of dipalmitoyl
2003), poly(dimethylsiloxane) (Cronin etal. 1998; phosphatidylcholine, linoleic acid, and tetradec-
Du Plessis etal. 2002; Farinha etal. 2003; Frum ane showed an order of magnitude improvement
etal. 2007), polyvinylidene difluoride (Olivella in transport resistance when compared to IPM
etal. 2006), polyvinyl chloride, polyether sulfone membrane (Hadgraft and Ridout 1988). Matsuzaki
(Farinha etal. 2003), ethyl vinyl acetate (Farinha etal. developed a model skin membrane by fixing
etal. 2003), multimembrane laminates (Scheuplein liposomes composed of SC lipids: ceramides, pal-
and Bronaugh 1983; Houk and Guy 1988), and a mitic acid, cholesterol, and cholesterol-3-sulfate
mixture of isopropyl myristate and silicone oil onto a supporting filter, Biodyne B (Matsuzaki
(Ottaviani etal. 2006) have been used to this end. etal. 1993; Miyajima etal. 1994). Drug permea-
In spirit of the fluidmosaic model of the skin, bility through this system correlated very well
organic solvents such as 1-octanol, alkanes, ether, (r=0.88) with that through guinea pig skin
chloroform, esters, and paraffins have also been although permeability through the model system
used to model diffusion through skin (Houk and was an order of magnitude higher. Moghimi etal.
Guy 1988). Relatively less studied synthetic mem- constructed a model lipid matrix from cholesterol,
brane systems are porous materials. In diffusion water, and free fatty acids of the SC and their
studies across model biomembranes, filter sup- sodium salts (Moghimi etal. 1996). This model
ports have typically gained prominence as support matrix was shown to be a good representation of
membranes. A few studies, however, have used the SC barrier based on the permeability of a
filter supports or filter supports filled with organic model hydrophobic drug, 5-fluorouracil. Using a
liquid to study diffusion of topical agents (Tanaka similar approach, de Jager etal. created a SC sub-
etal. 1978; Demeere and Tomlinson 1984; stitute (SCS) by applying a mixture of synthetic
Turakka etal. 1984; Viegas etal. 1986). Schramm- SC lipids, free fatty acids, and cholesterol on a
Baxter etal. have used polyacrylamide gels to porous substrate. The composition, organization,
model human skin and study the energetics of liq- and orientation of lipids in the SCS bore high
uid jet penetration into skin (Schramm-Baxter resemblance to that of the intercellular barrier lip-
etal. 2004). Dyer etal. have tested zeolites as ids in SC (de Jager etal. 2006a, b). Other groups
model systems (Dyer etal. 1979). Although such have reported studies on membranes reconstituted
models are simplistic and lack all the functional from porcine SC lipids or porcine brain ceramides
and structural complexity of skin, they provide on porous substrates. These models have been
several other advantages such as uniformity of shown to reproduce the permeability of water and
structure, sample-to-sample reproducibility, and some other permeants across intact SC (Abraham
ease of procurement. and Downing 1989; Friberg and Kayali 1989;
Friberg etal. 1990; Kittayanond etal. 1992;
Lieckfeldt etal. 1993; Kuempel etal. 1998).
9.4.5 Lipids
of the chambers to create homogeneous compart- (Washitake etal. 1980), L-shape configuration
ments is critical in horizontal diffusion cells. In case (Dyer etal. 1979; Tojo etal. 1985a), glass con-
of vertical diffusion cells, mixing is not critical. ical flasks configuration (Wurster etal. 1979),
However, a homogeneous well-mixed receiver vertical membrane, equi-compartment diffu-
chamber better mimics invivo conditions and pre- sion cell with high area to volume ratio (Flynn
vents the formation of a static boundary layer of and Smith 1971), glass diffusion cells with
high-solute concentration in the receiver chamber. steel mesh (Southwell and Barry 1983), Valia
Formation of unmixed zones is especially critical Chien cells (Tojo etal. 1985a, b), and flow
when assessing the permeability of a hydrophobic through system with central inlet and periph-
solute in an aqueous receiver compartment (Tsuruta eral effluent ports (Astley and Levine 1976). In
1977; Bronaugh and Stewart 1984, 1986). Efficient recent years, several modified versions of these
mixing can be obtained using small magnetic stir early designs have been used (Morell etal.
bars. Flow-through diffusion cells in which the 1996; Aramaki etal. 2003; Bakand etal. 2006;
receptor fluid is continuously refreshed to mimic Soni etal. 2006; Tas etal. 2007).
invivo sink conditions (i.e., metabolism and diffu- (b) Vertical cells
sion into the subdermal vasculature) have also been Vertical diffusion cells are closer to
used successfully (Ainsworth 1960). Temperature invivo situation in obtaining permeability
control of the diffusion cell can be attained using data across the skin (Friend 1992). The
water jackets or simply submerging the entire cell Coldman cell represents the earliest of all
assembly into a water bath. For studying perme- vertical diffusion cells (Coldman etal. 1969).
ation of highly hydrophobic compounds, solubiliz- A glass cell with a side arm for sampling and
ing solvents can be added to the aqueous receiver stir bar for mixing forms the receiver cham-
chamber. These include Triton X-lOO (Bronaugh ber. Skin is sandwiched between the donor
and Stewart 1984), bovine serum albumin (Brown and receiver using a clamp. Whitton etal.
and Ulsamer 1975), Poloxamer 188 (Hoelgaard and studied a similar cell with the sampling arm
Mollgaard 1982), PEG 400 (Valia etal. 1984), and located at the bottom of the receiver (Whitton
ethanol (Scott etal. 1986). Caution needs to be exer- and Everall 1973). The Franz diffusion cell
cised when using solubilizing agents that they do remains the most widely studied vertical dif-
not alter inherent membrane properties. Hydration fusion cell today (Franz 1978). The original
effect on membrane integrity needs to be consid- design had poor mixing properties which
ered when assessing solute permeability over have been addressed in subsequent modifica-
extended periods of time. Some studies have tions (Nacht etal. 1981; Loftsson 1982; Kao
reported that long-term hydration in rodent skin, etal. 1983; Dugard etal. 1984; Hawkins and
and in particular hairless mouse skin, can lead to Reifenrath 1986; Gummer etal. 1987;
changes in permeation rates (Whitton and Everall Tiemessen etal. 1989). In the past two
1973; Bond and Barry 1988a, b, c, d; Hinz etal. decades several modifications of the Coldman
1989). Finally, the area of diffusion of the donor cell have been used successfully. These
chamber has been shown to have some effects on include the release cells (Morell etal. 1996),
the permeation rates (Karande and Mitragotri enhancer cells (Bosman etal. 1996), Kelder
2003). Water penetration into the skin introduces a cells in combination with the Automatic
lateral strain at the edges of the donor chamber due Sample Preparation with Extraction Columns
to swelling. This scales as the strain edge available system (Bosman etal. 1996), Oak Ridge
per unit area and results in higher observed perme- National Laboratory Skin Permeability
ation rates in smaller donor chambers. Chamber (Holland etal. 1984), and Ussing
type chambers (Li etal. 2006; Ito etal. 2007).
(a) Horizontal diffusion cells (c) Skin Flaps
Several designs have been suggested and In addition to the conventional diffusion
used successfully for this type of diffusion cell. systems discussed above, novel invitro sys-
These include the T-shape configuration tems that measure effect of perfusion rates on
9 Models, Methods, andMeasurements inTransdermal Drug Delivery 167
solute permeation have also been designed. where x=0 corresponds to the SC surface and
Isolated Perfused Porcine Skin Flap (IPPSF) x=L corresponds to the end of the SC, K is the
is a model system of porcine skin flap per- average solute partition coefficient in the SC, and
fused by the caudal superficial epigastric C0 is the donor concentration of the solute. The
artery and its associated veins and mounted resulting equation for solute concentration in the
on a diffusion cell (Williams etal. 1990; SC, Cs is given as follows (Crank 1975):
Riviere etal. 1991). Bovine udder is used in
a similar fashion for permeation studies
Cs ( t ) - D ( 2n + 1) p t
2 2
(Kietzmann etal. 1993). 8 1
C
= 1-
p2
n = 0 ( 2n + 1)
2
exp
L2
(9.6)
9.5.2 Tape Stripping
where C is the solute concentration in SC at
Tape stripping is a technique that has been found steady state ( C = KC0 ). For short times, i.e.,
useful in dermatopathological and dermatophar- 2
macological research for selectively or at times Dt
exhaustively removing the SC (Surber etal. low values of L2 , Eq. (9.6) can be simplified as
1999). Typically, an adhesive tape is applied to
Cs ( t ) 4 DKt
the skin and removed abruptly. This application (9.7)
can be repeated between 10 and 100 times (Sheth C0 L2
etal. 1987; Ohman and Vahlquist 1994). The
observation that skin may serve as a reservoir for Using the definition of permeability P, the above
chemicals was first reported in 1955 (Malkinson Eq. (9.7) further simplifies to
and Ferguson 1955). Drugs like corticosteroids
4 Pt
were shown to localize within the SC (Vickers Cs ( t ) = C0 (9.8)
1963; Carr and Wieland 1966). These observa- L
tions led to the use of the tape stripping tech- Equation (9.8) shows that the solute concentra-
nique in investigating the barrier and reservoir tion in the SC measured at short times is propor-
function of the skin (Rougier etal. 1983; Tojo tional to its steady-state permeability.
and Lee 1989). This technique is now being Accordingly, solute concentration can be mea-
increasingly used in measuring drug concentra- sured via tape stripping in the SC to infer its
tion and its concentration profile in the SC steady-state permeability. An analytical tech-
(Pershing etal. 1990; Pellett etal. 1997; Shah nique such as high-performance liquid chroma-
etal. 1998). tography (HPLC) or an immunoassay or
radioimmunoassay is required in conjunction to
9.5.2.1 Theory accurately determine solute concentration.
Solute diffusion in the SC can be described by Several studies have successfully applied this
Ficks law (Crank 1975) as follows method to determine the skin permeability of a
wide range of solutes (Stinchcomb etal. 1999;
Cs 2C
= D 2s (9.5) Alberti etal. 2001a, b, c; Moser etal. 2001).
t x
where D is the average solute diffusion coeffi-
cient in the SC, Cs is the solute concentration in 9.5.3 Impedance Spectroscopy
the SC, and x is the distance from the SC surface.
Eq. (9.1) can be solved with the following bound- Methods based on measuring solute diffusion
ary conditions: across skin may not always provide the sensitiv-
ity required to measure small perturbations in
Cs ( x = 0 ) = KC0
skin permeability or follow permeability
Cs ( x = L ) = 0 changes over short intervals of time. Electrical
168 D.M. Cropek and P. Karande
measurements across the SC provide improved Kalia etal. 1996; Kumar and Lin 2008), and
sensitivity (Dugard and Scheuple 1973). chemical enhancers on skin permeability
Electrical properties of SC parallel those of per- (Karande etal. 2004; Karande etal. 2006a, b).
meability and play a dominant role in the con- In comparison to diffusion measurements of
trol of current flow (Lawler etal. 1960; Tregear solute permeability in diffusion cells, electrical
1966). A review of factors governing the pas- impedance measurements are relatively simpler,
sage of electricity across skin has been pre- faster, and more sensitive. Impedance-based per-
sented by Tregear (Tregear 1966). meability assessment provides direct readout of
barrier integrity and does not require subsequent
9.5.3.1 Theory analysis, which may be tedious, time consuming,
Flow of ions across skin under an electric field is and expensive. Also, since these measurements
analogous to diffusion of solutes under a chemi- can be performed rapidly and by use of auto-
cal gradient. Current across skin can thus be mated systems the throughput of impedance-
related to permeability of skin. Skin and its based assays is significantly larger compared to
appendages can be represented by an equivalent diffusion cells. Karande etal. have described the
circuit containing a resistance R shunted by design of an impedance based high-throughput
capacitance C (Lackermeier etal. 1999). The assay to determine the effect of chemical perme-
impedance, Z, of this equivalent skin model can ation enhancers on skin permeability (Karande
be represented as etal. 2004, 2006a, b). This system, invitro
1
impedance Guided High Throughput (INSIGHT)
1 2 screen, is capable of assessing thousands of for-
Z = R2 + (9.9) mulations per day for their ability to modulate
( 2p fC )
2
skin permeability. The authors discovered syner-
gistic formulations of permeation enhancers
where f is the frequency of the applied alternating using this screen, which were capable of deliver-
current (AC) signal. A formal porous pathway ing a biologically active hormone across the skin
theory based on NernstPlanck flux equations at therapeutically relevant doses. One downside
and the NernstEinstein relations for ideal solu- of using impedance to assess skin permeability is
tions has been developed that relates skin imped- that impedance serves only as a surrogate mea-
ance to skin permeability (Lakshminarayanaiah sure of actual skin permeability. The actual flux
1965; Srinivasan and Higuchi 1990; Li etal. of a solute needs to be assessed by conventional
1998, 1999; Tezel etal. 2003). diffusion methods.
A simplified correlation between skin resistiv-
ity, R, and skin permeability, P, is provided by
Tang etal. (2001) as 9.5.4 Infrared Spectroscopy
molecular vibrations. The integrated absor- mation in the superficial 12m layer of the
bance of these vibrations is directly propor- skin. Repeated tape stripping can be used to
tional to the amount of solute present in the scan successive layers of the skin for solute
skin. Depth-dependant profiling of the solute penetration. Transport properties for the solute
in the skin can be achieved by attenuated total can then be obtained from an unsteady state
reflectance (ATR)-FTIR spectroscopy. ATR- solution for Eq. (9.5) given as (Pirot etal.
FTIR spectroscopy generally provides infor- 1997)
x 2 np x - Dn 2p 2 t
C ( x ) = C0 1 - - C0 sin exp (9.11)
L n =1 np L L2
where C(x) is the concentration of solute at a 2001; Rocha etal. 2007; Rossi etal. 2008).
depth x from skin surface and C0 is the concentra- Remittance spectroscopy and photothermal
tion of the solute at the skin surface. L is the total deflection are a few of the other methods that
thickness of skin and D is the effective diffusivity have been suggested in this area (Gotter etal.
of the solute in skin. The rate per unit area at 2008).
which the solute diffuses out of the skin can be
obtained by differentiation of Eq. (9.11) as
9.5.5 Trans Epidermal Water Loss
C
-D . Further, integration of this rate
x x = 0 Mammalian skin has evolved to regulate the
with time yields the total amount of solute diffus- transport of material into and out of the body.
ing across skin in a given time. This information One of the primary functions of skin is to regu-
can be used to obtain the permeability profile of late the loss of water from the body. It follows,
the solute and hence its permeability across the then, that the quantitative measure of water loss
skin. Several assumptions have been made in from the skin is indicative of its barrier integrity.
deriving this correlation that is discussed in Pirot A device, such as an evaporimeter, that can ade-
etal. (1997). One downside of using ATR-FTIR quately and accurately measure the water vapor
spectroscopy is that the solute to be monitored flux at the skin surface, and hence the rate of
needs to be IR active and have a signature distinct trans-epidermal water loss (TEWL), can be used
from those of the SC components. This limitation to assess barrier integrity of the skin. A quantita-
can, in theory, be overcome using spectral correc- tive correlation between TEWL and skin perme-
tion techniques (Naik etal. 2001). ability has been reported (Rougier etal. 1989)
Several other methods have been used to study and is found to be consistently reproducible
permeation and absorption of material into and invivo and invitro (Pinnagoda etal. 1989, 1990).
across skin. Xiao etal. have used Raman micros- Several studies have reported the use of TEWL to
copy and imaging to track penetration of phos- assess the effect of permeation enhancers on skin
pholipids in skin (Xiao etal. 2005a, b). Williams permeability (Loden 1992; Kanikkannan and
etal. provide a critical comparison of different Singh 2002; Luzardo-Alvarez etal. 2003;
Raman spectroscopy techniques and their appli- Tokudome and Sugibayashi 2004). A compre-
cation invivo (Williams etal. 1993). Sonavane hensive review of their findings is provided by
etal. have used a combination of UV-vis spec- Levin and Maibach (Levin and Maibach 2005).
troscopy, X-ray spectroscopy, and inductive cou- This review also sheds light on possible reasons
pled mass spectroscopy to track permeation of why two particular studies did not observe
gold nanoparticles in the rat skin (Sonavane etal. quantitative correlation between percutaneous
2008). Other groups have used photoacoustic absorption and TEWL (Tsai etal. 2001; Chilcott
spectroscopy for studying permeation of etal. 2002). Recent literature continues to pro-
Carbopol 940 and transdermal gels (Christ etal. vide opposing views on the use of TEWL as a
170 D.M. Cropek and P. Karande
measure of skin barrier integrity (Fluhr etal. and pharmacodynamics than the parent com-
2006; Netzlaff etal. 2006a). Nevertheless, sev- pound and can give misleading results.
eral commercial devices based on this principle
are readily available today for laboratory work. 9.6.1.2 Surface Loss
Alternate approach to determine invivo percuta-
neous absorption is to measure the loss of solute
9.6 valuation ofSkin
E from the surface as it penetrates the skin. This
Permeability InVivo can be achieved when all residual material, con-
tained in a reservoir, can be recovered. Difference
In vivo methods for quantification of solute per- between concentration or amount at time zero
meation across the skin serve as a gold standard and at time of recovery provides an estimate of
in transdermal drug delivery. Such methods can amount absorbed. Generally, the method used to
potentially eliminate variables associated with detect the residual amount needs to be sensitive
using excised human or animal skin, surrogate enough, especially in supersaturated systems, as
endpoints as well as faithfully reproduce meta- the reservoir can be infinitely large when com-
bolic, pharmacokinetic, and pharmacodynamic pared to the amount absorbed. Also, this method
behavior of the drug molecule. Wherever possi- assumes that the skin does not act as a reservoir,
ble, and practically feasible, invivo measure- which in itself can be a poor assumption.
ments are considered reliable and superior to
measurements made on model systems invitro.
9.6.2 Pharmacological Response
for invivo measurements with little or no is thus a truly noninvasive technique. Stamatas
modification at all. Tape stripping and TEWL etal. have used invivo confocal Raman micro-
measurements in particular are attractive for spectroscopy to study the uptake of vegetable oils
invivo measurements as they are relatively and paraffin oil in infants (Stamatas etal. 2008).
straightforward and minimally invasive or nonin- Pudney etal. have demonstrated the use of
vasive. The amount of solute that penetrates the Raman spectroscopy to obtain depth profiles of
SC can be quantified invivo, in humans, by tape trans-retinol in the epidermis for 10h after appli-
stripping with an appropriate adhesive tape (Tsai cation in an invivo setting (Pudney etal. 2007).
etal. 1991). In case of animal studies, invivo, it Caspers etal. have used confocal Raman spec-
is possible to quantify penetration in deeper skin troscopy to detect dimethyl sulfoxide in the SC
layers by sacrificing the animal and harvesting its (Caspers etal. 2002). For both techniques, ATR-
skin. The concentration profiles can then be used FTIR and Raman spectroscopy, the molecule of
to determine solute permeability as suggested in interest should have an active IR signature of suf-
Eq. (9.8). Umemura etal. have used tape strip- ficient intensity that is distinct from the signa-
ping invivo in healthy human subjects to deter- tures of skin components. An additional drawback
mine the pharmacokinetics of topically applied of using Raman spectroscopy is that it can only
maxacalcitol from an ointment and lotion provide relative concentrations as against abso-
(Umemura etal. 2008). Puglia etal. have used lute amounts of the diffusing solute in different
tape stripping to quantify skin penetration of layers of the skin. Impedance spectroscopy is a
lipid nanoparticles (Puglia etal. 2008). A review highly sensitive and relatively straightforward
of tape-stripping methods in determining percu- technique that can be used invivo to assess skin
taneous absorption invivo is provided by barrier integrity. Curdy etal. have used imped-
Herkenne etal. (Herkenne etal. 2008). Several ance spectroscopy to follow barrier recovery
reports have documented the use of TEWL to after the application of iontophoresis (Curdy
assess the barrier integrity after treatment with etal. 2002). Dujardin etal. describe the use of
physical or chemical enhancers of skin permea- impedance measurements to assess effects of
bility (Atrux-Tallau etal. 2008; Kolli and Banga electroporation on barrier function invivo in rats
2008). Maibach etal. have documented the effect (Dujardin etal. 2002). Kalia etal. have looked at
of successive tape strippings on TEWL invivo in the effect of surfactant treatment and iontophore-
human subjects as well as compared two differ- sis on skin impedance invivo (Kalia and Guy
ent configurations for devices measuring TEWL 1997).
(Zhai etal. 2007). Xhauflaire-Uhoda etal. have In addition, several other techniques have
used TEWL measurements to study barrier repair been utilized to quantify invivo transdermal
after the application of miconazole nitrate and delivery (Herkenne etal. 2008). These include
tape stripping (Xhauflaire- Uhoda etal. 2006). creation of suction blisters and punch biopsies.
Among the various spectroscopic techniques, Although relatively straightforward, these are
ATR-FTIR spectroscopy has been used exten- painful and invasive procedures that are less pop-
sively in tracking solute permeation in the skin ular in studying percutaneous absorption of sol-
(Ayala-Bravo etal. 2003; Tsai etal. 2003; Curdy utes, especially in human subjects. Recently,
etal. 2004; Escobar-Chavez etal. 2005; Remane microdialysis has been suggested as a novel tech-
etal. 2006). A comprehensive review of ATR- nique to measure the diffusion of solutes across
FTIR spectroscopy and its use in invivo studies the skin (Kreilgaard 2002; Mathy etal. 2005). A
appears in Naik etal. (Naik etal. 2001). Raman thin probe perfused with a physiological solution
spectroscopy, similar in principle to FTIR spec- is implanted under the dermis where, on equili-
troscopy, has also been used in studying transder- bration, it can exchange material with the extra-
mal solute penetration invivo. A big advantage cellular tissue components by passive diffusion.
of Raman spectroscopy is that it does not require The perfusate from the probe can then be ana-
tape stripping of the skin for depth profiling and lyzed for the solute diffusing across the skin. A
172 D.M. Cropek and P. Karande
time-based concentration profile of the solute dif- across rabbit and human skin invitro. JPharm Sci
93(10):24312438
fusing across the skin into the probe can be used
Astley JP, Levine M (1976) Effect of dimethyl-sulfoxide
to determine the pharmacokinetics of the solute. on permeability of human skin invitro. JPharm Sci
A practical challenge associated with this method 65(2):210215
is the careful and reproducible insertion of the Atrux-Tallau N, Huynh NTT etal (2008) Effects of physi-
cal and chemical treatments upon biophysical
probe in the deeper layers of the skin. Also,
properties and micro-relief of human skin. Arch
extremely sensitive detection methods are Dermatol Res 300(5):243251
required to analyze the small amounts of material Audus KL, Bartel RL etal (1990) The use of cultured epi-
obtained from the perfusate. thelial and endothelial-cells for drug transport and
metabolism studies. Pharm Res 7(5):435451
Ayala-Bravo HA, Quintanar-Guerrero D etal (2003)
Conclusion Effects of sucrose oleate and sucrose laureate on
The field of transdermal drug delivery research invivo human stratum corneum permeability. Pharm
has come of age and is rich with opportunities Res 20(8):12671273
Bach M, Lippold BC (1998) Percutaneous penetration
and promises. A combination of improved
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quantification methods, as well as innovative
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skin permeabilization strategies will expedite study of the physiochemical properties of human kera-
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Human Native andReconstructed
Skin Preparations forInVitro 10
Penetration andPermeation
Studies
Abbreviations
HPLC High-performance liquid chromato- major drawback of invitro skin absorption studies
graphy is the absence of peripheral blood flow which can-
Jpeak Peak flux not be simulated completely. Furthermore, concern-
Jss Steady state flux ing risk assessment the acceptance of invitro skin
Jss(max) Maximum flux absorption data by the authorities are different. In
LC Liquid chromatography the European Union, dermal absorption data for
K P
Apparent permeability coefficient many pesticides has been estimated by invitro
MS Mass spectrometry experiments (EC 2003a, b; EFSA 2012). In con-
NAFTA North American Free Trade Agreement trast, NAFTA countries (the USA, Canada, and
REACH Registration, Evaluation, Authorisation Mexico (NAFTA 2009)) do not accept invitro skin
and Restriction of Chemicals absorption data alone for risk assessment. However,
SC Stratum corneum most of the skin absorption data published in the
SCCNFP Scientific Committee on Cosmetic field of cosmetic and pharmaceutical sciences is
Products and Non-Food Products based on invitro skin absorption studies.
SCCS Scientific Committee on Consumer
Safety
TEWL Transepidermal water loss 10.1.2 Rate Determining Processes
tlag Lag-time Involved inSkin Absorption
USEPA US Environmental Protection Agency
WHO World Health Organisation In general, skin invasion is predominantly gov-
erned by the stratum corneum, which is accepted
as the main barrier of the skin. Passive diffusion
10.1 Introduction according to Ficks law is considered the rate
determining kinetic process for skin absorption.
10.1.1 Needs forInVitro Skin Modulation of this diffusion process therefore
Absorption Studies allows controlling skin invasion.
tion, especially if small and dissolved molecules example, the lipid bilayer structure is partly lost
are concerned. However, for nanoparticles or or the number of SC layers may be reduced.
submicron-sized drug delivery systems, for Therefore, from studies with healthy skin only
example, liposomes and nanoparticles, the limited conclusions can be drawn for diseased
appendageal pathway may be predominant skin. However, there are nowadays also invitro
(Lademann etal. 2006, 2007; Patzelt etal. 2011). models available to mimic the situation of dis-
An additional mechanical rubbing, e.g., a mas- eased skin (see Sect.10.2.1.5).
sage, will improve the appendageal delivery.
Moreover, for healthy skin it is reported that
nanoparticles >10nm are unlikely to overcome 10.2 I n Vitro Barriers forSkin
the SC barrier (Prow etal. 2011). Absorption Studies
Governed by the anatomical structure of the
intact SC the following absorption pathways are 10.2.1 Excised Human Skin
feasible: the intercellular route and the transcel-
lular route. The intercellular route is associated Excised human skin from surgery is the best
with the lipid bilayer structures in between the available invitro model for skin absorption
corneocytes and is considered to be the predomi- studies, it is therefore considered as the gold
nating invasion route, particularly if steady state standard. When using skin from surgery, care
conditions are assumed. Due to the liquid crystal- has to be taken during transport and preparation.
line structures of the lipid matrix surrounding the In any case, transport and preparation condi-
corneocytes, the intercellular route provides tions have to be standardized and must be con-
hydrophilic as well as lipophilic domains offering sidered when interpreting the results. The
the possibility that both lipophilic and hydrophilic following fundamental points have to be noted.
entities diffuse via this pathway. Although the As several cycles of freezing and thawing influ-
intercellular route is very tortuous and therefore ence the absorption behavior of the skin, freeze-
much longer, the diffusion is relatively fast in this thaw cycles should be avoided at all cost. Thus,
region due to the enhanced diffusion coefficient in transport at reduced temperature, e.g., on ice
comparison to the corneocytes. Furthermore, this packs with no direct contact to the skin, is
pathway can easily be modulated by penetration appropriate. During transport and skin prepara-
enhancers. The transcellular route is normally tion, care must be taken to prevent the skin sur-
regarded negligible because diffusion in the solid face from contamination by lipids originating
corneocytes is low and the necessity to partition from the subcutaneous fatty tissue which will
several times between the lipoidic bilayers and the change the permeability characteristics of the
more hydrophilic corneocytes. Both phenomena SC (Wertz 1996). It has been shown that the
result in a reduced absorption with the exception storage period in a freezer (20 to 26C)
if penetration enhancers are used which increase should be limited to 6 months (Swarbrick etal.
the permeability of the corneocytes, e.g., by appli- 1982; Harrison etal. 1984; Hawkins and
cation of urea due to keratolyic actions (Williams Reifenrath 1984; Bronaugh etal. 1986; Schaefer
and Barry 2012). and Loth 1996) as otherwise the barrier function
As a result of the various possibilities for sub- may be affected. Furthermore, evaporation
stance invasion through and into the SC invitro should be prevented during storage by using
skin absorption experiments must take into tightly sealed bags.
account these different pathways.
10.2.1.1 Full Thickness Skin
10.1.2.2 Pathways Full thickness skin consisting of SC, viable
ThroughtheDiseased Skin epidermis, and dermis (Fig.10.1a) is usable for
In diseased skin the barrier function is reduced permeation studies (diffusion through the skin,
because of structural changes in the SC.For see also Sect.10.4) as well as penetration studies
188 U.F. Schaefer et al.
a b
c d
Fig. 10.1 Different human skin membranes for absorption studies: (a) full thickness skin, (b) dermatomed skin,
(c) heat separated epidermis, and (d) enzyme split stratum corneum (According to Schaefer etal. 2008)
(see Sect.10.5), the latter addressing the sub- the stratum corneum as well as parts of the
stance distribution in the different skin layers. dermis (Fig. 10.1b). Normally, a slice thickness
For skin permeation studies, the following of 200400/500m is recommended for human
aspects are noticeable: Hydrophilic substances skin (OECD 2004b; USEPA 2004). In doing so,
often cause very long lag-times and may thus the hair follicles will be cut, but the resulting
require extremely long observation periods. This holes will rapidly close during incubation with
holds true also for lipophilic substances because the acceptor medium due to swelling of the tis-
of reduced partitioning at the interface between sue, especially if aqueous donor and receptor
stratum corneum and viable epidermis. However, fluids are used. During dermatomization care
long observation periods enhance the problem of must be taken to avoid mechanical damage of the
microbial contamination and change of skin SC, the main barrier of the skin. To overcome
integrity (due to epidermal separation) and should this problem, protection of the skin surface by a
therefore be avoided. To overcome these prob- plastic foil is indicated (Brain etal. 1998).
lems, further segmentation of the skin will be Typically, dermatomed skin is used for perme-
needed. ation studies, especially if the influence of the
hydrophilic layers of the epidermis and dermis
10.2.1.2 Dermatomed or SplitSkin should be considered on skin absorption (influ-
Dermatomed skin can be prepared by means of a ence of partition step between SC and viable epi-
dermatome which performs surface parallel sec- dermis/dermis). Moreover, dermatomed skin
tions, composed of the epidermis and including might be of particular interest to study the fate of
10 Human Native andReconstructed Skin Preparations forInVitro Penetration andPermeation Studies 189
cumulative permeated
epidermis and dermis. full thickness skin
160 reconstructed skin
methods, e.g., by determining the amount of SC methods such as animal skin form sources of
removed (Hahn etal. 2010) or measuring the food supply (pig ear, cow udder) or bioengi-
transepidermal water loss (TEWL) (Kalia etal. neered skin surrogates can be used.
1996; Russell etal. 2008). Complete removal of
stratum corneum and epidermis can best be
achieved preferably by rigorous dermatomization 10.2.3 Bioengineered Skin
rather than by heat separation or trypsinization.
The latter two methods may compromise the pro- For some time bioengineered skin has been intro-
tein structure of the epidermal/dermal tissue due duced in the field of skin research due to the limited
to precipitation or digestion of proteins. supply of human skin, some ethical or cultural
restraints to use human skin, and the 3R-initiative
concerning animal welfare (Refinement,
10.2.2 Animal Skin Replacement, Reduction) (Russell and Russell
1957). Firstly, skin corrosion (Kandrov etal.
As human skin has only limited availability, 2006a, b), sensitization, (Bernard etal. 2000;
alternatives are necessary. Besides, due to wide- Spielmann etal. 2000), metabolic phenomena
spread demands in skin absorption studies based (Gysler etal. 1999), and phototoxicity (Liebsch
on regulatory aspects (e.g., REACH program of etal. 1999) were addressed and afterwards skin per-
the EU (EC 1907/2006, 2006) the pressure to meability was evaluated (Dreher etal. 2002).
establish alternative models was enforced. In Meanwhile various commercial reconstructed epi-
addition, some researchers have ethical and cul- dermis models are available (Netzlaff etal. 2005)
tural restraints to use human skin (Hikima etal. and also accepted for skin corrosion (OECD 2004c,
2012). Skin from mouse, hairless rat, hamster 2006) and irritation testing (OECD 2010).
(cheek pouch), snake (shed skin), pig (ear, flank, Moreover, many attempts have been made to com-
abdomen, or back), and cow udder has been sug- pare in detail bioengineered skin to human skin and
gested as alternatives (Haigh and Smith 1994). animal skin (Schreiber etal. 2005; Netzlaff etal.
However, depending on the animal species used, 2005, 2007). In two large studies sponsored by the
differences in SC thickness, number of corneo- German BMBF (Federal Ministry of Education and
cyte layers, hair density, water content, lipid pro- Research), the usefulness of the commercially
file and morphology, and different permeabilities available reconstructed epidermis models EpiskinTM
in comparison to human skin have been found. (LOreal, France), Epiderm (MatTek corporation,
Typically the permeability of animal skin was USA), SkinEthic (SkinEthic Laboratories, France)
enhanced in comparison to human skin (Bronaugh were tested with nine substances in aqueous solu-
etal. 1982; Netzlaff etal. 2006b). In general, por- tion covering a wide range of lipophilicities and
cine skin is reported to match human skin best. In molecular weights (Schfer-Korting etal. 2006,
addition, porcine skin, especially the ear, is easily 2008). As a result, it could be clearly shown that all
available from slaughter houses. However, it has reconstructed epidermis models were more perme-
to be kept in mind that porcine skin must not be able than human heat separated epidermis and der-
scalded. Moreover, there are references pointing matomed porcine skin; however, the rank order of
also to the alternative of cow udder skin permeability agreed reasonably among these mod-
(Kietzmann etal. 1993; Netzlaff etal. 2006b; els. Besides, it could be shown that reconstructed
Pittermann and Kietzmann 2006). By the 7th epidermis provides less variability in comparison to
Amendment to the Cosmetic Directive for cos- human and animal skin which is consistent with
metic products (EC (2003c) Directive 2003/15/ results from the EDETOX (Evaluations and
EC; Rossignol MR 2005) a ban of tests on ani- Predictions of Dermal Absorption of Toxic
mals and of the marketing of products/ingredi- Chemicals) project (van de Sandt etal. 2004). A
ents tested on animals came into action in review about morphology, biochemical character-
September 2009. Since then only alternative ization, and application of the bioengineered mod-
10 Human Native andReconstructed Skin Preparations forInVitro Penetration andPermeation Studies 191
els EpiskinTM, Epiderm, and SkinEthic is reported human skin (see Fig.10.2). However, the actual
by Netzlaff etal. (2005). Besides, a list of materials value of such reconstructed skin might be in the
tested with Epiderm is provided by MatTek, USA development of disease models to simulate spe-
(www.mattek.com/pages/products/epider/ cific situations (Semlin etal. 2011).
materials-tested).
Additional reports are available for other,
sometimes self-made, bioengineered skin mod- 10.2.4 Artificial Skin Surrogates
els. Suhonen etal. reported permeability experi-
ments for 18 compounds in buffered aqueous Artificial skin surrogates have a long lasting his-
solutions with a cell culture model based on kera- tory. Starting from simple membranes of dialysis
tinocytes from rat skin (Suhonen tm etal. 2003). tubing and polymeric membranes for example of
A close relationship to isolated human cadaver regenerated cellulose, cellulose derivatives, poly-
skin was found; however, again the permeability carbonate, polyolefines, silicon, etc. more and
in the cell culture model was enhanced. The more complex systems became available (Haigh
group of Mueller-Goymann developed an artifi- and Smith 1994). It was recognized early that
cial skin construct based on collagen and HaCaT these very simple membranes do not resemble
(human adult low calcium high temperature kera- the skin characteristics and therefore their recom-
tinocytes) cells cultivated at the air-liquid inter- mended use was to determine the release of active
face (Savic etal. 2009). Investigating the compounds only (FDA 1997). However, by load-
permeation of caffeine and diclofenac from vari- ing of porous filter materials with lipids, these
ous formulations containing natural surfactant/ surrogates better resemble the skin properties
fatty alcohols mixed emulsifiers they reported especially if native SC lipids are used. Jaeckle
that the vehicle skin interaction was mirrored in etal. used systematic variation of lipid composi-
the artificial skin construct. tion to establish suitable membranes which pro-
A full-thickness commercial skin model, the vide properties similar to heat-separated
Phenion model (Henkel AG & Co.KGaA, epidermis (Jaeckle etal. 2003). Recently, the
Germany) was tested by Ackermann etal. (2010) Bouwstra group published different methods to
for percutaneous absorption testing of the OECD establish SC model membranes (De Jager etal.
(Organisation of economic Co-operation and 2004, 2005). For benzoic acid, they could nicely
Development) reference compounds caffeine and demonstrate that the flux is comparable to human
testosterone. They concluded that the results SC (Groen etal. 2008). Based on the PAMPA
closely paralleled human skin (Ackermann etal. technique (parallel artificial membrane permea-
2010). One of the major problems with bioengi- bility Assay) that has been successfully applied
neered skin is that the sheets must be transferred to predict gastrointestinal and bloodbrain bar-
to standard diffusion cells after the cell layer has rier absorption (Avdeef 2005) Ottavianai etal.
been punched out from the cultivating support. reported on the use of a PAMPA-membrane
To overcome this drawback, Grgoire etal. loaded with an optimized mixture of silicone
improved the experimental setup for screening (70%) and isopropyl myristate (30%) to address
skin absorption with the reconstructed epidermis skin permeation (Ottaviani etal. 2006). They
model Episkin (Grgoire etal. 2008). By modi- could show for 31 substances of a diverse data set
fying the culture conditions, it was possible to a good correlation to kp-values of human skin.
omit bypass diffusion in the inserts. In contrast to Recently, two commercial models based on syn-
studies in static diffusion cells the cell layer no thetic lipid mixtures, Strat M (Joshi etal. 2012)
longer has to be punched out. In summary, and Skin-PAMPA (Sink etal. 2012), have
although there are references suggesting the become available.
helpfulness of bioengineered skin in skin absorp- The Skin-PAMPA assay mimics the lipid phase
tion studies, the basic problem is the lack of of the SC using an optimized lipid mixture com-
building up a barrier at the same level as natural posed of certramides, free fatty acids, and choles-
192 U.F. Schaefer et al.
terol. It can be considered as a refined approach of 28 (OECD 2004a). In general, finite dosing mim-
the method of Ottavani etal. mentioned above. The ics the in use conditions where normally a lim-
method was validated comparing the permeation ited dose of the formulation is applied to the skin.
(kp) of various solutes through three different kinds In contrast by infinite dosing a disproportionally
of skin preparations (isolated SC, isolated epider- high amount of formulation is applied. Typically
mis, and full thickness skin). Moreover, it could be a maximum of 15mg/cm2 for powders, up to
shown that the assay is capable to predict the per- 10l/cm2 for solutions, and up to 10mg/cm2 for
meation in human skin with a reasonable correla- semisolid preparations are considered finite
tion between Skin-PAMPA permeability and Franz doses. One problem with finite dosing is to spread
cell permeability (Sink etal. 2012). When it the formulation evenly across the skin surface.
comes to mimic different skin preparation, the best Hahn etal. could demonstrate that this effect may
correlation was found for full thickness skin. Since influence the results of invitro skin absorption
Skin-PAMPA is a rather new approach, it should be studies depending on the applied drug (Hahn
investigated further to prove its reliability and etal. 2012). Also, the finite dose kinetics of drug
applicability to various scenarios. concentration over time are usually quite com-
The Strat M approach uses a synthetic mem- plex due to multifactorial overlapping processes
brane sandwich composed of polyether sulfone such as the depletion of active in the donor phase
treated with synthetic lipid materials to mimic (which should usually lead to a decrease in the
the human skin. The method was tested using rate of absorption), changes in the donor compo-
various substances in different formulations in sition which affect the skin permeability, and
comparison to human cadaver skin in a Franz cell elimination of the active due to metabolism and
setup. For aspirine, nicotine, and hydrocortisone, systemic clearance to name but the most relevant.
the usefulness of the model could be d emonstrated The complexity of the problem shall be illus-
showing comparable fluxes in comparison to trated by the following example. If the formula-
human skin. Furthermore, comparable vehicle tion is applied without an occlusive dressing, the
effects, such as penetration enhancement, could donor concentration may increase due to evapo-
be observed for different caffeine formulations. ration of components of the vehicle and conse-
The authors of the study state that the model was quently increase the rate of absorption. This may
investigated against a diverse data set but unfor- even lead to over-saturation or recrystallization
tunately did not state exactly which compounds of formulation components or the active on the
were tested and what kind of human skin prepa- skin which further complicates matters. Normally
ration was used in the study (Joshi etal. 2012). true finite dosing is assumed if the donor concen-
Besides, a definition of the lipid mixture compo- tration depletes to at least 10% of the initial con-
sition is missing. Since this is also a novel centration of the applied formulation after about
approach its reliability and versatility should be 2024h (WHO 2006).
shown in greater detail in further studies. By infinite dosing, the amount of formulation
should be large enough to assure that practically
no donor depletion occurs. From that it follows
10.3 I n Vitro Experimental Setups that a maximum rate of absorption is gained and
forSkin Absorption Studies maintained over the whole experimental period.
Typically the steady state flux is determined and
10.3.1 Finite Versus Infinite Dosing based on that the apparent permeability coeffi-
cient is calculated. In between of finite and infi-
For skin absorption studies one differentiates nite dosing all transient cases are possible. For
usually between two different modes of more details on the effects of finite, semifinite,
application, finite and infinite dosing. Both terms and infinite dosing on skin invasion see Chapter 1
are defined in the OECD Guideline 428 (OECD Basic mathematics in skin permeation of drugs
2004b) and more specifically in OECD Guidance in this volume.
10 Human Native andReconstructed Skin Preparations forInVitro Penetration andPermeation Studies 193
As stated before finite dosing is normally In principal two different types of test cham-
related to in use conditions. However, the con- bers are available. These are either upright/verti-
ditions during bathing or wearing closely fitting cal (Fig.10.3a) or side-by-side/horizontal cells
clothes are more realistic depicted by infinite (Fig. 10.3b) with receptor volumes from 0.5 to
dosing (WHO 2006). 15ml and application areas of about 0.22cm2
(Brain etal. 1998). As by manufacturing pro-
cesses dimensions may vary in certain limits cal-
10.3.2 Open Versus Occluded Dosing culation of data has to be related to individual
cells. Vertical cells are preferable for testing
Occlusion during invitro skin absorption experi- semisolid formulations which can be spread on
ments will influence the results in different ways. the incubation area analogously to the invivo
Normally these effects are limited to the finite situation. In addition, it is easy to realize both
dose case, because for infinite dosing practically occlusive effects by capping the donor chamber
no changes of the donor compartment will take and in-use condition (nonoccluded) by leaving
place. In the open system, due to evaporation of the donor chamber open. Usually, in vertical cells
vehicle compounds, the concentration of active the receptor phase is mixed by stirring with a
increases and may even achieve supersaturation. magnetic bar. Side-by-side cells consist of two
This changes the conditions completely depend- chambers separated by a membrane. Both sides
ing on the magnitude of evaporation. Surber and are stirred leading to highly controlled diffusion
Smith have reported this phenomenon as meta- conditions, including continuous concentration
morphosis of the formulation (Surber and Smith equilibrium in both chambers and well-defined
2005). On the other hand, if the active is volatile unstirred layers. Therefore, side-by-side diffu-
the concentration will decrease during sion cells are preferable for kinetic studies, espe-
experimental time. In this case, it is essential to cially if solutions are tested. For testing semisolid
determine the rate of evaporation during the preparations, the design of side-by-side diffusion
experiment by collecting the volatile substance, cells has to be changed by replacing the donor
for example, by using an adsorption trap (adsorp- side by a static system without stirring (Feldmann
tive powder) mounted on top of the diffusion cell and Maibach 1969; Bronaugh and Stewart 1984,
(Rauma etal. 2013). This is essential if mass bal- 1985). For vertical as well as side-by-side diffu-
ance is addressed (Kasting and Miller 2006). sion cells, the static and the flow-through mode
are available. Using the static mode the receptor
phase is not replaced continuously except for the
10.3.3 Diffusion Cells volume exchange during sampling. This leads to
a constant increase of substance in the receptor
In the past, many different protocols have been fluid, and depending on the saturation concentra-
used to address skin invasion (ECETOC 1993; tion of compound diffusion may be affected. In
EC 2003a, b; SCCNFP 2003; SCCNFP/0690/03 general it is agreed upon that until reaching a
2003; EC 2004; WHO 2006; SCCS/1358/10 maximum of 10% saturation concentration in the
2010; EFSA 2012). This results in problems of acceptor, diffusion will not be influenced (known
comparability of results. Meanwhile in 2004 the as sink conditions). Therefore, care must be taken
OECD Guideline 428 and OECD Guidance 28 not to exceed this limit. One possibility to over-
became effective and provide general rules for come this problem is to exchange the receptor
conducting invitro tests for dermal absorption medium completely at each sampling point.
(OECD 2004a, b). In principal special protocols However, it has to be considered that problems
are allowed; however, deviations from the with compound quantification, due to quantifica-
recommended procedures should be restricted tion limits, and air bubbles may occur.
and have to be rationally motivated on a case-to- Because of the easy handling the most widely
case basis. used static diffusion cell is the Franz diffusion
194 U.F. Schaefer et al.
a c
b d
Fig. 10.3 Sketch of a static vertical Franz diffusion-cell (a), static side-by-side diffusion cell (b), after static side-
by-side diffusion cell, flow-through cell (c), and Saarbruecken penetration model (d)
cell which was introduced in 1975 (Franz 1975). diffusion cells too, for example, described by
As an alternative a flow-through version can be Solich etal. (2001, 2003). In general several
used (Fig.10.3c). In this mode the receptor vol- comparative studies have shown the equivalence
ume is continuously replaced by means of a of static and flow-through cells (Bronaugh and
pump, which more or less mimics the vivo condi- Maibach 1985; Bronaugh and Stewart 1985).
tions of drainage by the systemic circulation and Due to the easy handling and realization of in-use
facilitates maintaining sink conditions. A typical application conditions, static Franz diffusion
flow-through cell, based on a side-by-side diffu- cells are generally preferred. However, for stud-
sion cell, has been designed by Bronaugh and ies where metabolic effects are involved flow-
Steward (1985). It has to be kept in mind that through systems like the Bronaugh cell are
flow rate, chamber volume, and detection limit superior because the continuous solvent replace-
are related variables. The flow rate has to be ment better mimics the invivo situation
adjusted in a way that during the entire experi- (Bronaugh 2004a, b).
mental time sink conditions apply in the receptor
chamber, the amount can be detected, and proper
mixing exists. In addition adsorption of analytes 10.3.4 Barrier Integrity Check
to the tubing used to pump the receptor medium
may influence the results. A real benefit of flow- Barrier integrity has to be checked prior or after
through systems especially for higher numbers of invitro skin invasion experiments. Common phys-
tests such as in an industrial setting is that con- ical methods are measurement TEWL or transcu-
tinuous replacement of the receptor fluid can eas- taneous resistance or alternatively the measurement
ily be automated (Moody 1997, 2000). of the permeation of a marker substance, mostly
Automation of sampling is available for vertical tritiated water. TEWL measurements are well
10 Human Native andReconstructed Skin Preparations forInVitro Penetration andPermeation Studies 195
established for invivo studies. For invitro experi- 10.3.6 Selection ofReceptor Fluid
ments, it is strongly recommended to use a closed
chamber measurement setup (e.g., Aquaflux The selection of the right receptor fluid is essen-
AF200, Biox, London, UK; VapoMeter, Delfin tial for the results. The following points have to
Technologies, Kuopio, Finland). In contrast, be considered: the receptor fluid should enable
Netzlaff etal. reported that devices using the sufficient solubility of the active compound under
open chamber principle (e.g., the Tewameter investigation (see also Sect.10.3.3 for explana-
TM 300, Courage-Khazaka Electronic, Koeln, tion of sink conditions), allow unhindered parti-
Germany) are not sensitive enough to detect tioning of the active from the skin to the receptor
small defects of the skin which nonetheless influ- fluid, and not disturb the barrier integrity of the
ence skin permeation (Netzlaff etal. 2006a). skin. For water soluble compounds, normal saline
However, it has to be kept in mind that TEWL or isotonic buffer systems of pH7.4 are appropri-
threshold values that might indicate a violation of ate. To prevent microbial influences on skin
barrier integrity depend on the TEWL instrument integrity preservatives, preferentially sodium
used as well as the skin preparation and therefore azide (0.05% wt/V), should be added. To main-
no general limits can be provided (Elkeeb etal. tain viability of fresh skin cell culture medium is
2010). Using tritiated water as a marker is also a feasible. For lipophilic substances additives to
well-established method although the exclusion enhance the solubility are needed (OECD 2004a).
criteria for damaged skin are nowadays under For example, for essential oils ethanol water mix-
discussion. Too many samples may be rejected tures of different concentrations may be used
using a permeability coefficient of water of 2.5 x (Reichling etal. 2006). Furthermore, the addition
103 cm/h as the upper limit to reject cells with of surfactants (Challapalli and Stinchcomb 2002;
damaged barrier (Wilkinson etal. 2004). Another OECD 2004a), bovine serum albumin (Dal Pozzo
problem is the use of tritiated water before doing etal. 1991), or cyclodextrines (Sclafani etal.
the experiment. During the removal of the 1995) are reported. In all these cases, the influ-
remaining tritiated water barrier damage may ence of the additives on permeability must be
occur. Therefore, the use of tritiated water must clarified, e.g., by testing different concentrations
be evaluated critically. of a solubilizer. Besides, it has to be considered
that the analytical procedure may be affected by
the additives.
10.3.5 Temperature
concerned a sufficiently large number of sam- within feasible experimental time periods (<48h,
pling points should be planned so that a represen- in well-founded exceptional cases up to 72h).
tative number of data points are available Therefore, it is generally recommended to use
especially during steady state (rule of thumb: split-thickness human skin for permeation stud-
preferentially at least five sampling points which ies, if possible (Van De Sandt etal. 2000;
are evenly spread out during steady state) to SCCS/1358/10 2010). If full thickness skin is
describe the permeation kinetics properly. needed, rational has to be provided for that.
Especially concentration changes between sam- Moreover, the use of epidermal membranes must
pling points should be large enough to be detected be justified. Due to their fragility, heat separated
by the analytical procedure. epidermis may cause problems with tape strip-
ping which is needed for establishing a mass bal-
ance to address the content inside the SC in finite
10.3.8 Quantification Methods dose experiments especially if skin absorption of
pesticides etc. is addressed. In this case, the
For risk assessment frequently radioactive amount in the SC is often considered as not
labeled compounds are used (Poet and McDougal absorbed depending on substance properties and
2002). In this context, it has to be demonstrated application (EFSA 2012).
that the intact molecule is measured by scintilla-
tion counting. In addition one cannot fully
exclude that labeling may result in a change of 10.3.10 Number ofExperiments/
permeability properties. Meanwhile new highly Replicates
sensitive and selective quantification methods are
available, e.g., high-performance liquid chroma- As basic criteria concerning the number of sam-
tography (HPLC), Liquid chromatography-mass ples to assess invitro absorption of cosmetic
spectrometry (LC-MS-MS), etc. With these ingredients eight samples from four different
methods simultaneous quantification of the donors are recommended by (SCCS/1358/10
mother compound as well as metabolites or deg- 2010). In addition a minimum of 0.64cm2 skin
radation products is possible. Therefore, it is rec- area should be covered by the formulation
ommended to use such methods preferentially. (SCCS/1358/10 2010). Furthermore, the total
recovery should be in the range of 85115%
(SCCS/1358/10 2010). However, for risk assess-
10.3.9 Influence ofThickness ofSkin ments of chemicals the recovery limits are
Preparation 90110% but may be extended in special justi-
fied cases (OECD 2004b).
It is well known that the thickness of the skin
preparation may influence the results of invitro
skin absorption experiments in various manners 10.3.11 Segmentation ofSkin
depending on compound, formulation, and dos- inDifferent Skin Layers
ing (finite/infinite) (Wagner etal. 2002; Veccia
and Bunge 2003; EDETOX 2004; Wilkinson To localize the active compound in different skin
etal. 2004, 2006; Henning etal. 2008, 2009). layers, e.g., to determine depot effects, a segmen-
Due to the complexity of the skin invasion pro- tation of the skin is needed. Incubation of the skin
cess and lack of data, relations to physico- can be done either in diffusion cells or in a spe-
chemical characteristics are difficult to establish. cial apparatus, the so-called Saarbruecken pene-
However, in general if using full thickness skin tration model (SB-M, Fig.10.3d) (Wagner etal.
steady state conditions may often not be achieved 2000). In contrast to diffusion cells, the SB-M
10 Human Native andReconstructed Skin Preparations forInVitro Penetration andPermeation Studies 197
does not have a liquid receptor phase and there- c orresponding skin depths the removed amount
fore the original hydration state of the skin is pre- of stratum corneum has to be determined. This
served. Normally full thickness skin is used for can be done, e.g., by weighing (Russell and Guy
this model, and experiments are done for a series 2012) or by a more advanced method such as
of incubation times to obtain drug-concentration infrared densitometry (Hahn etal. 2010; Franzen
skin depth profiles. For more details, please refer etal. 2012) protein extraction and quantification
to (Wagner etal. 2000; Melero etal. 2011). (Dreher etal. 2005) or other optical methods
Skin dissection can be done first by tape strip- (Weigmann etal. 2003). For human skin and pig
ping to remove the stratum corneum layer-by- skin, it could also be demonstrated that infrared
layer using adhesive tapes and afterwards by densitometry offers the possibility to determine
segmentation of epidermis and dermis into sur- the point of total removal of the stratum corneum
face parallel slices by means of a cryomicrotome. (Hahn etal. 2010; Franzen etal. 2012). For more
Skin segmentation in invitro absorption experi- detailed instructions see (Melero etal. 2011).
ments is only meaningful if skin of a certain
thickness, e.g., dermatomed or full thickness 10.3.11.2 S egmentation oftheDeeper
skin, is used. Skin Layers Consisting
ofViable Epidermis
10.3.11.1 R emoval oftheStratum andDermis (Cryosectioning)
Corneum byTape Stripping The best way for segmentation of the deeper skin
Many protocols exist for tape stripping invitro as layers is surface parallel cryosectioning. After
well as invivo (Shah etal. 1998; Surber etal. removal of the SC the skin specimen is mounted
1999; Voegeli etal. 2007; Lademann etal. 2009; on a metal bloc, the surface is adjusted horizon-
Melero etal. 2011). Normally tape stripping is tally, and rapidly frozen, for example, by expand-
not possible for epidermis sheets due to their fra- ing carbon dioxide. Subsequently, the sample is
gility. Furthermore, for longer incubation times transferred into a cryomicrotome and the skin is
or strong interaction of excipients with the SC segmented into horizontal slices. The corre-
tape stripping may be altered. sponding skin depth can easily be related to the
To obtain valid results, the following points weight of the slice. For details see reference
have to be considered: (Melero etal. 2011).
The surface of the skin must be properly
cleaned, for example, by cotton swabs or by a
washing procedure. The washing step removes the 10.4 Results Obtained
remaining formulation from the skin surface and fromPermeation Studies
thus assures that the tape strips may stick to the
skin surface. Especially, organic solvents should be Permeation studies are normally conducted to
avoided due to the effect of substance extraction at determine the transdermal/systemic availability
least from the outermost stratum corneum layers. of a compound. Therefore, the main experimental
For homogeneous tape stripping, the problem parameter which is addressed is the amount per-
of wrinkles has to be eliminated and a constant meated per cm2 into the receptor compartment
pressure has to be applied. That can be done, for over time. Two different dosing regimens are
example, by mounting the skin disk under stretch- available. The infinite dosing regimen serves to
ing on cork disks in a special apparatus (Wagner determine kinetic constants such as the steady
etal. 2000) and charging each strip with a con- state flux or the apparent permeability coefficient
stant weight for a predetermined time period or and lag time, whereas finite dosing evaluates the
using a special roller (Lademann etal. 2009). amount absorbed after a certain time. Additionally
Furthermore, to be able to calculate the by applying the technique of mass balance the
198 U.F. Schaefer et al.
fate of a compound can be evaluated (i.e., the per- experiment a mass balance is inevitable. Total
centage that stays on the skin surface, enters the recovery should be in the range of 85115%
SC or other parts of the skin, etc.). according to guidelines or references
(SCCNFP/0690/03 2003; SCCS/1358/10 2010).
However, for risk assessments of chemicals the
10.4.1 Infinite Dosing Studies recovery limits are set to 90110% but may be
extended in special justified cases (OECD 2004b).
Permeation studies using infinite dosing address
the steady state flux of a compound from a
formulation through a given membrane, e.g., SC, 10.5 Results Obtained
epidermis, dermatomed skin, or full thickness fromPenetration Studies
skin, according to Ficks law of diffusion. The
steady state flux value Jss is calculated from the The scope of penetration studies is to determine
linear part of a diagram of the amount of com- the distribution of a compound within the differ-
pound permeated into the receptor phase per area ent skin layers. Therefore, skin segmentation is
versus time. At the beginning of the experiment, needed (see Sect.10.3.11). Within each skin seg-
the membrane will first be filled up with perme- ment the amount of drug must be determined
ant until saturation (steady state) is reached. The selectively and completely. For doing that an
extrapolation of the linear steady state intersects extraction procedure has to be established and
with the time-axis at the lag time tlag which can be validated which allows the determination of very
used to calculate the apparent diffusion coeffi- low concentrations with a high accuracy and
cient. Moreover, based on the steady state flux recovery. This may lead to complications if quan-
(Jss) the apparent permeability coefficient Kp=Jss/ tification techniques like HPLC, LC-MS, etc. are
Cv, with Cv=concentration in the vehicle can be used. Furthermore, due to quantification limits
calculated. Permeability coefficients may be used consecutive segments may need to be pooled
for the comparison of different formulations. (Wagner etal. 2000; Hansen etal. 2008).
Furthermore, the maximum flux (Jss(max)) of the However, this reduces the precision of the results.
solute, which denotes the maximal possible der- Besides, for the construction of concentration
mal delivery from a given vehicle, can be esti- skin depth profiles the exact segment position in
mated by Kp(max)=Jss(max)/Css, with Css=saturation the skin has to be known (Hahn etal. 2010).
concentration of the solute in the vehicle (Roberts Examples for SC profiles of infinite dosing and
etal. 2002; Magnusson etal. 2004). For more finite dosing are shown in Fig.10.4.
details see Chap. 1 Basic Mathematics in Skin Due to potential contamination by not com-
Permeation of Drugs in this volume (Selzer pletely removed formulation often the two first
etal. 2013). strips are discarded. Based on such profiles diffu-
sion parameters according to Ficks law can be
calculated. For more details, please refer to refer-
10.4.2 Finite Dosing Studies ence (Selzer etal. 2013). Apart from that addi-
tional information can be gained. Wagner etal.
Based on the permeated amount of compound into demonstrated by applying a model based on a
the receptor phase per cm2 versus time dependency Michaels Menten kinetic that the time to reach
the amount permeated can be calculated for 50% of saturation within the SC can be related to
selected time points. These values can serve for the permeability coefficients for heat-separated
comparison of different formulations. Moreover, epidermis sheets (Wagner etal. 2002). Moreover,
the highest flux value (Also: peak flux, Jpeak) during calculation of total amounts in different skin lay-
the experimental time period can be determined. ers, e.g., SC and deeper skin layer, may allow the
For finite dosing studies after the end of the identification of skin depots.
10 Human Native andReconstructed Skin Preparations forInVitro Penetration andPermeation Studies 199
8000
3h
6000 6h
References
4000
Ackermann K, Lombardi Borgia S, Korting HC, Mewes
2000 KR, Schfer-Korting M (2010) The phenion full-
thickness skin model for percutaneous absorption test-
0 ing. Skin Pharmacol Physiol 23:105112
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Stripping Procedures
forPenetration Measurements 11
ofTopically Applied Substances
JrgenLademann, SabineSchanzer,
HeikeRichter, MartinaC.Meinke, Hans-
JrgenWeigmann, andAlexaPatzelt
Hair shaft
Intercellular penetration
Transcellular penetration
Stratum
corneum
Epidermis
J. Lademann (*) S. Schanzer H. Richter
M.C. Meinke H.-J. Weigmann A. Patzelt
Charit Universittsmedizin Berlin,
Department of Dermatology, Venereology and Allergy, Follicle penetration
Center of Experimental and Applied Cutaneous
Physiology, Berlin, Germany Fig. 11.1 Schematic representation of the penetration
e-mail: juergen.lademann@charite.de pathways of topically applied substances
was found to be a second essential pathway hair follicles are somewhere cut in their middle.
(Patzelt etal. 2011; Schroeter etal. 2010). The This would imply, if the hair follicles were acces-
third pathway is the transcellular penetration sible and not plugged by the contraction process,
pathway, where the substances pass through both that the topically applied substances would pass
the corneocytes and the lipid layers; this pathway directly through the hair follicle stump into the
seems to be of minor importance, yet. receptor medium. Since, however, the hair folli-
While the intercellular penetration process has cles are plugged due to contraction, intercellular
been recognized for decades, the existence of fol- penetration can be investigated more or less prop-
licular penetration could be demonstrated not erly by this method whereas follicular penetra-
before a few years ago. Follicular penetration tion is not measurable. Thus, follicular penetration
experiments require measurements at high spatial must be investigated either invivo or exvivo on
resolution (Jung etal. 2006). Only in recent porcine ear model skin, which strongly resembles
years, suitable techniques for such measurements human skin in terms of composition and struc-
could be provided. These systems are based ture. The advantage of the porcine ear skin model
mainly on optical methods specifically developed is that the skin remains tightly attached to the car-
or optimized for that purpose (Tfayli etal. 2012; tilage even after truncation of the ear and does
Lademann etal. 2012; Mura etal. 2012; Forster not contract. However, it has to be taken into con-
etal. 2011; Haag etal. 2011). A further challenge sideration that the porcine hair follicles extend
is that while intercellular penetration can be usu- deeper into the skin and are larger in diameter
ally investigated invitro or exvivo, follicular than human hair follicles (Lademann etal. 2010;
penetration must be analyzed invivo or ex vitro Hutton etal. 1978).
on porcine ear model skin (Patzelt etal. 2008). The present chapter will summarize the easy
This is due to the fact that skin contracts immedi- and wide application possibilities of the different
ately after excision during surgical intervention. stripping methods, which can be applied for
Although the interfollicular fibers can be investigating intercellular and follicular penetra-
expanded by stretching the excised skin to its tion pathways.
original size for the measurements, the very
dense network of fibers surrounding the hair fol-
licles remains contracted, which is an almost irre- 11.2 Tape Stripping
versible process (Patzelt etal. 2008). The effect
of contraction of the hair follicles in excised One of the oldest methods used in penetration
human skin was initially demonstrated by Patzelt studies is the tape stripping method. Briefly, tape
etal. (2008), who could moreover show that the stripping comprises the successive removal of
follicular penetration of a fluorescent dye- adhesive films pressed onto the skin after the top-
containing formulation was reduced by 90% in ical application and penetration of the substance
the case of ex vitro investigations on excised skin under investigation. With each adhesive tape, a
in comparison to invivo investigations, whereby certain amount of corneocytes is removed includ-
methods and skin models were identical. ing the topically applied substances contained
As a consequence, the follicular orifices are within this layer. The tape stripping method is
not accessible for the penetration process in illustrated in Fig.11.2 in detail. After applica-
exvivo models, which has to be considered when tion with a syringe Fig.11.2a and homogeneous
certain methods are employed such as the diffu- distribution by means of a saturated rubber
sion cell experiment. Diffusion cell experiments glove finger Fig.11.2b, the applied formulation
(Okuda etal. 2011; Baert etal. 2010) are widely is then allowed to penetrate for a determined
used for penetration investigations on split or time. Thereafter, the individual adhesive strips
full-thickness skin. However, the hair follicles are pressed one by one onto the skin by a roller
reach deeply into the subcutaneous tissue, which Fig. 11.2c or stamp leading to a smoothing of
is mostly removed before the skin probes are the skin surface and are then removed quickly
clamped onto the diffusion cell meaning that the Fig. 11.2d. The smoothing of the skin surface
11 Stripping Procedures forPenetration Measurements ofTopically Applied Substances 207
a b
c d
Fig. 11.2 Images presenting the tape stripping method. (a) Application of the formulation with a syringe. (b)
Distribution of the formulation using a saturated rubber glove finger.(c) Adhesive tapes are pressed onto the skin by
using a roller. (d) Adhesive tapes are rapidly removed from the skin
Fig. 11.3 Influence of the roller smoothing the skin surface. (a) Skin furrows before rolling. (b) Smoothed skin surface
after rolling (Lademann etal. 2005)
represents an important step in the tape stripping (Fig. 11.3a) has been converted into a flat sur-
protocol as by pressing the adhesive film onto the face (Fig.11.3b). During the rolling process, the
skin, the influence of furrows and wrinkles can be adhesive film is permitted to contact the surface
avoided or at least minimized (Lademann etal. of the respective skin area completely. The adhe-
2005). In Fig.11.2c, a roller is used for smooth- sive films pressed onto the skin are subsequently
ing (Mohammed etal. 2012; Bettoni etal. 2012; removed and analyzed for the amount of stratum
Lademann etal. 2006, 2009) the skin surface. corneum and the amount of the topically applied
The principle of smoothing the skin surface by substance. In Fig.11.4, the image of a typical
a roller is represented in Fig.11.3 using opti- tape strip after removal from the human skin is
cal coherence tomography (OCT). After rolling shown. The corneocyte cover is clearly recogniz-
the skin, the previously structured skin surface able on the adhesive film.
208 J. Lademann et al.
Thus, tape stripping is a well-established method initially been considered as a measure for the
to investigate the penetration of topically applied stratum corneum depth at which the tape strip
substances into the complete stratum corneum; how- was removed. Applying the tape stripping method
ever, it is not suited to remove cells from the viable with growing expertise and experience, it became
epidermis located beneath the stratum corneum. In evident that the tape number is not a reproducible
Fig. 11.5, a biopsied human skin sample prior to value characterizing the depth of the stratum cor-
(Fig.11.5a) and after tape stripping (Fig.11.5b) is neum at which the tape strip is removed. This is
shown, demonstrating that this procedure is capable attributable to the fact that different formulations
of removing the stratum corneum completely. Prior influence the amount of stratum corneum adher-
to tape stripping, the wrinkles and furrows in the ing to the tape strip differently. Whereas greasy
skin surface structure are clearly recognizable, yet, formulations may reduce the adhesive strength of
whereas a flat skin surface is visible after tape strip- a tape strip, meaning that less stratum corneum is
ping. This is due to the tape stripping procedure removed per tape strip, an ethanolic formulation
causing a light swelling of the skin surface. may increase the amount of stratum corneum
When the tape stripping method was intro- removed. In addition, also the pressure used for
duced, the number of the individual tapes had pressing the adhesive film onto the skin and the
type of adhesive film decisively influences the
amount of stratum corneum removed by each
tape strip. The literature reports of a variety of
methods to quantify the amount of stratum cor-
neum on the respective tape strips which further
allow the determination of the depth at which the
tape strips were removed. This can be performed,
for instance, by weighing the tape strips prior to
and after their application to the skin (Weigmann
etal. 2005). A decisive shortcoming of this
method is, however, that the weight of the first
tape strips is not only determined by the corneo-
cytes, but also by the topically applied substances
penetrated into the uppermost layers of the cor-
neocytes. Moreover, in deeper layers of the stra-
Fig. 11.4 Microscopic image of a tape strip removed tum corneum interstitial fluid may escape, which
from the skin could also increase the weight. As an alternative
Fig. 11.5 Histological sections from biopsies removed prior to (a) and after (b) tape stripping, demonstrating that the
stratum corneum can be removed completely (Lademann etal. 2005)
11 Stripping Procedures forPenetration Measurements ofTopically Applied Substances 209
method, Lindemann etal. (2003) proposed to use stratum corneum is neglected in contrast to the
the pseudoabsorption at 430nm as a measure for extended method where this essential part of
the amount of stratum corneum on the tape strips. information is likewise considered. In this way,
The pseudoabsorption characterizes the attenua- it can be clearly seen that although the amount of
tion of the light penetrating through the tape strip. corneocytes may vary between the individual
It is determined by the absorption, reflectance, tape strips, on average it is declining with the
and scattering of the corneocytes. These signals increasing tape strip number. Therefore, based
are known to depend on the wavelength, and the on the investigations of the amount of removed
shorter the wavelength, the stronger the signal, stratum corneum, the tape strips were used to
indicating that the scattering effect is more pro- calculate the profile of the horny layer. Here, the
nounced in shorter wavelength. However, wave- space between two horizontal lines corresponds
lengths below 400nm are unsuitable for these to the amount of stratum corneum removed with
investigations as a variety of formulations, the specific tape strip. In the deeper layers of the
including sunscreens, have absorption bands in stratum corneum the distances between the hori-
this spectral range. A third method for the deter- zontal lines are getting smaller. The top line of
mination of the amount of stratum corneum on the horny layer profile shown in this figure repre-
the tape strips, which is meanwhile commercially sents the surface of the stratum corneum, while
available, was suggested by Voegeli etal. (2007, the bottom horizontal line represents the bound-
2009). This method uses the infrared absorption ary to the living cells.
of the corneocytes for determining the amount of The amount of topically applied substance
stratum corneum. The measuring device is based removed with a single tape strip can be deter-
on an infrared densitometer permitting indirect mined by classical analytical methods like high-
measurements of the stratum corneum protein performance liquid chromatography (HPLC) and
content on the removed tape strip by means of mass spectroscopy. If the information on the
absorption. This measuring method is a nonde- amount of topically applied substance detected
structive technique so that the tape strips can sub- on the respective tape strips is added to the horny
sequently be used for other analyses. It provides layer profile, a penetration profile is obtained.
a clear advantage as it is not influenced by the This is illustrated in Fig.11.7 showing part of a
absorption of applied formulations in the ultravi- histological section. The adjacent penetration
olet (UV) spectral range, and it is not influenced profile of a UV filter exhibits the distribution of
by nanoparticles. A further method suggested by this substance in the stratum corneum.
Dreher etal. is based on staining the corneocytes As already mentioned above, the tape stripping
after removal so that the amount of stratum cor- procedure is unsuitable for the investigation of
neum on the tape strips can be determined by transdermal penetration as tape strips only remove
absorbance measurements using a UV/VIS spec- the stratum corneum. However, it could be shown
trometer (Dreher etal. 2005). that tape stripping can moreover be used for ana-
Taking into consideration the above-lyzing the dermatopharmakinetics of topically
mentioned facts, it became established as a stan- applied substances intended for penetration
dard that instead of the number of tape strips, the through the cutaneous barrier, such as steroids
amount of stratum corneum is now used for the (Pelchrzim etal. 2004). In a previous study, two
calculation of penetration profiles of drugs into different formulations of 0.05% clobetasol propri-
the stratum corneum. A comparison of the tradi- onate were topically applied and the penetration
tional and the extended method is schematically profile was determined 2 h after topical applica-
shown in Fig.11.6, highlighting the advantages tion. It was observed that clobetasol proprionate in
of the extended method. The traditional tape the Temovate emollient (Glaxo Wellcome Inc.;
stripping procedure only considers the amount Research Triangle Park, NC, USA) was predomi-
of substance in relation to the tape strip number, nantly located on the skin surface (Fig.11.8b).
whereas the information about the amount of Contrary to that, clobetasol proprionate in the
210 J. Lademann et al.
0,05 1
0,5
0 0
1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9
Tape number Penetration profile Tape number
0 1
Amount of corneocytes [%]
10
Tape number
30
40
100 70
0 100
Fig. 11.6 Principle of the calculation of the penetration X-ray fluorescence (XRF), atomic absorption spectros-
profile. The amount of corneocytes on each tape strip is copy (AAS), high-performance liquid chromatography
determined by UV/VIS spectroscopy. The concentration (HPLC), or gas chromatography in combination with
of the applied substance on each tape strip is determined mass spectroscopy (GC/MS). The combination of both
by traditional methods such as UV/VIS spectroscopy, results allows the calculation of a penetration profile
0
1
10
ca. 10 m
20 5
30
10
40
Tape number
Concentration [%]
50
15
60
20
70
25
80 30
35
90 40
45
50
100
1,8 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8
Fig. 11.7 Distribution of a UV filter substance (sunscreen) in the stratum corneum determined by tape stripping
11 Stripping Procedures forPenetration Measurements ofTopically Applied Substances 211
Temovate cream penetrated significantly deeper other authors (Gorne etal. 2007). Six hours after
into the stratum corneum (Fig.11.8a). The amount application of the two formulations, only weak
of cream detected on the skin surface was essen- blanching effects were visible for the Temovate
tially reduced compared to the emollient, indicat- emollient, whereas intensive blanching occurred
ing that the cream is penetrating better through the after application of the Temovate cream indicat-
skin barrier. As clobetasol does not vaporize after ing its higher local concentration.
topical application, the various steroid concentra- Thus, tape stripping is not only a suitable and
tions in the stratum corneum permit conclusions simple method for the detection of topically
about the efficiency of drug penetration through applied substances in the stratum corneum, but it
the cutaneous barrier (Pelchrzim etal. 2004; provides also information about the dermato-
Weigmann etal. 2001). Additionally, differences pharmakinetics of these substances when they
in the reservoir formation depending on the formu- are passing through the cutaneous barriers. The
lation applied could be correlated to a biological tape stripping procedure can be applied both
response in the form of a blanching effect invivo and exvivo on human skin and on porcine
(Fig.11.9), which has already been described by
0 1 0 1
5 5
Horny layer thickness [%]
20 10 20 10
Tape number
Tape number
40 20 40 20
60 60 30
30
40
80 40 80
50 50
60 60
100 79 100 85
0 5 0 5
Concentration of clobetasol propionate [mg/cm2], Concentration of clobetasol propionate [mg/cm2],
correlated to the absorbance at 430 nm correlated to the absorbance at 430 nm
Fig. 11.8Penetration profiles of two commercially emollient both were purchased from Glaxo Wellcome,
available clobetasol propionate preparations into the stra- Inc. (Weigmann etal. 2001)
tum corneum. (a) Temovate cream and (b) Temovate
a b
Fig. 11.9 The blanching effect as a biological response vasoconstriction (blanching effect) (a), while this effect is
of the skin to the steroid application corresponds to the considerably lower in the case of the Temovate emollient
penetration profiles. The formulation, which shows the (b). Temovate cream and Temovate emollient were both
best penetration (Temovate cream), produced the highest purchased from Glaxo Wellcome Inc
212 J. Lademann et al.
ear skin. It is suited to investigating intercellular, acrylate surface biopsy. For this purpose, cyano-
but not follicular penetration. acrylate (super glue) is placed on the stripped
skin areas and is then covered with a glass slide.
After polymerization, the glass slide is removed
11.3 Differential Stripping quickly and contains follicular casts and contents
that can be likewise analyzed quantitatively for
The method of differential stripping was devel- the substance applied by conventional methods.
oped to distinguish between the amounts of topi- During the development of differential strip-
cally applied substances that penetrated into the ping, the method has been also evaluated by his-
stratum corneum and into the hair follicles, tological sections (Teichmann etal. 2005). It
respectively (Teichmann etal. 2005). The hair could be demonstrated histologically that after
follicles represent an important target structure as tape stripping, the stratum corneum can be
they are surrounded by a dense network of blood removed completely, whereas the hair follicles
capillaries and dendritic cells necessary for drug remained intact. Only after cyanoacrylate skin
delivery and immune responses (Meinke etal. surface biopsy, the follicular content was removed
2010; Teichmann etal. 2005). Additionally, they completely.
are host of the stem cells in the bulge region Unfortunately, this procedure lends itself only
(Nagao etal. 2012; Blume-Peytavi and Vogt for invivo investigations on human skin. As
2011), which are likely to become an important already explained, excised human skin immedi-
factor in regenerative medicine. ately contracts; the follicular orifices being con-
The differential stripping procedure combines tracted and not open for penetration. Porcine ear
the above-described classical tape stripping pro- skin also proved unsuitable for such investiga-
cedure with cyanoacrylate surface biopsies tions, as the hair follicles extend considerably
(Teichmann etal. 2005). This procedure is sche- deeper into porcine skin than into human skin, so
matically represented in Fig.11.10. After topical that the cyanoacrylate glue either cannot pene-
application of a substance, it is spreading in the trate into the hair follicles completely or gets
stratum corneum and in the hair follicle. stuck there during removal. In this context, Knorr
Subsequently, the stratum corneum is removed etal. (2009) developed a method which resem-
by tape stripping as described above. Thereafter, bles the differential stripping procedure but can
the amounts of penetrated substance adhering to be also utilized exvivo on porcine ear skin. Using
the individual tape strips are quantified. Once the this method, the stratum corneum is likewise
stratum corneum has been completely removed removed by tape stripping after topical drug
by tape stripping, the topically applied substance application. Subsequently, a biopsy is removed,
is only contained in the hair follicles, yet. These which is subsequently extracted in ethanol and
hair follicle contents are then removed by cyano- then analyzed for the amount of penetrated sub-
stances by fluorescence microscopy. This method ments as would be the case if optical spectro-
is based on the fact that once the stratum corneum scopic measurements were applied.
has been removed, the substances are exclusively
located in the hair follicles; thus, the extraction of
the biopsies only reveals the amount penetrated
into the hair follicles. The amount of topically
References
applied substances, which may have penetrated Baert B, Boonen J, Burvenich C, Roche N, Stillaert F,
through the cutaneous barrier using the intercel- Blondeel P etal (2010) A new discriminative criterion
lular or follicular pathways, is almost less than for the development of Franz diffusion tests for trans-
1%. Therefore, it can be neglected compared to dermal pharmaceuticals. JPharm Pharm Sci 13(2):
218230
the amount of substances penetrated into the hair Bettoni CC, Felippi CC, de Andrade C, Raffin RP, Jager
follicles (Feldmann and Maibach 1969). A, Guterres SS etal (2012) Isotretinoin-loaded nano-
capsules: stability and cutaneous penetration by tape
stripping in human and pig skin. JBiomed Nanotechnol
8(2):258271
11.4 Summary Blume-Peytavi U, Massoudy L, Patzelt A, Lademann J,
Dietz E, Rasulev U etal (2010) Follicular and percuta-
In summary it can be stated that stripping meth- neous penetration pathways of topically applied min-
ods are simple and low-cost procedures for ana- oxidil foam. Eur JPharm Biopharm 76(3):450453.
S0939-6411(10)00159-1 [pii]. doi:10.1016/j.ejpb.2010.
lyzing the penetration of topically applied 06.010
substances into the stratum corneum and into the Blume-Peytavi U, Vogt A (2011) Human hair follicle: res-
hair follicles. Using these procedures correctly, ervoir function and selective targeting. Br JDermatol
the following is to be taken into consideration: 165(Suppl 2):1317. doi:10.1111/j.1365-2133.2011.
10572.x
both tape stripping and differential stripping are Dreher F, Modjtahedi BS, Modjtahedi SP, Maibach HI
noninvasive methods. For tape stripping the skin (2005) Quantification of stratum corneum removal
surface must be stretched during application of by adhesive tape stripping by total protein assay in
the adhesive film so that the influence of furrows 96-well microplates. Skin Res Technol 11(2):97
101. SRT103 [pii]. doi:10.1111/j.1600-0846.2005.
and wrinkles can be neglected. This can be easily 00103.x
performed by pressing the adhesive film onto the Essa EA, Bonner MC, Barry BW (2002) Human skin
skin surface with a roller. In addition, the depth sandwich for assessing shunt route penetration during
from which the corresponding tape strip is passive and iontophoretic drug and liposome delivery.
JPharm Pharmacol 54(11):14811490
removed should not be related to the tape strip Feldmann RJ, Maibach HI (1969) Percutaneous penetra-
number, but to the amount of stratum corneum tion of steroids in man. JInvest Dermatol 52(1):6
removed. In this way, the penetration profile Forster M, Bolzinger MA, Montagnac G, Briancon S
becomes reproducible, independent of the inves- (2011) Confocal Raman microspectroscopy of the
skin. Eur JDermatol 21(6):851863. ejd.2011.1494
tigator and the type of adhesive film applied. [pii]. doi:10.1684/ejd.2011.1494
Also for differential stripping it is essential Gorne RC, Greif C, Metzner U, Wigger-Alberti W, Elsner
that the skin surface is stretched by pressing the P (2007) Assessment of topical corticosteroid activity
adhesive film onto the skin surface by using a using the vasoconstriction assay in healthy volunteers.
Skin Pharmacol Physiol 20(3):133140
roller. Only in this case the stratum corneum can Haag SF, Fleige E, Chen M, Fahr A, Teutloff C, Bittl R
be removed completely also from the furrows etal (2011) Skin penetration enhancement of core-
and wrinkles. Otherwise, parts of the stratum cor- multishell nanotransporters and invasomes measured
neum remain on the skin surface and will conse- by electron paramagnetic resonance spectroscopy. Int
JPharm 416(1):223228. S0378-5173(11)00597-7
quently be removed by the cyanoacrylate biopsy. [pii]. doi:10.1016/j.ijpharm.2011.06.044
This falsifies the measuring results in terms of Hutton RD, Kerbs S, Yee K (1978) Scanning electron
follicular penetration. Notwithstanding their microscopy of experimental Trichophyton mentagro-
many advantages, the stripping methods have phytes infections in guinea pig skin. Infect Immun
21(1):247253
also limitations insofar as a removed skin area Jung S, Otberg N, Thiede G, Richter H, Sterry W, Panzner S
can be analyzed only once. Consequently, this etal (2006) Innovative liposomes as a transfollicular
skin area is not available for kinetic measure- drug delivery system: Penetration into porcine hair fol-
214 J. Lademann et al.
StefanF.Haag, JrgenLademann,
andMartinaC.Meinke
Contents
12.1 Introduction
12.1 Introduction 215
12.2 Nanocarriers 216 Drug delivery to the skin requires intensive
12.2.1 Invasomes 216 research into the synthesis of new carrier sys-
12.2.2 Core Multishell Nanotransporters 217
tems, which promote an effective and selective
12.2.3 Nanostructured Lipid Carriers 217
delivery of pharmaceutics to the site of interest.
12.3 lectron Paramagnetic Resonance
E For a variety of active compounds, incorporation
Spectroscopy 218
into a carrier system is important as (a) the com-
12.4 table Nitroxide Spin Probes
S pound could be poorly soluble in water or tradi-
Aminoxyl Radicals 220
tional formulations and (b) to increase skin
12.5 Applications 220 penetration of the compound while avoiding
12.5.1 Partitioning ofaDrug WithinaCarrier 220 absorption into systemic circulation, which mini-
12.5.2 Skin Penetration Enhancement 222
12.5.3 Stabilization ofNitroxides mizes adverse effects, or to achieve a sustained
Determination ofSustained Release release of the compound from the carrier to the
ofTEMPO 223 skin. Therefore, it is important to create new car-
12.5.4 Further Applications 226 rier systems by means of a technique that corre-
Conclusion 226 sponds to the therapeutic needs (Martini and
References 227 Ciani 2009). To achieve these goals, the physico-
chemical properties of the drug-loaded carrier
systems must be fully understood.
Besides optical methods, electron paramag-
netic resonance (EPR) or electron spin resonance
spectroscopy is a unique method that provides
information about the localization of a drug
within a carrier system, and it offers dynamic and
S.F. Haag J. Lademann M.C. Meinke (*) structural information concerning a carrier sys-
Department of Dermatology, Venereology and tem and lends itself excellently for the observa-
Allergy, Charit Universittsmedizin Berlin, Center
tion of drug release processes. In order to observe
of Experimental and Applied Cutaneous Physiology,
10117 Berlin, Germany these processes, paramagnetic material has to be
e-mail: martina.meinke@charite.de added to the systems. Spin probes, mainly
12.2.1 Invasomes
penetration, thus leading to increased skin per- The terminal shell provides a good solubility
meability. However, treatment with rigid vesi- of the particle in water as well as in organic sol-
cles did not affect stratum corneum ultrastructure vents and a high degree of biocompatibility.
or permeability (van den Bergh etal. 1999). The Furthermore, as well as the core, it serves as a
second mechanism postulates that ultraflexible matrix for encapsulation of hydrophilic agents.
vesicles can penetrate the skin unfragmented on The nonpolar inner shell allows incorporation of
areas with low penetration resistance, e.g., hydrophobic guest molecules (Radowski etal.
between neighboring corneocyte clusters with 2007).
no lateral overlapping of the corneocytes CMS nanotransporters comprise of small
(Schtzlein and Cevc 1998; Cevc etal. 2002). unimers, 5nm in size, but if the critical aggrega-
Moreover, it was found that ultraflexible vesicles tion concentration of 0.1g/l is reached, larger
penetrate intact into deeper stratum corneum aggregates are formed with a diameter of approx-
layers; however, only small amounts were found imately 3050nm. -Carotene loaded CMS
in the deepest stratum corneum layers. These nanotransporters form aggregates up to 144nm
findings could not be observed after the applica- in size (Radowski etal. 2007).
tion of rigid gel- state vesicles (Honeywell- CMS nanotransporters were shown to enhance
Nguyen etal. 2002). skin penetration of the lipophilic dye Nile red
(Kchler etal. 2009b) and the hydrophilic dye
rhodamine B (Kchler etal. 2009a) and thus
12.2.2 Core Multishell appear to be of outstanding versatility.
Nanotransporters Furthermore, CMS nanotransporters are used for
tumor targeting (Quadir etal. 2008), cellular cop-
Core multishell (CMS) nanotransporters are per uptake by yeast cells (Treiber etal. 2009), or
chemical chameleons, which, simular to lipo- as stabilizers for catalytic platinum nanoparticles
somes, can encapsulate both hydrophilic and (Keilitz etal. 2010).
lipophilic guest molecules. Additionally, they
can be dissolved in a variety of solvents, ranging
from water to toluene. The unimolecular archi- 12.2.3 Nanostructured Lipid Carriers
tecture of CMS nanotransporters contains a
hydrophilic core, followed by a hydrophobic Nanostructured lipid carriers (NLCs) consist of a
middle layer, and a hydrophilic outer shell. This solid lipid matrix formed by, e.g., glycerol tri-
architecture mimics the structure of liposomes, stearate (Dynasan 118; Cognis, Monheim,
which are lipid vesicles that comprise of an aque- Germany) and are loaded with liquid lipids of,
ous inner phase, enclosed by a phospholipid e.g., caprylic/capric triglycerides (medium chain
membrane and surrounded by an exterior aque- triglycerides, Miglyol 812, Caelo, Hilden,
ous phase. As described above, the liposome Germany). NLCs are dispersed in water and sta-
architecture was found to have a penetration bilized by an emulsifying agent. However, the
enhancing effect. It has been suggested that lipo- structure of NLCs is described differently in the
somes adhere to the skin surface and destabilize, literature. One theory describes NLCs as spheri-
fuse, or mix with the lipid matrix, resulting in a cal nanoparticles with droplets of liquid lipid
loosening of the lipid structure, which lowers the within the solid lipid matrix (Mller etal. 2002),
barrier strength of the skin and enhances skin whereas another group describes them as lipid
penetration of loaded drugs or agents (Elsayed platelets with oil spots located on the surface of
etal. 2007; Kirjavainen etal. 1996). Due to their the solid lipid matrix (Jores etal. 2004).
liposome-like architecture, CMS nanotransport- Preparation of NLCs is performed by high-
ers might exhibit a similar penetration enhancing pressure homogenization (HPH), applying two to
efficiency and additionally, as polymer-based three cycles at 500bar. HPH can be performed at
nanotransporters, an improved stability. high temperatures, e.g., 80C but also cold HPH
218 S.F. Haag et al.
Besides detection and quantification of nitrox- frequencies as shown in Fig.12.4 for Q- and
ides or other paramagnetic molecules, EPR spec- W-band measurements. The g-factor increases
troscopy can provide information on the with increasing lipophilicity of the microenvi-
physicochemical properties of the molecule as ronment. A further indicator of the polarity is
well as information about its immediate sur- the hyperfine coupling constant, aiso, which
rounding, i.e., polarity and viscosity. This infor- reflects the polarity independent of the applied
mation can be derived from the line shape of the frequency. Low values of aiso represent a lipo-
EPR spectrum. EPR spectra of nitroxides in solu- philic environment, whereas high values a
tion comprise of three resonant lines (Fig.12.3), hydrophilic one, e.g., the nitroxide 2,2,6,6-tet-
which are due to the magnetic moment of the ramethyl-1-piperidinyloxy (TEMPO; Sigma
neighboring nitrogen nucleus. Interactions of Aldrich, Steinheim, Germany) has an aiso of
free, unpaired electrons with the magnetic 1.74mT in water and 1.59mT in a mix of phos-
moment of the nitrogen nucleus are described as phatidylcholine and lysophosphatidylcholine
14
N hyperfine coupling, which splits the EPR (Haag etal. 2011c).
spectrum in three lines. The 14N hyperfine cou- An indicator of the microviscosity is the rota-
pling constant (aiso) is strongly influenced by tional correlation time in seconds, which is
hydrogen bonds between solvent molecules and
the oxygen of the nitroxide and therefore, gives
information about the polarity of the immediate
surrounding of the paramagnetic molecule. It was
found that aiso increases proportionally with the
concentration of hydrogen donor groups of the
solvent (Gagua etal. 1978).
The g-factor indicates the position of the
resonant lines within the magnetic field.
Moreover, it also reflects the polarity of its Fig. 12.4 Chemical structure of selected nitroxide spin
immediate surrounding when measuring at high probes: TEMPO (left) and PCA (right)
Fig. 12.3 EPR spectrum of the nitroxide PCA that illustrates typical EPR parameters: I: signal amplitude, aiso: hyper-
fine coupling constant, lw: peak to peak line width
220 S.F. Haag et al.
defined as the time it takes for the spin probe to 12.5 Applications
rotate once around its own axis. This is one of the
main features of EPR spectroscopy when study- 12.5.1 Partitioning ofaDrug
ing drug release processes, e.g., a spin probe or WithinaCarrier
spin labeled drug that is incorporated in a carrier
system can be differentiated from a released one, EPR spectroscopy allows distinguishing between
especially if it is covalently bound to a nanopar- environments differing in polarity (Kempe etal.
ticle (see spectra in Fig.12.1). 2010). Smirnov etal. (1995) could show TEMPO
Additionally, in the case of highly concen- partitioning between the aqueous and lipid
trated paramagnetic substances within carrier domains of liposomes. Previously, Haag etal.
systems a broadening of the signals appears due (2011a) studied ultraflexible liposomes (inva-
to spinspin coupling. In the case of release, the somes) and nanostructured lipid carriers, which
broadening disappears. had also been prepared with TEMPO.Partitioning
The four described EPR parameters can be of TEMPO between lipid and aqueous phases
derived from the EPR spectrum by using simula- could be observed and quantified. Changes in
tion software, e.g., the EasySpin toolbox (Stoll partitioning after application to the skin resulted
and Schweiger 2006). in a sustained release of TEMPO to the skin. In
Fig.12.5, EPR spectra of TEMPO-loaded inva-
somes (solid, black line) recorded at different
12.4 Stable Nitroxide Spin microwave frequencies are shown. Sensitivity is
ProbesAminoxyl Radicals directly related to the square of operating fre-
quency, which means the higher the frequency,
Nitroxides are versatile spin probes for various the higher the measuring sensitivity of the spec-
applications in biology, medicine, and pharma- trometer. Due to the low magnetic field strength
ceutics. Nitroxides are paramagnetic species, at L-band frequency (1.3GHz), spectra from
possessing a free, unpaired electron in the outer phases originating from different polar environ-
shell of the molecule and are therefore detect- ments appear as a single three-line spectrum,
able by EPR spectroscopy. While all have a because the spectra from the different environ-
free electron at the nitrogen in common, the ments overlap. By increasing the frequency to X-
chemical structures and thus physicochemical and Q-band, spectra from the different phases are
properties can differ significantly. Most com- separated more efficiently. Measurements at
monly applied nitroxides are six-ring systems W-band frequency (94GHz) can completely sep-
(piperidines) and five-ring systems (pyrro- arate the spectra from environments differing in
lidines). The stability of the free radical arises polarity due to the high magnetic field strength,
from the delocalized electron from the NO which results in two spectra. In this case one
bond and steric hindrance by the four methyl from the phosphatidylcholine/lysophosphatidyl-
groups. choline phase and one from the aqueous phase of
Two representative spin probes are 3-carboxy- the invasomes. Since we can separate the spectra
2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PCA; at W-band, the magnetic parameters can be fully
Sigma Aldrich, Steinheim, Germany), derived from the spectra in each phase (i.e.,
(logP=1.7; MW=186 g/mol) and TEMPO g-value indicates the position of the spectra
(logP=2.3; MW=156 g/mol) (Fig. 12.4). The within the magnetic field), 14N hyperfine cou-
stability of the nitroxide is mainly determined by pling (aiso, distance between low field and central
its ring structure, PCA with its five-ring system line, high values indicate a hydrophilic environ-
is significantly more stable than TEMPO with ment and vice versa), and the rotational correla-
its six-ring system (Fuchs etal. 1993). Both spin tion time (the time it takes for a spin probe to turn
probes can be applied invivo (Fuchs etal. 1997). once around its own axis). This is a prerequisite
12 Application ofEPR-spin Probes toEvaluate Penetration Efficiency, Storage Capacity 221
Fig. 12.5 Multifrequency measurement of the spin probe TEMPO within an invasome dispersion. The asterisk indi-
cates signals of TEMPO from the lipophilic membrane and the arrows from the aqueous phase
for spectral simulation of spectra recorded at lipid-based carrier systems for a specific purpose,
lower frequencies. By entering these parameters e.g., in the surfactant shell of the nanoparticle for
into computer software, e.g., EasySpin the spec- burst release or in the core of a liquid lipid emul-
tra can be simulated at each frequency as shown sion for sustained release. At W-band frequency,
in Fig.12.5 (gray, dashed line). the distribution can be easily determined and
The L-band low frequency measurement quantified..
shows a broadened low and high-field line. The The use of different spectrometers operating
spectrum at X-band frequency shows a parti- at diverse microwave frequencies allows compre-
tioned high-field line. When measuring at Q-band hensive studies of pharmaceutical formulations.
frequency, two lines of the lipophilic TEMPO At high frequency W-band (94GHz) the localiza-
EPR spectrum already appear and are shifted tion of a spin probe or a spin-labeled drug within
down-field due to higher g-values. The measure- a carrier matrix becomes feasible allowing the
ment at W-band clearly resolves three lines from observation of drugcarrier interactions. This can
each of the two phases. As demonstrated in be the localization in a certain polar environment
Fig.12.5, the resolution increases with increasing in a two-phase system like invasomes, or the
frequency. At W-band frequency the distribution localization of a spin probe in the core of a lipid
between phases of different polarity can be deter- nanoparticle or within the surfactant shell.
mined, and most importantly quantified. This is Especially interesting for the investigation of
especially important for the development of topical dermatics is the low frequency L-band
222 S.F. Haag et al.
the spin probe within the carrier and/or the spin 12.5.3 Stabilization ofNitroxides
probe itself only penetrate the upper stratum Determination ofSustained
corneum. Applying PCA solution, the spin label Release ofTEMPO
penetrates the stratum corneum only poorly. The
signal is low from start on, and after tape 4 it is The next application of nitroxide stabilization is
below the detection limit. related to the possible sustained release of
TEMPO incorporated into NLCs, i.e., the possi-
bility that the formulation/carrier could act as a
depot system. In this context NLCs were investi-
gated (Haag etal. 2011a). Repetitive EPR spectra
were recorded after the application of the TEMPO
containing NLC formulations onto porcine ear
skin. The change of aiso, as a measure of the polar-
ity of the immediate surrounding of the spin
probe, over time was determined and is repre-
sented in Fig.12.8. High values of aiso represent a
hydrophilic and low values indicate a lipophilic
microenvironment. The aiso of free TEMPO
remains almost stable over time, indicating that
TEMPO that interacts with the skin is immedi-
ately reduced to the EPR-silent hydroxylamine
by skin antioxidants. For TEMPO in NLCs a
slow decline is shown for approximately 15min
Fig. 12.7 Spin label intensity of tape stripped porcine followed by a strong decline. This means that the
skin 30min after application of PCA formulations. Tape polarity of the environment of the spin probe
number 0 corresponds to the intensity after penetration within the NLCs after skin application changes
and removal of remaining liquids, number 15 correspond with time, from high values corresponding to a
to the PCA-related signal intensity of the skin after each
tape strip is taken hydrophilic environment to low values,
corresponding to a lipophilic environment. That TEMPO is reduced much faster than protected
indicates that TEMPO has a higher surviving TEMPO.The slower reduction rate of TEMPO
probability in lipophilic environments, for exam- containing NLCs compared to solution indicates
ple, in the liquid lipid droplets of the NLCs. a fast reduction of unprotected TEMPO from the
In Fig.12.9 the EPR intensity decline of aqueous compartments of the suspension and
TEMPO measured at L-band is shown for solu- first a stabilization of TEMPO in the liquid lipid
tion and NLCs. As soon as TEMPO penetrates compartments. After a longer penetration time,
the skin it reacts with antioxidants and is reduced the liquid lipid compartments get to interact with
to the EPR-silent hydroxylamine, which results the skin and TEMPO turns EPR silent. This indi-
in intensity decline of TEMPO.EPR intensity cates a delayed reduction of lipid-associated
rapidly decreases when applied in solution and is TEMPO and, therefore, slow release of TEMPO
below the detection limit after 17min, indicating from the NLCs.
that TEMPO penetrates the skin well and is For TEMPO in NLCs a change of aiso toward a
quickly reduced. NLCs delayed TEMPO interac- more lipophilic microenvironment could be
tion with the skin resulting in prolonged signal observed over time. The exponential aiso decline
detection by 12min, when compared to solution. can be explained by the distribution profile of
Thus, NLCs increase the measurement time TEMPO within the nanocarrier dispersion. NLCs
exvivo 1.7-fold, indicating that NLCs prevent had 35% of TEMPO associated with the liquid
TEMPO from reacting with the skin antioxidants lipid droplets and 65% with the aqueous phase,
and act therefore as slow release depot systems as illustrated in Fig.12.10. This becomes obvious
for TEMPO penetration into the skin. Statistical when plotting intensity versus aiso as shown in
analysis revealed significance between solution Fig.12.10.
and NLCs (p0.05). After the application of TEMPO-loaded nano-
The decrease in EPR spectra intensity carriers to the skin and upon interaction of unpro-
(Fig. 12.9) of TEMPO and the change in aiso tected TEMPO of the aqueous phase with the
(Fig.12.8) give information about the interaction skin, it is reduced quickly to the corresponding
of the NLCs with the skin. TEMPO penetrating EPR-silent hydroxylamine, which results in a
the skin reacts with reducing agents, which turn rapid change of signal intensity with primarily a
the spin probe EPR silent that unprotected slow decline of aiso. With decreasing amounts of
Fig. 12.10 Plot of EPR intensity versus aiso of the NLCs and solution during penetration into porcine ear skin exvivo,
TEMPO, NLC.The insert is the plot for invasomes of intensity versus aiso
TEMPO in the hydrophilic compartments aiso is a low molecular weight drug. In order to investi-
strongly influenced by lipid protected gate the release mechanisms, they incorporated
TEMPO.The reaction of TEMPO from both the lipophilic spin probe HD-PMI (2-heptadecyl-
phases leads to a somewhat linear decrease of aiso 2,3,4,5,5-pentamethyl-i midazoline-1 -oxyl)
with intensity. Fig.12.10 clearly indicates that and the hydrophilic spin probe 15N-PCM
the decline of aiso with intensity depends on the (3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolidin-
distribution of TEMPO within the carrier. Since 1-oxyl-115N) into the gelling emulsion. The spin
NLCs had 35% of TEMPO associated with the probes could be distinguished from each other
lipid phase, the decline is less steep in the begin- due to the 15N-PCM, which possesses a nitrogen
ning. As soon as more TEMPO is associated with isotope that exhibits two resonant lines only, in
lipid compartments, as shown by Haag etal. contrast to HD-PMI that has a typical three-line
(2011c) for invasomes, which had 56% TEMPO spectrum. Analysis of the micropolarity revealed
in the lipid phase, the decline of aiso with intensity that the hydrophilic 15N-PCM is located in the
turns less steep (see insert in Fig.12.10) (Haag outer aqueous phase and the lipophilic HD-PMI
etal. 2011a; Haag etal. 2011c). The different is located in the oil droplets of the gelling emul-
intensity decline between solution and the nano- sion. Although the gelling emulsion was rather
carriers was also observed invivo (Haag etal. viscous, the spin probes were not hindered in
2011a). This shows that EPR spectroscopy also motion in their microenvironment. This could be
allows the determination of sustained release on derived from the sharp spectral lines of the spec-
the skin of human volunteers. trum. Subsequently, the release of 15N-PCM from
In order to distinguish between different phases the aqueous phase was analyzed and it was found
in a system, dissimilar spin probes that possess that after 3h the spin probe was completely
different EPR spectra can be used. Kempe etal. released invivo, whereas invitro the release
(2010) have shown with an oil and water chito- was completed after 6h. This could be observed,
san-based gelling emulsion the distribution and because the intensity of the spectral lines of the
release of a spin probe that served as model for two-line spectrum of 15N-PCM decreased over
226 S.F. Haag et al.
time. The three-line spectrum of HD-PMI did not localization in a human body. Burks etal. (2011)
change in intensity, which indicates that the spin investigated the clearance and distribution of
probe is protected by the oil droplets of the gell- nitroxides encapsulated in different liposomes.
ing emulsion. The use of different spin probes The encapsulation in sterically stabilized lipo-
that possess different spectra allows distinguish- somes allowed the transport to different organs
ing between environments differing in polarity like spleen, kidney, liver, and a breast tumor in a
and/or viscosity. Moreover, the localization of mouse model to be followed. By characterizing
a spin probe within a carrier system is possible. the pharmacological properties of nitroxides in
The possibility of determination of the aforemen- liposomes, it could be demonstrated that only
tioned parameters makes EPR spectroscopy a modest improvements are required to enable
powerful tool in drug release research. high-contrast EPR images of a mouse breast
tumor (Hc7 tumors) invivo. This shows that by
the use of liposomes sufficient amounts of spin
12.5.4 Further Applications probe can be delivered to the site of interest.
label can be distinguished. Drug release can characterization and invitro skin penetration studies.
also be triggered by external or internal trig- JControl Release 127(1):5969
Dragicevic-Curic N, Scheglmann D, Albrecht V, Fahr A
gers such as pH, temperature, etc., allowing (2009) Development of different temoporfin-loaded
the observation of release by EPR invasomes-novel nanocarriers of temoporfin: charac-
spectroscopy. terization, stability and invitro skin penetration stud-
ies. Colloids Surf B Biointerfaces 70(2):198206
El Maghraby GM, Williams AC, Barry BW (2001) Skin
AcknowledgmentThis work was funded by the Freie delivery of 5-fluorouracil from ultradeformable and
Universitt (FU) Berlin, Focus Area Functional Nanoscale standard liposomes in-vitro. JPharm Pharmacol
Materials. Furthermore, we thank Ming Chen and Alfred 53(8):10691077
Fahr (Department of Pharmacy, Friedrich-Schiller- Elsayed MM, Abdallah OY, Naggar VF, Khalafallah NM
Universitt Jena) for providing the invasomes, Emanuel (2007) Lipid vesicles for skin delivery of drugs:
Fleige and Rainer Haag (Department of Chemistry, reviewing three decades of research. Int JPharm
FU-Berlin) for providing the CMS-nanotransporters, and 332(12):116
Daniel Peters and Cornelia Keck (Department of Fuchs J, Freisleben HJ, Podda M, Zimmer G, Milbradt R,
Pharmacy, FU-Berlin) for providing the nanostructured Packer L (1993) Nitroxide radical biostability in skin.
lipid carriers. We also thank Robert Bittl and Christian Free Radic Biol Med 15(4):415423
Teutloff (Department of Physics, FU-Berlin) for Q- and Fuchs J, Groth N, Herrling T, Zimmer G (1997) Electron
W-band measurements as well as their valuable support paramagnetic resonance studies on nitroxide radical
regarding spectra analysis, and Monika Schfer-Korting 2,2,5,5-tetramethyl-4-piperidin-1-oxyl (TEMPO)
(Department of Pharmacy, FU-Berlin) for fruitful redox reactions in human skin. Free Radic Biol Med
discussions. 22(6):967976
Fuchs J, Herrling T, Groth N (2001) Detection of free
radicals in skin: a review of the literature and new
developments. Curr Probl Dermatol 29:117
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Confocal Raman Spectroscopy
asaTool toInvestigate theAction 13
ofPenetration Enhancers Inside
theSkin
StphanieBrianon, Marie-AlexandrineBolzinger,
andYvesChevalier
etal. 2008). The exact mechanisms by which same time structural information on the confor-
enhancers promote or delay drug diffusion mation changes of the molecules in the skin
through the skin are not fully understood, and induced by the absorption of a drug, a penetration
even the different penetration paths of transder- enhancer, or any physical parameter like temper-
mal permeation are still under debate. One pre- ature. The Raman spectrometer is associated with
sumed mechanism is related to the disordering or a confocal microscope, providing three-
fluidizing the SC structure upon mixing the pen- dimensional spectroscopic information with the
etration enhancers with the intercellular lipids spatial resolution of an optical microscope.
(Benson 2005). However, the molecular organi- In this chapter, we report on absorption mea-
zation of the SC lipids is still not firmly estab- surements of drugs and penetration enhancers
lished. Three main structures have been disclosed: inside skin using this vibrational spectroscopic
(i) the orthorhombic phase, a dense organization technique, and then on the structural modifica-
with crystalline hydrocarbon chains that are not tions induced by such compounds as measured
equally distributed in the lattice, resulting in two by CRM.After a brief description of the tech-
different distances between lattice planes nique, the second part gives detailed examples of
(0.37nm and 0.41nm); (ii) the hexagonal phase applications to dermatological research.
with equally spaced crystallized hydrocarbon
chains; and (iii) finally the fluid lamellar phase
where the lipids are in a fluid (molten) state. The 13.2 C
onfocal Raman Microscopy
current view of the lipid organization in the SC at forCutaneous Absorption
32C is the orthorhombic structure coexisting Experiments
with the hexagonal packing (Pilgram etal. 1999;
Norln 2001; Babita etal. 2006; Bouwstra and CRM is based on the combined use of spectro-
Ponec 2006; Bouwstra etal. 2007). An open scopic analysis and structural observation by
question with respect to lateral packing of lipids optical microscopy. The characteristic features of
(Norln 2001) is whether the lipid matrix is made this technique are the chemical analysis by spec-
of a single gel phase or several coexisting fluid troscopy and the three-dimensional mapping of
and solid (crystalline and/or gel phases). Another the sample by microscopy. CRM brings about
question which has recently been addressed is new possibilities and definite benefits over the
whether the barrier efficiency of SC is related to classical methods for assessing skin absorption.
the extent of the orthorhombic and lamellar struc- The several classical methods for assessing
tures (Boncheva etal. 2008; Damien and skin penetration of drugs (or any other kind of
Boncheva 2010; Groen etal. 2011). molecule) from a formulation containing them
New insights into the mechanisms of action of rely on invitro measurements of passive diffu-
penetration enhancers have been gained by sion of drugs through excised skin (Bronaugh
implementation of noninvasive physicochemical and Maibach 1999) in Franz cells. The diffusion
techniques into skin absorption experiments. of drugs through excised skin from a donor com-
Among them, confocal Raman microspectros- partment containing the formulation to a receptor
copy (CRM) is becoming a major technique compartment containing a receiver fluid is mea-
because of its nondestructive character, and the sured. The permeation profile is a plot of the
possibility of monitoring the penetration of the cumulated amount of drug that reached the recep-
drug and/or penetration enhancer provided that tor fluid as function of duration of exposure to the
characteristic Raman bands can be associated formulation. The distribution of drug molecules
with the compounds (Frster etal. 2011a). The inside the skin layers is more difficult to measure.
spectroscopic method allows obtaining at the In a classical Franz cell experiment, the cell is
13 Raman Scattering Probing Penetration Enhancers in Skin 231
dismantled at the end of the experiment, the skin Raman spectroscopy is closely related to
layers are separated, and an analysis of the drug infrared (IR) spectroscopy since in both tech-
is performed. This method is open to several niques the frequencies of molecular vibrations
drawbacks since all manipulations (separation of are measured. However Raman spectroscopy is
skin layers, solvent-based extraction of drug) sensitive to polarizable chemical bonds, whatever
may cause strong bias of the drug concentration. their polarity, while IR absorption takes place for
Such a method gives the distribution of drug in polar bonds for which the molecular vibration
the vertical direction (z-direction). Information changes the dipole moment. Therefore, the spec-
pertaining to the spatial distribution in the hori- troscopic selection rules are different for both
zontal direction (x,y plane) and the possible mod- techniques. Symmetric molecules have Raman
ification of the skin structure induced by spectra, but not many IR bands. Most molecules
absorption of compounds are missing. The usual present in the skin have both absorption bands in
method to obtain depth-dependent profiles of the Raman and IR spectroscopy, but the intensities of
drug content is the so-called tape-stripping their bands are different.
method; it is restricted to analysis in the IR spectroscopy is a classical absorption tech-
SC.Chemical or spectroscopic analysis along nique where the absorbance or the attenuated
histological sections of skin provide the distribu- reflectance of the sample is measured. IR and
tion along the depth and one lateral direction. All Raman spectroscopy both measure the vibra-
these methods (tape-stripping, histological sec- tional energy of molecules, but the interaction
tioning) are destructive and involve hard manipu- between the incident radiation and the compound
lations of the skin samples that cause several differs. IR requires a change in the dipole moment
artifacts (Touitou etal. 1998). Moreover, time- of the molecule, whereas Raman relies on a
resolved experiments (i.e., study of dynamic pro- change in the polarizability of the molecule.
cesses) are not possible and information on the Raman spectroscopy measures inelastic light
possible perturbations of the structure of endog- scattering by the sample. This makes an impor-
enous skin components is lacking, except if it is tant difference between Raman and IR spectros-
measured by attenuated total reflectance-infrared copy (Caspers etal. 2001). When using Raman
(ATR-IR) spectroscopy before tape-stripping spectroscopy, the sample is irradiated with an
(Francoeur etal. 1990; Naik etal. 1995; Zhang intense beam of monochromatic light from a
etal. 2007a). laser; a very small part of the light (~ 1 photon
CRM overcomes the limitations reported out of 108) undergoes inelastic scattering from
above because it is not destructive. Time-revolved molecular vibrations, resulting in Stokes and
3D pictures of the distribution inside skin are col- anti-Stokes scattered light at frequencies differ-
lected. CRM measures vibration frequencies of ent from the incident radiation. The frequency
chemical bonds and parts of molecules. It allows shifts correspond to the frequencies of the molec-
distinguishing chemical species through their ular vibrations; they are expressed as wave num-
characteristic vibrations and provides finger- bers (cm1) as in IR spectroscopy.
prints for the identification of substances without The identification of peaks in the resulting
requiring any labeling or dyeing. spectra can be complex. There are many lists of
Measurement of drug penetration invivo is a Raman frequencies and assignments in the litera-
much more difficult task. Adaptation of the clas- ture (Barry etal. 1992; Anigbogu etal. 1995;
sical invitro techniques to invivo measurements Frster etal. 2011a; Tfayli etal. 2012a). The
faces major limitations (Herkenne etal. 2008). Raman frequency assignments of the major
The CRM could be adapted for allowing invivo vibrational modes for the SC of mammalian skin
measurements with good accuracy. are given in Table13.1.
232 S. Brianon et al.
Table 13.1 Raman assignments of the major vibrational The strong absorbance of water limits the
modes for stratum corneum
application of IR spectroscopy to tissues con-
Raman frequency taining water. Only thin samples can be analyzed
(cm1) Assignment in transmission mode, and small penetration
526 (SS) depths are available in attenuated reflection
600 (CH)
mode. On the contrary, Raman absorbance of
623 (C=S)
water is weak, and the frequency of the light can
644 (C=S); amide IV
be chosen independently of the frequency range
746 (CH2) in phase
of the spectroscopic analysis. This is a definite
827 (CCH) aliphatic
advantage of a method relying on a scattering
850 (CCH) aromatic
phenomenon. Therefore, the wavelength of the
883 (CH2); (CC); (CN)
incident laser light for Raman analysis is selected
931 (CH3) terminal; (CC) of
proteins -helix such that the sample is transparent and there is
956 (CH3); (CCH) alkenic no fluorescence that would disturb the analysis.
1002 (CC) aromatic ring In the modern equipment, several laser wave-
1031 (CC) skeletal cis conformation lengths are available (from green light to infra-
1062 (CC) skeletal trans red) as excitation light to match the various
conformation sample properties. The depth of penetration in
1082 (CC) skeletal random the sample can reach several 10m. Since visi-
conformation ble light is used, a confocal optical microscope is
1126 (CC) skeletal trans associated for collecting 3D maps of Raman
conformation
spectra with the spatial resolution of optical
1155 (CC); (COH)
microscopy (0.20.5m in x,y plane; 0.51m
1172 (CC)
in z-axis).
1244 (CH2) wagging; (CN) amide III
disordered A definite advantage of CRM is its ability to
1274 (CN); (NH) amide III of provide spatially resolved concentration and
proteins -helix molecular structure information about skin com-
1296 (CH2) ponents (protein conformation, lipid organiza-
1336 Not assigned tion) while keeping the sample intact. Analyses
1385 (CH3) symmetric are possible in skin up to 100m depth.
1421 (CH3) Measurements can be made invitro on excised
1438 (CH2) scissoring skin samples as well as invivo.
1552 (NH); (CN) amide II The intensities of Raman bands are propor-
1585 (C=C) alkenic tional to the concentration of species, so that
1602 not assigned quantitative analyses can be done (Tfayli etal.
1652 (C=O) amide I of proteins 2012a). A close selection of appropriate condi-
-helix
tions is achieved to study the skin, especially the
1743 (C=O) amide I of lipids
excitation wavelength of the laser source (Tfayli
1768 (COO)
etal. 2012b). Various excitation wavelengths in
2723 (CH) aliphatic
the visible or near-IR (from 532nm to 1064nm)
2852 (CH in CH2) symmetric
are available as commercial sources; they are
2883 (CH in CH2) asymmetric
safe regarding invivo measurements.
2931 (CH in CH3) symmetric
2958 (CH in CH3) asymmetric
Wavelengths of 532, 633, and 785nm were
3060 (CH) alkenic
widely used to record Raman spectra inside the
3280 (OH) of H2O
skin. This parameter affects the scattered Raman
intensity, possible fluorescence background,
From Frster etal. (2011a), with permission
deformation, stretch, rock and signal attenuation along skin depth (Tfayli
13 Raman Scattering Probing Penetration Enhancers in Skin 233
etal. 2012b). The actual z-depth in the sample is quency difference between the two lights
different than the optical path length because of matches the frequency of the molecular vibra-
the different refractive indices of the skin layers. tion. A signal-to-noise ratio enhancement by
A correction factor of 20% is often applied to several orders of magnitude is achieved when
calculate the actual depth. The correction actu- the resonance condition is met. However, such
ally depends on the z-value; it is larger at deep resonance methods are not spectroscopic meth-
locus of analysis (Xiao etal. 2004; Tfayli etal. ods because a single Raman frequency is mea-
2008). sured at once. Coherent anti-Stokes Raman
The conformational disorder of SC lipids was scattering (CARS) (Evans and Xie 2008) and
extensively studied using IR spectroscopy. stimulated Raman scattering (SRS) (Downes
Utilization of classical Raman spectroscopy may and Elfick 2010; Slipchenko etal. 2010) are
suffer a lack of sensitivity in the spectral regions of the two major techniques. Several improve-
interest for lipid analysis. Different Raman spec- ments of these techniques were recently intro-
tral manifestations of conformational order of lip- duced in order to increase the sensitivity and
ids can be drawn from experiments performed on decrease the nonresonant background by epi-
thin films of lipids, such as in recent works invitro detection (Li etal. 2005); high speed confocal
on ceramide films prepared from seven classes and microscopy data can be collected (Saar etal.
subclasses of ceramides (Tfayli etal. 2010, 2012c). 2010). Equipment for two-color microscopy
For example, information on intra-chain confor- has been designed (Lu etal. 2012). SRS
mational order is obtained from the bands of C-C appears more sensitive and accurate than
skeletal optical mode and the band of the twisting CARS (Nandakumar etal. 2009; Freudiger
mode of CH2 groups, whereas the CH2 scissoring etal. 2008). Such techniques allow time-
and stretching modes reflect the lateral packing of resolved confocal microscopy measurements
the lipid molecules, and the amide I band is used to with high sensitivity at the selected vibration
evaluate the strength of H-bonds (Tfayli etal. frequencies of the penetrant molecules.
2012c). In vivo determination of lipids ordering Determination of band shifts that are charac-
by Raman spectroscopy is much more difficult teristic of interactions of penetrant molecules
(Tfayli etal. 2012d). and skin components requires several measure-
There are several variants of classical ments at different specific frequency and merg-
Raman spectroscopy besides the spontaneous ing of all in a full spectrum. A recent SRS
Raman scattering technique where the spec- technique making use of modulation of excita-
trum acquisition is a direct measurement of the tion Raman frequencies and signal demodula-
scattered light. Surface-enhanced Raman scat- tion by Fourier transform allows acquisition of
tering (SERS) shows enhanced intensity for several Raman bands at the same time, and
molecules located in the vicinity of metal par- possibly parts of Raman spectra (Fu etal.
ticles. The sensitivity improvement requires 2012). SRS has been used for investigating the
the deposition of metal nanoparticles at the skin absorption of trans-retinol, DMSO, and
surface of the sample. SERS allows a specific propylene glycol (Freudiger etal. 2008; Saar
analysis of the species present at the sample etal. 2010, 2011).
surface. Resonance Raman spectroscopy pro- The determination of the thickness of stra-
vides a selective spectrum of a molecule with tum corneum by CRM analysis of the water
high sensitivity when the frequency of the laser absorption band is shown as an illustration of
light (of the Raman spectrometer) is tuned to a the power of CRM.Figure13.1 shows a typical
UV-VIS absorption band of the molecule. series of Raman spectra measured at 6 depths
Coherent Raman spectroscopy (Min etal. inside an excised pig skin sample. The Raman
2011) relies on nonlinear effects of two-photon spectra were collected at different depths from
absorption. A resonance occurs when the fre- 0m to 20m, as indicated in the picture of the
234 S. Brianon et al.
Intensity (a.u.)
(OH)
600
400
200
)
m
2
(
6
th
8
ep
D
10
in
14
Sk
20
2800 3200 3600 4000
Wavenumber cm1
Fig. 13.1 In depth Raman profiles of untreated pig skin indicating skin depths (magnification 40) (From Frster
between 2800 and 4000cm1. At the right side, the histo- etal. (2011a), with permission)
logical image of the skin is illustrated with the arrows
histological section of skin (right side of structure. Several mechanisms have been pro-
Fig.13.1). The intensity of the stretching band posed to explain their effect: lipid fluidization,
of O-H bond of water, (OH) at 3280cm1, lipid extraction, interaction with keratin, and
increases upon going deeper from the skin sur- pore formation (Benson 2005).
face (left side of Fig.13.1). A sharp increase of
the Raman band occurs when passing from the
SC to the viable epidermis because the viable 13.3.1 Water asPenetration
epidermis contains much more water (~70%) Enhancer
than SC (an average value is 13%). The thick-
ness of the SC can be easily measured by CRM Many chemicals show skin penetration enhanc-
in this way. However, quantitative analysis is ing properties. Well-known examples are
not a straightforward reading of the spectra. Azone, dimethylsulfoxide, alcohols, fatty acids
Conversion of the Raman intensity into water such as oleic acid, surfactants, solvents, and ter-
concentration requires a calibration which has penes. However, the first major penetration
often been made using the CH3 band at enhancer is water. The permeability of skin for
2935cm1 as a reference (Caspers etal. 2000). hydrophilic molecules increases dramatically as
skin is hydrated. Hydration by occlusion or
exposition to high humidity level results in an
13.3 C
onfocal Raman Microscopy increase of the SC water content from 13%
forStudying theMechanism (standard value) to near 400% of the dry tissue
ofAction ofPenetration weight (Williams and Barry 2004). In the SC,
Enhancers the water can be bound to structural elements
(natural moisturizing factor (NMF), functional
The role of penetration enhancers to increase or groups) or free. The free water acts as a solvent
modify the permeability of drugs through the for hydrophilic molecules in the SC, thus
skin has been thoroughly studied (William and increasing the solubility of drugs in the tissue.
Barry 2004; Benson 2005). However, fewer stud- Drug partition between the formulation and the
ies have been reported on the use of CRM for SC is then modified, and the increase of solubil-
studying the mechanism by which penetration ity leads to higher transdermal fluxes of hydro-
enhancers improve the penetration of different philic substances in hydrated skin. It is not
compounds. It is commonly accepted that pene- clearly established whether water can enhance
tration enhancers are able to alter the barrier the skin penetration of lipophilic drugs. It has
properties of the SC by disrupting temporarily its been proposed that swelling of the polar head
13 Raman Scattering Probing Penetration Enhancers in Skin 235
group regions of the lipid bilayers by water could 13% in the SC to 6070% in the viable epider-
disrupt the lipid domains and cause higher per- mis. Several analysis methods used to determine
meability for lipophilic permeants such as ste- the water depth profiles were proposed to deter-
roids. However, several researchers have shown mine the boundary between SC and viable epi-
that water does not cause modification of the dermis (Caspers etal. 2000, 2003; van der Pol
lipid bilayer packing (Bouwstra etal. 1991; Gay etal. 2007; Crowther etal. 2008; Egawa and
etal. 1994; Bouwstra etal. 1996; Bouwstra etal. Kajikawa 2009; Darlenski etal. 2009; Hancewicz
2003). van Hal etal. (1996) incubated human etal. 2012). The values of water content in SC
skin explants with a saline phosphate buffered reported by various authors are rather scattered,
solution for 24h under occlusive or nonocclu- which is mainly due to the intrinsic variability of
sive conditions. They proved that water pene- different types of skin samples (Hancewicz etal.
trated in the corneocytes inducing their swelling, 2012), variation with respect to body site (Egawa
but also between the cells in the intercellular lip- etal. 2007), whether the skin is dry or freshly
ids region, mainly located in separated water hydrated (Egawa and Kajikawa 2009; Hancewicz
pools. The images revealed these water pools etal. 2012), and also may come from different
and some vesicle-like structures occasionally CRM data treatments. Raman microspectroscopy
found. However, the majority of the lipid bilay- data correlated with values obtained from other
ers exhibited a smooth fracture plane and no methods such as Karl Fisher analysis (Wu and
change in appearance. The amount of water in Polefka 2008) or optical coherence microscopy
these smooth regions was low, and swelling of for evaluating SC thickness (Crowther etal.
lamellae or lateral swelling of lipids was not 2008). Based on the same methodology, NMF
observed. content and depth profiles of NMF can also be
Transepidermal water loss (TEWL) and elec- determined. CRM is a useful tool for the mea-
trical impedance (corneometry) measurements surements of skin hydration levels, moisturizing
are two standard methods used invivo to get product efficacy, and the influence of skin hydra-
information about the skin barrier integrity and tion on drug penetration enhancement. Egawa
the skin hydration level. Both methods are nonin- and Kajikawa (2009) addressed by using CRM
vasive and they can be considered as indirect the effect of skin hydration by measuring water
measurements of the skin water content. CRM profile after applying water or steam on the fore-
enables to monitor the water content in the tissue arm of volunteers. The incubation time of the
at various depths, enabling the water concentra- skin with water and the water temperature were
tion profiles through the skin depth to be drawn varied to study the influence of these parameters
(Caspers etal. 2000). The water content in the on water profile changes. Water content in the SC
tissue can be determined from the ratio of Raman increased with increasing the duration of water
intensities of the OH stretch vibration of water at application from 1 to 10min. Heating the applied
3390cm1 and the CH3 stretch of protein at water from 22 to 32C also resulted in an increase
2935cm1. To improve the signal-to-noise ratio, of the penetration depth, penetrated amount, and
intensities were integrated from two bands (IOH: holding time of water. Furthermore, application
33503550cm1 and ICH: 29102965cm1). water-steam increased the water content in the
Caspers etal. (2000, 2001) proposed a quantifi- upper part of the SC.However, this study revealed
cation method to determine absolute water con- an increase of the water content by comparing the
tent by calibration of the ratio of the water profile before and after water application,
abovementioned areas using spectra of concen- but it was not possible to differentiate the endog-
trated aqueous solutions of proteins. The profiles enous water from that penetrated in the skin.
of water content can also be used to estimate the Direct measurement of water penetration in the
SC thickness, as it is well known that the water skin by CRM requires replacing water of natural
content increases sharply at the separation sur- isotopic abundance by deuterated water D2O,
face between SC and viable epidermis from about which shows a Raman peak corresponding to the
236 S. Brianon et al.
O-D stretching vibration at 2500cm1, different out on thin films of ceramides, the main lipid class
from endogenous water and clearly distinguish- of SC lipids, differing by their hydrocarbon chain
able in the spectra (Frster etal. 2011b). By length, polar head group, and the presence of a
applying emulsions and surfactant solutions lateral group (ester or hydroxyl groups). The
made with deuterated water, Frster etal. showed DMSO penetration enhancer affected mainly the
that water penetrated from the formulations into polar interaction between lipid chains and the lat-
the SC with a profile similar to that of endoge- eral packing of lipids, and its effect was depen-
nous water. However, no significant differences dent on the structure of the ceramides. For
were observed between D2O ingress from all for- example, adding DMSO on ceramides IIIb
mulations, and no correlations could be found resulted in the increase of the band at 1633cm1
between water penetration and the penetration of indicating weak H-bonds at the expense of the
trans-retinol present in the formulations. Amide I band at 1614cm1 characteristic of
Penetration of deuterated water in the SC after strong H-bonds. DMSO also induced a slight
application of pure D2O on skin sample under change of the peak height ratio of the bands at
occlusive conditions was recently confirmed by 2850cm1 (CH2 sym) and 2880cm1 (CH2
Ashtikara (2013). asym) that reflected a looser lateral packing of all
ceramides studied, indicating a decrease of com-
pactness which could be associated with a higher
13.3.2 DMSO asaPenetration permeability.
Enhancer Finally, DMSO had a weak influence on the
lipid chain conformations. Caspers etal. (2002)
Several studies aimed at explaining the penetra- recorded the distribution of DMSO in the SC by
tion enhancement mechanism of dimethylsulfox- CRM after application of an 80/20 mixture of
ide (DMSO), one of the most common penetration DMSO and a 5% aqueous solution of propylene
enhancers. DMSO enhances the penetration of glycol. The mixture presented some characteris-
both hydrophilic and lipophilic drugs; its action tic bands, a doublet at 671 and 702cm1 assigned
in the skin is complex and remains largely an to the (CSC) modes and a broad band at 1030
open question. Upon interaction with keratin, 1048cm1 assigned to (S=O) stretching. After
DMSO changes the keratin conformation, from application to the skin of volunteers, there was a
-helical to a -sheet structure. DMSO can also shift of these bands, indicating the interaction of
interact with the polar head-groups of intercellu- DMSO with water and possibly other components
lar lipids, resulting in a perturbation of their of SC.The SC depth profile was calculated as a
packing. Finally, DMSO can alter the drug parti- function of time from the ratio of DMSO/proteins
tion between the formulation and the skin layers bands ((CSC)677cm1/(CH2)1450cm1). They
due to its good solvent properties for a large num- showed that most of the applied DMSO perme-
ber of molecules (Williams and Barry 2004). ated through SC within 20min and reached the
Most studies on the behavior of SC lipids in viable epidermis where a small fraction was
skin have been carried out using IR spectroscopy. detected. Data collected for a longer time showed
The poor signal-to-noise ratio in Raman spectros- evidence of a progressive penetration of DMSO
copy has often limited its use for precise mecha- through the SC.DMSO could still be detected
nistic investigation inside skin. Experiments using inside skin 72h after application. Other conclu-
thin films of SC lipids allow devising what could sions about DMSOlipids or DMSOprotein
be expected from measurements of skin. Tfayli interactions could not be drawn during this study.
etal. (2012c) studied the modification of the lip- However, in a study by Zhang etal. (2007b),
ids conformational order, lateral packing, and reversible alteration of keratin secondary struc-
polar interactions induced by three penetration ture was shown using CRM when DMSO was
enhancers. The Raman investigation was carried applied in keratinocytes culture. They reported an
13 Raman Scattering Probing Penetration Enhancers in Skin 237
appearance of -sheet structures in the cellular in the epidermis, which appeared both in lipids
keratin after treatment with DMSO; this change and water images, that is, when the incident laser
was reversible, and the original -helix structure frequency was tuned to the lipids (CH2 stretch-
was recovered upon rehydration. This result was ing) or water (OH stretching) vibrations. The
explained by strong complex formation between sebaceous gland appeared clear when the fre-
DMSO and water, which displaced the bound quency was tuned to that of lipids vibration and
water that stabilizes the secondary structure of dark when it was tuned to the water vibration. It
keratin. was possible to image the ear of living mice by
Saar etal. (2010) used stimulated Raman scat- SRS frequency-tuned to the protein vibration
tering (SRS) microscopy for invivo skin optical (CH3 stretching) (Fig.13.2c). SRS was also used
imaging in mice, first focusing on 3 vibration by the authors to explore the skin penetration of
bands to image skin structure: lipids (CH2 stretch- drugs and excipients (Saar etal. 2010, 2011).
ing, 2845cm1), water (OH stretching, They explored DMSO penetration on human skin
3250cm1), and proteins (CH3 stretching, by SRS imaging using a specific vibration of deu-
2950cm1). This allowed imaging the water, terated DMSO-d6 at 2120cm1 (2010). As shown
lipid, and protein distribution of the SC and the in Fig.13.2d, DMSO was found in the area sur-
viable epidermis (Fig13.2a, b image of the pro- rounding the hair follicles, confirming the initial
tein distribution in the SC and viable epidermis). rapid penetration of DMSO into hair follicles.
The corneocytes and the intercellular spaces However, DMSO was not found to penetrate the
clearly appeared, as well as the sebaceous glands hair itself.
238 S. Brianon et al.
1500
1585 cm1 20 m
(C=C) olefinic
1000
Intensity (a.u.)
500
0
)
4
m
(
6
th
8
p
14 De
10
in
Sk
400 800 1200 1600 2000 20
Wavenumber cm1
Fig. 13.3 Raman spectra of pig skin after application of Arrows indicate skin depths in the image of histological
a surfactant solution containing 0.5% trans-retinol for section (magnification of 40) (From Frster etal.
24h. The Raman profile was measured at the surface (2011a), with permission)
(0 m), and at 2, 6, 8, 10, 14, and 20m skin depth.
reflecting the lateral packing of the lipids. A (TritonX100), both enhancers were introduced
low I2880/I2850 ratio corresponds to a disorga- at 1%. Comparison was done with a very effi-
nized lipid bilayer, that is, a fluid medium. cient delivery formulation of trans-retinol in pro-
Fluidization of lipids was observed when apply- pylene glycol/ethanol (30/70). Raman spectra
ing surfactant solution, especially PEG6 mono- provided depth concentration profiles of trans-
oleate in dodecane, pure dodecane also, retinol and Myritol on the volar forearm of two
resulting in a significant decrease of the male volunteers with the different formulations.
I2880/I2850 ratio. The strong decrease of the Retinol solutions were spread gently on the skin
I2880/I2850 ratio by PEG6 monooleate was corre- surface, and measurements began 10min after
lated with the highest penetration of trans-reti- application. Raman spectra were collected from
nol into the deeper skin layers (epidermis and the skin surface to 3040m below with a step
dermis). This ratio was not correlated with the of 2m at different locations on both treated and
total quantity of trans-retinol penetrated into untreated skin area. Measurements were per-
the skin (including the SC). CRM analyses of formed each hour up to 6h after treatment. An
trans-retinol absorption were in agreement with improved delivery of trans-retinol was obtained
skin penetration experiments performed in when adding penetration enhancers in the for-
Franz diffusion cells. mulation; oleic acid was found to be more effi-
The first invivo study was reported by Mlot cient than TritonX100. In the absence of a
etal. (2009) who monitored the effect of two penetration enhancer, the Raman signal of trans-
penetration enhancers on the delivery of trans- retinol was mainly confined in the first 5m
retinol through human skin. Three formulations depth. Trans-retinol penetrated the skin deeper
based on 0.3% trans-retinol in caprylic/capric in the presence of both penetration enhancers.
triglyceride (Myritol 318, a nonpenetrating oil) Oleic acid allowed the penetration to the same
were tested: the first one (used as control) con- depth as the propylene glycol/ethanol formula-
tained no enhancer, the second one contained a tion; however, the amount of trans-retinol pene-
lipid fluidizer, oleic acid, the third trated into the skin was tenfold higher from the
contained a lipid extractor octoxynol-9 propylene glycol/ethanol system. Oleic acid
240 S. Brianon et al.
caused a phase separation in SC lipids because it attributed to the penetration of Myritol 318
is not miscible with the SC lipids. The phase which interacted with the SC lipids and formed
separation was proved by spectroscopic studies an internal barrier to water loss by an occlusion
(Ongpipattanakul etal. 1991; Naik etal. 1995) mechanism. This hypothesis was confirmed by
and by two-photon fluorescence microscopy electrical impedance measurements showing an
showing quite large patches containing oleic increased water uptake in the outer SC.The effect
acid within the SC (Yu etal. 2003). Such liquid- of oleic acid in the skin lasted for a longer time
like inclusions are supplementary penetration than that of TritonX100 (7h vs. 5h). The effect
pathways through the SC lipids that may be the of oleic acid was explained by the interaction
origin of enhanced penetration of trans-retinol. with the SC lipids and the formation of oleic
TritonX100 is a surfactant which can be incor- acidrich domains by phase separation, enhanc-
porated in the lipid membrane, leading to a lipid ing the Myritol 318 penetration for a longer time
solubilization (or extraction). When the surfac- compared to that induced by the surfactant that
tant concentration exceeds the CMC, lipid- extracts the SC lipids temporarily.
surfactant mixed micelles form, so that lipids are Boncheva etal. (2008) used IR spectroscopy
extracted off the skin. The selective extraction of to evaluate the intercellular lipid conformation in
lipids causes a modification of the composition the SC and the effect of the penetration of the
of the lipids remaining in the skin and an altera- penetration enhancer oleic acid and propylene
tion of their organization in the SC results. glycol. The lateral packing of lipids is related to
However, this mechanism is less efficient to pro- the density of the lipids within the lipid lamellae,
mote the penetration of trans-retinol than the and therefore to their barrier properties prevent-
phase separation caused by the penetration of ing skin penetration. Lipids in human SC are
oleic acid. Mlot etal. (2009) also showed that organized as an orthorhombic lateral packing,
penetration enhancers are able to promote the which is a very dense structure. The coexistence
penetration of oil (Myritol 318) which was of the orthorhombic structure with a liquid phase
detected at 10m depth, whereas it all remained is still under debate (Bolzinger etal. 2012).
at the skin surface when no penetration enhanc- Boncheva etal. (2008) developed a method to
ers were used. evaluate the chain conformation and the lateral
These results were recently complemented by chain packing from IR spectra collected in the
an invivo spatially resolved NMR also named attenuated reflectance mode (ATR). The position
magnetic resonance imaging (MRI) measure- and the bandwidth of the CH2 symmetric stretch-
ment of the penetration of skin moisturizers and ing band (2850cm1) and of the splitting of the
the effect of penetration enhancers (Ciampi etal. CH2 rocking (~720cm1) and scissoring modes
2011). The NMR relaxation parameters T1, T2 (~1468cm1) into two separate bands are indeed
and the self-diffusivity D were measured at 3h, sensitive to the lateral packing of the hydrocar-
5h15, and 6h40 after application of same formu- bon chains. After incubation of the skin with
lations of trans-retinol in Myritol 318 contain- oleic acid, a shift of the CH2 symmetric stretch-
ing 1% oleic acid or TritonX100. After exposure ing peak was observed together with the appear-
to penetration enhancers solutions, both T2 and D ance of a shoulder at 2858cm1 (Fig.13.4e),
decreased compared to untreated skin and to skin indicating a minor conformational disordering of
treated with neat Myritol 318 showing the skin a fraction of the SC lipid chains and the forma-
penetration of Myritol 318 induced by oleic acid tion of a new, disordered phase. Moreover, the
and TritonX100. After a period of 5h with scissoring mode of the treated samples contained
TritonX100 and 7h with oleic acid, T2 and D the two components characteristic for ortho-
recover their initial (preapplication) values. A rhombic phase centered around 1473 and
moisturization of the skin by an internal occlu- 1464cm1, together with the component charac-
sive mechanism was proposed as the origin of teristic for hexagonal or liquid phase centered at
such changes in the skin properties. This was 1468cm1. The treatment with oleic acid led to
13 Raman Scattering Probing Penetration Enhancers in Skin 241
a d
Absorbance [AU]
Absorbance [AU]
1120 1080 1040 1000 960 1780 1760 1740 1720 1700
Wavenumber [cm1] Wavenumber [cm1]
b e
d 2A/d2
d 2A/d2
2860 2856 2852 2848 2844 2860 2856 2852 2848 2844
Wavenumber [cm1] Wavenumber [cm1]
c f
FW-15 FW-15
d 2A/d2
d 2A/d2
FW-50 FW-50
1476 1472 1468 1464 1460 1476 1472 1468 1464 1460
Wavenumber [cm1] Wavenumber [cm1]
Fig. 13.4 Influence of propylene glycol (ac) and oleic ment with propylene glycol (ac) and oleic acid (df).
acid (df) on the molecular organization of the lipids in Dotted lines were acquired with control skin samples
human SC.Absorbance and second-derivative spectra (From Boncheva etal. (2008), with permission)
collected at 32C from excised human skin after treat-
an increase of the intensity to 1468cm1, show- keratin conformation from -helix to -sheets
ing the formation of a new fluid phase (Fig.13.4f). and the lipid bilayer from a crystalline phase with
In a previous study, Anigbogu etal. (1995) alkyl chains of lipid molecules in their all-trans
also showed using Raman spectroscopy that conformation (called gel) to a liquid crystalline
DMSO penetrated the SC and altered both the phase where the alkyl chains of lipids undergo
242 S. Brianon et al.
equilibrium between gauche and trans conforma- to 2324m in the skin after 1 h diffusion and
tions. After the immersion of the SC obtained between 15 and 40m after 2 h. These results
from human abdominal skin into aqueous solu- were confirmed by Raman images obtained on
tions of DMSO or deuterated DMSO-d6 for 1 h, thin slices which were cut in the skin samples
CRM was performed and spectra showed an at the end of the experiment. Spectral images
alteration of the keratin conformation: -helix were reconstructed by integration of the metro-
progressively converted into -sheets as the con- nidazole vibration (1191cm1) intensity. They
centration of DMSO increased. A plateau was showed that metronidazole was present in the
reached at a concentration of DMSO above 60% hair follicles at 4045m and at 25m in the
for which the protein conformation was approxi- SC.Furthermore, the authors used CRM to study
mately 50% -helix and 50% -sheets. Lipid the effect of metronidazole- transcutol solu-
bilayer organization was only altered at high tion on skin structure. A decrease in the inten-
DMSO concentrations (70% to 100%). This sity of the 1084cm1 peak was observed after
behavior can be related to the penetration applying the metronidazole-transcutol solution,
enhancement power of DMSO, which is gener- corresponding to a decrease of the lipid chains
ally significant when used at concentrations organization. This fluidization of the lipid chains
above 60% (Williams and Barry 2004). in the skin can be related to the drug or the trans-
Saar etal. (2010) used SRS to follow trans- cutol penetration (Tfayli etal. 2007).
retinol skin penetration by tuning the excitation The same team applied their methodology to
wavelength to the vibration of trans-retinol follow caffeine penetration in human skin sam-
(C=C stretch at 1596cm1). Images showed that ples. Caffeine is widely used in cosmetics as an
penetration of trans-retinol occurred through the active substance; it is also one of the most fre-
hair shaft. SRS was also used to image human quently used compounds in transdermal delivery
skin invivo up to a depth of 50m showing the studies. Due to its low logP, caffeine highly pen-
localization of trans-retinol around the hair and etrates through the skin and generates high fluxes
in the top of the sebaceous gland. (Bolzinger etal. 2008). Raman analysis of caf-
feine is a challenge due to its rapid penetration
and low storage in the SC.Tfaili etal. (2013)
13.4.2 Penetration Enhancement applied solutions of caffeine at two concentra-
ofVarious Drugs Followed tions, 2.57101mg.mL1 and 5.15102mg.
byConfocal Raman mL1 on human skin samples. Raman spectra
Microscopy were collected every half hour in the skin sam-
ples from the surface to 50m depth by 6m
Tfayli etal. (2007) studied the penetration of increments. The total duration was 4 h after caf-
metronidazole, a drug used in the treatment feine solution application. Raman analysis was
of rosacea, on excised human skin samples. not possible with the most concentrated solution
The drug was applied on the skin as a solution due to the crystallization of caffeine on the skin
in transcutol (diethylene glycol monoethyl, surface, caffeine crystals generating high inten-
Gattefoss, France), a penetration enhancer. sity signals which hide the Raman skin signal.
They first determined specific vibrations which The diluted solution allowed to monitor the caf-
can be detected in the skin in the Raman spec- feine in the skin, using two vibration bands at
trum of metronidazole dissolved in transcutol: 555cm1 ( C=O-N) and 1360cm1 ( CN). The
two vibration bands were selected at 1191 and maximum of caffeine signal intensity at 12m
1369cm1. Metronidazole was applied at the under the skin surface was recorded after 2 h35
concentration of 18g.mL1 in transcutol, and after exposure. After 4h, caffeine was no more
Raman spectra were acquired at several depths detected in the skin by Raman analysis. Several
in the skin after, respectively, 1 and 2 h diffu- causes were proposed by the authors: a too low
sion time. Metronidazole was detected down concentration in the skin due to caffeine diffu-
13 Raman Scattering Probing Penetration Enhancers in Skin 243
sion, a heterogeneity of the diffusion which may ibuprofen crystal at the skin surface before the
result in the absence of caffeine in the analyzed first measurement, less than 30min after applica-
volume, and a too low penetration depth (50m) tion. This was explained by a rapid penetration of
enabled by the Raman analysis. propylene glycol which increased the ibuprofen
Saar etal. (2011) used SRS to study the pen- concentration at the skin surface above its solu-
etration of two nonsteroidal anti-inflammatory bility, leading to its crystallization.
drugs, ketoprofen and ibuprofen, applied as solu- The penetration of -carotene, a lipophilic
tions in propylene glycol on skin samples. 3D molecule dissolved in DMSO, was studied by
images of the skin during drug penetration and Ashtikara (2013). Three main bands were used to
drug concentration profiles in the skin depth identify -carotene in the skin, the C=C bond
were obtained as a function of time. Ketoprofen stretching at 1510 and 1153cm1 and the C-CH3
presents a specific vibration band at 1599cm1 bond rocking at 1004cm1. Raman analysis
corresponding to the aromatic CH bond stretch- showed that -carotene hardly penetrated the
ing, whereas no specific bands could be detected skin; it was essentially found in the first 10m of
for ibuprofen and propylene glycol. These two the SC and presented a heterogeneous distribu-
molecules were used in deuterated form to cre- tion. This result was quite expected with such a
ate a specific vibration band around 2120cm1 lipophilic molecule which possesses poor skin
corresponding to the CD2 bond stretching. The permeability (Ashkitara 2013). DMSO may have
incident laser wavelength was tuned either to enhanced the penetration of -carotene, but this
1599cm1 to observe ketoprofen penetration was not proved by the authors.
or to 2120cm1 in order to image ibuprofen All these studies showed the potential of CRM
and propylene glycol in the skin. First ketopro- for imaging the skin with regards to its composi-
fen in deuterated propylene glycol solution was tion (lipids, proteins, water), for identification
applied on skin samples. Raman analysis at the and localization of drug and/or excipients such as
skin surface and at 12m and 15m in the SC penetration enhancers in the SC and epidermis in
revealed the penetration of ketoprofen and pro- order to improve the knowledge on penetration
pylene glycol, signal intensities increasing with mechanism.
time until 158min after solution application.
Integration of the signals obtained at each posi- Conclusion
tion in the skin allowed drawing depth profiles CRM is a very promising tool for the investi-
of propylene glycol and ketoprofene concen- gation of the penetration enhancement mecha-
tration at each measurement time. The results nisms and the effect of penetration enhancers
showed that propylene glycol penetrates the skin on the SC structure, lipid chain organization,
more rapidly and more efficiently than keto- and protein conformation. However, there are
profene. Furthermore, it was shown that pro- still few studies reporting such investigations.
pylene glycol concentration at 15m under the Development of such a technique for evalua-
skin surface increased continuously with time tion of penetration enhancers requires that
until reaching its surface value after 120min. Raman microscopy techniques pass from the
Focusing on a hair shaft area, the authors showed academic research laboratory to the routine
that the propylene glycol concentration was the analysis. The current skin absorption method
same throughout the experiment time (158min), based on extraction and HPLC analysis is well
whereas it increased continuously in another area established and described in Organization for
of the SC.This was explained by a rapid penetra- Economic and Cooperation Development
tion of propylene glycol in the hair shaft which (OECD) guidelines. In contrast to conven-
was saturated before the first measurement time. tional methods used for the determination of
Finally, the authors applied a solution of deuter- the drug distribution in skin layers, which
ated ibuprofen in normal propylene glycol (i.e., require separation of various skin layers (tape-
nondeuterated). They observed the formation of stripping) and extraction by solvents, CRM
244 S. Brianon et al.
may bring about a definite improvement Bolzinger MA, Brianon S, Pelletier J, Chevalier Y (2012)
because it allows measurement of depth con- Penetration of drugs through skin, a complex rate-
controlling membrane. Curr Opin Colloid Interface
centration profiles without separation of skin Sci 17:156165
layers. Additionally, CRM gives access to Boncheva M, Damien F, Normand V (2008) Molecular
information on skin lipids and proteins that organization of the lipid matrix in intact Stratum cor-
the classical methods do not provide. The neum using ATR-FTIR spectroscopy. Biochim
Biophys Acta 1778:13441355
methodology has to be further improved for Bouwstra JA, Ponec M (2006) The skin barrier in healthy
invivo analysis for which there is no standard and diseased state. Biochim Biophys Acta
technique available. Raman techniques are 1758:20802095
also very promising for academic research Bouwstra JA, Gooris GS, van der Spek JA, Bras W (1991)
Structural investigations of human stratum corneum
because these methods allow for the first time by small angle X-ray scattering. JInvest Dermatol
the nondestructive and simultaneous evalua- 97:10051012
tion (in the same experiment) of the skin pen- Bouwstra JA, Cheng K, Gooris GS, Weerheim A, Ponec
etration of several molecules and their effect M (1996) Phase behavior of isolated skin lipids.
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ATR-FTIR Spectroscopy
andtheSkin Barrier: Evaluation 14
ofPenetration-Enhancement
Effects
JuliaCovi-Schwarz, VictoriaKlang,
andClaudiaValenta
0.04
Amid 1
0.03
c=o
Amid 2
C-N
CH2
Absorbance units
0.02
CH2
Vas CH2
0.01
Vs CH2
0.00
c=o
-0.01
Wavenumber cm1
Fig. 14.1 ATR-FTIR spectrum of porcine ear skin recorded at 32C, showing the typical skin bands
arranged in two suborganisational patterns: the etal. 2008; Rodriguez etal. 2009). The lipid
lamellar organisation, that is, the arrangement in organisation is an important factor to determine
layers parallel to the skin surface, and the lateral skin permeability (Boncheva etal. 2008; de
organisation, which describes the lipid arrange- Sousa Neto etal. 2011). In addition to lipid com-
ment in the lipid lamellae themselves. Regarding position, temperature and skin hydration, sol-
the lamellar organisation, long-periodicity phases vents or penetration enhancers can likewise
and short-periodicity phases are present (Caussin influence the lateral packing of the stratum cor-
etal. 2009; Obata etal. 2010; Groen etal. 2011). neum lipids and thus the skin barrier (Naik and
Recent findings suggest that the long-periodicity Guy 1997).
phase is essential for the skin barrier function Regarding the stratum corneum, the most
(Caussin etal. 2009; Groen etal. 2011). informative lipid absorbances in the IR spectrum
Regarding the lateral lipid organisation, the originate from the hydrophobic alkyl chains
domain mosaic model is consistent with most (Potts and Francoeur 1993). In particular, the car-
experimental data. This model proposes that the bonhydrogen stretching vibrations with peaks at
lipids are organised in ordered domains and con- 2850 and 2920cm1, that is, the asymmetric and
nected by lipids in a disordered phase (Forslind symmetric stretching modes, provide informa-
1994). The ordered domains, also referred to as tion about the conformational order of the hydro-
gel phase, contain lipids in the orthorhombic or carbon lipid chains of the horny layer (Naik and
hexagonal phase. The disordered domain con- Guy 1997; Babita etal. 2006; Hathout etal.
sists of lipids in the liquid-crystalline phase 2011). Although both bands describe the three-
(Boncheva etal. 2008; Rodriguez etal. 2010; de dimensional arrangement of the lipids, the sym-
Sousa Neto etal. 2011). Lipids in the ortho- metric stretching mode is more susceptible to
rhomic phase are tightly packed, and the alkyl changes (Moore etal. 1997; Gooris and Bouwstra
chains are organised in an all-trans conformation. 2007; Rodriguez etal. 2010). The peak width as
The packing in the hexagonal phase is less tight, well as its wavenumber allow a classification of
and the lipids have more rotational freedom. The the lipids. The symmetric stretching band s
lipids in the liquid- crystalline phase have the appears at a wavenumber of about 2849
greatest freedom of movement and their alkyl 2850cm1 if the majority of the lipids are
chains exhibit a high percentage of gauche con- arranged in an orthorhombic pattern. During the
formation (Bouwstra and Ponec 2006; Boncheva transformation to a more fluid structure and a
250 J. Covi-Schwarz et al.
mainly hexagonal lipid arrangement, the peak drugs (He etal. 2009). Moreover, it is possible to
width increases and is shifted to higher wave- analyse the absorption of water within the stra-
numbers (28512852cm1). When most lipids tum corneum by analysing the amide 1 and 2
are arranged in the liquid-crystalline phase, the bands. While the intensity of the amide 2 peak
symmetric stretching peak appears at a wave- remains stable, the intensity of the amide 1 band
number of about 28532854cm1 (Boncheva increases upon water absorption. By calculating
etal. 2008; Caussin etal. 2008). the ratio of these intensities, the water content in
In addition, the CH2-scissoring band at about the skin can be determined (Prasch etal. 2000;
1460cm1 characterises the lateral packing of the He etal. 2009).
lipid chains in the stratum corneum. Depending
on the arrangement of the lipid domain, either
one or two peaks are visible in the skin spectrum 14.2.2 Influence ofSelected
(Moore etal. 1997; Boncheva etal. 2008; Penetration Enhancers and
Rodriguez etal. 2010). When two separate bands Vehicles ontheSkin Barrier
appear in the spectrum with a distance of approx-
imately 10 wavenumbers, the orthorhombic The characterisation of the skin barrier by bio-
phase dominates. These two separate peaks are physical methods is constantly exploring new
caused by the interaction between closely packed levels as sophisticated methods of analysis are
lipid chains, and appear between 1460 and continuously evolved and refined. Among other
1470cm1 (Hasanovic etal. 2011). Moreover, as methods, ATR-FTIR spectroscopy has success-
shown by Boncheva and coworkers, the scissor- fully been employed to determine the influence
ing bandwidth, especially when consulting sec- of substances such as solvents, penetration
ond derivative spectra, provides a suitable enhancers, excipients, or even of complex drug
measure for the presence and extent of the ortho- delivery systems on the stratum corneum (Babita
rhombic and hexagonal phases (Boncheva etal. etal. 2006). With this method, the enhancer effi-
2008). The more lipids pass into the hexagonal cacy can be evaluated, while the mode of action
phase, the more the distance between the two can be elucidated as well (Naik and Guy 1997).
peaks decreases, until they merge (Caussin etal. In the following section, examples of recent
2008). The C=O-stretching band represents the ATR-FTIR studies of interest are discussed in
lipid ester carbonyl and is mainly indicative of which the penetration enhancement effect and
the presence of sebum in and on the SC (Machado mechanism of certain excipients and vehicles
etal. 2010). were investigated.
While around 1650cm1, the stretching band Regarding the stratum corneum lipids, there
of the C=O group of keratin (amide 1) is present are two main effects that can be observed when
in the spectrum, the stretching band of the CN the skin is treated with penetration enhancers.
bond of the amino group of keratin appears at Some enhancers combine both modes of action,
around 1550cm1 (amide 2) (Moore etal. 1997; while others only make use of one of them. On
Babita etal. 2006; Rodriguez etal. 2009; He the one hand, a fluidisation of the stratum cor-
etal. 2009). The amide 1 band describes the sec- neum lipids can be induced and observed most
ondary structure of keratin (He etal. 2009). prominently on the CH2-stretching peaks in the
Wavenumbers 16151638cm1 suggest a -sheet spectra. In this case, the lateral packing of the lip-
arrangement, 16381645cm1 a random coil ids becomes less tight, and substances can pene-
structure, 16451662cm1 -helix arrangement trate the stratum corneum more easily. On the
and 16621695cm1 -turning structure (He other hand, penetration enhancers can also cause
etal. 2009). Excipients or penetration enhancers a lipid extraction. Due to the lower amount of lip-
can affect the secondary structure of keratin. ids in the stratum corneum, the barrier is weak-
They may induce a larger degree of freedom and ened (Naik and Guy 1997). Furthermore,
may thus enhance the penetration of co-applied penetration enhancers may also affect the skin
14 ATR-FTIR Spectroscopy andtheSkin Barrier: Evaluation ofPenetration-Enhancement Effects 251
barrier by changing the secondary structure of and does not alter the stratum corneum lipid
keratin. This leads to a loose accumulation of organisation (Mak etal. 1990). It does not show
stratum corneum proteins with a larger degree of an effect on the symmetric CH2-stretching band,
carbon movement (He etal. 2009). which indicates a preservation of the conforma-
The effect of ethanol, one of the most promi- tional order of the lipids (Panchagnula etal.
nent permeation enhancers and a simple vehicle, 2001; Boncheva etal. 2008, 42). However, a
on the skin barrier has been investigated by decrease in height and area of the CH2-stretching
numerous groups (Kurihara-Bergstrom etal. peak suggests a mild extraction of stratum cor-
1990; Bommannan etal. 1991; Krill etal. 1992; neum lipids (Babita etal. 2006).
Panchagnula etal. 2001). An invitro study on Dimethyl sulfoxide (DMSO) is a well-known,
human skin showed that ethanol induced lipid but rather aggressive, penetration enhancer. Results
extraction and could thereby enhance the flux of of ATR-FTIR studies suggested lipid extraction,
salicylate (Kurihara-Bergstrom etal. 1990). These protein denaturation and lipid fluidisation to be
results were confirmed invivo (Bommannan etal. among the enhancers mode of action (Naik and
1991). Interestingly, in this study as well as in Guy 1997; Babita etal. 2006). DMSO effectively
another study employing the stratum corneum of enhances the skin permeation of both hydrophilic
hairless mouse skin, a shift of the symmetric CH2- and lipophilic drugs, but concentrations of more
stretching band to lower wavenumbers was than 60% w/w are required for this effect. However,
observed. These findings indicate an increase in such high concentrations lead to erythema on skin,
lipid order after treatment with ethanol, which and thus DMSO is rarely used nowadays (Babita
represents a rather surprising observation. Based etal. 2006). In lower concentration, DMSO may be
on these findings, ethanol seems to enhance drug employed as a drug solubiliser in the stratum cor-
penetration by stratum corneum lipid extraction neum (Remane etal. 2006).
and does not, even at high concentrations, fluidise The analysis of the effect of dermal or trans-
the lipids (Babita etal. 2006). dermal drug delivery systems on the skin barrier
Fatty acids are natural constituents of the stra- properties is a rather complex task. Where possi-
tum corneum lipids. Therefore, compounds of ble, employing deuterated compounds is advanta-
this group, such as oleic acid, have a natural abil- geous to distinguish the skin bands from the
ity to intercalate within the lipid bilayer and thus formulation peaks in the spectra (Naik and Guy
modulate the barrier function (Babita etal. 2006). 1997). Otherwise, very careful data interpretation
Oleic acid is a penetration enhancer that is widely is necessary. However, reliable results can be
used in the field of transdermal drug delivery. obtained, especially if control spectra are
Regarding its mode of action, it is assumed that recorded. For instance, the influence of natural
the molecules create a highly permeable, fluid- sucrose esters on the stratum corneum was ana
like phase that coexists with the endogenous stra- lysed by ATR-FTIR.Sucrose oleate and laurate
tum corneum lipids, but disrupts their tight showed no effect on the skin bands or drug per-
packing (Ongpipattanakul etal. 1991; Babita meation when applied in aqueous solutions
etal. 2006; Boncheva etal. 2008). This hypothe- (Ayala-Bravo etal. 2003). However, a permeation-
sis was supported by experimental data which enhancing effect could be determined when the
showed a shift of the symmetric CH2-stretching sucrose esters were dissolved in Transcutol.
vibration to higher wavenumbers (Mak etal. Especially sucrose laurate induced a decreased
1990; Boncheva etal. 2008). Moreover, the scis- absorbance and a frequency shift to higher wave-
soring bandwidth confirmed that oleic acid forms numbers of the CH2-stretching bands (Ayala-
a separate phase within the stratum corneum lip- Bravo etal. 2003). Another group investigated the
ids (Boncheva etal. 2008). effect of sucrose laurate on the permeation of ibu-
Propylene glycol, a vehicle for penetration profen from a hydrogel (Csizmazia etal. 2012).
enhancers and a cosolvent in many dermal drug Besides an increase in drug permeation and a
delivery systems, interacts mostly with keratin more pronounced skin-hydrating effect as shown
252 J. Covi-Schwarz et al.
by the amide bands, the sucrose laurate-loaded Babita K, Kumar V, Rana V, Jain S, Tiwary AK (2006)
hydrogel resulted in the same slight lipid extrac- Thermotropic and spectroscopic behaviour of skin:
relationship with percutaneous permeation enhance-
tion and fluidisation as the control hydrogel. The ment. Curr Drug Deliv 3:95113
latter effects were reversible, and thus, sucrose Bello D, Smith TJ, Woskie SR, Streicher RP, Boeniger
laurate may be used as a mild penetration and MF, Redlich CA, Liu Y (2006) An FTIR investigation
hydration enhancer (Csizmazia etal. 2012). In of isocyanate skin absorption using invitro guinea pig
skin. JEnviron Monit 8:523529
another study, microemulsions based on natural Bommannan D, Potts RO, Guy RH (1991) Examination of
surfactants, namely sucrose laurate, lecithin and the effect of ethanol on human stratum corneum
alkylpolyglucoside, were investigated (Schwarz invivo using infrared spectroscopy. JControl Release
etal. 2012a). In skin diffusion experiments, leci- 16:299304
Boncheva M, Damien F, Normand V (2008) Molecular
thin showed the strongest skin-enhancing proper- organization of the lipid matrix in intact Stratum cor-
ties for the model drugs flufenamic acid and neum using ATR-FTIR spectroscopy. Biochim
fluconazole. Accordingly, the CH2-stretching Biophys Acta 1778:13441355
indicated that the stratum corneum lipids had Bouwstra J, Ponec M (2006) The skin barrier in healthy
and diseased state. Biochim Biophys Acta 1758:
undergone a phase transition to liquid-crystalline 20802095
organisation. Also, the CH2-scissoring and the Caussin J, Gooris GS, Janssens M, Bouwstra JA (2008)
amide bands suggested a more permeable skin Lipid organization in human and porcine stratum cor-
structure (Schwarz etal. 2012a). In contrast, the neum differs widely, while lipid mixtures with porcine
ceramides model human stratum corneum lipid orga-
application of solid lipid nanoparticles and nano- nization very closely. Biochim Biophys Acta 1778:
structured lipid carriers based on alkylpolygluco- 14721482
sides seemed to exert a barrier- strengthening Caussin J, Rozema E, Gooris GS, Wiechers JW, Pavel S,
effect on the stratum corneum (Schwarz etal. Bouwstra JA (2009) Hydrophilic and lipophilic mois-
turizers have similar penetration profiles but different
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Contents
15.1 Introduction
15.1 Introduction 255
15.2 Confocal Laser Scanning Microscope 256 Over the years, various methods such as diffusion
15.2.1 Principle ofCLSM 257 experiments (Addicks etal. 1987; du Plessis etal.
15.2.2 Major Advantages ofCLSM 259
15.2.3 Major Disadvantages ofCLSM 259 1994), microdialysis (Benfeldt 1999; Fang etal.
15.2.4 CLSM Used forTracking Liposomal 1999; Murakami etal. 1998; Schnetz and Fartasch
Formulations intheSkin 260 2001), visualization by microscopic techniques
15.2.5 Tracking thePenetration of like conventional fluorescence microscopy
Fluorescence Labels intoHair Follicles 266
15.2.6 The Efficacy ofDermaroller toEnhance (Kriwet and Mller-Goymann 1995; Yarosh etal.
Penetration intotheSkin 267 1994), electron microscopy (Bouwstra and
15.3 Two-Photon Fluorescence Microscopy 268
Honeywell-Nguyen 2002; Hashimoto etal. 1991;
15.3.1 Principle ofTwo-Photon Fluorescence Hofland etal. 1995; Kanerva 1990; Schreiner
Microscopy 268 etal. 2000; van den Bergh etal. 1999), confocal
15.3.2 Application ofTwo-Photon Microscopy laser scanning microscopy (Betz etal. 2001;
inSkin Penetration Experiments 270
Kirjavainen etal. 1996; Schatzlein and Cevc
15.4 Confocal Raman Microscopy 272 1998; van Kuijk- Meuwissen etal. 1998a, b;
15.4.1 Principles ofRaman Microscopy 272 Vardaxis etal. 1997; Veiro and Cummins 1994),
15.4.2 Confocal Raman Microscopy
forSkin Penetration Experiments 273 etc., have been exploited by researchers for study-
ing percutaneous penetration and to gain a better
15.5 Coherent Raman Microscopy 275
understanding of the skin barrier. Tape stripping
Conclusion 277 and microdialysis experiments, though exten-
References 278 sively used to measure the amount and rate of
penetration of the model compound, are lacking
M.A. Ashtikar in providing information about the effect of the
Lehrstuhl fr Pharmazeutische Technologie, model drug on cells and lipid organization or par-
Friedrich-Schiller-Universitt Jena, Jena, Germany
ticular pathways for the penetration of the model
e-mail: Mukul-arun.ashtikar@uni-jena.de
drug. These techniques also do not give much
D.D. Verma
information about spatial distribution of the model
Novartis Pharmaceutical Corp., East Hanover, NJ, USA
e-mail: ddverma@yahoo.com drug inside the tissue or comment on the mecha-
nism of penetration and more importantly, these
A. Fahr (*)
Friedrich-Schiller-Universitt Jena, Institut fr are destructive methods where tissue has to be
Pharmazie, Lehrstuhl fr Pharmazeutische carefully removed or homogenized to quantify the
Technologie, Lessingstrae 8, 07743 Jena, Germany amount of drug.
e-mail: alfred.fahr@uni-jena.de
Springer-Verlag Berlin Heidelberg 2017 255
N. Dragicevic, H.I. Maibach (eds.), Percutaneous Penetration Enhancers Drug Penetration
Into/Through the Skin, DOI10.1007/978-3-662-53270-6_15
256 M.A. Ashtikar et al.
Electron microscopy has been extremely use- pinhole aperture in front of the detector which
ful in understanding the structure of the stratum limits the out of focus light or by utilizing a non-
corneum (SC) and intercellular lipid organiza- linear technique such as 2-photon fluorescence,
tion. Bouwstra and coworkers have made impor- which excites the specimen only in a very small
tant contributions to the understanding of the focal plane at a time. Confocal microscopes cre-
interactions between vesicles and human skin ate quite sharp images compared to conventional
(Hofland etal. 1994, 1995; Schreiner etal. microscopes where light coming from different
2000). They employed freeze fracture electron planes of the specimen is not differentiated,
microscopy and small angle X-ray scattering to resulting in blurry and low resolution images.
study the effects of vesicular formulations on the Confocal microscopy on a broad basis can be
SC.Their results indicated increased diffusion of classified as an imaging and spectroscopic tech-
the drug due to adsorption and fusion of drug- nique. Confocal techniques that are discussed in
loaded vesicles to the surface of the skin. They this chapter are confocal laser scanning micros-
also reported perturbations in the ultrastructure copy (CLSM), two-photon microscopy, and
of the SC intercellular lipid domains due to mix- Raman microspectroscopic techniques. Although
ing of liposomal components with SC lipids, these techniques are based on different princi-
which could have also enhanced penetration of ples, they all have something in common and that
the model drug. Although freeze fracture elec- is confocality, ability to image one small focal
tron microscopy provides rather useful informa- volume at a time to construct a high-resolution
tion about the effect of liposomal vesicles on the image. Thanks to the modern electronics and
ultrastructure of the skin barrier, it lacks in pro- optics, we are able to image deeper into a thick
viding information of penetration pathways and biological specimen like skin at high resolution,
depth up to which the model drug has penetrated. high speed, and in-vitro as well as in-vivo condi-
Another widely appreciated technique is the tions. Sophisticated lasers and detection systems
conventional fluorescence microscopy. have enabled us to exploit nonlinear excitation
Evaluation of skin treated with fluorescently phenomena such as 2-photon fluorescence,
labeled liposomes by fluorescence microscopy coherent anti-stokes Raman scattering, stimu-
has demonstrated that the fluorescent marker lated Raman scattering, etc. The following chap-
remained in the SC (Kriwet and Mller-Goymann ter reviews these techniques and discusses some
1995) or penetrated deeper in the epidermis of the results obtained pertaining to dermal
mainly along the hair shaft (Yarosh etal. 1994). delivery.
However, during the sample preparation the tis-
sue needs to be (cryo-) fixed which might lead to
the alteration of the original specimen by the 15.2 C
onfocal Laser Scanning
change in skin lipid organization, redistribution Microscope
of the marker, etc.
Confocal microscopy has evolved over the last In the last two decades, CLSM has been exten-
five to six decades and today it is an essential tool sively used as a tool to visualize the fluorescent
for a life-sciences laboratory; it has emerged as a model compounds in the skin. CLSM examina-
sophisticated tool for tracking and studying tion revealed valuable additional morphological
transport phenomena of different compounds information of porcine skin in wound healing
with a high degree of precision. Confocal micros- studies which was not obtained by conventional
copy offers significant advantages over other microscopy (Vardaxis etal. 1997). CLSM was
microscopic techniques in evaluation of thick also used to understand the mechanism by which
specimens due to their inherent ability to distin- nanoparticulate systems facilitate skin transport.
guish photons generated from different focal The surface images revealed that (a) polystyrene
planes of the specimen. This ability is mainly nanoparticles accumulated preferentially in the
achieved in two ways, either by introducing a follicular openings, (b) this distribution increased
15 Confocal Microscopy forVisualization ofSkin Penetration 257
in a time-dependent manner, and (c) the follicular time resolved diffusion of a lipophilic dye into
localization was favored by particles of smaller the hair follicle of fresh and unfixed piece of
size (Alvarez-Roman etal. 2004). Simonetti etal. human scalp skin. The authors claimed that this
visualized diffusion pathways across the SC of technique was able to visualize the diffusion of a
native and invitro reconstructed epidermis by dye into the upper hair follicle at different time
using CLSM (Simonetti etal. 1995). points (Grams etal. 2004). In another study, they
Many researchers used CLSM to evaluate the also report that follicular accumulation of lipo-
penetration of liposomes into human or animal philic dyes increased when applied in combina-
skin using a fluorescent compound or a fluores- tion of surfactant-propylene glycol (Grams etal.
cent model drug. It was demonstrated that flexi- 2003).
ble liposomes penetrated deeper into the skin in
non-occlusive conditions than after occlusive
application (van Kuijk-Meuwissen etal. 1998a) 15.2.1 Principle ofCLSM
as measured by a fluorescent liposome marker.
Touitou and coworkers examined the penetration Confocality is achieved in CLSM by using a point
of fluorescent probes into fibroblasts and nude illumination and point detection. Marvin Minsky
mice skin by CLSM and showed that ethosomes constructed the first confocal microscope in 1957
facilitated the penetration of all probes into the at Harvard (Semwogerere and Weeks 2008). He
cells, as evident from the high-intensity fluores- used a zirconium arc lamp and two pinholes, one
cence as compared to the hydroethanolic solution in front of the lamp and another in front of the
or conventional liposomes (Touitou etal. 2001). detector to achieve the confocality, while the sam-
Many research groups examined different ple was scanned through the light beam to gener-
lipid combinations for their potential to influ- ate the image. Todays confocal microscopes
ence the skin. Zellmer etal. used CLSM to although different are based on the same principle
demonstrate that vesicles made of native human of point illumination and detection, and their abil-
SC lipids interact rapidly with phosphatidylser- ity to image thick samples with high resolution
ine liposomes, weakly with human SC lipid have enabled their use in studying skin penetra-
liposomes and do not interact with phosphati- tion of various molecules and delivery systems.
dylcholine (PC) liposomes (Zellmer etal. A CLSM can create a very sharp and high-
1998). Kirjavainen etal. reported that resolution image of a specimen compared to a
lipophilic fluorescent marker, L-- conventional microscope. This ability arises from
phosphatidylethanolamine-N-lissamine rhoda- the fact that CLSM is able to filter light coming
mine B sulfonyl (N-Rh-PE) was able to from parts of the specimen that are not in focus,
penetrate deeper into the SC from liposomal and in the process as much as 95% of the light
vesicles containing dioleylphosphatidyl- coming from the specimen can be blocked to
ethanolamine (DOPE) than from liposomes generate an image. As a result, very few photons
without DOPE.A pretreatment of the skin with actually make it to the detector therefore a very
unlabeled liposomes containing DOPE or lyso- high intensity light source in the form of laser is
phosphatidylcholine (lyso-PC) enhanced the used in modern CLSMs. One disadvantage of
subsequent penetration of the fluorescent mark- this is that more than one laser might be required
ers, N-Rh-PE, and sulforhodamine B into the in order to have multiple excitation wavelengths.
skin, suggesting a possible penetration enhanc- The principle of confocality is explained in
ing activity (Kirjavainen etal. 1996). the schematic diagram in Fig.15.1a where light
Procedures like iontophoresis which enhance coming from the specimen is focused on the
the percutaneous flux of model drugs like calcein detector with the help of two lenses. Due to the
(Turner and Guy 1998) and mannitol (Kirjavainen pinhole in front of the detector only the light
etal. 2000) were also examined by CLSM in ani- coming from a very small focal point reaches the
mal models. Grams etal. used CLSM to visualize detector and light from out of focus regions of the
258 M.A. Ashtikar et al.
specimen is blocked by the pinhole. The second light is provided by a laser which passes through
pinhole is employed in front of the light source, a pinhole onto a dichroic mirror which directs
which limits the amount of specimen that is illu- the light to a scanning mirror assembly and onto
minated at any time, and as a result reduces the the specimen through the microscope objective.
background scatter from out of focus region of The scanning mirrors scan the laser across the
the specimen. Two pinholes of the confocal specimen in order to generate the image. Light
microscope are able to significantly reduce the reflected from the specimen is de-scanned at the
background blur and produce very high- scanning mirrors and passes to the photo-multi-
resolution images (Hollricher and Ibach 2011; plier tube (PMT) detectors through the dichroic
Nwaneshiudu etal. 2012). mirror (Semwogerere and Weeks 2008). Light
Schematic representation of a CLSM is pro- coming from out of focus regions of the specimen
vided in Fig.15.1b. In a modern CLSM, a beam of is blocked at the detector by a pinhole. The image
a
Detector
Pinhole aperture
Dichroic mirror
Laser
Pinhole
aperture
Specimen
Fig. 15.1(a) Pinhole in front of the detector rejects light emerging from out of focus regions. (b) Schematic represen-
tation of CLSM optics (Semwogerere and Weeks 2008)
15 Confocal Microscopy forVisualization ofSkin Penetration 259
Laser
b
Pinhole aperture
Scanning
mirrors
Detector
Dichroic mirror
Microscope
Fluorescent specimen
Fig. 15.1(continued)
15.2.4 CLSM Used forTracking study confirmed the assumption that liposomes
Liposomal Formulations CFin-out were not under osmotic stress and will,
intheSkin therefore, transfer themselves more easily into
the SC.The results indicated further that phos-
Liposomes have been extensively studied and pholipid vesicles not only carry the entrapped
suggested as a vehicle for topical drug delivery hydrophilic substance, but also the non-
(Chen etal. 2010, 2011; Dragicevic-Curic etal. encapsulated hydrophilic substance into the SC
2008, 2009; Ntimenou etal. 2012). However, the and possibly to the deeper layers of the skin.
mechanism of penetration of liposomes as drug Although CLSM served as a useful tool to esti-
carriers into the intact skin is not fully under- mate skin penetration, CLSM images do not pro-
stood. In this section, we will discuss the interac- vide the visualization of single liposomes, so the
tions between liposomes, containing hydrophilic exact mechanism of penetration of liposomes
and lipophilic fluorescent probes, and human as still remains an unsolved question.
well as rat skin using CLSM.
15.2.4.2 E ffect ofVesicle Diameter
15.2.4.1 T racking Skin Penetration on Skin Penetration of
ofLiposomally Entrapped or Liposomally Entrapped
Un-entrapped Hydrophilic Drugs
Fluorescent Model Drug The influence of vesicle size on the penetration of
The mechanism of action of liposomes as pene- fluorescently labeled liposomes into the human
trable drug carriers in topical delivery is not com- skin was investigated using lipophilic
pletely understood. In order to evaluate if fluorescent label, 1,1-dioctadecyl-3,3,3,3-
liposomes are able to increase skin penetration of tetramethylindocarbocyanine perchlorate (DiI)
only entrapped hydrophilic drugs or also of unen- (Verma etal. 2003a). In all CLSM images
trapped hydrophilic drugs, three different liposo- (Fig.15.3) very high fluorescence was observed
mal formulations using carboxyfluorescein (CF) in the SC, which was expected due to the lipo-
as a fluorescent hydrophilic model drug were pre- philic nature of the fluorescent label, DiI.This
pared: CFin-out (non-entrapped CF was not study indicated that large vesicles with a
removed), CFin (non-entrapped CF was size600nm were not able to deliver their con-
removed), and CFout (a pre-calculated amount tents into the deeper layers of the skin. These
of CF was added to empty liposomes). All the liposomes probably stayed in/on the SC and after
liposomal formulations had a similar size of lipo- drying they formed a layer of lipid, which would
somes and the concentration of CF was the same have further strengthened the barrier properties
in all formulations (Verma etal. 2003b). After of the SC.The liposomes with size300 nm
6h incubation of the skin with different formula- were able to deliver their contents to some extent
tions, the skin was cryofixed and 7m thick sec- into the deeper layers of the skin. However, the
tions were cut using a cryo-microtome. These liposomes with size70nm were most promis-
cross-sections were investigated using CLSM for ing for dermal drug delivery into the deeper skin
the skin penetration of CF. layers as they showed maximum fluorescence
The penetration studies and CLSM images both in viable epidermis, as well as in dermis.
showed that liposomes CFin-out exhibited maxi-
mum deposition of CF in the SC, whereas lipo- 15.2.4.3 Synergistic Penetration
somes CFin showed higher penetration of CF into Enhancement Effect of
the deeper skin layers such as the viable epider- Ethanol andPhospholipids
mis (Fig.15.2), and through the skin to the accep- onTopical Drug Delivery
tor compartment of the Franz diffusion cell. It was observed that the composition of liposo-
These results were comparable to the data mal formulation had an appreciable effect on the
obtained from tape stripping experiments. This penetration of compounds into and through the
15 Confocal Microscopy forVisualization ofSkin Penetration 261
a b
c d
Fig. 15.2 Fluorescence micrographs of cross-sections of CFin; (b) liposomes CFin-out; (c) liposomes CFout, CF out-
human abdominal skin incubated on Franz diffusion cells side liposomes; and (d) CF dissolved in Tris buffer. Scale
with different formulations containing CF.Formulations bar represents 100m (Reprinted from Verma etal.
were applied without occlusion for 6h. (a) Liposomes (2003b) with permission from Elsevier)
skin (Hofland etal. 1994; Jimbo etal. 1983; Kobayashi etal. 1994; Simonetti etal. 1995). In
Loftsson etal. 1989; Tenjarla etal. 1999). Hence, a preliminary study, we have seen that not only
the effect of lipid vesicular systems embodying are the amount and the type of phospholipids
ethanol in relatively high concentrations on the important for skin penetration enhancement, but
percutaneous penetration of cyclosporin A (CyA) also the amount of ethanol has a significant role
was investigated using a standardized skin strip- in delivering the fluorescent model compounds
ping technique and CLSM (Verma and Fahr into the skin (Fig.15.4) (Verma 2002). We
2004). Ethanol has been widely reported as an hypothesized that ethanol and phospholipids
efficient skin penetration enhancer in the concen- might have synergistic skin penetration enhanc-
tration of 5100% (Bhatia and Singh 1999; ing effect.
262 M.A. Ashtikar et al.
a b
c d
Fig. 15.3 Fluorescence micrographs of cross-sections of tion of DiI; (b) NAT8539/ethanol (10/3.3); (c) NAT8539/
human abdominal skin incubated on Franz diffusion cells ethanol (10/10); (d) NAT8539/ethanol (10/20). Scale bar
with different formulations containing DiI.The vesicles represents 50m (Reprinted from Verma etal. (2003a)
were applied non-occlusively for 12h. (a) Ethanolic solu- with permission from Elsevier)
In order to evaluate synergistic effect of the no fluorescence was noticeable in the viable epi-
ethanol and phospholipids on penetration of DiI, dermis and dermis (Fig.15.5a). Formulation,
vesicles with composition of 10% (w/v) phos- NAT 8539/ethanol (10/3.3), produced a homo-
pholipid mixture NAT 8539 (Lipoid, Germany) geneous bright fluorescence throughout the SC,
and ethanol at different concentrations ranging but no fluorescence was observed in the viable
from 0 to 20%w/v were prepared. The results epidermis and dermis (Fig.15.5b). Formulation,
of the CLSM studies are represented in Fig.15.5. NAT 8539/ethanol (10/10), produced a bright
Ethanolic solution of the DiI dye was able to fluorescence throughout the SC with very weak
deliver only weak fluorescence into the SC, and to weak fluorescence observed in the viable
15 Confocal Microscopy forVisualization ofSkin Penetration 263
a c e g
b d f h
Fig. 15.4 Fluorescence micrographs of cross-sections of and CF (d). (e, f) PL 90H-liposomes with N-Rh-PE (e)
human skin incubated with vesicles with different lipid and CF (f); (g, h) PL25 liposomes with N-Rh-PE (g) and
content for 12h. (a, b) Ethanolic solution of N-Rh-PE (a) CF (h)
and CF (b). (c, d) Flexible liposomes with N-Rh-PE (c)
a b
c d
Fig. 15.5 Fluorescence micrographs of cross-sections of solution of DiI; (b) NAT8539/ethanol (10/3.3); (c)
human abdominal skin incubated on Franz diffusion cells NAT8539/ethanol (10/10); (d) NAT8539/ethanol (10/20).
with different formulations containing DiI.The vesicles Scale bar represents 50m (Reprinted from Verma and
were applied without occlusion for 12h. (a) Ethanolic Fahr (2004) with permission from Elsevier)
CLSM images of cross-sections of rat skin and weak fluorescence in the epidermis, suggest-
incubated for 6h with Fl-CyA liposomes pre- ing diffusion of the Fl-CyA from SC to the epi-
pared with and without terpenes are compared in dermis (Fig.15.6c). Ethanolic solution of Fl-CyA
Fig.15.6. A bright fluorescence was observed in showed a very weak fluorescence in the SC and
the SC (Fig.15.6b) for skin treated with Fl-CyA no fluorescence was observed in the viable epi-
liposomes without terpenes, but only a negligible dermis and dermis (Fig.15.6a). Based on the
or no fluorescence was observed in the viable fluorescence scores obtained from CLSM images,
epidermis and dermis. The skin treated with results indicate that penetration enhancers do
Fl-CyA liposomes containing 1 % terpenes play an important role in the penetration of fluo-
showed a relatively higher fluorescence in the SC rescent labels into the skin.
15 Confocal Microscopy forVisualization ofSkin Penetration 265
a b
Fig. 15.6 Cross-sections of rat skin incubated with dif- penes, and (c) liposomes containing 1% terpenes. The
ferent formulations containing Fl-CyA.The vesicles were length bar represents 10m. Arrows represent hair folli-
applied non-occlusively for 6h on rat skin. (a) Ethanolic cles in the dermis
solution of Fl-CyA, (b) Fl-CyA liposomes without ter-
Cross-sections of human skin treated with viable epidermis. Samples treated with hydro-
double-labeled (DiI and Alexa Fluor 488) lipo- alcoholic solutions of the dyes exhibited fluores-
somes with and without terpenes are represented cence for both DiI and Alexa Fluor-488 only in
in Fig.15.7. Incubation period of formulation the SC.Increasing the incubation period from 6h
with the skin was 6h (Fig.15.7ac) and 12h to 12h only increased the fluorescence intensities
(Fig.15.7df). Control was provided by skin for both the labels (Fig.15.7a, d). In samples
samples treated with hydro-alcoholic solution treated with dual labeled liposomes without ter-
containing both dyes mentioned above. In all the penes, fluorescent markers after 6h diffusion are
samples, the fluorescence was restricted mainly restricted to the SC only. DiI penetrated up to
to the SC and to a smaller or larger extent, to the deeper layers of the SC however no fluorescence
266 M.A. Ashtikar et al.
a b c d
e f
Fig. 15.7Cross-sections of human abdominal skin (df). (a, c) Hydro-alcoholic solution of DiI and Alexa
treated with formulations containing two fluorescent Fluor 488, (b, e): double-labeled liposomes without ter-
markers, DiI and Alexa Fluor 488. The formulations penes (c, f): double-labeled vesicles containing 1% ter-
were applied without occlusion for 6h (ac) and 12h penes. The bar represents 10m
was observed in the viable epidermis and dermis able to deliver relatively higher amounts of fluo-
(Fig.15.7b). Increasing the incubation period to rescent labels into the SC, epidermis, and to a
12 h did not have marked improvement in pene- small extent the dermis. In this study, we clearly
tration depths. CLSM images (Fig.15.7e) show demonstrated the ability of CLSM to simultane-
higher intensities and slightly higher penetration ously image two fluorescent dyes.
up to viable epidermis achieved for both the dyes.
Although distribution of Alexa Fluor-488
appears uniform for both 6 and 12h diffusion 15.2.5 Tracking thePenetration
samples, distribution of lipophilic dye DiI was ofFluorescence Labels
not uniform for 12h samples. For skin treated intoHair Follicles
with liposomal formulations containing 1% ter-
penes, much higher intensities were observed for 15.2.5.1 T racking ofFluorescently
both the fluorescent dyes in the SC (Fig.15.7c, f). Labeled Cyclosporin Ainto
For sample with 6h incubation, very weak fluo- Rat Hair Follicles
rescence for Alexa Fluor-488 while weak fluo- CLSM has been successfully used to track the
rescence for DiI was observed in the viable follicular penetration of drugs. Topical applica-
epidermis. For 12h incubation samples, fluores- tion of the CF-loaded liposomes has been
cence intensity scores were bright-fluorescence reported to significantly increase the accumula-
at SC for both the dyes and moderate fluores- tion of CF in the pilosebaceous units compared to
cence intensities were observed in the viable epi- other non-liposomal formulations (Lieb etal.
dermis. Surprisingly, the reddish fluorescence 1992). Using CLSM, Patzelt etal. demonstrated
continued from the epidermis toward the dermis, that follicular penetration for nanoparticles was
indicating a diffusion of the lipophilic marker. It size dependent (Patzelt etal. 2011).
was observed that liposomal formulations con- The effect of terpenes as penetration enhanc-
taining terpenes as penetration enhancers were ers in liposomes for targeting hair follicles was
15 Confocal Microscopy forVisualization ofSkin Penetration 267
investigated using Fl-CyA vesicles (as men- 15.2.5.2 A ccumulation ofCF and
tioned above), with and without terpenes, in rat N-Rho-PE Loaded Liposomes
skin. Figure15.6 also depicts the role of the pilo- intheHuman Hair Follicles
sebaceous unit in the penetration of drugs into In this study, liposomal formulations contain-
the skin. A bright fluorescence was observed in ing CF and N-Rh-PE were prepared (Verma
the pilosebaceous units (bright green fluorescent 2002). Liposomal formulations were able to
regions in dermis in Fig.15.6b, c identified as deliver both the hydrophilic and lipophilic fluo-
the hair shaft, a part of the pilosebaceous unit) rescent labels to the human hair follicles as seen
for both formulations, with and without PE.The in Fig.15.9a, b. Studies represented in Figs.15.6,
fluorescence was also visualized in the outer root 15.8, and 15.9 demonstrated that the presence of
sheath of the hair shaft (Figs.15.6bc and 15.8) hair follicles plays a significant role in the skin
(Verma 2002). The images presented here dem- penetration of drugs, as well as that the presence
onstrate that the liposomal vesicles can enter the of penetration enhancers helps the formulation in
pilosebaceous unit and deliver their content to enhancing the follicle delivery of drugs.
the hair follicle and possibly to the hair bulb. The
importance of this was stressed upon by the fact
that ethanolic solution of Fl-CyA failed to 15.2.6 The Efficacy ofDermaroller
deliver any fluorescence into the skin. These toEnhance Penetration
CLSM results were supported by invivo studies intotheSkin
with Dundee Experimental Bald Rats model
(Verma etal. 2004) and other published reports CLSM was also used to investigate the efficacy
(Agarwal etal. 2000; Bohm and Luger 1998; of the micro-perforation devices, Dermaroller
Lieb etal. 1992; Niemiec etal. 1995). The (Horst Liebl ETS, France), in increasing the skin
CLSM investigations enabled us to visualize the penetration of a fluorescent lipophilic model
accumulation of fluorescent label in the pilose- compound DiI encapsulated in liposomes (Verma
baceous units, which is not possible with tape 2002). Three types of Dermarollers were tested
stripping. in this study, namely, Dermaroller C8 0.1315,
a b
Fig. 15.8 Fl-CyA liposomes treated rat skin cross-sec- 10 m. (a) Fluorescence micrograph and (b) transmis-
tion demonstrating the role of pilosebaceous units in the sion-mode image of the cross-section
penetration of substance into the skin. The bar represents
268 M.A. Ashtikar et al.
a b
Fig. 15.9 Fluorescence micrographs showing (a) CF delivery to hair follicles and (b) high-resolution micrograph
showing rhodamine delivery to hair follicle following topical application of fluorescent liposomes
M8 1.515, and M8 1.530. Control was pro- get a better understanding of the mode of action
vided by the skin not treated with Dermarollers. of the micro-perforation device, Dermaroller.
Bright intensity of fluorescence was observed
in SC for all samples treated with Dermaroller
and for control (Fig.15.10). For control samples, 15.3 Two-Photon Fluorescence
however, fluorescence intensities in the viable Microscopy
epidermis and dermis were very low. For
Dermaroller C8 0.1315, highest fluorescence 15.3.1 Principle of Two-Photon
was observed in the SC and fluorescence intensi- Fluorescence Microscopy
ties rapidly decreased in the viable epidermis and
dermis (Fig.15.10b). Dermaroller C8 0.1315 As seen from aforementioned studies, CLSM is
was intended for delivering drugs to the SC a very good technique for the imaging of bio-
whereas Dermaroller M8 1.515 and M8 1.5 logical samples; however, it suffers from some
30 were optimized for delivering drugs to the disadvantages. One of the most important limita-
deeper regions of skin. Both M8 Dermarollers tions of the CLSM is that because only a small
did show very high fluorescence intensities in the fraction of the incident light reaches the detec-
dermis region (Fig.15.10c, d). We observed that tor, relatively high intensities of light are
samples treated with both M8 Dermarollers required to achieve high signal to noise ratios.
showed only a weak fluorescence in the viable This results in high total energy transfer to the
epidermis region and showed weak lateral diffu- specimen during imaging. Also, the laser beam
sion of the fluorescent label from the pores. This excites the fluorophores in the specimen above
might be because Dermaroller was able to and below the focal plane that is under investiga-
deliver the liposomes into the deeper layers tion and this can cause severe photo bleaching,
quickly during application of the Dermaroller but photo damage, and even dehydration of the bio-
after the application, liposomes probably were logical specimen. Many fluorophores generate
not able to diffuse to a great extent from the sur- free radicles or singlet oxygen when excited
face of the skin to the deeper layers in the skin. In which can be toxic if imaging living cells or tis-
this study, we emphasized on the spatial distribu- sue. CLSM also suffers from low axial resolu-
tion of the fluorescent label which helped us to tion especially when imaging deep within a thick
15 Confocal Microscopy forVisualization ofSkin Penetration 269
a b
c d
Fig. 15.10 Fluorescent micrographs of cross-sections of were applied without occlusion for 3h. (a) Control; (b)
human abdominal skin pretreated with Dermaroller and Model C 8 0.1315; (c) Model M 8 1.515; (d) Model
incubated on a Franz diffusion cell with liposomes con- M 8 1.530
taining lipophilic fluorescent label DiI.The liposomes
tissue. The poor axial resolution results from the Due to limitations in deep tissue imaging,
scattering of light by the specimen itself, which when adopting a CLSM for imaging skin, the
can lead to detection of light that is not gener- sample must be cryo-sectioned and this is not
ated from the observed focal plane. Also CLSM always possible and desirable.
employs short wavelength excitation sources Multiphoton excitation phenomenon was pro-
and shorter wavelengths are scattered stronger posed in 1931; however, Kaiser and Garret were
(scattering intensity is inversely proportional to able to validate it only in 1961 after the develop-
fourth power of the wavelength) hence these ment of lasers which were sufficiently powerful
excitation sources are not very efficient at deep to generate the high photon flux required for two-
tissue imaging. photon excitation. Figure15.11a explains the
270 M.A. Ashtikar et al.
huem
Energy
epidermis they used Laurdan generalized polar- the SC.Yu etal. used a high-speed two-photon
ization (Laurdan-GP) values as an indicator of microscope with dual channels capable of simul-
polarity. As expected, the Laurdan-GP values taneous monitoring of autofluorescence of skin
were highest for SC and decreased for deeper and fluorescence from rhodamine as a model
layers of the skin indicating an increase in fluid- drug (Yu etal. 2003). Treatment of human skin
ity or hydration. The authors report an interesting with oleic acid as a penetration enhancer was
observation in the intercluster region (canyons found to induce increased intra-corneocyte diffu-
or wrinkles as in human skin), the Laurdan-GP sion of the hydrophilic model drug (sulforhoda-
values in the canyons were quiet high, compa- mine- B), while causing localization of the
rable to that of SC, and they did not change with hydrophobic model drug (Rhodamine-B hexyl
depth (from surface of SC to dermis), indicating ester) to the intercellular region. Hence, Yu etal.
that canyons might show environment similar to provided evidence that for chemical penetration
SC.To study the penetration of liposomal for- enhancers, intra-corneocyte diffusion is an
mulations, Lissamine rhodamine B 1,2-dihexad important enhancement mechanism along with
ecanoyl-sn-glycero-3-phosphoethanolamine was fluidization of intercellular lipid domains, and
incorporated in the liposomal membrane. After physicochemical properties of the drug would
16-h diffusion, image stacks were collected and determine which pathway predominates in the
penetration was measured in the form of ratio of penetration enhancement.
fluorescence intensity of rhodamine and auto- In 2002, Jenlab GmbH introduced a dual-
fluorescence of the skin. Authors report that the channeled noninvasive multi-photon tomograph
liposomes with lower lipid content (10mg/ml) capable of invivo optical biopsies with subcel-
were able to deliver more fluorescent label to the lular spatial resolution based on near-IR femto-
lower parts of viable epidermis compared to the second pulsed laser. Knig etal. reported the
formulations with higher lipid content (25 and potential of this microscope to acquire high-
50mg/ml). resolution images from deep within the skin
van den Bergh etal. elucidated the mechanism invitro and invivo (Konig etal. 2006;
of interactions of elastic and rigid liposomal ves- Schenke-Layland et al. 2006). The microscope
icles with human skin (van den Bergh etal. utilized the second harmonic generation (SHG)
1999). Using 2-PFM and different electron signal to monitor the position of the microscope
microscopic techniques they studied interactions in the skin, while the fluorescence channel was
between skin and various elastic liposomal for- used to detect either autofluorescence from the
mulations to understand the modulation of the NAD(P)H, flavins, melanin, etc., or to detect
skin barrier. They observed that fluorescently exogenous fluorophores. In vivo imaging capa-
labeled elastic vesicles primarily affected the bilities of 2-PFM promises great advances in can-
intercellular lipid lamellae of the SC, while the cer diagnostics and skin diseases, as well as in
underlying layers of the viable epidermis understanding the mechanisms involved in der-
remained relatively unchanged. This effect was mal drug delivery.
visualized using 2-PFM, in the form of small 2-PFM is, as already mentioned, a relatively
penetration pathways that were confined to the young technology. Bio-Rad Laboratories
SC only. Authors also reported that the penetra- (USA) introduced the first commercial two-
tion enhancing ability of elastic vesicles was photon microscope in 1996 and since then this
dependent on whether vesicles were applied with technology has seen exponential growth in its
or without occlusion. use and applications. Its ability to take high-
Oleic acid is a good penetration enhancer and resolution optical biopsies of the intact skin or
has been found to increase transdermal penetra- even do live imaging invivo holds a great
tion of both hydrophilic and hydrophobic drugs potential to unlock the secrets that were kept in
by modulating the intercellular lipid domains in the dark by nature.
272 M.A. Ashtikar et al.
15.4 Confocal Raman Microscopy tered by the sample such that the scattered light
has the same wavelength as the incident light;
15.4.1 Principles ofRaman this phenomenon is known as elastic scattering
Microscopy or Rayleigh scattering. However, a small frac-
tion of photons do interact with the molecules
Fluorescence microscopy has seen a lot of evolu- of the sample and as a result, scattered photons
tion in the last couple of decades and modern have a different energy compared to the inci-
microscopic techniques, like CLSM, multi- dent photons which manifests in the form of a
photon microscopy, and various imaging modes shift in the wavelength of the scattered pho-
such as fluorescence life-time imaging, Frster tons. This phenomenon is known as inelastic
resonance energy transfer, etc., have increased scattering or Raman scattering. Raman effect
the applications and flexibility of the fluores- was first observed by C.V.Raman in 1928
cence imaging. However, fluorescence micros- (Raman and Krishnan 1928). It is a very weak
copy still has certain disadvantages; mainly, phenomenon, about one photon undergoes
fluorescence microscopy still utilizes exogenous Raman scattering in a million Rayleigh scat-
dyes to label the drug or formulation. These tered photons. Due to low signal to noise ratio,
labels are often toxic or could cause perturba- Raman spectroscopic techniques were not very
tions in the cells or other physiological processes. practical as they needed an intense source of
The biggest limitation for studying skin penetra- light and a sophisticated detection system.
tion is that often the fluorescent label and the for- After the discovery of lasers and charged cou-
mulation or the drug do not travel together and pled detectors (CCD), however, Raman spec-
consequently fluorescence imaging could give troscopy has seen tremendous increase in its
incorrect interpretation of the observations. application (Hollricher 2011).
Due to the disadvantages of fluorescent label- To understand the Raman effect, we shall first
ing to monitor the penetration depth in the skin, have a look at the Rayleigh scatter. According to
label-free vibrational spectroscopic techniques the classical theory, when photons are incident
have gained importance. Vibrational spectros- on the sample, they are absorbed by the mole-
copy is an identification technique based on the cules of the sample and are excited to a virtual
principle that it is possible to interfere with the state. When molecules relax back, if they relax
vibrational state of a molecule by irradiating it to the same energy level as they started in, the
with light of certain frequencies. Such interac- emitted photons have the same energy as the
tions can be recorded in the form of spectra which incident photons and this type of scattering is
are representative of the bonds present in the called Rayleigh scattering. This effect is
molecule. Because each molecule has its unique explained schematically in Fig.15.12. In Raman
atomic structure and as a result a characteristic scattering, a molecule relaxes back to a higher
vibrational state, the vibrational spectra can pro- energy level after absorbing a photon, and as a
vide a host of information about the structure of result the emitted photon has lower energy by an
the molecule. Vibrational spectroscopy involves amount required to vibrationally excite the mol-
different techniques such as mid infrared (IR) ecule. Reemitted photon exhibits red shift and
spectroscopy, NIR spectroscopy, Raman spec- hence this effect is also called Stokes Raman
troscopy, etc., but for this chapter we shall only scattering. If after absorbing the photon the
review the Raman spectroscopic techniques. In molecule loses a fraction of vibrational energy,
addition, because water content in the biological i.e., if the molecule relaxes back to a lower
specimen interferes strongly with IR spectros- energy state compared to its energy state before
copy, Raman techniques are more suitable for absorbing the photon, then the emitted photon
studying the drug penetration into the skin. has higher energy compared to the incident pho-
When a monochromatic beam of light is ton. This phenomenon is called the anti-stokes
incident on the sample, most of the light is scat- Raman scattering. Raman spectrum is a plot of
15 Confocal Microscopy forVisualization ofSkin Penetration 273
Electronic
excited state
portional to the fourth power of the frequency of
incident photons. Hence, excitation at 400nm
would give a 16 times more intense Raman signal
compared to excitation at 800nm (Hollricher
Virtual
excited state 2011). However, skin has a very strong autofluo-
rescence in the range of 400700nm and hence
lower wavelengths are not ideal for Raman map-
ping of the skin. Although Longer wavelengths
are useful for imaging thick specimen, in a dif-
fraction-limited system they are also associated
with lower spatial resolution. As a result, select-
Electronic ing the right wavelength of excitation is very
ground state
important in order to maintain a good balance
Rayleigh Stokes Anti-Stokes
Scattering Raman Scattering Raman Scattering between resolution and signal quality. For col-
lecting Raman spectra from skin, usually lasers
Fig. 15.12 Jablonski diagram representation of Rayleigh with wavelength of 633, 660, or 785nm are
and Raman scattering
preferred.
tored using the Phenylalanine-ring breathing vertex component analysis, which decomposes a
mode at 1004cm1 and the amide-I band at given dataset into fractions of most dissimilar
1652cm1. The results indicated that the liquid spectral information and reconstructs the image
state phospholipid P-d31OPC penetrated by plotting their individual abundances (Du etal.
4048m deep into the skin as compared to the 2008; Winter 1999). P-d31OPC penetrated to a
gel state phospholipid DPPC-d62, which was depth of ~1015m after application of flexible
found to penetrate only 1015m deep. invasomes; also it was interesting to note that the
We reported a method for recording depth penetration profile of the phospholipid was not
profiles using CRM in excised human skin biop- uniform throughout the stratum corneum.
sies to study diffusion patterns of drugs and com- Retinol is a common ingredient of the anti-
ponents of the formulation system (Ashtikar aging creams, which is highly lipophilic and does
etal. 2012). Figure15.13 shows a XZ-penetration not penetrate the skin very well. Raman micros-
profile, where every pixel represents intensity of copy is widely employed for studying penetra-
Raman signal obtained from intact human skin tion profiles of retinol as it has a characteristic
treated with deuterated invasomes (P-d31OPC). Raman peak at 1594cm1, which can easily be
The depth profile scans (XZ-scans) were col- traced through the various layers of the skin. In
lected using WITec Alpha 300R CRM.Excitation vitro penetration profile of retinol in human skin
was provided by 785nm laser and step size for biopsies was studied by Failloux etal. using
collecting the depth profiles was 1m with illu- CRM.They have reported that retinol penetrated
mination time of 0.5s/spectra. In Fig.15.13b, the the SC faster when it was applied in the form of
penetration profile of deuterated phospholipid is microspheres as compared to an oil in water
plotted as a gradient from red to yellow, where emulsion (Failloux etal. 2004). Frster etal.
the red color corresponds to the highest concen- studied the penetration of retinol from various
tration of P-d31OPC.The protein distribution of emulsions and also monitored penetration of oil
the skin is plotted in blue. Spectra corresponding and aqueous components of the emulsion by
to the invasomes and stratum corneum are repre- using D(26)-n-dodecane and deuterated water,
sented in Fig.15.13c. The penetration profile was respectively. Their results show that retinol pen-
constructed using spectral un-mixing algorithm, etration from an emulsion depends on the nature
a c
Fig. 15.13XZ-profiles recorded on a full thickness C-D signal and while-blue represents signal from endog-
human skin treated with deuterated invasomes. (a) Raman enous skin tissue. (c) The red spectra on the right are rep-
map generated by integrating C-H stretching intensities resentative of the applied P-d31OPC invasomes while blue
from 2800 to 3100cm1. (b) False color image showing spectra are representative of the skin. Peaks at 2100 and
dissimilar spectral mixing. Red color represents highest 2170cm1 represent C-D stretching vibrations
15 Confocal Microscopy forVisualization ofSkin Penetration 275
of the surfactant used and that an emulsified reti- was ~25m in the SC.Several changes were
nol can penetrate deeper in the skin, while micel- noted in the Raman shifts for C-H vibration
lar retinol is localized in the SC (Forster etal. bands associated with skin endogenous proteins
2011). Mlot etal. studied the effect of penetra- and lipids. Zhang etal. took advantage of differ-
tion enhancers to deliver retinol into human skin ences in the Raman spectra of 5-fluorouracil
invivo. The invivo measurements were carried (5-FU) and the prodrug of 5-FU (1-ethoxycarbon
out using an inverted Raman microscope custom- yl-5-fluorouracil) to determine the spatial distri-
ized for fast invivo spectral acquisition from bution and the transformation of prodrug to 5-FU
volar forearm. Formulations containing penetra- in skin (Zhang etal. 2007).
tion enhancers like oleic acid, polyethylene gly-
col showed to have improved penetration of
retinol into SC after 6 h of application time 15.5 Coherent Raman Microscopy
(Mlot etal. 2009).
Caspers etal. used CRM to study the interac- In spontaneous Raman microscopy, images are
tions between human skin and dimethyl sulfox- recorded using the mapping mode where Raman
ide (DMSO) invivo (Caspers etal. 2002). DMSO spectra from each point on the sample are col-
shows strong C-S-C symmetric and asymmetric lected. Due to the inherent weak nature of the
vibration modes at 671 and 702cm1, while the Raman scattering, acquisition of such images can
position of the microscope in the skin was deter- take several hours. This is a problem for biologi-
mined using the natural moisturizing factor to cal samples as they can undergo dehydration or
protein ratio (NMF, 885 & 1415cm1). Authors photo damage due to high exposure to the intense
reported that DMSO penetrated the SC within 20 laser radiation. Today several variants of Raman
min, however traces of DMSO could be found in microscopy are available, such as coherent anti-
the SC even after 72 h. Caspers etal. also reported Stokes Raman scattering microscopy (CARS),
the use of CRM for monitoring invivo water pro- stimulated Raman scattering microscopy (SRS),
files in the SC.Water content at various depths surface-enhanced Raman scattering (SERS), etc.,
was determined by calculating the ratio of the which have an advantage of higher S/N ratio. In
water intensities (33503550cm1) and protein this part, we would like to introduce CARS and
intensities (CH3 stretching mode 2910 SRS in brief with some examples of their appli-
2965cm1). Chrit etal. also reported application cation in transdermal delivery research, but it is
of CRM for invitro and invivo measurement of out of scope of this book to go in depth into the
water in human skin, and they were able to dem- principles and instrumentation of these tech-
onstrate the hydration efficiency of a cosmetic niques; further references can be found elsewhere
product containing hydrating polymers in micro- (Chabay etal. 1976; Cheng 2007; Eesley 1981;
capsules (Chrit etal. 2007). Nandakumar etal. 2009; Scholten and Scholten
CRM was used to follow the penetration of 1989; Scholten etal. 1989).
metronidazole dissolved in diethylene glycol CARS and SRS are nonlinear enhancement
monoethyl (Transcutol, Gattefoss, France) in techniques, which enhance the weak Raman sig-
human skin (Tfayli etal. 2007). Metronidazole nal and as a result reduce the imaging duration to
was tracked in the skin by monitoring ( C-N) a few minutes or even seconds (Hanson and
stretching vibrations at 1191 and 1369cm1, Bardeen 2009). In both techniques the molecular
while the skin proteins were tracked from the vibrations are excited by using a Stokes and
amide-I, amide-III, Phenylalanine-ring breathing pump laser beams. CARS is a complex process in
mode and other weaker spectral features. Spectra which three laser beams stokes (s), pump (p),
were collected from full thickness skin 1 and 2 h and probe (pr) interact with the sample to gener-
after the application of metronidazole solution at ate anti-stokes emission. When difference in the
different z-planes with 4m steps. Maximum s and p frequencies approach the vibrational
penetration depth achieved for metronidazole frequency of a molecular bond (v), such that
276 M.A. Ashtikar et al.
v=p s, the electrons in the electron cloud stretching mode at 2850cm1, which are mainly
around the molecular bond are in the virtual generated by the intercellular lipids and adipo-
excited vibrational state. Such vibrational excita- cytes. Figure15.14b shows the SHG signal,
tions only take place in small focal volume where which is generated mainly from the collagen
both stokes and pump beams are coherently in fibers of the dermis. Figure15.14c shows
phase. To probe the virtual excitations, a third 2-photon fluorescence detected at 435485nm,
beam (pr) is applied, which gets scattered off and Fig.15.14d shows a false color image gener-
and generates anti-stokes radiation (as) modu- ated by overlapping all the signals, where CARS
lated at difference frequency such that as = signal is represented as green, SHG as white, and
pr+(p s) (Min etal. 2011). The emitted two-photon fluorescence as red.
photon is blue shifted and as a result can be easily In vivo real-time video-rate epi-CARS (back-
separated from the incident laser beams. Due to scattered CARS) imaging was demonstrated by
the nature of the excitation source of CARS Evans etal. on anesthetized female mice (Evans
microscope, it also generates 2-photon fluores- etal. 2005). The excitation source was tuned in
cence and second harmonic generation (SHG) order to excite the CH2 stretching vibrations and
signal, and a multimodal detection system could anti-Stokes photons were detected with the help
be employed to detect these signals (Le etal. of a sensitive PMT detector. The microscope had
2007; Potma etal. 2001; Wang etal. 2008). lateral resolution of 0.3m and axial resolution
The capability to identify endogenous and of 1.5m. Authors were able to image the mouse
exogenous components by a multimodal-imaging skin at various depth up to 100m and identified
mode is demonstrated in Fig.15.14, which shows lipid rich structures such as sebaceous glands and
CARS, SHG, and 2-photon fluorescence signals adipocytes. Real time diffusion of the topically
collected from a human skin cross-section applied mineral oil was also observed in the
(Heuke etal. 2012). Figure15.14a shows the mouse skin. The mineral oil penetrated the SC
anti-stokes Raman shifts recorded for C-H within 20 min, however it remained confined to
a c
b d
Fig. 15.14 Multimodal CARS image of an untreated rescence image. (d) False color image showing overlap of
skin cross-section to demonstrate the scope of multimodal CARS (green), SHG (white), and two-photon fluores-
microscopy. (a) CARS map for CH2 symmetrical stretch- cence (red) signals
ing vibrations, (b) SHG image, and (c) two-photon fluo-
15 Confocal Microscopy forVisualization ofSkin Penetration 277
the viable epidermis and was not able to pene- and faster than ketoprofen into the skin. In addi-
trate the dermis. Zimmerley etal. used CARS tion, penetration of propylene glycol via hair fol-
microscopy to generate quantitative concentra- licle was much faster and reached a steady state
tion maps of deuterated water and d-glycine in in less than 30min compared to ~2 h via intercel-
human hair (Zimmerley etal. 2009), while lular lipid penetration in the SC to reach simi-
Breunig etal. demonstrated application of invivo lar levels. Penetration profiles of ibuprofen were
multimodal CARS microscopy in healthy and similar to that of ketoprofen. Ibuprofen crystals
diseased human skin (Breunig etal. 2012). In on the surface of the SC during penetration also
healthy human skin, they were able to follow a indicated faster penetration of the solvent pro-
topically applied emulsion cream, while in skin pylene glycol compared to ibuprofen. Freudiger
affected with psoriasis they were able to identify etal. reported application of SRS microscopy to
differences in the SC intercellular lipid domains construct three-dimensional diffusion profiles of
compared to the healthy skin. DMSO and retinoic acid in mouse skin. From the
CARS microscopy is more sensitive than spon- diffusional profiles they were able to show that
taneous Raman microscopy; however, it has some DMSO mainly penetrated the SC via the protein
drawbacks such as the presence of non-resonant phase while retinoic acid penetrated mainly via
background and more importantly anti- Stokes the lipid rich intercellular spaces (Freudiger etal.
shifts are slightly different from stokes Raman 2008).
shifts, which makes spectral interpretation more Coherent Raman microscopy is a label-free
complicated. In SRS, pump beam and stokes and more sensitive approach of tapping into the
beam coincide the sample such that difference in advantages of Raman effect compared to the
the frequencies between Stokes and pump-beams spontaneous Raman microscopy. Due to the
matches a molecular vibration. This results in an enhancement of the Raman effect, the image
increase in the rate of molecular vibrations. The acquisition is much faster and hence more suit-
energy required for this excitation is taken up able for biological samples compared to sponta-
from the pump laser field and as a result pump neous Raman microscopy. Also the nonlinear
beam experiences loss in intensity (stimulated excitation approach in the generation of CARS
Raman loss). Stokes beam experiences a gain and SRS signal is limited to a very small focal
in intensity due to the emission of stokes pho- volume, which increases the resolution of these
tons as the molecular vibrations return to ground techniques without using the confocal detection
state (stimulated Raman gain). In SRS micros- setup. CARS and SRS microscopic techniques
copy, either stimulated Raman loss or gain can be are relatively young compared to other tech-
used as a contrast mechanism (Min etal. 2011; niques discussed here. Today these microscopes
Nandakumar etal. 2009). Using SRS micros- are mainly confined to the photonics labs; how-
copy, Saar etal. were able to determine penetra- ever, both the techniques hold a great promise for
tion profiles of ibuprofen and ketoprofen from mainstream applications.
solutions in propylene glycol applied topically
onto mouse skin (Saar etal. 2011). Ketoprofen Conclusion
was tracked in the skin by following the aromatic Microscopic techniques are very important in
C-H stretching vibrations at 1599cm1, ibuprofen understanding the mechanisms involved in
and propylene glycol were deuterated and were skin penetration as they enable spatial visual-
monitored at 2120cm1, while skin lipid architec- ization of the drug in the skin. In this chapter,
ture was imaged from CH2 stretching vibrations we report almost 50 years of development in
at 2845cm1. Ketoprofen and propylene glycol the microscopic techniques and at least today,
penetrated the SC mainly via intercellular lipid no technique holds superiority in all the aspects
pathways and through hair follicles. From time of imaging such as resolution, signal quality,
resolved images at various depths, the authors specificity, speed, sample preparation, sample
showed that propylene glycol penetrated deeper stability, etc. These are rather complementary
278 M.A. Ashtikar et al.
techniques, which are able to provide a host of Bohm M, Luger TA (1998) The pilosebaceous unit is part
information according to the requirements and of the skin immune system. Dermatology
196(1):7579
objectives of the experiments. For skin pene- Bouwstra JA, Honeywell-Nguyen PL (2002) Skin struc-
tration experiments, 2-PFM was first to set ture and mode of action of vesicles. Adv Drug Deliv
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AcknowledgmentAuthors would like to acknowledge (200008/09)31:8/9<813::AID-JRS573>3.0.CO;2-7
Prof. Dr. Benjamin Dietzek and Prof. Dr. Jrgen Popp Caspers PJ, Williams AC, Carter EA, Edwards HG, Barry
from Institut fr Photonische Technologien, Jena, BW, Bruining HA etal (2002) Monitoring the penetra-
Germany, for their cooperation in acquiring multimodal tion enhancer dimethyl sulfoxide in human stratum
CARS images on the human skin cross-sections. corneum invivo by confocal Raman spectroscopy.
Pharm Res 19(10):15771580
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Clinical Cutaneous Drug Delivery
Assessment Using Single 16
andMultiphoton Microscopy
AnthonyP.Raphael andTarlW.Prow
Although single and multiphoton microsco- current invivo CLSM systems are limited with
pies have been well characterized within exvivo the two major instruments being the Optiscan
and invivo animal models, their use is yet to be Stratum, Optiscan Ltd., Australia (designed
established within clinical studies. The lack of for fluorescence imaging), and the Vivascope
clinical microscopy is due partly to ethical con- 1500 and 3000 series from Lucid Inc., USA,
siderations such as the inability to apply for eth- designed for reflectance imaging but also with
ics (studies require clinician involvement) and fluorescence capabilities (Fig.16.1). What distin-
shortage of volunteers. Even though many of the guishes these clinical microscopes from those
microscope systems being developed can be used in the laboratory for exvivo and invitro
modified for invivo work, it does not make them analysis are their small size and portability (both
clinically suitable. Investigators are required to companies have hand-held scanning systems).
use independent microscope systems one being Although the microscope size limits the room
for animal work and the other for human work. available for complex optics (i.e. the excitation
Another limitation to confocal microscopy is that and collection ability), it is expected that the con-
the techniques often require fluorescent dyes that tinual development of compact optics and lasers
when conjugated to a drug change the drugs will result in greater functionality and down-
physical and chemical properties. stream use of such CLSMs.
Even with the issues limiting the clinical anal-
ysis of percutaneous enhancers, there is much
interest in the area. With advancement in micros- 16.2.1 C
haracterization
copy and multimodal imaging techniques, it is ofPercutaneous Drug Delivery
expected that the clinical assessment of percuta-
neous drug delivery will become standard prac- Percutaneous drug delivery encompasses many
tice and important in determining the effectiveness approaches from the simple passive diffusion of
of novel percutaneous drug delivery systems. small compounds through to the active delivery
This chapter focuses on single photon and multi- of much larger macromolecules. The need for
photon approaches that have been utilized invivo enhanced percutaneous penetration has been
within human skin addressing their advantages well-established, with a range of chemical and
and limitations as potential techniques for the physical techniques being developed. CLSM has
analysis of percutaneous penetration enhancers. played and is continuing to play an important role
in validating these techniques visualizing the
delivery and distribution within the skin (Prow
16.2 Cutaneous Applications etal. 2010; Fernando etal. 2010). For example,
ofSingle Photon Microscopy in Fig.16.2 we show the difference in delivery
profiles between topical and microneedle
The most relevant technique in relation to percu- enhanced delivery of sodium fluorescein within
taneous penetration is the use of CLSM for volunteers. By averaging the signal intensity per
invitro, exvivo and invivo models. CLSM has optical section we were able to map the delivery
the ability to restrict out-of-focus light, collecting profiles in a cross-sectional manner. This is a
only fluorescence signal from a single optical powerful technique because it clearly shows the
plane. Each plane is then processed computation- influence of enhanced percutaneous delivery giv-
ally to construct three-dimensional images of the ing insight into distribution and penetration of the
fluorescent signal. In particular, CLSM is payload.
extremely useful for observing spatial properties Lademann etal. (2003) investigated the use of
of molecules within cells and/or tissues, which is a hand-held CLSM designed for dermatology
relevant to the field of pharmaceutical science applications (Optiscan Stratum, Optiscan Ltd.,
and drug delivery (Agarwal etal. 2008; White Australia) (Lademann etal. 2003). The device
and Errington 2005). However, the availability of was designed to be relatively simple and portable
16 Clinical Cutaneous Drug Delivery Assessment Using Single andMultiphoton Microscopy 285
for ease-of-use within a clinical setting. The exci- microneedles within volunteers (Mukerjee etal.
tation source consisted of an argon ion laser with 2004; Bal etal. 2010a, b). Mukerjee etal. devel-
a wavelength of 488nm specific for invivo dyes oped hollow and solid microneedles up to 200m
curcumin or fluorescein. The two dyes were in length with various diameters (Mukerjee etal.
applied to male and female volunteers in either 2004). Solid microneedles were applied to the
ethanol or sunscreen emulsion. Fluorescein skin, near the first knuckle of the thumb followed
application resulted in visualization of the differ- by application of a 0.1% sodium fluorescein
ent skin layers stratum corneum, stratum granu- solution. The application of the sodium fluores-
losum, stratum basale and although of low cein solution without microneedles resulted in no
resolution, the papillary dermis (Fig.16.3). dye solution penetrating through the stratum cor-
Lademann etal. hypothesized that using their neum. CLSM assessment of microneedle appli-
CLSM system in conjunction with the fluores- cation resulted in the visualization of successful
cein/ethanol solution, different skin layers could microneedle penetration to a depth of 160m.
be distinguished between a healthy or diseased Although no investigation was done on dye dis-
state (Lademann etal. 2003). tribution and diffusion within the skin, the study
The curcumin/sunscreen emulsion was used showed that the application of Optiscan
to investigate potential penetration pathways and Stratum was a suitable technique for investi-
distribution of sunscreens within the skin. It was gating the microneedle enhanced delivery of
determined that the sunscreens accumulated fluorescein.
around the edges of the corneocytes. Initially In 2010, Bal etal. conducted a more in-depth
only the skin surface could be detected, however microneedle study in volunteers using the delivery
over time (20min) three cell layers of the stratum of fluorescein as a visual guide for characterizing
corneum became visible. Additionally, the cur- the microneedle conduits and their closure over
cumin solution accumulated around the hair fol- time (Bal etal. 2010a). Bal etal. also used the
licles and within the sweat glands. Interestingly, Optiscan Stratum. The microneedle array con-
Lademann etal. were able to distinguish between sisted of 5 microneedles, 300m in length, 120m
active and passive sweat glands based on the dis- in diameter and a spacing of 2mm. Bal etal. inves-
tribution of the dye in or around the gland tigated the two techniques of fluorescein
(Lademann etal. 2003). This study demonstrated microneedle application by either applying the dye
that hand-held CLSMs have the potential for to the skin prior to or after microneedle adminis-
non-invasive clinical imaging. tration. After each application CLSM was used to
The Optiscan Stratum has also been used as characterize the microneedle conduit. Dye appli-
a validation tool assessing the penetration of cation post microneedle piercing resulted in
286 A.P. Raphael and T.W. Prow
Fig. 16.2Images
showing the invivo
human delivery of sodium
fluorescein (NaF) with
and without microneedles
(MN) using dermoscopy
(top panels) and confocal
laser scanning microscopy
(middle panels). The
bottom panels are a
cross-sectional heat map
representation of the
average delivery profile
(represented by rectangu-
lar box). Arrow indicates a
microneedle deposition
site
significantly higher signal than dye application However, TEWL measurements can be influ-
prior to microneedles. Bal etal. concluded that the enced by internal and external factors such as
conduits closed within 1015min after micronee- humidity, temperature and air convection.
dle application and that a depth of 150m was Additionally, TEWL is not reliable post drug
achieved (suitable for macromolecules to reach the application where the drug or chemical enhancer
viable epidermis (Fig.16.4a, b)). solution is still on the skins surface. Vergou etal.
More recently, Vergou etal. compared the use assessed the barrier integrity prior to and post
of transepidermal water loss (TEWL) and CLSM treatment of dry skin (without disease) with gel
for the invivo characterization of the stratum and oil products (Valeo Holundergel and Valeo
corneum barrier in volunteers (Vergou etal. Dehnungspflegeoel, Lesttic GmbH, Germany)
2012). Once again, this study was done using the designed to rehydrate the skin following radio-
Optiscan Stratum. TEWL is a relatively stan- therapy treatment (Vergou etal. 2012). The gel
dard technique used to characterize the integrity was designed to penetrate the skin increasing
of the epidermal barrier following disruption hydration followed by the oil which formed a
with percutaneous penetration enhancers. protective layer on the skins surface to maintain
16 Clinical Cutaneous Drug Delivery Assessment Using Single andMultiphoton Microscopy 287
Fig. 16.3 Confocal laser scanning microscopy of sodium basale, and (d) papillary dermis (Adapted from Lademann
fluorescein within the different layers of volunteer skin: etal. (2003))
(a) stratum corneum, (b) stratum granulosum, (c) stratum
hydration. The products were applied twice result in information relating to drug distribu-
daily on the volar forearm over a 4-week period. tion within skin and insight into their mecha-
No treatment was done to the other arm. nism of action. A benefit of using the Optiscan
Assessment was done prior, 2 weeks after and 4 Stratum in relation to the investigation of per-
weeks after application. The initial TEWL values cutaneous enhanced penetration is that tissue
were standardized to 100% between volunteers and cellular disruption can be visualized.
and it was determined that for all volunteers the Understanding the change in skin integrity is
TEWL for untreated skin remained constant important for characterizing the chemical and/
(9917%). The TEWL reading for the treated or physical methods used to deliver drugs.
arm increased by 2127% suggesting disruption However, the limitation of the current technique
of the epidermal barrier. However, stratum cor- is that a fluorescent solution is required to visu-
neum hydration and elasticity experiments resulted alize the skin layers (in the previous cases,
in an average increase of 20% and 10%, respec- sodium fluorescein). Depending on the depth
tively, indicating improved integrity of the epider- required, the fluorescent solution is injected into
mal barrier. CLSM showed a similar trend to the the skin or topically applied, potentially influenc-
hydration and elasticity studies, where a distinct ing the drug of interest.
improvement in the integrity of the stratum cor-
neum was observed. Prior to treatment the corneo-
cytes showed an irregular mountainous structure. 16.2.2 Reflectance Microscopy
After the 4-week treatment, the corneocytes fortheAssessment
formed a flat honey-comb pattern surrounded by ofTherapeutic Changes
intact lipid layers. Vergou etal. hypothesized that intheSkin
even though TEWL is non-invasive, when placed
on the skin surface it disrupted the protective oil Reflectance confocal microscopy (RCM) is an
layer resulting from the treatment. Accumulated alternate approach to fluorescence-based CLSMs.
water from the gel treatment was then detected by RCM can especially be useful to characterize
TEWL (Vergou etal. 2012). This resulted in higher skin integrity and disruption as well as the change
TEWL readings, which inferred a greater disrup- in disease state of the skin following therapeutic
tion of the stratum corneum, when in reality visual treatment (Ardigo etal. 2010; Segura etal. 2011;
microscopic inspection showed that the stratum Ulrich etal. 2009). Various studies have been
corneum was intact. published characterizing the morphological
Overall Vergou etal. and others have shown changes seen in the skin over time, including
the utility and importance of visual methods studies looking at wound healing.
when characterizing topically applied formula- RCM utilizes a near-infrared diode laser as a
tions invivo. The application and instrumenta- monochromatic and coherent light source. The
tion of the described invivo fluorescence light source is reflected (backscattered) off the
microscopes are relatively simple to use and sample and passes through a pinhole aperture to
288 A.P. Raphael and T.W. Prow
Fig. 16.4 Microneedle-enhanced delivery of sodium flu- surface). (b) Comparison maximum depth of dye diffu-
orescein in volunteers. (a) Confocal laser scanning sion over time between sodium fluorescein application
microscopy of microneedle delivery 5, 10 and 15min post before or after microneedle insertion (Adapted from Bal
application at two depths (surface and 20m below the etal. (2010a))
the detector. RCM results in quasi-histological structures. RCM can be used for assessing
resolution because backscattering occurs at the structural changes within the skin in addition to
edges of two biological materials with different serial imaging of the same site over time (Wurm
refraction indexes thus distinguishing cellular etal. 2012). Furthermore, the use of near-infrared
16 Clinical Cutaneous Drug Delivery Assessment Using Single andMultiphoton Microscopy 289
laser sources results in relatively deep imaging a 830nm diode laser and a 30 water immersion
down to the dermo-epidermal junction. The high lens facilitating a horizontal optical resolution of
cellular and structural resolution images obtained 2m and vertical resolution of 5m. It was deter-
from RCM make it a valuable tool when assess- mined that RCM analysis was suitable for charac-
ing percutaneous penetration enhancers. RCM terizing whether lesions underwent partial or
has the potential of providing information on the complete remission. In these cases not all treated
change in skin integrity post chemical and/or lesions underwent complete remission. However,
physical enhancement and unlike the previous those that did were characterized by the presence
fluorescence-based technique described it does of normal dermal papilla and collagen fibres
not require a dye solution. (Fig.16.6).
RCM is also used as a qualitative and quantita- Torres etal. published a similar study treating
tive technique where differences can be observed basale cell carcinomas (BCCs) with 5% imiqui-
relating to the cellular and extracellular structures mod (Torres etal. 2004). Patients applied imiqui-
within skin. For example, we have investigated in mod 5 times daily for 2, 4 or 6 weeks, with RCM
the Prow group, the differences in skin from two analysis at the 6-week follow-up. Torres etal. also
age groups, 2030 years and 5060 years, respec- used a Lucid Inc., USA, based RCM system
tively, using RCM (Fig.16.5). The RCM images designed for invivo investigations. Whereas
in Fig.16.5bd, fh correspond to three distinct Segura etal. characterized the BCCs based on the
skin layers of interest (stratum corneum, stratum skin morphology within the dermo-epidermal
spinosum and dermo-epidermal junction). We junction and dermis, Torres etal. focused on the
have reported in detail and scored the RCM fea- keratinocyte structure and cytoplasm-to-nucleus
tures associated with photo-ageing on the forearm ratio within the epidermis (Torres etal. 2004).
of the two age groups (Wurm etal. 2012). We BCCs were characterized as having elongated
concluded that 15 statistically significant RCM keratinocytes with a high cytoplasm-to-nucleus
features, such as furrow width/shape, keratinocyte ratio compared to normal skin, which consisted of
irregularity/disarray and dermal papillae mor- a lower ratio and regular honey-comb pattern of
phology could be used to quantify photo-ageing the cells (Fig.16.7). Even though in this study,
in forearm skin (Wurm etal. 2012). RCM was seen as a useful technique evaluating
One particular area that has seen much atten- tumour regression invivo without the need of sur-
tion using RCM is the characterization and pre- gery, there were still some limitations. The inves-
and post-topical treatment of pre-cancerous and tigators felt that the technique was time-consuming
cancerous skin lesions (actinic keratosis [AK], (although far quicker than an invasive surgery
basale cell carcinoma [BCC], squamous cell car- approach). There were also technical difficulties
cinoma [SCC] and melanoma). Segura etal. due to movement of the relatively large scanning
investigated the efficacy of photodynamic therapy head, skin contact angle and maintaining contact
(PDT) using methyl-aminolevulinate (MAL) on with the skin during imaging. These limitations
the treatment of BCCs (Segura etal. 2011). Six can be overcome with practice, however it does
patients were treated with 13 cycles of MAL- prevent implantation of such invivo systems
PDT with each cycle consisting of two sessions of especially in laboratories that are familiar primar-
MAL-PDT 7 days apart. To enhance MAL pene- ily with invitro and exvivo investigations.
tration into the skin, debridement of the BCC sur- However, it should be noted that the RCM used
face was done with a curette followed by a 3h was a much earlier model available from Lucid
application of MAL.The patients were assessed 1 Inc. (Vivascope 1000) and since then Lucid Inc.
week and 3 months post treatment using RCM have developed small hand-held systems with
with consecutive follow-ups (without RCM) greater ease-of-use and flexibility (Fig.16.1).
every 6 months for 3 years. The RCM used was a In summary, CLSM systems are increasing in
commercially available instrument designed spe- their use as non-invasive techniques for percuta-
cifically for invivo applications (Vivascope neous drug delivery. Although fluorescence-
1500, Lucid Inc., USA). The system consists of based systems are limited mainly to invitro and
290 A.P. Raphael and T.W. Prow
Fig. 16.5 Clinical photographs and their corresponding RCM images of volunteers (a) and (e), respectively. (b)
reflectance confocal microscopy (RCM) images. (a) and and (f) stratum corneum, (c) and (g) stratum spinosum,
(e) show representative clinical photographs of volunteers (d) and (h) dermo-epidermal junction (Prow, Raphael
from the two age groups, 2030 years and 5060 years, and AR Soyer, unpublished 2012)
respectively. (bd) and (fh) show the corresponding
exvivo applications, RCM is being utilized clini- MPM can additionally be used for three-photon
cally much more due to its ability to resolve cel- excitation as well as second and third harmonic
lular and structural morphology within the skin generation of biological structures. These fea-
pre and post drug application. With an increase in tures, combined with the fact that MPM can
biocompatible fluorophores, user-friendly equip- result in single photon sensitivity with submicron
ment and cost-effective instruments, it is expected resolution has led it to being an important micro-
that invivo CLSM systems will continue to be scopic technique with the highest resolution in
incorporated into pharmaceutical and percutane- the area of non-invasive clinical imaging
ous research. (Fig.16.8) (Konig etal. 2006; JenLab GmbH).
Two-photon excitation works on the theoretical
principal developed by Maria Gppert-Mayer,
16.3 A
pplications ofMultiphoton where two photons of similar energy interact with a
Microscopy molecule (fluorophore) resulting in excitation
equivalent to the absorption of a single photon with
Multiphoton microscopy (MPM) is a deep tissue twice the energy (Fig.16.9) (Zipfel etal. 2003;
imaging technique that is predominantly used for Prow 2012). The probability of successful two-
non-linear two-photon excitation of fluorophores. photon interaction (absorption) can be measured
16 Clinical Cutaneous Drug Delivery Assessment Using Single andMultiphoton Microscopy 291
Fig. 16.6Clinical,
dermoscopy and RCM a d
images of a 4mm
pigmented basal cell
carcinoma on the abdomen
of a 25-year-old woman. (a)
Clinical and dermoscopy
(inset) images of the lesion
before photodynamic
therapy. (b) A 44mm
reflectance confocal
microscopy image (RCM)
of the lesion prior to d e
treatment. (c) Magnified
image (0.50.5mm) of the
lesion at the dermo-
epidermal junction. (d)
Clinical and dermoscopy
(inset) images of the lesion
after two cycles of
photodynamic therapy. (e)
A 44mm RCM image of
the lesion after treatment
showing reduction in
tumour size. (f) Magnified
c f
image (0.50.5mm) of the
lesion after treatment at the
dermo-epidermal junction
showing the presence of
normal dermal papilla
(Adapted from Segura etal.
(2011))
quantitatively by the two-photon cross- section tracers for analysis of percutaneous penetration
(2p). The two-photon cross-section is referred to as with multiphoton microscopy it is important to
Gppert-Mayer (GM) where 1 GM is equivalent to have an understanding of F 2p.
1050 cm4s. Instead of measuring 2p, which is quite For successful excitation to occur, the photons
complex, the product of the fluorescence quantum need to interact with the molecule almost simul-
yield (F) and absolute two-photon cross-section is taneously (approx. 1016 s). Therefore, the prob-
calculated, which is the two-photon action cross- ability of successful two-photon excitation is
section (F 2p) (Zipfel etal. 2003). To determine greatest within the beam area within the focal
the optimal wavelength for two-photon excitation, plane. Outside of the focal plane the probability
it is useful to have an understanding of the change drops significantly with negligible fluorescence
in F 2p values with wavelength (two-photon exci- emission. To further increase the probability of
tation spectra). This is because a molecules maxi- two-photon excitation and generate sufficient
mum single photon excitation does not always fluorescence for imaging, high laser powers are
correspond to two photon excitation with similar required. However, high laser powers are detri-
energy (in particular symmetrical and intrinsic mental to biological samples. To address this
molecules). Therefore, when selecting fluorescent problem, MPMs utilize a pulsed laser (most
292 A.P. Raphael and T.W. Prow
Fig. 16.7Reflectance confocal microscopy (RCM) to-nucleus ratio (nucleus low signal, cytoplasm high sig-
images of a basal cell carcinoma (BCC) before and after nal). (b) BCC morphology 2 weeks after treatment showing
treatment with imiquimod. (a) BCC morphology prior to regular honey-comb pattern and normal cytoplasm- to-
treatment showing elongated cells with a high cytoplasm- nucleus ratio (Adapted from Torres etal. (2004))
Single photon
Tunable titanium Scanning
fluorescence
fluorescence
Two-photon
sapphire laser galvanometers
Selectable
detectors PMT
Second harmonic
Dichroic
Energy transfer
mirror
upconversion
generation
Objective
Specimen
Fig. 16.9 Simplified schematic of the optical layout for excitation beam to the sample. The emission is collected
multiphoton microscopy and energy transfer diagrams. and passed to one or more detectors. Photomultiplier tubes
Tuneable 80MHz titanium sapphire laser sources are most and time-correlated single-photon counting detectors can
commonly used for MPM.The light source is then raster be used depending on application. Single, two-photon and
scanned over the imaging area using scanning galvanom- second-harmonic generation Jablonksi diagrams are
eters. The dichroic mirror and objective lens then pass the showed on the right (Reprinted from Prow (2012))
visualize individual nanoparticles) and ability to etal. applied fluorescein or 300nm fluorescein
distinguish nanoparticles extra/intra- cellularly embedded nanoparticles to the surface of the skin
makes it a powerful technique in relation to (Dromard etal. 2007). The nanoparticles accu-
nanoparticle cell co-localization (Prow etal. mulated preferentially around the edges of the
2008). However, for samples to be imaged using corneocytes defining their boundaries. This
TEM, they must be at a thickness that allows the approach provided a relatively simple and quick
electrons to transmit through the sample. This is a analysis of nanoparticle interaction with the sur-
time-consuming highly technical process that face of the skin.
requires sectioning and embedding of the sample MPM is one particular technique that is show-
within resin prior to imaging. Additionally, TEM is ing great promise as a tool for defining the risk of
an exvivo microscopy technique and therefore nanoparticle exposure (Fig.16.10). In particular,
inherent to the limitations of using an exvivo MPM results in high-resolution deep tissue imag-
model system to describe what is occurring invivo. ing within biological tissues (ex vivo or invivo)
Single photon microscopy is another tech- with the ability to separate nanoparticle signals
nique that has been used for nanoparticle imag- from endogenous fluorophores (although MPM
ing both exvivo and invivo (Prow etal. 2006, is not able to resolve individual nanoparticles)
2008, 2012). As discussed earlier, the major (Fig.16.10) (Prow 2012; Labouta etal. 2011; Liu
advantage of this technique is its ease of use and etal. 2012). Furthermore, when coupled with
availability of relatively inexpensive equipment. fluorescence lifetime imaging (FLIM) and time-
However, for the nanoparticles to be detected correlated single photon counting (TCSPC)
they need to be intrinsically fluorescent or be detectors, MPM systems have the capability of
fluorescently labelled. distinguishing between the simultaneous excita-
Dromard etal. conducted a study investigating tion of nanoparticles and endogenous fluores-
the use of invivo corneocyte imaging using a cence within biological tissues without the need
customized fibered confocal fluorescence micro- of fluorescent dyes (not possible with other
scope technique (Dromard etal. 2007). Briefly, techniques).
an optical fibre bundle was attached to a fluores- We utilized the advantages of MPM-FLIM to
cence microscope set-up and used to image the investigate the use of TCSPC for simultaneous
surface of the skin. Although not assessing the monitoring of zinc oxide nanoparticles and the
delivery of nanoparticles to the skin, Dromard metabolic state of volunteer skin (without the use
294 A.P. Raphael and T.W. Prow
Fig. 16.10 En face multiphoton microscopy fluorescence yellow) (Adapted from Liu etal. (2012)) The samples
lifetime imaging (MPM-FLIM) showing the layers of consisted of a (a-d) saline control, (e-h) sodium citrate
freshly excised human skin after 24h treatment with gold control, (i-l) 10 nm gold nanoparticle treated skin, (m-p)
nanoparticles. Nanoparticles are pseudocoloured based on 30 nm gold nanoparticle treated skin and, (q-t) 60 nm
signal 95100% (orange to red), skin autofluorescence is gold nanoparticle treated skin.
pseudoloured based on signal 095% (blue/green to
16 Clinical Cutaneous Drug Delivery Assessment Using Single andMultiphoton Microscopy 295
of fluorescent dyes) (Lin etal. 2011) (Fig.16.11). Overall MPM provides an invivo microscopy
The study consisted of a single healthy volunteer technique that is well suited for the high resolution
and eight others with psoriasis or atopic dermatitis and deep imaging or nanoparticles within the skin.
(five with active psoriasis lesions and three with The majority of the studies using these systems
atopic dermatitis). Zinc oxide nanoparticles were within humans have focused on zinc oxide and tita-
applied to the forearm of healthy, tape-stripped nium dioxide nanoparticles. This is because these
and lesional skin. The application time for the nanoparticles are clinically relevant being utilized
healthy and tape-stripped skin was 4 and 24h, but in numerous daily lotions such as sunscreens and
for the lesional skin only 2h. In vivo imaging was cosmetics. It is expected that with the continual
done using a DermaInspect MPM (JenLab increase in nanoparticles as a percutaneous deliv-
GmbH, Germany) with a pulse mode- locked ery enhancer, MPM will play an important role in
80MHz titanium sapphire MaiTai laser. The assessing nanoparticle penetration and distribution
excitation wavelength was 740nm and the emis- within skin potentially providing information aid-
sion was filtered through a band pass filter (350 ing in their clinical approval and use.
450nm) prior to the TCSPC 830 FLIM detector.
The multiphoton-excited photoluminescence
(derived from the fluorescence lifetime amplitude) 16.3.2 A
nalysing theSkins Metabolic
was used to separate the zinc oxide nanoparticle Response toDrugs Using
signal from the endogenous nicotinamide adenine Multiphoton Microscopy
nucleotide (NADH), also expressed as NAD(P)H Fluorescence Lifetime Imaging
signal. The microscopy set up clearly showed the
zinc oxide nanoparticle distribution within the dif- The combination of MPM with FLIM provides an
ferent skin types and layers (Fig.16.11). invivo microscopy technique which could poten-
Roberts etal. used a similar MPM set up ana- tially provide information on the progression of
lysing xenobiotic transport invivo (fluorescein disease and metabolic changes within skin post
and zinc oxide nanoparticles within rat liver and drug administration (Prow 2012; Labouta etal.
human skin) (Roberts etal. 2008). It was also 2011; Liu etal. 2012; Lin etal. 2011). MPM itself
determined that zinc oxide nanoparticles do not is useful because it provides detailed resolution on
penetrate the skin and this was further verified the morphological changes of the cells (in particu-
using scanning electron microscopy coupled with lar keratinocytes) and also any changes to the col-
energy-dispersive x-ray (SEM-EDX) of treated lagen network. However, one endogenous
exvivo human skin. Zvyagin etal. resulted in fluorophore of particular interest is NAD(P)H (i.e.
similar conclusions using MPM and SEM-EDX NADH and NADPH). NAD(P)H is localized
of both zinc oxide and titanium dioxide nanopar- within living cells, either unbound in the cytoplasm
ticle penetration in exvivo and invivo human or bound to mitochondrial enzymes, and plays an
skin (Zvyagin etal. 2008). important role in cellular metabolism (Sanchez
Knig etal. conducted a study using MPM to etal. 2010). Depending on the cellular microenvi-
investigate the delivery and distribution of ronment (i.e. cellular metabolism), the NAD(P)H
poly(lactic-co-glycolic acid) (PLGA) nanoparti- fluorescence lifetime changes, which can be
cles (approx. diameter of 200nm) loaded with the detected by FLIM.Simply put, an increase in oxi-
antirheumatic drug flufenamic acid (Konig etal. dation within the skin may result in an increase in
2006). The drug was specifically chosen because unbound NAD(P)H and a decrease in bound as
it fluoresces at 420nm. The excitation wavelength well as their corresponding lifetimes (unbound has
was tuned to 720nm to distinguish between the a short lifetime and bound has a longer lifetime).
loaded nanoparticles compared to the unloaded As discussed in the previous section, we used
controls. It was determined that like the zinc oxide MPM-FLIM for simultaneous monitoring of zinc
and titanium dioxide nanoparticles the PLGA oxide nanoparticles and the metabolic state of vol-
nanoparticles remained on the surface of the skin. unteer skin (Lin etal. 2011). It was determined that
296 A.P. Raphael and T.W. Prow
Fig. 16.11In vivo multiphoton microscopy (MPM) signal 90100% (green to red), skin autofluorescence is
images of intact and tape-stripped skin at different layers pseudoloured based on signal 085% (blue). Grayscale
within the skin after 24h treatment with zinc oxide insets show overall intensity only (Reprinted from Lin
nanoparticles. Nanoparticles are pseudoloured based on etal. (2011))
the free NAD(P)H signal increased significantly in lesional skin (Fig.16.12). This corresponded to
tape-stripped skin treated for 4h with zinc oxide what was reported by Knig etal. where the appli-
nanoparticles compared to the control. However, cation of FLIM was found to be technically chal-
no significant changes were detected for the lenging in thicker stratum corneum (i.e. lesional
16 Clinical Cutaneous Drug Delivery Assessment Using Single andMultiphoton Microscopy 297
Fig. 16.12In vivo multiphoton microscopy (MPM) signal 90100% (green to red), skin autofluorescence is
images of intact and tape-stripped skin at different layers pseudoloured based on signal 085% (blue) (Reprinted
within the skin after 2h treatment with zinc oxide from Lin etal. (2011))
nanoparticles. Nanoparticles are pseudoloured based on
areas) (Knig etal. 2011). However, Knig etal. skin compared to healthy skin. The lesional coeffi-
observed a greater variance in the amplitude of cient of variation was largest in the stratum spino-
bound and unbound NAD(P)H lifetimes in lesional sum, being 8.1 times greater than non-lesional
298 A.P. Raphael and T.W. Prow
areas. It is proposed that these differences in FLIM obtained by measuring the amplitude and the
output, combined with the high resolution visual echo time delay of backscattered light. Knig
images from MPM, can be used to assess therapeu- etal. used two OCT systems (EX1301,
tic or toxic affects following topical exposure. Michelson Diagnostics, UK and HSL-2000-10
MDL, Santec Corp., Japan) in conjunction with
the DermaInspect MPM (JenLab GmbH,
16.4 A
dvancements inNon- Germany) (Konig etal. 2009). To image the same
invasive Analysis ofDrug area of skin between instruments, a custom metal
Delivery totheSkin Using O-ring and custom glass coverslip was developed
Multimodal Imaging to fit into the MPM, OCT and dermoscope
(Fig.16.13ad). The locator was reversibly fixed
Non-invasive multimodal imaging is the combi- to the skin using double-sided adhesive tape. The
nation of two or more imaging techniques used OCT provided fast low resolution wide-field
within a single examination. The origins of mul- cross-sectional images 52mm2 in size. This
timodal imaging can be largely attributed to the was a contrast to the high cellular resolution of
development of combined nuclear medicine, pos- the MPM images. However, MPM has a small
itron emission tomography and magnetic reso- acquisition volume approximately
nance imaging. These multimodal instruments 0.30.30.2 mm3. It is their differences that
were developed due to the need for more accurate make the two techniques complimentary, where
and localized imaging of drugcell/tissue interac- OCT provides a wide-field image within a lesion
tion in relation to neurology and oncology, in showing potential areas of interest that can be
particular the longitudinal assessment of tumour further analysed at a much higher resolution
progression/regression after treatment. using MPM.Knig etal. investigated three dif-
There is much interest in the areas of pharma- ferent skin states being hemangioma, Pemphigus
ceutical science and percutaneous drug delivery vulgaris and melanocytic lesions using the com-
for the incorporation of multimodal imaging, bined MPM-OCT systems. It was observed that
resulting in the development and introduction of OCT was capable of identifying features of inter-
various multimodal invivo skin imaging tech- est that when examined by MPM was shown to
niques (Konig etal. 2009, 2010; Graf and Boppart be significant. For example, optical biopsies of
2012; Zhang etal. 2011). One common technique Pemphigus vulgaris resulted in regions of low
as discussed in the previous section is the use of signal or black cavities within the skin using
MPM-FLIM.The significance of MPM and FLIM OCT (Fig.16.13e). These cavities (blisters) were
is that the same area of skin can be imaged with also observed in the MPM images showing
MPM and analysed by FLIM concurrently provid- regions absent of cells and signal (Fig.16.13f).
ing information-specific drugcell/tissue interac- The study by Knig etal. showed the signifi-
tions. However, a major challenge when combining cance of combining MPM and OCT analysis
different imaging techniques for skin analysis is (Konig etal. 2009). However, the two systems
that each instrument often has a different field-of- were independent being two different instru-
view and optical sectioning ability. Additionally, ments, limiting its utility and ease of use. Graf
the region-of-interest within skin studies is often and Boppart addressed this issue by integrating
quite small (being limited to the size of the objec- the two systems into a single instrument (Graf
tive lens) making it difficult to image the exact and Boppart 2012). In addition, Graf and Boppart
same area of skin with different techniques. utilized optical coherence microscopy, which is a
Although not technically multimodal, Knig high resolution variation of OCT.The initial
etal. investigated the use of sequential optical challenge faced when integrating the two systems
coherence tomography (OCT) and MPM (Konig was choice of laser. MPM most commonly uti-
etal. 2009). OCT is a reflectance-based tech- lizes a wave-length tuneable titanium sapphire
nique where optically sectioned images are laser with a narrow 1020nm optical bandwidth.
16 Clinical Cutaneous Drug Delivery Assessment Using Single andMultiphoton Microscopy 299
Fig. 16.13 Images of interface for alignment of dermo- with locator markings. (e) OCT image showing subcor-
scope, multiphoton microscopy (MPM) and optical coher- neal (*) and subepidermal (arrow) blistering, and (f) cor-
ence tomography (OCT) and representative images from responding MPM optical section showing subcorneal
MPM and OCT.Interface consisting of (a) cover lens, (b) blistering (Adapted from Knig etal. (2009))
double-sided adhesive, (c) metal O-ring, and (d) coverslip
This is in contrast to OCM, which utilizes a broad contrast providing complimentary information
optical bandwidth (100200nm) laser source. about the state of the skin.
Graf and Boppart achieved excitation using a The combination of MPM and OCT/OCM
commercially available titanium sapphire dual provided a novel imaging modality assessing the
spectrum laser source based on supercontinuum cellular and extracellular structure within skin.
generation. The laser was integrated into the The multimodal MPM-OCM system benefited
MPM-OCM system and described in detail by from the high-resolution capabilities of MPM in
Graf and Boppart (2012). combination with the wide-field imaging of
The next challenge was that MPM requires a OCM.Such MPM-based multimodal imaging
high numerical aperture (NA) lens compared to techniques have potential clinical cutaneous drug
OCM which relies on the coherence length and delivery applications, in particular the ability to
not the NA.By separating the laser source into resolve cellular and structural morphology within
two beams (one for MPM and the other for OCM) the skin pre and post nanoparticle drug applica-
Graf and Boppart were able to independently tion. The continual development of multimodal
control the effective NA for both the MPM and systems has the potential to provide novel invivo
OCM while maintaining a single objective lens analytical techniques resulting in information on
(Graf and Boppart 2012). Finally the reconstruc- drug distribution and interaction within cells and
tion of the collected OCM data had to be repro- tissues.
cessed to compensate for the coherence gate
curvature so as to utilize the full field of view and Conclusion
integrate with the MPM images. To validate the This chapter introduces and discusses the
MPM-OCM system, multiple focal areas from major imaging techniques used for the non-
the dorsal hand of a human volunteer was imaged. invasive invivo analysis of percutaneous drug
The system resulted in high cellular resolution delivery. Single photon microscopy tech-
within the skin attributed to the MPM in addition niques to characterize percutaneous drug
to wide area mosaics 11mm in size (Graf and delivery have been well established within
Boppart 2012). The combined system provides invitro, exvivo and invivo animal models.
data on both scattering and fluorescence-based With non-invasive clinical analysis of drug
300 A.P. Raphael and T.W. Prow
distribution and diffusion being a major goal functional changes within the skin following
in the area of percutaneous drug delivery, cutaneous drug delivery. For example, investi-
CLSM systems are being developed to address gations with MPM-OCT-based systems have
this need, resulting in small portable fluores- benefited from MPMs resolution and OCTs
cence and reflectance microscopes for clinical wide-field and imaging depth providing detailed
use. Fluorescence CLSMs have the potential information on structural abnormalities within
to provide information on drug pathways and diseased skin. With advances in optics and
skin barrier integrity post percutaneous lasers it is expected that the range of diverse
enhancement. Not being restricted to fluores- multimodal systems will increase providing a
cence, reflectance CLSM utilizes backscatter- powerful tool set for the invivo analysis of
ing of cellular structure within the skin to percutaneous drug delivery.
obtain structural images of tissue. RCM has Overall, visualization of percutaneous
been shown to be a clinically important tech- delivery is a significant experimental tech-
nique being able to resolve structural nique providing clear information on drug dis-
changes within skin before and after cutane- tribution and optimal delivery strategies.
ous drug delivery. The ability to assess the Downstream computational image analysis
change in cellular morphology during treat- extends the qualitative visualization of drug
ment is an important tool, providing insight delivery to a semi-quantitative and quantita-
into drug/enhancer efficacy and the mecha- tive analytical method resulting in information
nism of action within the skin. on drug trafficking, concentration and diffu-
An alternate technique that has shown sivity within tissues all important factors in
much promise in the area of non-invasive understanding percutaneous delivery of drugs.
invivo analysis is the use of multiphoton
microscopy (MPM) for deep tissue imaging.
A significant feature of MPM is that it can be References
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Additionally, MPM results in single photon research. Curr Drug Targets 9:895898
Ardigo M, Cameli N, Berardesca E, Gonzalez S (2010)
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it one of the highest resolution techniques in tion and response to therapy in melasma using invivo
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Corneoxenometry: ABioassay
Exploring Skin Barrier Breaching 17
ClaudinePirard-Franchimont,
TrinhHermanns-L, andGraldE.Pirard
Contents
17.1 Introduction
17.1 Introduction 303
17.2 SPBF Modulation and One of the outmost functions of the epidermis is
Corneoxenometry 304 to guarantee a continuous permeability barrier
17.3 Dose-Response Corneoxenometry preventing the body from the ingress of poten-
withChemical Penetration Enhancers 305 tially noxious xenobiotics. The toxic, irritant and
17.4 Corneoxenometry andOrganic caustic compounds are various. The skin perme-
Solvents 305 ability barrier function (SPBF) is therefore essen-
Conclusion 306 tial for maintaining a regulated and constant
internal milieu. In recent decades, much research
References 306
was undertaken to modulate or keep intact the
SPBF (Notman etal. 2013) which is located in
the stratum corneum (SC). The concerns are mul-
tifaceted. On the one hand, some formulations
are designed for protecting or restoring the SPBF
(Xhauflaire-Uhoda etal. 2008a). On the other
hand, chemical penetration enhancers, also
named absorption enhancers or accelerants, are
offered for overcoming the genuine SPBF in
order to increase specific drug penetration
through the SC.In fact, penetration enhancers act
in a number of distinct ways to induce a tempo-
rary and reversible failure in the SPBF (Woodford
and Barry 1986; Hadgraft and Walters 1994;
Keerthi etal. 2012; Seto etal. 2012). Some of
these compounds alter or disrupt the epidermal
lipids in their solubility properties and ordered
C. Pirard-Franchimont T. Hermanns-L structure (Notman etal. 2013). Other penetration
G.E. Pirard, MD, PhD (*) enhancers impede the corneocytes cohesiveness
Laboratory of Skin Bioengineering and Imaging, and the tidy SC structure.
Lige University Hospital, CHU Sart Tilman,
BE-4000, Lige, Belgium The desirable attributes for penetration enhanc-
e-mail: Gerald.Pierard@ulg.ac.be ers are varied (Woodford and Barry 1986;
Hadgraft and Walters 1994). The compounds with confidence on most invitro models using
must be pharmacologically inert without any reconstructed epidermis or whole skin. Usage of
activity at cell receptor sites. In addition, the pen- excised human skin is subject to ethical concerns
etration enhancer must be compatible, both chem- and necessitates both a surgical setting and an
ically and physically, with the drugs and vehicles experienced laboratory. Skin of animals is often
in the relevant dosages. Its onset of action has to irrelevant due to prominent interspecies
be rapid with a predictable duration of activity. differences.
The effects are expected to be completely and rap- In vivo testing with penetration enhancers was
idly reversible upon removal of the material or claimed to be performed safely by some investi-
formulation from the skin. Furthermore, the gators in contrast to others who reported severe
effects should ideally be unidirectional, allowing cell damage in the epidermis and even skin
only the ingress of specific xenobiotics without necrosis (Lavrijsen etal. 1994). Such potential
any loss of endogenous compounds from the hazards call for exvivo predictive bioassays on
internal tissues. Penetration enhancers should be human SC (Abrams etal. 1993; Goffin etal.
cosmetically acceptable, odorless, inexpensive, 1997a; Welss etal. 2004; Kandrov etal. 2009;
tasteless, colorless and spreading smoothly over Macfarlane etal. 2009; Engelbrecht etal. 2012;
the skin with a suitable feel. The risk for irrita- Kojima etal. 2012; Ochalek etal. 2012). This
tion, allergy and systemic toxicity must be mini- chapter focuses on the value of corneoxenometry
mal or absent. Despite the diversity of penetration and corneosurfametry in predicting the value of
enhancers, none of them combines all of the desir- chemical penetration enhancers. The corneoxen-
able above-mentioned attributes. ometry bioassay named after corneocyte, xenobi-
Some penetration enhancers are chemicals spe- otic and metry was introduced as a convenient
cifically designed for this purpose. An example is and simple approach to explore the effect of some
given by the 1-dodecylazacycloheptan-2-one (lau- xenobiotics on human SC (Goffin etal. 1997a). It
rocapram, Azone). Other compounds, such as is a variant of corneosurfametry which was spe-
surfactants and solvents, are more regular constit- cifically designed for testing neat or diluted sur-
uents of any topical formulation (Som etal. 2012). factants (Pirard etal. 1994; Goffin etal. 1995,
The efficacy of penetration enhancers toward vari- 1996; Pirard and Pirard-Franchimont 1996;
ous drugs were thoroughly explored and compared Uhoda etal. 2003).
(Williams and Barry 1992). Synergistic effects Corneoxenometry is used for investigating the
were reached after combining different classes of effects of chemicals potentially harmful to the SC
penetration enhancers such as solvents and lipid (Goffin etal. 1997a, b, 1998, 2000; Xhauflaire-
fluidizers (Wotton etal. 1985; Ward and Du Reau Uhoda etal. 2008b).The bioassay entails a col-
1991). Some binary and ternary mixtures were lection of cyanoacrylate skin surface strippings
reported to be more active than single penetration (CSSS) from normal human skin. The harvested
enhancers (Rojas etal. 1991). In complex formula- SC sheet which is uniform in thickness is sub-
tions, each component possibly acts in many dif- jected to the exvivo action of the selected xeno-
ferent ways, precluding the determination of the biotics. CSSS covered in excess with each
actual operative interactions. chemical are kept for 2h at room temperature in
a close environment in order to limit any evapora-
tion from the test solution. Samples are thereafter
17.2 SPBF Modulation thoroughly rinsed under running tap water, air
andCorneoxenometry dried and stained for 3min with a toluidine blue-
basic fuchsine solution at pH3.45. Any lipid dis-
There is a need for accurate assessments of the ruption and protein denaturation is responsible
alterations in the SPBF because any effect quan- for an increased dye binding on corneocytes
tification of penetration enhancers should allow (Fig.17.1). Harsh compounds to the skin consid-
to design safe, reliable and effective formulations erably increase the staining intensity of the CSSS
(Diembeck etal. 1999). SPBF is hardly explored (Goffin etal. 1997a, b, 1998, 2000; Uhoda etal.
17 Corneoxenometry: ABioassay Exploring Skin Barrier Breaching 305
(40.7), ethanol (26.5), methanol (23.5), hexane- solvents and the SC.In previous studies (Goffin
ethanol (23.3), chloroform (20.8), chloroform- etal. 1997b), the time range between 1 and
methanol (15.5) and hexane-methanol (7.8). 120min was selected following available infor-
CIM values showed that the effect of hexane- mation about the kinetics of lipid extraction from
methanol on SC was significantly higher (p < human SC [13]. The corneoxenometry data were
0.01) than those of all other solvents with the in line with previous experiments using other
exception of chloroform-methanol. There was methodological approaches (Deffond etal.
no significant difference between ethanol, meth- 1986; Imokawa etal. 1986; Abrams etal. 1993;
anol and hexane-ethanol, but each of them was Lavrijsen etal. 1994). However, it does not
significantly (p < 0.05) more aggressive than explore the effects of solvents on the living epi-
hexane. dermis and on the nature and intensity of inflam-
The influence of exposure time of solvents mation that is present in irritant dermatitis.
with the SC showed some inter-product differ-
ences. However, all correlations reached signifi- Conclusion
cance (p < 0.01) and best fitted as logarithmic Corneoxenometry appears as a relevant and
relationships. For each solvent, most of the CIM predictive bioassay for assessing the overall
changes were reached within 10min. effect of single and combined penetration
The organic solvents under consideration are enhancers. It is cheap, rapid, minimally inva-
known to extract lipids (Bligh and Dyer 1959; sive and relevant to human skin. In addition,
Scheuplein and Ross 1970; Deffond etal. 1986; the reproducibility, specificity and sensibility
Imokawa etal. 1986; Abrams etal. 1993; Lavrijsen are reasonably high. Corneoxenometry is
etal. 1994). In addition, SC alterations other than therefore a valuable screening test proposed as
lipid extraction are likely (Abrams etal. 1993). an alternative to animal and invitro testings.
Large interindividual CIM differences were found
for each solvent or mixture (Goffin etal. 1997b)
References
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Part III
The Retardation of Percutaneous Drug
Penetration
Retardation Strategies
forSunscreen Agents 18
KatharinaBohnenblust-Woertz
andChristianSurber
Contents
18.1 Introduction
18.1 Introduction 311
18.2 Cyclodextrins andPhotostability 312 There is overwhelming evidence indicating that
18.3 Transcutol
312 human skin is damaged in different ways by expo-
sure to sunlight. Of the solar radiation reaching the
18.4 Encapsulation Structures 313
earths surface, the ultraviolet (UV) component
18.5 P
hysical Properties ofOrganic (290400 nm) is a major factor leading to skin
Particulate UV Absorbers 315 pathologies that range in severity from inflamma-
18.6 Inorganic Materials 315 tory responses, cutaneous photoaging, dendritic
18.7 Penetration Retarders 316 keratitis to various types of skin cancer (Pathak
1991; Ziegler etal. 1994; Hochberg and Enk 1999;
18.8 Vehicle Effects 317
Surber etal. 2012). The increasing knowledge of
Conclusions 317 the deleterious effects of sunlight has promoted
References 318 the widespread use of topical sunscreen prepara-
tions (Hayden etal. 1998; Green etal. 1991),
which contain chemicals that absorb, reflect, or
scatter UV radiation (Patel etal. 1992) and are
thereby highly effective skin protectants. Organic
sunscreen agents are compounds that decrease the
intensity of UV light reaching the epidermal strata
by absorbing radiation (typical electron promotion
from a lower- to a higher- energy molecular
orbital). The activated sunscreen molecule dissi-
pates the excess energy in the form of heat, by
K. Bohnenblust-Woertz fluorescence, phosphorescence, interaction with
Galderma Spirig, Egerkingen, Switzerland
neighboring molecules, or by undergoing photo-
C. Surber (*) chemical modifications (Broadbent etal. 1996).
University Hospital Zrich, Department of
Particulate sunscreens present a physical barrier
Dermatology, Zrich, Switzerland
between the incident radiation and the epidermis,
University Hospital Basel, Department of
scattering or reflecting the radiation. However, to
Dermatology, Petersgraben 4,
CH-4031 Basel, Switzerland be effective, these agents must remain on or in the
e-mail: christian.surber@unibas.ch outermost layers of the skin, the stratum corneum
(SC). One major drawback of current sunscreen decreasing their penetration into or permeation
formulations is that they are constantly lost from through the skin (Rajewski and Stella 1996;
the skin surface by abrasion from clothing, sweat- Loftsson and Masson 2001). Butyl-
ing, or swimming, requiring frequent reapplica- methoxydibenzoylmethane (BM-DBM) is a
tion for continued effectiveness. Moreover, several widely used filter that provides protection against
of the chemical sunscreens currently on the market UVA radiation in the 320400nm range. However,
exhibit irritancy and sensitization reactions after BM-DBM experiences marked photodegradation
absorption into the dermal strata in predisposed (Schwack and Rudolph 1995; Scalia etal. 1998;
individuals, often causing immunological prob- Tarras-Wahlberg etal. 1999; Chatelain and Gabard
lems (Deflandre and Lang 1998; Dromgoole and 2001; Damiani etal. 1999; Scalia etal. 2002),
Maibach 1990; Mariani etal. 1998). A significant forming highly reactive photolytic products that
improvement in sunscreen technology would be are exposed to the living tissues of the epidermis
the development of systems that retard the pene- and dermis following percutaneous permeation.
tration of the chemical into the skin and bind the Scalia etal. (1998, 2002) have demonstrated that
agent in the stratum corneum so that minimal loss the degree of decomposition and free radical for-
occurs by diffusion or abrasion. The degree of sun- mation upon exposure of BM-DBM to simulated
screen penetration depends strongly on the physi- sunlight were reduced by complexation with
cochemical properties of the active compound and hydroxypropyl-13-cyclodextrin (HP-13-CD). The
of the nature of the vehicle in which the sunscreen effects of HP-13-CD and sulfobutylether-13-cy-
is applied, that is, polarity of the ingredients that
clodextrin (SBE7-13-CD) on invitro human skin
form the vehicle and the filters particle size penetration and retention of the sunscreen agent
(Benech-Kieffer etal. 2000). Therefore, the devel- BM-DBM were investigated by Simeoni etal.
opment of suitable formulations that prevent pen- (2004). They reported that approximately 1416%
etration of the sunscreen into the skin is a challenge
of the applied dose of BM-DBM penetrated into
for developers. Some of the few vehicular penetra- the skin tissue; however, no sunscreen was detected
tion retardation strategies being researched for in the dermis and in the receptor phase. The greater
sunscreens are reviewed below. proportion (84.695.5%) of the absorbed UV fil-
ter was localized in the SC with no significant dif-
ferences between uncomplexed and complexed
18.2 Cyclodextrins BM-DBM.Notable levels (2.3% of the applied
andPhotostability dose) of the sunscreen agent accumulated in the
epidermis from the preparation containing free
Cyclodextrins are cyclic, toroidal-shaped oligo- BM-DBM.The epidermal concentration of the
saccharides with a hydrophilic external surface UV filter was markedly reduced (0.7% of the
and a hydrophobic central core. They are capable applied dose) by complexation with SBE7-13-CD,
of incorporating appropriately sized, nonpolar whereas HP-13-CD had no effect. The results
compounds or some lipophilic moiety of a mole- demonstrated that complexation of BM-DBM
cule into their apolar cavities, forming non- with SBE7-13-CD attained high sunscreen levels
covalent inclusion complexes (Rajewski and Stella at the skin surface where its action is most desir-
1996; Loftsson and Brewster 1996). This type of able and produced lower concentrations of the
molecular encapsulation can lead to changes in active in the epidermis.
some of the physical and chemical properties of
the included substance, such as the enhancement
of stability to air and light and apparent aqueous 18.3 Transcutol
solubility (Rajewski and Stella 1996; Loftsson and
Brewster 1996; Uekama etal. 2003). Moreover, Ethoxydiglycol (Transcutol CG, Gattefosse,
cyclodextrin complexation can affect the topical France) is a hydgroscopic liquid that is freely
availability of applied drugs, either increasing or miscible with both polar and nonpolar solvents.
18 Retardation Strategies forSunscreen Agents 313
Transcutol has been recognized as a potential not only in the epidermis but also in deeper layers
transdermal permeation enhancer due to its non- of the skin the potential bioavailability remains
toxicity, biocompatibility with skin, and excel- unknown.
lent solubilizing properties (Godwin etal. 2002).
However, Transcutol has also been reported to
increase the skin accumulation of topically 18.4 Encapsulation Structures
applied compounds without a concomitant
increase in transdermal permeation (Ritschel Colloidal drug carriers, including submicron
etal. 1991; Panchagnula and Ritschel 1991). It is emulsions, nanospheres, nanocapsules, lipo-
theorized that this depot effect is created by a somes, and lipid complexes, have been attracting
swelling of stratum corneum intercellular lipids, increasing interest as drug delivery vehicles.
without alteration of their multiple bilayer struc- These encapsulation systems have been evalu-
ture. The expanded lipid domain is then able to ated for the intravenous administration of lipo-
retain drugs (especially lipophilic compounds) to philic drugs, as improved parenteral formulations,
form the depot, with a simultaneous decrease in and as systems for site-specific drug delivery
transdermal permeation. such as tumors (Allemann etal. 1993). In gen-
Godwin etal. (2002) studied the influence of eral, two techniques have been used for the prep-
Transcutol CG concentrations in sunscreen aration of nanocapsules based on biodegradable
formulations on the transdermal permeation and polymers: the emulsification-diffusion technique
skin accumulation of the UV absorbers 2-hydroxy- (Quintanar-Guerrero etal. 1996) and the solvent
4-methoxybenzophenone (oxybenzone) and displacement procedure (Fessi etal. 1988; AI
2-octyl-4-methoxycinnamate (cinnamate). Khouri Fallouh 1986). The ideal medium in
When formulated alone, both these lipophilic which an active ingredient is incorporated must
sunscreens have been shown to permeate through provide not only the necessary solubility but also
the skin and enter the systemic circulation maintain contact between the active ingredient
(Treffel and Gabard 1996). In their study, the and the skin. The nature of the colloidal carrier
concentration of the UV absorber was held con- and the effects of size and surface charge influ-
stant at 6% (w/w) for all vehicle systems while ence penetration and permeation of UV filters
the concentration of Transcutol CG was varied into the skin (Zeevi etal. 1994; Treffel and
from 0 to 50% (w/w). The data demonstrated that Gabard 1996; Gupta etal. 1999).
both UV absorbers exhibited an increase in skin Alvarez-Roman etal. (2001) investigated the
accumulation with increasing concentrations of optimization of a solvent displacement method
Transcutol CG.Skin accumulation of oxyben- for poly(-caprolactone) nanocapsules, using
zone was significantly (P<0.05) greater than that the lipophilic UV filter, octyl methoxycinna-
of cinnamate for all formulations investigated. mate (OMC) as the oil core. In addition, these
However, no significant differences were found researchers evaluated the influence of polysor-
in the transdermal permeation of oxybenzone or bate 85 (Tween 85) and poloxamer 188 (Pluronic
cinnamate for any of the formulations tested. The F 68) as filter-stabilizing agents, the OMC load-
results of this study demonstrate that the inclu- ing capacity, and the photoprotective potential
sion of Transcutol CG in sunscreen formula- of the formulations. The OMC-nanocapsule-gel
tions appears to increase the skin accumulation preparation resulted in a significantly better
of the UV absorbers oxybenzone and cinnamate, (P<0.05) protection against UV-induced ery-
without a concomitant increase in transdermal thema than a simple OMC gel. Sunscreen effec-
permeation. Their data support the theory of the tiveness implies that the sunscreens adhere to
formation of an intracutaneous depot for both the skin more efficiently as a protective film.
oxybenzone and cinnamate when formulated These results suggest that the nanoparticles are
with Transcutol CG.These observations have to able to cover more efficiently the skin surface
be confirmed invivo. As the accumulation occurs due to their high specific surface area. Sunscreen
314 K. Bohnenblust-Woertz and C. Surber
nano-capsules, therefore, show good potential Nanocapsules (NC) have been introduced as a
as improved skin retention vehicles. new generation of carriers for cosmetics and UV
Liposomes and emulsions are formulated blockers for use on human skin and hair. Jimenez
from biocompatible excipients and can easily be etal. (2004) compared the porcine skin permeation
produced on a large scale. Compared to lipo- of a lipophilic sunscreen, OMC, from different
somes and emulsions, solid particles afford pro- emulsions and encapsulated sunscreen-poly(-
tection of incorporated active compounds against caprolactone) nanocapsules. Their results showed
chemical degradation and allow more flexibility that the use of NC emulsions decreases the perme-
in modulating the release of the compound. The ation of OMC through pig skin when compared
advantages of solid particles, emulsions, and with equivalent w/o and o/w emulsions. NC emul-
liposomes were, therefore, combined by the sions are, therefore, novel vehicle-type dispersion
development of solid lipid nanoparticles (SLNs) systems and can be used advantageously as sun-
(Mller etal. 2002), produced by simply exchang- screen carriers to lower permeation of the active
ing the liquid lipid (oil) of the emulsions by a through the skin.
solid lipid. A very promising approach is to entrap the
Wissing and Mller (2002a) compared an SLN organic UV absorbers in a sol-gel silica glass
and a conventional o/w emulsion carrier system for shell. The product is commercialized under the
the sunscreen oxybenzone, by studying the invitro trade name of Eusolex UV Pearls (Merck,
rate of release with a membrane-free model and Germany) and is promoted under the slogan
static Franz diffusion cells. They reported that the Sunglasses for the Skin. The UV absorber,
release rate could be decreased by up to 50% with which is usually an oil or oil soluble compound,
the SLN formulation. Penetration of oxybenzone constitutes about 80% of the capsule weight. The
into stratum corneum on the forearm invivo was products are manufactured as an aqueous disper-
also investigated by a tape stripping method. In sion, containing 35% (by weight) UV absorber(s).
congruity with the invitro data, it was shown that The particle size is about 1 micron on average and
the active release rate could be decreased by 30 they do not tend to agglomerate; therefore, they
60% with SLN formulations. In all test models, give a pleasant skin feeling. The refraction
oxybenzone penetrated into the skin less rapidly index of the particles is small enough that they
and to a lesser extent than from conventional emul- appear transparent when applied topically. The
sions. The authors concluded that using SLN as a glass microcapsules were shown to effectively
carrier system offers two main advantages. SLNs retain the encapsulated UV absorbers in a series
act as physical sunscreens on their own; therefore, of stress experiments. These include temperature,
the concentration of molecular sunscreen agents drying, pressure (e.g., spraying), and incubation
can be decreased while maintaining the formula- with emollients, tensides, and other raw materials,
tion sun protection factor. Moreover, SLNs are able which could have the potential to extract the cap-
to provide a sustained release carrier system, sules content. All experiments clearly demon-
enabling the sunscreen to remain longer at its site strated the stability of the capsules. Additionally,
of action on the surface of the skin. different skin penetration experiments showed the
Similar results were obtained by Wissing etal. maintenance of sunscreen actives on or near the
(2002b) when they compared the efficacy of a surface of the skin compared to non-encapsulated
conventional o/w emulsion and crystalline lipid material.
nanoparticles (CLN) incorporating the sunscreen The encapsulated sunscreens obtained through
benzophenone-3. This invitro study based on the the Sol-Gel glass-making technology is currently
TransporeTM test (3M, USA) (Diffey and Parr the most promising technology for significantly
1996) showed that the amount of molecular sun- improved product characteristics. The direct con-
screen can be decreased by up to 50% while tact between the sunscreen filters and the body
maintaining the UV protection efficacy, simply tissues is suppressed the filter is no longer bio-
because of the particulate nature of the CLN available. Products with UV Pearls are therefore
structures. suited for products for sensitive skin. In addition,
18 Retardation Strategies forSunscreen Agents 315
the UV Pearls give the product a pleasant sensory hair follicles, and sweat glands. This penetration
feel (Pflcker etal. 2002). is undesirable because of the risk of damage to
DNA and RNA by the photocatalytic effects of
the TiO2 after absorption of UV light (Dunford
18.5 Physical Properties etal. 1997). Furthermore, the particles can acti-
ofOrganic Particulate UV vate the immune system and accumulations of
Absorbers these particles in the skin can decrease the thresh-
old for allergies (Granum etal. 2001). The func-
Most of the UV filters in use are oil-soluble and, tion of the stratum corneum as a barrier against
consequently, are incorporated into the oil phase dermal uptake of ultrafine particles was the sub-
of sunscreen emulsions; however, even solubility ject of several investigations, which came to dif-
in the oil may be problematic. UV absorbers that ferent conclusions concerning the penetration
are poorly soluble in oils and relatively insoluble depth of the particles (Pflcker etal. 2001; Tan
in water may be micronized to form aqueous dis- etal. 1996). Researchers have investigated a
persions of ultrasmall particle size. The protec- number of other ultrafine, inorganic particles,
tive performance of these particles depends on including ceria (CeO2) (Yabe and Sato 2003) and
size, as both absorption and scattering play a role zinc oxide, for efficacy as UV-protectants. Most
in the attenuation of UV light. To this end, it is of these agents are ideal for cosmetic applica-
desirable to achieve particle sizes in the submi- tions because they are relatively transparent to
crometer range. visible light, but have excellent ultraviolet radia-
Herzog etal. (2004a) generated microparticles tion absorption properties, and appear transpar-
of a benzotriazole derivative in the 0.1640 m ent on the skin. However, many of these chemicals
range by milling particles in the presence of a dis- exhibit high photocatalytic potential after UV
persing agent. The UV absorption increases with activation (Cai etal. 1991). This reactivity can be
a decrease in particle size, while the light scatter- lowered by coating of the particles (with amor-
ing shows a maximum at a certain particle size. phous silica for example) or by doping with a
These researchers investigated the UV-attenuating metal ion possessing lower valence and larger
properties of particulate organic absorbers as a ionic size.
function of particle size, with special emphasis on Menzel etal. (2004) investigated the percuta-
the differentiation between absorption and scat- neous penetration of coated TiO2 through pig
tering functionalities of the particles (Herzog skin and observed a penetration of particles
etal. 2004b). The efficiency of the UV extinction through the stratum corneum into the underlying
of the dispersion increases with decreasing parti- stratum granulosum via the intercellular spaces,
cle size down to a maximum extinction at a parti- but the TiO2 particle concentration in the stratum
cle size of 80 nm, and the UV extinction decreases spinosum was negligible. Hair follicles did not
for particles smaller than 80nm indicating an seem to be major penetration pathways as TiO2
optimum at 80nm. It was found from reflection was not detected inside these appendages. These
spectroscopic measurements that scattering findings show the importance of coating the TiO2
accounts for about 10%, and absorption 90%, of particles as a mechanism to reduce genetic dam-
the UV-attenuating effect of the particles. age in the skin. Alternatively, any formulation
mechanism that would retard the penetration of
the particles below the outermost SC layers
18.6 Inorganic Materials would be highly advantageous. One possible
mechanism to achieve this may be the chemical
Micronized TiO2 particles with a diameter of sequestration of the organic active. To minimize
about 15nm are used in sunscreens as physical absorption of sunscreen agents through the skin,
UV filters. These particles are suspected to be organic materials may be incorporated in the
absorbed through the stratum corneum into the nanospaces of inorganic materials, thereby avoid-
epidermis or dermis via intercellular channels, ing direct exposure to the stratum corneums bio-
316 K. Bohnenblust-Woertz and C. Surber
its activity as a retardant with ethanol, but behaved with increasing viscosity for the finite dosed for-
as enhancer with water, PG and PEG 400. mulation (simulating in use situation). The epi-
The investigations of Hadgraft and Michniak- dermal membrane retention of benzophenone
Kohn are significant since they underline the role also decreases with viscosity for the infinite dose.
of pharmaceutical formulations and show that In contrast, the epidermal membrane retention
ingredients termed as enhancers can become a for the finite dose appears to be unaffected by the
retardant or vice versa depending upon the vehi- viscosity of the formulation used. The absorption
cle in which they are applied to the skin. and retention profiles with viscosity are similar
Very obvious is the penetration retarding for HDPE membranes to that observed for human
effect produced by the lipophilicity and the high epidermal membranes. However, even the con-
molecular weight of filters. An illustrative exam- cepts of finite and infinite dose have been
ple was the design and development of Bis- open to wide interpretation by researchers
Ethylhexyloxyphenol Methoxyphenyl Triazine (Surber and Davis 2002).
(Tinosorb S, BASF, Germany) with a molar The discrepancy in the infinite and finite dos-
mass of 627.81 g/mol and a log partition coeffi- ing results is likely to arise from the differing dif-
cient n-octanol/water: >5.5 at 20C, a molecular fusion of benzophenone in the formulations, and
feature that predicts no permeation into deeper skin hydration arising in the two situations.
parts of the skin (Bos and Meinardi 2000). Slower water evaporation is likely to result in a
higher water content in the residual vehicle film
and an increase in skin penetration due to a higher
18.8 Vehicle Effects diffusivity in a more hydrated membrane (Roberts
and Walker 1993). It is unlikely that the formula-
Similarly, there are relatively few studies exam- tions have affected drug partitioning into the skin
ining the effect of vehicle viscosity on cutaneous as epidermal retention for the four vehicles was
penetration following the application of finite or similar. Hence, the flux of benzophenone-3
small in use doses of topical drug formulations. through both human epidermal and HDPE mem-
Cross etal. (2001) compared the effect of viscos- branes decreases with increasing formulation vis-
ity on the invitro penetration of benzophenone cosity. The clinical implications from the study is
from four different types of emulsion formula- that caution should be exercised in assuming that
tions. The researchers maintained the same ther- more viscous formulations applied to the skin
modynamic activity in all test modes, using both may retard the penetration of topically applied
epidermal and high density polyethylene (HDPE) sunscreens. Viscous formulations impede the
membranes, and allowed for control of any pos- skin penetration of benzophenone under infinite
sible vehicle-skin interactions. In addition, the dosing conditions, but appear to cause faster skin
change in percutaneous absorption and retention penetration using finite dosed in use formula-
kinetics of benzophenone from the emulsions tions. The interesting aspect from a formulation
following infinite and finite dose application was viewpoint is that it may be possible to modulate
determined in an attempt to define the effects of absorption by simple changes in the vehicle
viscosity on actual in use conditions. In the lat- matrix, a conclusion that corroborates early find-
ter situation, factors such as formulation evapora- ings of Haigh etal. (1992).
tion (estimated from the rate of vehicle water
loss) would be expected to make a significant Conclusions
contribution to release kinetics (metamorphosis A number of strategies have been individually
of the vehicle) (Surber and Smith 2005). The evaluated to limit the absorption of sunscreens
results from the human epidermal permeation after topical application. It would be interest-
flux data indicate that while the flux of benzophe- ing to judiciously combine these scientific con-
none decreased with formulation viscosity for the cepts that have been investigated individually,
infinite dosed formulation, the flux was increased into an integrated topical delivery system of
318 K. Bohnenblust-Woertz and C. Surber
chemical composition, formulation micro- cal fluid chromatography combined with atmospheric
structure and specific penetration retarder such pressure chemical ionisation mass spectrometry for
the investigation of photoproduct formation in the sun-
that the delivered sunscreen chemical is held in screen absorber 2-ethylhexyl-p-methoxycinnamate.
a bound reservoir in the layers of the stratum JChromatography 732:101110
corneum, preventing penetration into the lower Cai R, Hashimoto K, Itoh K, Kubota Y, Fujishima A
strata and dermis, and minimizing surface loss. (1991) Photokilling of malignant cells with ultrafine
Ti02 powder. Bull Chem Soc Jap 64:12681273
In this manner, the sun-protecting agent would Cavani F, Trifiro F, Vaccari A (1991) Hydrotalcite-type
be held at its optimal site of action the upper- anionic clays: preparation, properties and applica-
most layer of the skin for a prolonged period, tions. Catalysis Today 11:173301
and would induce minimal sensitizing or irri- Chatelain E, Gabard B (2001) Photostabilization of butyl
methoxydibenzoylmethane (Avobenzone) and ethyl-
tancy activity because of exposure to the sys- hexyl methoxycinnamate by bis-ethylhexyloxyphenol
temic circulation. The design and the methoxyphenyl triazine (Tinosorb S), a new lN broad-
development of the Eusolex UV Pearls and band filter. Photochem Photobiol 74:401406
Bis-Ethylhexyloxyphenol Methoxyphenyl Cross SE, Jiang R, Benson HAE, Roberts MS (2001) Can
increasing the viscosity of formulations be used to
Triazine (Tinosorb S) a high molecular lipo-
reduce the human skin penetration of the sunscreen
philic, photostable, broad- spectrum UV oxybenzone? JInvest Dermatol 117:147150
absorber efficiently covering both the UVA Damiani E, Greci L, Parsons R, Knowland J(1999)
and UVB range impressively demonstrate Nitroxide radicals protect DNA from damage when
illuminated invitro in the presence of dibenzoylmeth-
retardation strategies for sunscreen agents. ane and a common sunscreen ingredient. Free Radical
Perhaps the addition of a specific keratin-bind- Biol Med 26:809816
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terms of maintaining the active protectant at its UVB, UVA and blue light: an invivo and invitro com-
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sun-induced skin cancer protection vehicles. contact urticaria. JAm Acad Dermatol 22:10681078
Dunford R, Salinaro A, Cai L, Serpone N, Horikoshi S,
Hidaka H, Knowland J(1997) Chemical oxidation and
DNA damage catalysed by inorganic sunscreen ingre-
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Retardation ofDermal Release
byFilm Forming Emulsions 19
DominiqueJasminLunter andRolfDaniels
Reservoir
Fig. 19.1 Formulations for sustained dermal therapy; patch which exhibits a reservoir for the api (e.g., estrogen,
left: rapid release of the api from a semisolid formulation opioids, scopolamine)
and build-up of a reservoir in stratum corneum; right:
solids allow sustained release or permeation of Table 19.1 Formulation principles of dermal sustained
the api, about 90% of the semisolid formulation release formulations
is usually withdrawn from the skin by clothing or Type of
contact to other surfaces. Therefore, semisolids formulation Advantages Disadvantages
are unlikely to show the required substantivity Semisolid Convenient Insufficient
which is pivotal to ensure adequate contact to the formulation spreadability substantivity
Possibility to treat High application
skin for an extended treatment interval. Patches large areas of skin frequency
provide an alternative. The prolonged contact Patch High substantivity Limited area
time to the skin is a clear advantage but the size Low application Insufficient
of the patch limits the area that can be treated. frequency flexibility leading
Furthermore, most patches are not flexible to a feeling of
tension on the skin
enough to allow for unlimited movement of the
Film Convenient
patients skin. Therefore, patches may lead to an forming spreadability
uncomfortable feeling of tension on the skin. emulsion Possibility to treat
All these disadvantages give rise to a need for large areas of skin
High substantivity
a formulation concept which combines the
Low application
advantages of both formulation principles: sus- frequency
tained release, high substantivity and the possi-
bility to conveniently treat larger areas of affected
skin. We thus developed film forming emulsions, compares characteristics of semisolid, patches
which are easy to spread and therefore allow con- and film forming emulsions.
venient treatment of larger areas of affected skin. Important prerequisites for film forming emul-
These oil-in-water (O/W) emulsions form a water sions allowing sustained dermal therapy are:
insoluble film on the skin which ensures adequate
substantivity and sustains permeation of the api Sustained permeation of the api from the for-
(Lunter and Daniels 2012a, b). Table19.1 mulation at an adequate permeation rate
19 Retardation ofDermal Release byFilm Forming Emulsions 323
Delivery of an effective amount of the api to Evonik Rhm GmbH, Germany) and Ammonio
the tissue of interest Methacrylate Copolymer Dispersion, Type B
Film formation of the aqueous polymer dis- (Eudragit RS 30D (RS), Evonik Rhm GmbH,
persion which is incorporated in the aqueous Germany) are added to the aqueous phase.
phase of the emulsions at skin surface Therefore, the preparation of film forming emul-
temperature sions poses a number of challenges to the pro-
Strong adhesion to skin to ensure prolonged ducer. First, the drug needs to be incorporated
contact time of the formulation and the drug to into the inner oil-phase. Second, polymer disper-
the skin sions are sensible to shear stress, the dispersions
Sufficient flexibility to prevent rupture upon may only be added to the emulsion after homog-
movement of the patient enization. Third, the dispersions (NE and RS) are
Compatibility of all components within the not compatible with each other unless pH is
formulation adjusted to pH56 and polysorbate 80 is added
Adequate storage stability of the emulsions to both dispersions prior to mixing (Lehmann
and Dreher 1986). Fourth, the glass transition
Nonivamide was used as a model drug in our temperature of the polymers needs to be lowered
studies. It is a synthetic analogue of capsaicin to <20C by the addition of a plasticizer (Triethyl
which may be used in the treatment of chronic pru- citrate, Sigma-Aldrich Chemie GmbH, Germany)
ritus (Anand and Bley 2011; Ikoma 2010; Stnder to ensure complete film formation on the skin.
and Luger 2010), a symptom that accompanies Therefore, a three-step manufacturing process
various skin diseases, like atopic dermatitis or pso- needed to be applied. First, the emulsion itself
riasis. Conventional formulations containing cap- was made by homogenizing the drug solution in
saicinoids need to be applied several times a day. oil and the aqueous polymer solution. Second,
This has to be performed very carefully every time the pH of the dispersions (NE and RS) was
to avoid any unintended contact with the drug, adjusted to pH56 by addition of hydrochloric
which can induce an intense burning sensation. acid and the dispersions were plasticized. For
This makes therapy inconvenient and negatively that purpose, triethyl citrate was added to RS and
affects patient compliance, which is mandatory for the dispersion was stirred. Subsequently, poly-
effective treatment (Anand and Bley 2011). sorbate 80 was added to the plasticized RS and
Therefore, capsaicinoids are a good example the dispersion was again stirred. Similarly, poly-
where sustained release formulations could con- sorbate 80 was added to NE and the dispersion
tribute to an improvement in patient compliance was stirred. No triethyl citrate was added to
and thereby ensure therapeutic success. NE.Third, the dispersions were added to the
emulsion in order to obtain the final film forming
emulsion. Figure19.2 gives a scheme of the prep-
19.2 Manufacture and aration of film forming emulsions. Compositions
Characterization ofFilm of film forming emulsions are given in Table19.2.
Forming Emulsions Film forming emulsions were characterized
by laser diffraction to verify absence of floccula-
Film forming emulsions consist of an O/W emul- tion. The data show that droplet sizes of emul-
sion with the api dissolved in the dispersed oil sions before and after addition of Eudragit were
phase. The emulsions are stabilized by a water similar (Fig.19.3). The addition of the dispersion
soluble polymer (polyvinyl alcohol) which also of Eudragit resulted in a supplementary peak at
acts as a polymeric emulsifier. To allow the for- approx. 120nm which corresponds to polymer
mation of a water insoluble film on skin the dis- particles of the Eudragit dispersions. No addi-
persions of sustained release polymers, namely, tional peaks were detected. This indicates that no
Ethyl Acrylate and Methyl Methacrylate flocculation occurred and stable emulsions con-
Copolymer Dispersion (Eudragit NE 30D (NE), taining a combination of NE and RS were formed.
324 D.J. Lunter and R. Daniels
NVA 10 % PVA
HCl
50 C 80 C 80 C
1h 50 % PVA
3h 3h
eudragit
dispersion
NVA-solution 5 % PVA-gel 10% pH 5
PVA-gel 50 % TEC
PS 80
RT
homogenization 1h
plastified
emulsion mixing eudragit
dispersion
mixing Eudragit
- PVA-gel
film forming
emulsion
Fig. 19.2 Preparation scheme for film forming emulsions. HCl hydrochloric acid, MCT medium chain triglycerides,
NVA nonivamide, PS 80 polysorbate 80, PVA polyvinyl alcohol, TEC triethyl citrate
25
Glasstransition temperature [C]
20 *
15 *
10 n.s.
5 * n.s.
0
0/100 25/75 40/60 50/50 60/40 75/25 100/0
5
NE/RS
Fig. 19.4 Glass transition temperature of mixtures of alcohol; meanSD; n=3; * statistically different data;
Eudragit NE 30D+polysorbate 80+polyvinyl alcohol n. s. not statistically different data
and RS 30D+triethyl citrate+polysorbate 80+polyvinyl
according to our measurements), which is further droplets and thereby preserves the dispersed
reduced due to the addition of polysorbate 80. character in the dry state. The built-up of these
With regard to Tg, all formulations fulfilled the emulsion films was studied by confocal Raman
requirements and were therefore used in further microscopy (CRM). Figure19.5 shows color-
experiments. coded CRM images of dried emulsion films. It
In general, complete film formation is can be seen that the oil droplets are completely
achieved when Tg is lowered to a value approxi- embedded in a polymeric matrix of either polyvi-
mately 10C beyond process temperature. nyl alcohol and RS or polyvinyl alcohol, NE and
Therefore, the decrease of Tg to <20C allows RS, or polyvinyl alcohol and NE.This indicates
film formation at skin surface temperature (30 that the coalescence of the oil droplets upon dry-
32C). Upon drying, the polymers which are ing was successfully prevented and the dispersed
located in the continuous aqueous phase form a character is maintained in the emulsion films.
polymeric matrix which encapsulates the oil Retardation of drug release from the oil solution
19 Retardation ofDermal Release byFilm Forming Emulsions 327
a b
Fig. 19.5 Raman microscopic color-coded images of dry film forming emulsion containing NE; ( ) MCT, ( )
emulsion films. (a) Film forming emulsion containing RS, PVA; ( ) Eudragit NE, ( ) Eudragit RS; scale bar:
(b) film forming emulsion containing NE and RS, and (c) 3m
by diffusion through the polymeric matrix is taining a mixture of NE and RS is stronger com-
therefore rendered possible. pared to adhesion of films that contain only one
To allow substantivity, adequate adhesion to of the sustained release polymers and is indepen-
skin needs to be assured. Adhesion was tested dent of NE/RS ratio.
using glass as a surrogate for skin (Fig.19.6). Adhesion to polycarbonate is generally higher
Polycarbonate was used as a control. Adhesion to as both polymers (NE and RS) consist mainly of
polycarbonate was found to be significantly non-polar monomers. These non-polar mono-
higher than adhesion to glass. Emulsion films mers interact stronger with non-polar polycar-
containing NE (no RS) adhered weakly to glass bonate. One might have expected that adhesion
whereas films containing RS (no NE) adhered of emulsion films which contain mixtures of NE
weakly to polycarbonate. Adhesion of films con- and RS might be influenced by NE/RS ratio. The
328 D.J. Lunter and R. Daniels
fact that this is not the case may be explained by shows that the elongation of emulsion films is
a possible segregation of the components during largely influenced by NE content as NE itself is
the drying process and, possibly, the built-up of more elastic than RS and polyvinyl alcohol. The
the same structures at the bottom of the film, more NE the films contain, the more elastic they
independent of NE/RS ratio in the native film are. However, the relationship between NE frac-
forming emulsions. However, films containing tion and elongation is not linear. Films containing
both sustained release polymers show higher >40 parts NE show an elasticity of >30% and are
adhesion than films that contain only one of the therefore regarded as adequate with regard to an
polymers and are therefore regarded as superior. application onto skin.
Concerning elasticity, the results of research These results indicate that emulsion films are
on the extensibility of human skin indicate that a capable of (i) encapsulating the dispersed oil
reasonable value of elongation that the emulsion phase upon drying, (ii) adhering strongly to skin,
films should exhibit is approximately 30 % and (iii) following the normal movements of skin.
(Langer 1978; Sumino etal. 2009). Figure19.7 Adequate substantivity could thus be assumed.
30
25
force of adhesion [N]
20
15
10 *
0
0/100 25/75 40/60 50/50 60/40 100/0
5
NE/RS
glass polycarbonate
Fig. 19.6 Comparison of force of adhesion of free films meanSD; n=5; * statistically different data; n. s.
from emulsions containing varying amounts of Eudragit not statistically different data
NE30D and RS30D to (a) glass and (b) polycarbonate;
150
125 *
100
elongation [%]
75 *
Fig. 19.7 Comparison of
elongation at break of free 50 n.s.
films from emulsions n.s.
containing varying amounts
25
of Eudragit NE30D and
RS30D; meanSD; n=5;
* statistically different 0
0/100 25/75 40/60 50/50 60/40 100/0
data; n. s. not statistically
different data NE/RS
19 Retardation ofDermal Release byFilm Forming Emulsions 329
19.4 I n Vitro Performance ofFilm square root of time and permeation rate is equiva-
Forming Emulsions lent to that from HNC 0.05%. The permeation
coefficient from the emulsion films is 23 times
Emulsion films containing 40 parts NE (60 parts smaller compared to that from HNC 0.05%.
RS, respectively) were used in invitro perme- From emulsion films approx. 1% of nonivamide
ation and penetration experiments, as they show a permeated the skin after 48h, whereas from HNC
Tg <20C encapsulate the oil droplets in the dried approx. 30% permeated. Permeation rate and
emulsion film, adhere strongly to glass and show overall permeated amount from the emulsion
an elongation of approximately 30%. To investi- films containing 40 parts NE are slightly higher
gate the influence of the sustained release poly- than the respective parameters of the emulsion
mers on permeation from emulsion films, films containing only RS.However, the differ-
formulations containing only RS were also used ences are not statistically significant.
in invitro permeation experiments. The fact that the permeation coefficients of
nonivamide from HNC are independent of noni-
vamide content clearly shows that enhanced per-
19.4.1 In Vitro Skin Permeation meation rates solely depend on higher
thermodynamic activity of the api in more con-
To prove that emulsion films slow down and sus- centrated formulations (Hadgraft 2001). The dis-
tain dermal permeation of nonivamide, tinctly lower permeation coefficient, lower
permeation of nonivamide from dried emulsion cumulative amounts permeated and smaller
films was compared to permeation from proportion permeated from the emulsion films as
Hydrophilic Nonivamide Cream (HNC), which well as the correlation of cumulative amounts
releases nonivamide rapidly (Neues Rezeptur permeated to the square root of time prove that
Formularium 2010). To investigate nonivamide diffusion through the polymeric matrix instead of
permeation from film forming emulsions, formu- diffusion through skin is the rate limiting step in
lations containing 1% nonivamide and HNC con- drug delivery from emulsion films (Higuchi
taining nonivamide in concentrations used in the 1961, 1962). The permeation rates of nonivamide
therapy of chronic pain or pruritus were employed from emulsion films can be assumed to be appro-
(see Table19.3 for composition of HNCs). priate for therapy because they are equivalent to
An infinite dose set-up was used to be able to those of the hydrophilic creams which are fre-
calculate steady-state flux and allow parametrical quently used in the therapy of chronic pruritus.
comparison of permeation from different formu- Permeation rates and coefficients of nonivamide
lations. In this study, nonivamide permeation from both emulsion films are of the same magni-
from emulsion films was compared to noni- tude. Thus, it can be concluded that the nature of
vamide permeation from HNC in order to explore polymer does not largely affect permeation.
the capability of emulsion films to allow perme- Nevertheless, polymer composition does influ-
ation rates suitable for therapy. Additionally, ence mechanical properties of emulsion films.
finite dose experiments were performed to eluci- Emulsion films containing 40 parts NE exhibit
date the ability of emulsion films to sustain noni- superior properties with regard to adhesion and
vamide delivery to the skin. elongation and were therefore chosen for use in
Results of infinite dose permeation experi- finite dose experiments.
ments are shown in Fig.19.8a, b and Table19.4. Results of permeation experiments under a
It can be observed that after a lag time of 20h finite dose regime show that nonivamide perme-
nonivamide permeates the skin. The data clearly ates from HNC 0.05% for 69h only (Fig.19.9).
show that permeation rates from HNC increase In contrast, permeation from the optimized emul-
linearly with increasing nonivamide content sion films lasts for 24h and a constant perme-
whereas permeation coefficients remain constant. ation rate is maintained throughout the
Permeation from emulsion films is linear with the observation period. In contrast to the results of
330 D.J. Lunter and R. Daniels
a b
Fig. 19.8 In vitro skin permeation profiles of nonivamide of () film forming emulsion containing RS and () film
under infinite dose conditions: (a) comparison of () forming emulsion containing NE and RS; n=5; aver-
HNC 0.01%, () HNC 0.05%, () HNC 0.025%, () agestandard deviation
film forming emulsion containing RS and (b) comparison
Table 19.4 Permeation characteristics; n=5; meanstandard deviation; * statistically different data; HNC=hydro-
philic nonivamide cream
Cumulative amount Percentage
Permeation rate [g/ Permeation coefficient permeated after 48h permeated after 48h
cm2h1/2] [mL/cm2h1/2] [g/cm2] [%]
Film forming 1.50.5 1.50.5* 5.81.5 1.20.3*
emulsion containing
Eudragit RS
HNC 0.1% 4.20.3* 42.32.8 15.51.8* 32.63.8
HNC 0.05% 1.80.6 35.811.8 6.21.0 26.14.2
HNC 0.025% 0.90.2 38.07.1 3.10.3 26.12.5
Film forming 1.20.7 1.20.7 2.40.8 0.50.2
emulsion containing
Eudragit RS
Film forming 1.50.2 1.50.2 3.70.8 0.80.2
emulsion containing
Eudragit NE and
RS
HNC 0.1% 3.51.1* 34.511.4* 7.10.4* 12.05.1*
19 Retardation ofDermal Release byFilm Forming Emulsions 331
infinite dose experiments, the amount permeated of treatment intervals to 12 or even 24h by utili-
from the emulsion films is higher than the amount zation of emulsion films may therefore be
permeated from HNC 0.05% at any time point. possible.
The cumulative amount permeated from the
emulsion films is therefore five times higher than
that from HNC 0.05%. From the emulsion films, 19.4.2 In Vitro Skin Penetration
constant flux is achieved over a period of 24h.
After 48h merely 8.03.5% of the applied dose As the target structures of capsaicinoids are
permeated through skin. This indicates that the located within the viable epidermis (Anand and
amount of nonivamide in the formulation does Bley 2011; Ikoma 2010; Stnder and Luger
still suffice to maintain a constant flux. Extension 2010), it was of great importance to examine the
nonivamide concentration in the skin to ensure
that effective drug levels are obtained. As no dis-
tinct values of effective nonivamide concentra-
tions in skin exist, we decided to compare
nonivamide concentrations in skin achieved with
film forming emulsions to those obtained with
HNC 0.05%, because the effectiveness of this
formulation is proven clinically. Thus, we con-
cluded that film forming emulsions would also be
effective if they were able to produce drug levels
similar to those obtained with HNC 0.05%.
Results of penetration experiments
Fig. 19.9 In vitro skin permeation profiles of nonivamide (Fig.19.10a, b) reveal that nonivamide content in
under finite dose conditions comparison of () HNC
0.05% and () film forming emulsion containing NE and the stratum corneum is higher than in the viable
RS; n=5; averagestandard deviation skin, independent of formulation and incubation
a b
Fig. 19.10 In vitro skin penetration profiles of noni- NE and RS; drug amounts in () stratum corneum, ( )
vamide under finite dose conditions comparison of (a) epidermis, () dermis; n=3; averagestandard
HNC 0.05% and (b) film forming emulsion containing deviation
332 D.J. Lunter and R. Daniels
time. HNC 0.05% delivers only low amounts of rates and efficient api concentrations in skin
nonivamide to the skin within 1h. A maximum can be maintained for a period of 12h. This
concentration in all parts of the skin is apparent is a clear advantage over conventional semi-
after 4h. Although concentration in stratum cor- solid formulations as application intervals can
neum is still high after 12h, the amount in viable be extended. This results in enhanced compli-
skin decreases to a level similar to that obtained ance and ensures therapeutic success.
after 1h. The nonivamide amount that penetrated Furthermore, employment of film forming
into viable skin from the emulsion films after 1h emulsions is possible for a multitude of lipo-
is as small as for HNC 0.05%, but the amount philic substances using film forming emul-
present in stratum corneum is already 25 times sions as a drug delivery system which may be
higher. It increases substantially after 4 and 12h. tailored to specific needs of patients and will
Nonivamide concentrations in the viable skin are thereby allow sustained dermal therapy of
equivalent or even higher than those obtained various diseases.
with HNC 0.05%.
Substantially higher drug contents in stratum
corneum may be associated with the high lipo- AcknowledgmentsThe authors would like to thank
philicity of nonivamide (log PO/W: 3.74), which Martin Schenk and his team from the Department of
Experimental Medicine at the University of Tuebingen for
results in its accumulation in lipid rich tissue the supply of pig ears and Evonik Industries for the dona-
(Kasting 2001; Schlupp etal. 2010). The pres- tion of Eudragit dispersions.
ence of a maximum nonivamide concentration in
skin after 4h incubation with HNC 0.05% is in
accordance with the clinical experience, i.e., References
HNC has to be applied at least 4 times a day.
Results of penetration experiments employing Anand P, Bley K (2011) Topical capsaicin for pain man-
the optimized emulsion films clearly indicate that agement: therapeutic potential and mechanisms of
action of the new high-concentration capsaicin 8%
nonivamide concentrations achieved in epidermis patch. Br JAnaesth 107:490502
and dermis are of the same magnitude as those Grtzmann R, Wagner KG (2005) Quantification of the
observed with HNC 0.05%. In contrast to HNC leaching of triethyl citrate/polysorbate 80 mixtures
0.05%, the emulsion films maintain drug concen- from Eudragit RS films by differential scanning calo-
rimetry. Eur JPharm Biopharm 60:159162
tration in skin over a period of 12h. These find- Hadgraft J(2001) Skin, the final frontier. Int JPharm
ings suggest that therapy of chronic pain or 224:118
pruritus may be facilitated by film forming emul- Higuchi T (1961) Rate of release of medicaments from
sions as the treatment interval may be extended ointment bases containing drugs in suspension.
JPharm Sci 50:874875
substantially. Higuchi WI (1962) Analysis of data on the medicament
release from ointments. JPharm Sci 51:802804
Conclusion Ikoma A (2010) Neuroanatmy of itch. In: Misery L,
Stnder S (eds) Pruritus. Springer, NewYork, pp36
The results of this work elucidate the possi- Kasting GB (2001) Kinetics of finite dose absorption
bility of formulating sustained release semi- through skin 1. Vanillylnonanamide JPharm Sci
solid formulations with the innovative concept 90:202212
of film forming emulsions. We were able to Langer K (1978) On the anatomy and physiology of the
skin III.The elasticity of the cutis. Br JPlast Surg
formulate stable emulsions which form a 31:185199
film on skin that encapsulates the dispersed Lehmann K, Dreher D (1986) Mischbarkeit wssriger
oil phase. It is shown that the rate of perme- Poly(meth)acrylat-Dispersionen fr
ation of the active substance from its oily Arzneimittelberzge. Pharm Ind 48:11821183
Lunter DJ, Daniels R (2012a) New film forming emul-
solution is determined by diffusion through sions containing Eudragit NE and/or RS 30D for sus-
the polymeric matrix in which the droplets tained dermal delivery of nonivamide. Eur JPharm
are embedded. Thereby, constant permeation Biopharm 82:291298
19 Retardation ofDermal Release byFilm Forming Emulsions 333
Lunter DJ, Daniels R (2012b) In-vitro skin permeation three glucocorticoides. Skin Pharmacol Physiol
and penetration of nonivamide from novel film form- 24:199209
ing emulsions Stnder S, Luger TA (2010) Neuroreceptors and neurome-
Neues Rezeptur Formularium (2010) Hydrophile diators. In: Misery L, Stnder S (eds) Pruritus.
Capsaicinoid Creme 0,025 %/0,05 %/0,1 % (NRF Springer, NewYork, pp716
11.125). Govi Verlag, D-Eschborn Sumino H, Ichikawa S, Kasama S, Takahashi T, Kumakura
Schlupp P, Blaschke T, Kramer KD, Hltje H-D, Mehnert H, Takayama Y, Kanda T, Murakami M, Kurabayashi
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Part IV
Current Status of Dermal and Transdermal
Drug Delivery
Penetration-Enhancement
Strategies forDermal 20
andTransdermal Drug Delivery:
AnOverview ofRecent Research
Studies andPatents
SyedSarimImam andMohammedAqil
Contents
20.1 Introduction
20.1 Introduction 337
20.2 kin Penetration Enhancement
S The skin, the largest organ of the body, is com-
Techniques 338 posed of several layers: the stratum corneum
20.2.1 Microneedles 338
20.2.2 Sonophoresis 342
(SC) which is the uppermost layer, the viable
20.2.3 Electroporation 342 epidermis, the dermis, and the lower layers of
20.2.4 Magnetophoresis 343 adipose tissue (Schaefer and Redelmeier 1996;
20.2.5 Thermal Ablation 343 Carlos etal. 1999). Nevertheless, the SC is a
20.2.6 Photomechanical Waves 344
20.2.7 Inclusion Complex 344
remarkably efficient barrier, thus causing diffi-
20.2.8 Solid Lipid Nanoparticles (SLN) 344 culties for the transdermal delivery of therapeu-
20.2.9 Microemulsion 345 tic agents. The skin is a multilayered organ
20.2.10 Dendrimers 345 composed of many histological layers generally
20.2.11 Vesicles 345
described in terms of tissue layers, that is, the
20.3 ynergistic Use ofEnhancement
S epidermis and the dermis (Lai-Cheong and
Techniques 349
McGrath 2009). Epidermis, composed of kerati-
Conclusion 350 nocytes (95% cells bearing keratin), has been
References 350 used to conceptualize the barrier property of the
skin. The second layer beneath the epidermal
layer is the dermis, which is much thicker than
the epidermis (usually 14mm). The main com-
ponents of the dermis are collagen and elastic
fibers. Compared to the epidermis, there are
fewer cells and much more fibers in the dermis
(Igarashi etal. 2007). The components of trans-
dermal drug delivery systems (TDDS) are liners,
S.S. Imam
adherents, drug reservoirs, drug release mem-
Department of Pharmaceutics, Glocal School of
Pharmacy, The Glocal University, branes, etc. (Wokovich etal. 2006) that play an
Saharanpur 247121, Uttar Pradesh, India imperative role in the release of the drug from
e-mail: sarimimam@gmail.com the TDDS and in the drug permeation through
M. Aqil (*) the skin. It is considered that a well-designed
Department of Pharmaceutics, Faculty of Pharmacy, TDDS can supply the drug at a rate to sustain the
Hamdard University, M.B.Road,
required therapeutic plasma concentration with-
New Delhi 110062, India
e-mail: aqilmalik@yahoo.com out much fluctuation that may cause basic mani-
festation or therapeutic inefficacy (Thomas and the barrier function of the skin to allow easier
Finnin 2004). Lag times to reach steady-state passage of drugs into the dermal microcircula-
fluxes are in hours, as the transport of most drugs tion, as summarized in Table20.1 (Bogner and
across the skin is very slow. Attainment of a Wilkosz 2003; Rizwan etal. 2009b).
therapeutically effective drug level is, therefore,
difficult without enhancing skin permeation (Kai
etal. 1990). A number of techniques have been 20.2 Skin Penetration
developed to enhance and control transport Enhancement Techniques
across the skin, and enlarge the range of drugs
delivered. These involve chemical and physical An attempt was made to provide an overall over-
methods, based on two strategies: increasing view of information about transdermal patents
skin permeability and/or providing the driving and to summarize the different major penetration-
force acting on the drug (Foldvari 2000). There enhancement techniques used in cutaneous drug
have been many ingenious technologies devel- delivery systems (Table20.2).
oped to enhance percutaneous transport for ther-
apeutic and diagnostic purposes, ranging from
chemical enhancers to iontophoresis, electropor- 20.2.1 Microneedles
ation, and pressure waves generated by ultra-
sound effects or the synergistic mixtures of two Microneedles (MN) technology for enhanced
or more methods (Ahad etal. 2010). To enhance transdermal drug delivery was first reported by
transdermal absorption, different methodologies Henry etal (1998). Since then, there has been an
have been investigated and developed, including increasing interest in this technology. Increased
the use of drug derivatives, drug-saturated sys- skin permeability by using the microneedle tech-
tems, physical approaches, and chemical pene- nology involves creating larger transport path-
tration enhancers (sorption promoters) that ways (of micron dimensions) in the skin using
facilitate the diffusion of drugs through the SC arrays of microscopic needles. MN can penetrate
(Ahad etal. 2009; Asbill and Michniak 2000). the skin to create micron-sized pores that are big
The transdermal product has a promising future enough to permit the transport of macromole-
because of its noteworthy upward trend (Paudel cules and even microparticles (Prausnitz 2004a).
etal. 2010). The fate of effectiveness of a TDDS Safety studies performed with MN ranging in
lies on the drugs ability to invade the skin bar- length between 500 and 1500m showed that the
rier and how it reaches the targeted site (Prausnitz pain induced by the MN is significantly lower
etal. 2004b). The molecules permeate across the when compared to hypodermic needles (Gill and
SC by intercellular, intracellular (transcellular), Prausnitz 2007). Most drug delivery studies have
or follicular (appendageal) pathways. emphasized solid MN, which have been shown to
Transdermal drug delivery has been a subject of increase skin permeability to a broad range of
increasing research interest since the introduc- molecules. MN-mediated drug delivery has been
tion of the first transdermal product extensively investigated for transdermal delivery
TransdermScop patch developed by Alza Corp., of drug-loaded nanocarriers (Donnelly etal.
USA, for delivery of scopolamine, in 1981 2010; Gomaa etal. 2014). MN technology has
(Santus and Baker 1993). The patent literature is been disclosed in Patent No. 5879326 to Godshall
often a rich source of information that is unob- and Anderson (1999) and Patent No. 5250023 to
tainable and is an under-used resource mainly Lee and Shin (1993)). The use of micron-scale
due to legal style of writing. The result of the needles in increasing skin permeability has been
search of patents concerning transdermal skin proposed and shown to dramatically increase
penetration techniques shows to provide much transdermal drug delivery, especially of macro-
useful information. Several transport techniques molecules, such as insulin, proteins, oligonucle-
have been demonstrated to bypass or modulate otides, and vaccines (Henry etal. 1998; Prausnitz
20 Penetration-Enhancement Strategies forDermal andTransdermal Drug Delivery 339
Table 20.1 Transdermal drug delivery technologies and available commercial products
S.No Enhancement method Technology Manufacturer Drug
1 Iontophoresis E-Trans Alza Fentanyl
2 Iontophoresis Iontophoretic drug Iomed Lidocaine and
delivery system epinephrine
3 Microporation Microstructured 3M Hydrophilic
transdermal system molecules, large
molecules
4 Microporation Macroflux ZosanoPharma, Inc. Therapeutic protein
5 Needleless injector PowderJect PowderJect Insulin
Pharmaceuticals
6 Needleless injector IntraJect Weston Medical Vaccines
7 Microparticulate delivery SMP (solvent Atrix Labs Dapsone
microparticle system)
8 Thermal ablation Heat-aided drug delivery Zars, Inc. Lidocaine and
system tetracaine
9 Phonophoresis SonoPrep Echo Therapeutics, Inc Peptides, other
large molecules
etal. 2004a). Eppstein (2003) described a nonin- by Cho (2005). The base of microneedle was
vasive method for increasing the permeability of broad relative to height to minimize breakage
the skin, mucosal membrane through thermal during insertion. The microneedle device has few
microporation. Another approach that has been disadvantages, such as requiring a direct pushing
investigated more recently is an integrated deliv- motion against the skin and less efficient, com-
ery system comprising a hypodermic needle and pelling of drug fluid due to tiny openings.
a transdermal patch (Coulman etal. 2006a). Gartstein and Sherman (2006) suggested a micro-
Transdermal delivery of insulin invivo was stud- structured device (MN) for delivering the drug,
ied using solid MN, and it was demonstrated that which can provide efficient flow for some types
MN increased insulin delivery and thereby of fluid compounds through the SC.The micro-
decreased blood glucose levels in diabetic hair- structured device could penetrate the outer skin
less rats (Martanto etal. 2003). Patent of King layers by sliding or rubbing motions parallel to
and Walters (2003) claimed delivery of macro- the skin surface, rather than perpendicular to the
molecules into cells without employing a pres- skin surface. Wilkinson and Hwang (2006) pat-
surized fluid injection step. The macromolecules ented an invention entitled valved intradermal
in the electrode coating having a polynucleotide delivery device and method of intradermally
vaccine (deoxyribonucleic acid vaccine and/or delivering a substance to a patient. They
ribonucleic acid vaccine) or a protein-based vac- explained in their patent about controlled flow of
cine are administered using MN.Rosenberg pharmaceutical agents, such as a drug or vaccine,
(2003) provides a reliable way to deliver individ- through the device for the delivery into or below
ual and multiple pharmaceutical agents in small the SC to a depth sufficient for the pharmaceuti-
doses by an intradermal device (MN). The cal agent to be absorbed and utilized by the body.
multiple chambers of the delivery device enable US Patent No. 5,457,041 of Ginaven and Facciotti
the administration of multiple vaccines, adju- (1995) reported MN made up of silicon, which
vants, and pharmaceutical agents simultaneously carry a biologically active substance and are
without prior reformulation or combination of pierced into the target cells within the skin tissue.
pharmaceutical agents. A hollow microneedle of Thus, the biological substance is transferred from
the conical shaped body with beveled, noncoring the tip portion and deposited within the target
tip that is able to pierce tissue with broad base for cells of the skin tissue. Gertsek etal. (2003) dis-
transcutaneously conveying fluid was developed closed that MN have advantages of delivering a
Table 20.2 Different penetration enhancement techniques
340
reconstituted pharmaceutical agent, where the length of ultrasound pulse which is consistent with
dried substance is reconstituted by the dispensing a cavitation-based mechanism. Mitragotri etal.
diluent from the bladder within the device. MN (1998) used low-frequency (20 KHz) ultrasound
with suction pump (vacuum generator) were of intensity between 12.5 and 225mW/cm2 for
developed to provide painless, precised insertion, enhanced transdermal delivery of high-molecular
controlled depth penetration, and variable, pro- weight proteins. The application of ultrasound sig-
grammable delivery of active principles (Angel nificantly enhances permeation through human
etal. 2006). A vacuum generator was attached to cadaver skin, that is, by 3.3, 8, and 9.8 times for
MN, which creates suction at the site of applica- insulin, gamma interferon, and erythropoietin,
tion to penetrate the surface of the skin (Angel respectively. Therefore, it was possible to deliver
etal. 2006; Angel and Hunter 2008). This modi- an insulin dose of about 13 U/h from a patch hav-
fied device could be attached to the skin more ing an area of 100cm2 and providing an insulin
effectively than prior devices and can be made flux of 100mU/cm2/h. Weaver etal. (2012) dis-
with an extremely reproducible size and shape. closed in their patent that this method provides
Increased transdermal flux of polypeptides or rapid, controlled, and higher drug transdermal
proteins and improved attachment of the device fluxes, and allows drug delivery or analyte extrac-
to the skin with minimal to no skin irritation have tion at lower ultrasound intensities (f <100kHz)
been achieved by developing a device consisting than when ultrasound is applied in the absence of
of microblades (Cormier etal. 2007). These stud- an electric field. Shimada and Shapland (1993)
ies suggest that MN may provide a powerful new invented the use of multiple frequency sonophore-
approach for transdermal drug delivery of sis for enhancing the diffusion of substance to or
macromolecules, especially for the delivery of through the SC layer of the skin. This invention
proteins, DNA, and vaccines. used two different frequencies, that is, 1 and
3MHz. The method has many advantages such as
decreased required time to administer substances
20.2.2 Sonophoresis and increase in blood perfusion at the treated area,
which promote penetration of diffused substances
In 1990s, sonophoresis received a great attention across the skin membrane. Weimann (2007)
from researchers for the delivery of macromole- reported a device consisting of a suspension of
cules through the skin (Rizwan etal. 2009b). It encapsulated drug as microparticulate system in
uses low-frequency ultrasound (f <100kHz) that place of drug solution. The radiation frequency
enables the penetration of the drug. Commonly was applied for a short period of time at a distance
used frequencies for sonophoresis are generally from the skin area, effective to generate cavitation
separated into two: (i) low-frequency sonophore- bubbles. The penetration studies showed that mic-
sis (LFS) (range of 20100kHz), and (ii) high- roparticles of up to 25m penetrated into the skin
frequency sonophoresis (HFS), which includes under the conditions tested, whereas particles
frequencies in the range of 0.716MHz (Mitragotri larger than about 40m did not penetrate.
etal. 1995). The systemic absorption of drugs
through the transdermal route depends upon the
treatment duration, intensity, and frequency of 20.2.3 Electroporation
ultrasound (Rizwan etal. 2009b). Mitragotri and
associates reported in their two patents (US Patent This permeation enhancement technique involves
No. 6,002,961 (1999) and U.S. Patent No. the application of high-voltage pulse for a very
6,018,678 (2000)) about applications of low- short duration of time (<1s). The high-voltage
frequency (20 KHz) ultrasound for enhanced pulse forms aqueous pathway across the lipid
transdermal delivery of high-molecular weight bilayer of the skin (Prausnitz 1999; Rizwan
proteins. Their findings indicated that permeation etal. 2009b). The use of electroporation in
enhancement is dependent on the energy dose and transdermal delivery of macromolecules and
20 Penetration-Enhancement Strategies forDermal andTransdermal Drug Delivery 343
efficiency was evaluated. The developed formula- across the skin. The transdermal administration
tion showed improved effectiveness in skin protec- of oligonucleotides and nucleic acids offers
tion by reduced absorption of UV absorbers promise of simpler, easier, and less injurious
(Herzog. 2006). The patent of Constantinides administration without need for sterile proce-
etal. (2006) describes SLN suspensions for dures and their concomitant expenses.
mucosal and parenteral administration. However,
the developed formulation could also be adminis-
tered transdermally in the form of creams, oint- 20.2.10 Dendrimers
ments, etc. Muller and Olbrich (2004) described
SLN in their US patent No. 6,770,299 entitled The PAMAM dendrimers are widely used as
Lipid matrix-drug conjugates particle for con- transdermal carriers for drugs (Sonke 2009).
trolled release of active ingredient. The SLN These were proven to pass the cell membrane
was prepared by high-pressure homogenization barrier, and therefore they were used as transder-
in order to obtain particles of submicron size. mal carriers to transport several water-insoluble
Westesen and Siekmann (2001) used the emulsi- drugs into the cell membrane (Venuganti and
fying process to prepare SLN and reported about Perumal 2009; Borowska etal. 2010). They are
nonspherical particles showing controlled release three-dimensional nanosized (1100nm), highly
of poorly water-soluble drugs, primarily applied branched monodispersed macromolecules,
by i.v, and also by other administration routes obtained by chemical reaction and producing a
including the dermal route. unique structure (Tomalia etal. 1985). They con-
tain a dendritic core, which can accommodate
hydrophilic and hydrophobic drug molecules,
20.2.9 Microemulsion and they have been reported to release drugs in a
controlled manner (Umesh etal. 2006). The
Microemulsions (ME) are lipid-based drug deliv- PAMAM dendrimers themselves are biodegrad-
ery systems, which have been used to enhance able and nontoxic (Hans and Lowman 2002).
the transdermal permeation and bioavailability of Filipowicz and Woowiec (2011) prepared
poorly soluble drugs (Rizwan etal. 2009a). The riboflavin-loaded PAMAM dendrimers and con-
permeation enhancement largely depends on the cluded that the transdermal diffusion of den-
relative composition and concentration of the drimer complexes, both through PVDF
used oil, surfactant, and co-surfactant. Kreilgaard (polyvinylidenedifluoride) and PES (polyether-
etal (2000) studied the ME system loaded with sulfone) membranes was enhanced two to three
lipophilic (lidocaine) and hydrophilic (prilocaine times compared to control. The water-soluble
hydrochloride) drug molecules and compared the PAMAM dendrimers can be successfully used in
drug flux to the flux obtained with conventional cosmetic and dermatological emulsions
vehicles. The transdermal flux of both drugs was (Wollensak etal. 2003).
increased up to four and ten times, respectively,
as compared to a conventional formulation.
However, researchers also demonstrated the 20.2.11 Vesicles
enhancing effect of different individual constitu-
ents, such as oil and surfactant present in ME.US The most recent trend in dermal and transdermal
Patent No. 6,426,078 (Bauer etal 2002) described drug delivery is the use of vesicle formulations as
oil-in water ME for lipophilic vitamins composed delivery systems. Many vesicular carriers have
of diethylene glycol monoethyl ether or propyl- been reported in the literature which include lipo-
ene glycol as co-emulsifier. The co-surfactant somes (Mezei and Gulasekharam 1980), nio-
provided a considerable effect on the penetration somes (Niemiec etal. 1995), Transfersomes
of vitamins through the skin. The suitable combi- (Idea AG, Germany) (Cevc and Blume 1992), and
nation of surfactants has also influenced the flux invasomes (Dragicevic-Curic etal. 2008). These
346 S.S. Imam and M. Aqil
20.3 Synergistic Use reported that enhancers such as oleic acid and
ofEnhancement Techniques 1-
dodecylazacycloheptan-2-one (0.015%w/v)
in combination with iontophoresis provided
A number of synergistic techniques have been higher flux compared to the use of oleic acid or
used successfully to enhance transdermal drug iontophoresis alone. The use of liquid permeation
transport, and their combinations have been enhancers in an iontophoretic device is generally
hypothesized to be more effective in comparison limited by the occurrence of adverse interactions
to a single treatment (Table20.3). Many research- between the enhancer and drug, body surface, or
ers reported the use of binary combination tech- device components. Henley (1995) reported in his
niques; however, others used multiple techniques patent about the synergistic use of iontophoresis
simultaneously, including iontophoresis com- and ultrasonic transdermal delivery devices,
bined with chemical penetration enhancers; which can achieve greater skin penetration of
ultrasound combined with chemical penetra-
drugs and even larger peptide molecules such as
tion enhancers; iontophoresis combined with insulin for a prolonged period of time. Such
ultrasound; multiple combinational techniques device can be used to deliver cholecystokinin,
to achieve penetration enhancement of drugs nicotine, valium and naloxone, heparin (peptide),
(Henley 1995; Mitragotri etal. 1998; Rowe etal. fentanyl, and other therapeutic agents.
2001; Giammarusti 2004). Iontophoresis has been Seward (2006) and Kwiatkowski (2008)
synergistically used with chemical permeation employed iontophoresis/electroporation and an
enhancers, ultrasound, and liposomes, to further array of microneedles for enhanced delivery of
improve the drug penetration through the skin macromolecules. These combined systems have
(Kost etal. 2000; Francoeur and Potts 1991; the ability to deliver drugs in a controlled manner
Bommannan etal. 1992). for a prolonged period. Kwiatkowski (2008)
The combination of iontophoresis and developed a transdermal patch for therapeutic
chemical penetration enhancers has been agents that include two arrays of electrically con-
reported by Francoeur and Potts (1991). They ductive microprojections, and two reservoirs for
the same or different drugs. The microprojections and antiarthritic assessment. Drug Del Early Online
are adapted to provide a perforation of the epider- 16. doi:10.3109/10717544.2014.945130
Ahad A, Aqil M, Kanchan K, Hema C, Yasmin S, Mujeeb
mis to the depth of between about 50 and 150m. M, Sushama T (2009) Chemical penetration enhancers:
Rowe etal. (2001) disclosed a two-step noninva- a patent review. Expert Opin Ther Pat 19(7):969988
sive method which involves the application of Ahad A, Aqil M, Kohli K, Sultana Y, Mujeeb M, Ali
ultrasound to increase skin permeability followed A (2012) Formulation and optimization of nano-
transfersomes using experimental design technique
by the application of a chemical enhancer (alco- for accentuated transdermal delivery of valsartan.
hol, fatty acid, DMSO, and others) to further Nanomedicine 8(2):237249
increase the transdermal drug transport. Ahad A, Aqil M, Kohli K, Sultana Y, Mujeeb M, Ali
A technique to enhance the flux rate of bioac- A (2010) Transdermal drug delivery: the inherent
challenges and technological advancements. Asian
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microporation with different enhancement meth- Ahad A, Aqil M, Kohli K, Sultana Y, Mujeeb M (2013)
ods has also been reported (Ben-Amoz 1989; Enhanced transdermal delivery of an anti-hypertensive
Eppstein 2003; Giammarusti 2004). The low- agent via nanoethosomes: Statistical optimization,
characterization and pharmacokinetic assessment. Int
frequency ultrasound was used to enhance the JPharm 443:2638
transdermal delivery of high-molecular weight Ahmed S, Imam SS, Zafar A, Ali A, Aqil M, Gul A
proteins which was further enhanced and con- (2015) Invitro and preclinical assessment of factorial
trolled by the use of drug carriers, such as lipo- design based nanoethosomes transgel formulation of
an opioid analgesic. Art Cells Nanomed Biotech. doi:
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the combination of techniques is higher than the Allen Jr LV (2000) US6119036
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Perspectives onDermal Delivery
ofMacromolecular Drugs 21
MariannaFoldvari andP.Kumar
ucts including those intended for local tissue The combination of scientific and commercial
treatment (e.g. clonidine, estradiol, fentanyl, factors contributes to the timeliness to introduce
nitroglycerine, nicotine, testosterone, lidocaine, improvements in the delivery of biologics, which
diclofenac sodium). was previously not opportune.
However, the delivery of most biopharmaceu- So far, the only topically applied dermal or trans-
ticals is limited to parenteral administration dermal delivery systems available commercially
through IV, IM or SC injections. With increased is Regranex (becaplermin, recombinant human
number of macromolecules (DNA and recombi- platelet-derived growth factor, 25kDa) for lower
nant proteins) as drugs being discovered and extremity diabetic ulcers (OMJ Pharmaceuticals/
approved, new drug delivery technologies are Ortho-McNeil), which is applied on broken skin;
being explored to deliver these molecules through hence, localisation of the protein is the main
the skin (Prausnitz and Langer 2008). function of the delivery system, and permeability
One important area of progress involves pro- enhancement is not a factor in its efficacy.
tein pharmaceuticals. In the past several years, Another developing area is gene therapy.
therapeutic opportunities were greatly expanded Topical delivery of genetic material appears to be
by using biopharmaceuticals, such as proteins and promising, in that such delivery could be non-
peptides. Important classes of biopharmaceutical invasive and provide a more continuous supply of
compounds (biodrugs) include interferons and the protein within the skin. This approach could
cytokines, blood/clotting factors (erythropoietin), have further advantages over the delivery of pro-
growth hormones/factors, hormones (insulin), tein drugs: (i) the DNA is a more stable molecule
enzymes, monoclonal antibodies (mAbs) and vac- than the protein, (ii) the continuous expression of
cines (McCrudden etal. 2013). Biodrugs (new protein within the skin after topical administra-
biological entities or NBE) are becoming the most tion limits systemic exposure and (iii) topical
demanded medicines. Evaluate Pharma, for treatment can be self-administered by the patient.
example, shows that seven of the top ten drugs are However, these advantages are contingent upon
biopharmaceuticals in 2016. The PhRMA 2013 successful delivery of the DNA into the skin.
Annual Report (http://www.phrma.org/) lists over Current delivery and functional responses
900 biologics, including mAbs, vaccines, recom- after topical application of DNA are only possi-
binant proteins, cell therapy, gene therapy and ble on pretreated or damaged skin (use of depila-
antisense in development from Phase I to III trials tory lotion, scraping or stripping (Shi etal. 1999;
and submitted to the US Food and Drug Yu etal. 1999; Watabe etal. 2001)) to remove the
Administration (FDA) for more than 100 dis- stratum corneum, the main permeability barrier
eases. In addition to innovator biodrugs, the layer of the skin. For example, DermaVir, a DNA
generic versions, so-called biosimilars (FDA vaccine by Genetic Immunity, utilises a novel
recognises biosimilar and interchangeable bio- nanoparticle technology, consisting of mannosyl-
similar products) or follow-on biologics or bio- ated polyethyleneimine-plasmid complex, for
betters (a biologic with a structural modification topical HIV vaccination, which is applied on the
compared to the original and better performance) skin after dermabrasion (Lisziewicz etal. 2012).
are also emerging as important part of the biodrug Certain lipid-based delivery systems facilitate
pipeline. The global market size for biosimilars DNA uptake into intact skin, both invitro and
was $2.5B in 2011, which is expected to grow by invivo (Birchall etal. 2000; Delepine etal. 2000;
8% between 2012 and 2016. Currently, more than Xu etal. 1999), but the efficiency of these sys-
40 therapeutic mAbs are approved by EU and tems is still too low to produce therapeutic levels
FDA, and many more are under review. The mar- of protein. In the design of effective invivo sys-
ket for these is estimated at $58B by 2016 (BCC tems, the optimum invitro properties obtained in
Research). IMS Health estimated that by 2020, transfection experiments need to be extended to
most biologics that make up $64B of global sales also include optimum percutaneous permeation-
will be off patent. enhancer properties.
21 Perspectives onDermal Delivery ofMacromolecular Drugs 357
Currently, the most effective penetration into Birchall JC, Marichal C, Campbell L, Alwan A, Hadgraft
and permeation through the skin could be J, Gumbleton M (2000) Gene expression in an intact
ex-vivo skin tissue model following percutaneous
achieved by physical methods (e.g. microneedles delivery of cationic liposome-plasmid DNA com-
(Escobar-Chavez etal. 2011), thermal ablation plexes. Int JPharm 197:233238
(Lee etal. 2011)) and electrical methods (e.g. Bos JD, Meinardi MM (2000) The 500 Dalton rule for the
electroporation (Charoo etal. 2010), iontophore- skin penetration of chemical compounds and drugs.
Exp Dermatol 9:165169
sis (Gratieri etal. 2011)). Although the use of Cevc G, Gebauer D, Stieber J, Schatzlein A, Blume G
physical and electrical methods to enhance the (1998) Ultraflexible vesicles, Transfersomes, have an
drug permeation through the skin has shown extremely low pore penetration resistance and
some success in enhancing the delivery of both transport therapeutic amounts of insulin across the
intact mammalian skin. Biochim Biophys Acta 1368:
small and large molecules, there are still signifi- 201215
cant hurdles to overcome before approval. Charoo NA, Rahman Z, Repka MA, Murthy SN (2010)
Several non-invasive delivery vehicles, mostly Electroporation: an avenue for transdermal drug deliv-
lipid- or peptide-based (Baca-Estrada etal. ery. Curr Drug Deliv 7:125136
Delepine P, Guillaume C, Floch V, Loisel S, Yaouanc J,
2000a), have been evaluated for macromolecule Clement J, Des Abbayes H, Ferec C (2000) Cationic
delivery, such as liposomes (Schuetz etal. 2005; phosphonolipids as nonviral vectors: invitro and
Barry 2001), transfersomes (Benson 2006; Cevc invivo applications. JPharm Sci 89:629638
etal. 1998), niosomes (Schreief and Bouwstrab Elsabahy M, Foldvari M (2013) Needle-free gene delivery
through the skin: an overview of recent strategies.
1994; Shilpa etal. 2011) and solid lipid nanopar- Curr Pharm Des 19(41):73017315
ticles (Puglia and Bonina 2012). Peptide-targeting Escobar-Chavez JJ, Bonilla-Martinez D, Villegas-
ligands (Glogau etal. 2012) and biphasic vesicles Gonzalez MA, Molina-Trinidad E, Casas-Alancaster
(Biphasix) (Foldvari 2000; Foldvari etal. N, Revilla-Vazquez AL (2011) Microneedles: a valu-
able physical enhancer to increase transdermal drug
2010; King etal. 2002) developed in our labora- delivery. JClin Pharmacol 51:964977
tory were demonstrated to be effective in deliver- Foldvari M (2000) Non-invasive administration of drugs
ing several therapeutic proteins and vaccine through the skin: challenges in delivery system design.
antigens (Baca-Estrada etal. 2000b; Foldvari Pharm Sci Technol Today 3:417425
Foldvari M (2010) Biphasic vesicles: a novel topical drug
2010). delivery system. JBiomed Nanotechnol 6:543557
These delivery systems represent a trend Foldvari M, Badea I, Wettig S, Baboolal D, Kumar P,
towards potential protein and DNA delivery by Creagh AL, Haynes CA (2010) Topical delivery of
completely non-invasive techniques using some interferon alpha by biphasic vesicles: evidence for a
novel nanopathway across the stratum corneum. Mol
form of sophisticated carrier system made from a Pharm 7:751762
structurally and chemically synergistic mixture Glogau R, Blitzer A, Brandt F, Kane M, Monheit GD,
of chemical permeation enhancers and pharma- Waugh JM (2012) Results of a randomized, double-
ceutical excipients. blind, placebo-controlled study to evaluate the effi-
cacy and safety of a botulinum toxin type A topical gel
for the treatment of moderate-to-severe lateral canthal
lines. JDrugs Dermatol 11:3845
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Active Enhancement Methods
inTransdermal Drug Delivery: 22
Current Status andFuture
Perspectives
RyanF.Donnelly
Contents
22.1 Introduction
22.1 Introduction 359
22.2 Active Enhancement Strategies 360 Transdermal drug delivery has many advantages,
22.3 Moving Forwards 360 including:
Conclusion 364
References 365
Controlled delivery, achieving a steady-state
profile, thus reducing the likelihood of peak-
associated side effects and ensuring that drug
levels are above the minimal therapeutic
concentration
Reduced dosing frequency, with one transder-
mal patch delivering drug from typically
2472h
Avoidance of first-pass metabolism
Non-invasive means of drug delivery, putting
the patient in control (dosage form can be eas-
ily removed in the event of an adverse
reaction)
Less susceptible to bioavailability issues com-
pared to the oral route
Provides an alternative route when the patient
is unable to take drugs orally
to ensure delivery of pharmacologically effective have all gained immense attention for their prom-
doses of drugs not possessing the specific physi- ising applications, both in the pharmaceutical and
cochemical properties outlined above. Therefore, cosmeceutical industries. The ability of such
several active rate-controlled transdermal drug strategies to achieve therapeutically useful
delivery technologies (electrical-based, structure- plasma concentrations of hydrophilic drugs and
based, velocity-based, etc.) have been developed macromolecules and enhance vaccine efficacy
for the transdermal delivery of difficult drugs. whilst reducing dose is beyond doubt. However,
This is particularly so, given the high economic cost, safety and ease-of-use by patients remain
value of the transdermal delivery market, despite concerns.
the relatively small number of actives (currently
around 20) that can be delivered by this route.
Broadly, facilitated delivery falls into two catego- 22.3 Moving Forwards
ries: technological, of which microneedles, elec-
troporation, jet injectors, etc. are all good Given the ever-increasing evidence available
examples, and formulation approaches, most within the academic and patent literature that a
notably the focus on nanoscale delivery systems. wide variety of active enhancement strategies are
The latter is discussed elsewhere in this book. capable of achieving successful intradermal and
Here, we will concentrate on the current state of transdermal delivery of conventional drugs, bio-
play in active enhancement technologies used to pharmaceuticals, vaccines and active cosmeceu-
enhance transdermal drug delivery, possible tical ingredients, it is envisaged that the
future developments and key milestones that already-concerted industrial effort into develop-
must be achieved before patient benefit and com- ment of such devices will now intensify.
mercial return on investment can be realised. Furthermore, ever-more novel applications of
active enhancement technologies are likely to
come to the forefront. The ability of such devices
22.2 Active Enhancement to extract bodily fluids for drug and/or endoge-
Strategies nous analyte monitoring is particularly interest-
ing. For example, the use of microneedle-based
One of the most exciting developments currently devices to both deliver and extract molecules
ongoing in the transdermal drug delivery field is across the skin in a minimally invasive manner
the use of active delivery devices. Such devices opens up the possibility for the development of a
are minimally invasive in nature and can enhance closed-loop responsive device, with a
the permeability of a biological membrane by tar- microneedle-based delivery component deliver-
geting the shunt route (hair follicles and sweat ing a therapeutic molecule in response to infor-
glands) and creating transient aqueous transport mation provided by a microneedle-based
pathways of micron dimensions across that mem- monitoring component. As technological
brane. In transdermal drug delivery, these devices advances continue, active enhancement devices
force molecules across hair follicles and sweat may well become the pharmaceutical dosage
glands or create micropore pathways in the skins forms and monitoring devices of the near future.
stratum corneum barrier which, in turn, enables However, there are a number of barriers that will
delivery of a wide range of drug molecules. Often first need to be addressed in order for the various
hydrophilic small molecules, as well as macro- technologies in this field to progress.
molecules of biological origin (e.g. insulin, vac- The ultimate commercial success of active
cines), are investigated for delivery using such enhancement delivery and monitoring devices
strategies. Recently, such active devices, will depend upon not only the ability of these
namely, iontophoresis, laser, thermal ablation, devices to perform their intended function, but
electroporation, radiofrequency, ultrasound, also their overall acceptability by both health care
high-pressure jets and microneedle technology, professionals (e.g. doctors, nurses and pharma-
22 Active Enhancement Methods inTransdermal Drug Delivery: Current Status andFuture Perspectives 361
cists) and patients. Accordingly, efforts to ascer- itching, depending on the density of the applied
tain the views of these end-users will be essential current. Besides these uncomfortable but harm-
moving forward. A study by the Birchall Group in less effects, skin irritation is the most common
this regard on microneedle-based drug delivery local adverse effect of cutaneous iontophoresis
systems was highly informative (Birchall etal. (Roustit etal. 2014). It occurs at both the anode
2011). The majority of health care professionals and the cathode. Erythema is the most frequently
and members of the public recruited into this described adverse effect, with a variable fre-
focus group-centred study were able to appreciate quency according to the iontophoresis protocols.
the potential advantage of using microneedles, Skin irritation spontaneously and rapidly
including reduced pain, tissue damage, risk of resolves, does not lead to permanent skin damage
transmitting infections and needle-stick injuries, and does not disturb the barrier function of the
feasibility for self-administration and use in chil- skin. Although rare, burns have been observed,
dren, needlephobes and/or diabetics. However, mainly due to operator error and the incorrect
some concerns regarding effectiveness, means to choice of electrodes/formulation composition.
confirm successful drug delivery (such as a visual Indeed, the electrochemistry at bare metal or
dose indicator), delayed onset of action, cost of graphite electrodes involves electrolysis of water,
the delivery system, possible accidental use, mis- which induces changes in the pH of the skin by
use or abuse were also raised. Health care profes- generating H+ and OH at the positive and nega-
sionals were also concerned about interindividual tive electrodes, respectively. Variations in pH
variation in skin thickness, problems associated beyond the buffer capacities of the skin may lead
with injecting small volumes and risk of infection. to burns. High current density or prolonged appli-
Several other possible issues (accidental or errors cation, as well as positioning of the electrodes
based) and interesting doubts regarding micronee- over skin defects, increases the risk of burns.
dle use were discussed in this study. Overall, the Burns are generally more serious under the cath-
group reported that 100% of the public partici- ode, due to involvement of OH and rise in
pants and 74% of the health care professional par- pH.Indeed, an alkaline phase erodes the epider-
ticipants were optimistic about the future of mis and reduces skin resistance, making skin ero-
microneedle technology. Such studies, when sion worse. Therefore, an appropriate choice of
appropriately planned to capture the necessary buffer concentration in the formulation is needed
demographics, will undoubtedly aid industry in to reduce the risk of burns (Roustit etal. 2014). A
taking necessary action to address concerns and better solution is to use AgAgCl rather than bare
develop informative labelling and patient coun- metal or carbon-active electrodes, because they
selling strategies to ensure safe and effective use function at a lower potential and do not operate
of microporation-based devices. Marketing strate- by water electrolysis. Several simple recommen-
gies will, obviously, also be vitally important in dations can decrease the risk of skin injury, such
achieving maximum market shares relative to as avoiding pressure on the electrodes (not tap-
existing and widely used conventional delivery ing, binding or compressing either electrode) and
systems. ensuring that the electrode is uniformly wetted.
Of the most commonly investigated active Indeed, most commercially available electrodes
enhancement strategies, iontophoresis is the only are made of small sponges in contact with the
one that does not create transient aqueous micro- skin. After dampening the sponge with the drug
pores in the skin, but rather uses an electrical solution, it conducts the current. Therefore, het-
potential difference to force charged, polar or erogeneity in sponge dampening locally increases
neutral hydrophilic molecules of a wide range of current density and may lead to skin injury. In the
sizes (up to about 12,000 daltons) across the low same way, the adhesive seal should adhere uni-
resistance shunt route in the stratum corneum, formly to the skin to avoid leaks. Moreover,
consisting of hair follicles and sweat glands. cleansing the skin with alcohol and avoiding skin
Iontophoresis induces a sensation of tingling or defects and contact between metal components
362 R.F. Donnelly
and the skin are also recommended. Finally, cur- was 5.6- to 8.3-fold that of the s.c. route (Marbury
rent intensity should be <0.5mA cm2. etal. 2009). Given that it avoids patient exposure
Material defects are actually the main cause of to a rapid increase and high plasma concentrations
skin injuries, such as burns, usually resulting of sumatriptan in comparison to s.c. administra-
from direct contact between metal components tion, iontophoresis may reduce typical triptan-
and the skin. Another consequence of material related adverse events (i.e. chest tightness, chest
defects can be overdose, which is potentially heaviness, paresthesias and sedation/fatigue/mal-
harmful when drugs are delivered for a systemic aise) (Rapoport etal. 2010). A new drug applica-
action and have a narrow therapeutic index. The tion for an iontophoretic device containing
most striking recent example was the iontopho- sumatriptan (Zecuity, formerly Zelrix; NuPathe
retic delivery of fentanyl (Ionsys). After receiv- Inc., Conshohocken, PA, USA) has now been
ing marketing authorisation for the European approved by the US Food and Drug Administration
Union in 2006, corrosion of a component within (FDA) (Nupathe 2015). Other iontophoretically
the system was found in one batch. Although no delivered 5-HT1 agonists, such as zolmitriptan or
case of fentanyl overdose was reported, this almotriptan, have been studied recently; to date,
defect could have resulted in fentanyl release only preclinical data are available and suggest that
without activation by the patient. This could have these molecules are appropriate candidates for
exposed patients to fentanyl overdose, with a risk iontophoresis (Patel etal. 2009; Calatayud-Pascual
of severe respiratory depression. Ionsys has not etal. 2011).
been marketed in Europe since October 2008, Assessing the biological effects of ultrasound
and the marketing authorisation holder did not on tissues and cells has been investigated thor-
apply for renewal of authorisation. Ionsys has oughly. It has been commonly believed for
recently been acquired by another pharmaceuti- decades that ultrasound effects on tissues and
cal company (Incline Therapeutics Inc., Redwood cells are mainly through three mechanisms: cavi-
City CA, USA), currently developing new fea- tational (mainly inertial cavitation), thermal and
tures into the system in order to obtain regulatory acoustic streaming (Azagury etal. 2014). Recently
approval (Roustit etal. 2014). a new, non-thermal and non-cavitational mecha-
In most cases, iontophoretic devices are more nism for ultrasound effects on biological tissues
expensive than conventional topical and transder- has been proposed. There are innumerable devices
mal formulations. Although there are limited data in development that use sonophoresis technology.
about the cost effectiveness of iontophoresis, in Indeed, portable, pocket-size sonicators for drug
some cases, it has been shown to be in favour of injection and analyte monitoring are now avail-
iontophoresis compared with other formulations. able commercially (Azagury etal. 2014). In future
Among the drugs in the pipeline which could lead applications, ultrasound-based systems show
to new marketing applications soon, agonists of promise for immunisation with vaccines and in
the 5-HT1 family receptors used as antimigraine topical gene therapy. Some current applications
agents (triptans) have raised interest. Indeed, sub- consist of drug delivery, administration of tar-
cutaneous (s.c.) administration of sumatriptan geted therapeutic and diagnostic agents, detection
leads to a rapid but transient effect, whereas oral or and determination of analyte, termination of can-
nasal administration suffers from poor bioavail- cer tissues, fatty tissues or kidney and gall bladder
ability. The pharmacokinetics of iontophoretically stones (Azagury etal. 2014). Inovios electropor-
delivered sumatriptan (4mA for 1h followed by ation (high voltage, short duration) device also
2mA for 3h) in healthy subjects showed compa- creates transient aqueous pores in the stratum cor-
rable concentration over time areas under the neum and is currently under development for vac-
curve (AUC) to the s.c. route (Siegel etal. 2007; cine delivery (Inovio 2015).
Marbury etal. 2009), but the maximal concentra- In order to gain acceptance from health care
tion was about 30% of that by the s.c. route, professionals, patients and, importantly, regula-
whereas time to maximum plasma concentration tory authorities (e.g. the US FDA and the EMA in
22 Active Enhancement Methods inTransdermal Drug Delivery: Current Status andFuture Perspectives 363
Europe), it appears a strong possibility that cur- may be more inclined to remain on the more
rent safety concerns will need to be addressed. hydrophobic stratum corneum. Whether skin-
From a regulatory point of view, currently little is cleansing before microporation is necessary
known about the safety aspects that would be remains to be seen and is a vital question. Ideally,
involved with long-term usage of active enhance- this would not have to be done, so as to avoid
ment devices. In particular, studies will need to unnecessarily inconveniencing patients and mak-
be conducted to assess the effect that repeated ing the use of these products in the domiciliary set-
microporation or application of electrical energy ting appear more akin to a self- administered
or ultrasound has upon skin barrier function. injection than application of a conventional trans-
However, given the minimally invasive nature of dermal patch. Regulators will ultimately make the
the micropores created within the skin by key decisions based on the weight of available
microneedles or laser or thermal ablation, espe- evidence. Depending upon the application (e.g.
cially in comparison to the use of a hypodermic drug/vaccine/active cosmeceutical ingredient
needle, and the fact that statistically it is highly delivery or minimally invasive monitoring),
unlikely that micropores would be created at microporation-based devices may be classed as
exactly the same sites more than once in a drug delivery stems, consumer products or medi-
patients lifetime, it is envisaged that micropora- cal devices. From a delivery perspective, it will be
tion technologies will be shown to have a favour- important if microporation devices, such as
able safety profile. Indeed, skin barrier function microneedles or jet injectors are considered as
is known to completely recover within a few injections rather than topical/transdermal/intra-
hours of microneedle removal, for example, dermal delivery systems, since this will determine
regardless of how long the microneedles were in whether the final product will need to be steril-
place (Banks etal. 2010; Gomaa etal. 2010; ised, prepared under aseptic conditions or simply
Kalluri etal. 2011). Local irritation or erythema host a low bioburden. Any contained microorgan-
(reddening) of the skin may be an issue for some isms may need to be identified and quantified, as
patients. Since the skin is a potent immunostimu- may the pyrogen content. Should sterilisation be
latory organ, it would be interesting to know required, then the method chosen will be crucial,
whether repeated use of microporation devices to since the most commonly employed approaches
deliver biomolecules intradermally would ever (moist heat, gamma or microwave radiation, eth-
cause an immune reaction to the drug, especially ylene oxide) may adversely affect polymeric
considering the abundance of immune-presenting microneedles or electronic components of elec-
cells in this compartment (Al-Zahrani etal. troporation devices and/or any contained active
2012), and whether such an effect would be so ingredient (e.g. biomolecules).
significant as to cause problems for patients. Sterilisation and/or aseptic production will
Infection is an issue that has long been dis- add considerably to costs for manufacturers.
cussed in relation to use of microporation devices, Indeed, the practicalities involved in the large-
since they, by necessity, puncture the skins pro- scale production of microporation devices for
tective stratum corneum barrier. However, as we commercial applications will need to be carefully
and others have shown (Donnelly etal. 2009, considered. Currently, microporation devices are
2013; Wei-Ze etal. 2010), microbial penetration made by a wide variety of techniques, often in
through microneedle-induced holes is minimal. processes that are completely different to those
Indeed, there have never been any reports of used in the production of conventional dosage
microneedles or other microporation devices caus- forms. Often, multiple manufacturing steps are
ing skin or systemic infections. This may be required, particularly for coated microneedles,
because of the above-mentioned immune compo- for example, and those micromoulded from sili-
nent of the skin, or the skins inherent non-immune, con or metal masters. Silicon microneedles typi-
enzyme-based, defences. Alternatively, since the cally require clean room processing. As such, it
micropores are aqueous in nature, microorganisms would appear that any pharmaceutical or medical
364 R.F. Donnelly
devices firm wishing to commercialise micropor- Systeme AG, 3M, Theraject, Corium, Vaxxas and
ation technology would need to a make a signifi- Zosano Pharma; yet, no true microneedle array-
cant capital investment in order to design, develop based product has been marketed for drug deliv-
and optimise a cost-effective, reproducible, ery. Jet injectors, such as Bespaks SQ-Pen
method for mass production. Adaptation of stan- (Bespak 2015) and pellet-based skin delivery
dard quality control procedures will also be devices like Glide Pharmas SDI device (Glide
required. Ultimately, the regulatory specifica- Pharma 2015) already provide patients with a
tions applied to the first generations of micropor- level of assurance that they have been used appro-
ation products that reach the market may set the priately, and similar means of dosage confirma-
bar for all those that follow. Packaging of such tion could easily be built into radiofrequency,
devices will also be important, especially during iontophoresis, electroporation, sonophoresis,
transport and storage. Patient handling may only laser or thermal microporation devices. Some of
be controlled to a limited extent by effective these devices are, understandably, easier to use
product labelling and pharmacist-led counsel- by patients than others.
ling. Packaging should be sufficiently robust to
prevent damage, contamination or accidental Conclusion
release of active ingredients during storage. A wide variety of microporation strategies
Whilst a wide variety of microneedle applica- and devices have been shown to be effective
tor designs have been disclosed within the patient for the transdermal delivery of a diverse
literature, only a few, relatively crude, designs range of molecules, both invitro and invivo.
based upon high impact/velocity insertion, or The potential now exists to greatly expand
rotary devices have been described (Thakur etal. the range of types of drugs that can be deliv-
2011). Our own previous work has shown that ered effectively across the skin. This will sig-
application force has a significant role to play in nificantly enhance the value of the
microneedle insertion depth (Donnelly etal. transdermal delivery market and will be
2010). Clearly, patients cannot calibrate their increasingly important over the coming
hands and, so, will apply microneedles with dif- years, as the number of new drugs of biologi-
ferent forces. However, we have recently carried cal origin continues to increase. Small-scale
out a pilot clinical study showing consistent clinical trials have highlighted the minimally
depths of microneedle insertion into skin invivo invasive nature of microporation-based sys-
when applied by hand by human volunteers tems, causing no pain, minimal irritation if
counselled by a pharmacist and in receipt of a any and complete skin recovery within a few
patient information leaflet (Donnelly etal. 2014). hours. Vaccine-loaded microneedle arrays,
One of the key findings of this work was that for example, have been shown to elicit a
patients would like a level of assurance that the greater immune response in comparison to
microneedle device has actually been inserted conventional injections and have a number of
properly into their skin. This would be especially key advantages over the use of hypodermic
true in cases of global pandemics or bioterrorism needles. The ability of microorganisms to
incidents, where self-administration of traverse induced skin pores within the skin
microneedle- based vaccines becomes a neces- has been found to be minimal and with a
sity. Accordingly, a suitable means of confirming lower incidence for occurrence when com-
that skin puncture has taken place may need to be pared to the skin damage caused through
included within an applicator device or the hypodermic needle skin puncture.
microneedle product itself. Microneedle-based Microporation devices also have the poten-
products are currently being pursued by a num- tial for use in non-invasive therapeutic drug/
ber of companies, including Lhmann Therapie analyte monitoring, and the possibility for
22 Active Enhancement Methods inTransdermal Drug Delivery: Current Status andFuture Perspectives 365
closed-loop delivery systems may become are favoured most by patients, health care
important moving forward. Focus group professionals and regulators will be the
studies have identified key areas that need to one(s) that benefit patients and industry in
be addressed by the microneedles commu- the long term.
nity in order for the technology to progress.
These include a means to ensure reproduc-
ible microneedle application in every patient References
every time and confirmation of successful
device insertion. Similar studies are clearly Al-Zahrani S, Zaric M, McCrudden C, Scott C,
Kissenpfennig A, Donnelly RF (2012) Microneedle-
warranted for other microporation devices. A mediated vaccine delivery: harnessing cutaneous immu-
significant number of small and large indus- nobiology to improve efficacy. Expert Opin Drug Deliv
try players are presently engaged in clinical 9:541550
trials with the aim of commercialisation of Azagury A, Khoury L, Enden G, Kost J(2014) Ultrasound
mediated transdermal drug delivery. Adv Drug Deliv
their respective microporation-based devices. Rev 72:127143
Future studies will be needed to address Banks SL, Pinninti RR, Gill HS, Paudel KS, Crooks PA,
potential regulatory concerns over the use of Brogden NK, Prausnitz MR, Stinchcomb AL (2010)
such devices, as well as focussing on the Transdermal delivery of naltrexol and skin permeabil-
ity lifetime after microneedle treatment in hairless
design and development of processes to guinea pigs. Pharm Sci 99:30723080
enable a low-cost, efficient means for mass Birchall J, Clemo R, Anstey A, John D (2011)
production. Microneedles in clinical practice an explanatory
Overall, the future for the transdermal active study into the views and opinions of healthcare profes-
sionals and the public. Pharm Res 28:95106
delivery sector appears to be very bright, Calatayud-Pascual MA, Balaguer-Fernandez C, Serna-
with rapid expansion in fundamental new Jimenez CE, Del Rio-Sancho S, Femenia-Font A,
knowledge feeding industrial development. Merino V, Lopez-Castellano A (2011) Effect of ionto-
In due course, it is hoped that microporation- phoresis on in vitro transdermal absorption of almo-
triptan. Int JPharm 416:189194
based technological advancements will lead Donnelly RF, Thakur RRS, Tunney MM, Morrow DIJ,
to enhanced disease prevention, diagnosis McCarron PA, OMahony C, Woolfson AD (2009)
and control, with concomitant improvement Microneedle arrays allow lower microbial penetration
in health-related quality of life for patients than hypodermic needles in vitro. Pharm Res
26:25132522
worldwide. Of the active delivery strategies Donnelly RF, Garland MJ, Morrow DIJ, Migalska K,
currently available, microneedles appear the Thakur RRS, Majithiya R, Woolfson AD (2010)
most promising, in this authors opinion. Optical coherence tomography is a valuable tool in the
They are closest to conventional transdermal study of the effects of microneedle geometry on skin
penetration characteristics and in-skin dissolution.
patches in design and application, particu- JCont Rel 147:333341
larly if mechanical applicator devices are Donnelly RF, Singh TR, Alkilani AZ, McCrudden MT,
ultimately proven to be unnecessary. Other ONeill S, OMahony C, Armstrong K, McLoone N,
devices, while technologically more Kole P, Woolfson AD (2013) Hydrogel-forming
microneedle arrays exhibit antimicrobial properties:
advanced in some ways, may suffer from a potential for enhanced patient safety. Int JPharm
perceived closeness to conventional injec- 451:7691
tions (e.g. jet injectors), higher cost or patient Donnelly RF, Moffatt K, Zaid-Alkilani A, Vicente-Perez
confusion due to complex design and instruc- EM, Barry J, McCrudden MTC, Woolfson AD (2014)
Hydrogel-forming microneedle arrays can be effec-
tions (e.g. iontophoresis, sonophoresis, elec- tively inserted in skin by self-application: a pilot study
troporation or laser-based devices). centred on pharmacist intervention and a patient infor-
Ultimately, the market will decide which mation leaflet. Pharm Res 31:19891999
microporation technology proves to be most Gomaa YA, Morrow DIJ, Garland MJ, Donnelly RF,
El-Khordagui LK, Meidan VM (2010) Effects of
successful. Whichever strategy or strategies microneedle length, density, insertion time and
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tion: assessments by transepidermal water loss. Jackson D, Sebree T (2007) A unique iontophoretic
Toxicol In Vitro 24:19711978 patch for optimal transdermal delivery of sumatriptan.
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Marbury PM, ONeill T, Siegel C, Du S, Sebree W (2009) Recent Pat Drug Deliv Formul 5:1123
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Patel SR, Zhong H, Sharma A, Kalia YN (2009) ery applications. Int JPharm 389:122129
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Pharmacol 77:6371
Part V
Safety Issues and Ethics in Studies on
Dermal and Transdermal Delivery
Efficacy, Safety andTargets
inTopical andTransdermal Active 23
andExcipient Delivery
YousufH.Mohammed, HamidR.Moghimi,
ShereenA.Yousef, NavinC.Chandrasekaran,
CsaR.Bibi, SindujaC.Sukumar, JeffreyE.Grice,
WedadSakran, andMichaelS.Roberts
and skin atrophy after application of topical cor- pes simplex virus type 1 infections in hairless
ticosteroids in plaque psoriasis for the period mice. Figure23.2 shows a composite graph of
1980 to January 2011 found morning cortisol was their findings. It is evident that a similar concentra-
reduced in 048% of patients in 11 short-term tion response relationship exists for the 95%
studies (Castela etal. 2012). Thong etal. (2007) dimethyl sulfoxide (DMSO)+5% Azone for both
have listed a range of topical drugs and chemicals BDVU and ACV and for the 95% DMSO only
known to cause systemic effects. The agents formulation for ACV, whereas much higher
include anti-infectives, antihistamines, minoxi- BVDU C* values are needed for the BVDU in
dil, insecticides, solvents and steroids. 95% DMSO only formulation. They surmise that
The corollary measure to safety is cutaneous Azone could have two effects. Firstly, it may either
efficacy and, in general, the more potent the topi- enhance penetration of the active. This is evident
cal product used, the less skin flux required to from the 6.5 times higher C* for 5% ACV in 95%
achieve a desired local cutaneous effect after DMSO +5% Azone than in the 95% DMSO only
topical application. A commonly used approach, formulation. The effect is less for BVDU, where
analogous to that described earlier for toxicity, is the C* for 5% BVDU in 95% DMSO +5% Azone
to estimate the free drug concentration at the is 1.65 times that of 95% DMSO only formula-
local target site (C*), which, for antivirals, is tion. Secondly, Azone may have a synergistic
assumed to be the epidermal basal cell layer effect with the antiviral agents on viral kill, evident
(Patel etal. 1996). Afouna etal. (1998) analysed in the shift towards higher potencies of C* for
the receptor solutions for full thickness hairless BVDU but not for ACV (Fig.23.2).
mouse skin mounted in Franz diffusion cells. A key assumption made in this analysis is that
Assuming that receptor sink conditions applied, the dermal permeability coefficient (which is
C* was estimated as the ratio of the steady state normally expressed in the pharmacokinetic liter-
penetration flux (Js) to invivo dermis permeabil- ature as a dermal clearance (Cld) (Siddiqui etal.
ity coefficient (kp, d): 1989) (i.e. k p , d = Cld / A , where A is the surface
area of application) can be estimated by the ACV
Js
C* = (23.1) skin penetration flux that yields 50% topical effi-
k p,d cacy (J50) divided by the free plasma levels of
These C* values derived for various formulations ACV that inhibit 50 % of cutaneous lesions
of bromovinyldeoxyuridine (BVDU) and acyclo- treated systemically with ACV (C*50), i.e.
vir (ACV) were then compared with the invivo k p , d = J 50 / C *50 . It is also evident that systemic
efficacy of the formulations against cutaneous her- efficacy of various formulations is much less than
372 Y.H. Mohammed et al.
when applied topically, showing the enhanced the penetration flux divided by the clearance from
response that can be achieved by targeted topical the viable epidermis. Cordero etal. assumed that
application. this clearance, in turn, was related to the invitro
Davis has recast the free drug concentration dermis diffusion and thickness. In reality, as dis-
approach described above in Eq. (23.2), which cussed earlier in referring to the work of Afouna
describes the efficacy of the active in terms of both etal (1998), this should be invivo clearance. Our
potency of the active and how much penetrates to own work supports this, suggesting that, firstly,
a target site (Davis 2008; Jepps etal. 2013): uptake by the blood in the dermal capillaries
located just below the viable epidermis is likely to
Efficacy = Potency Delivery (23.2)
be the rate limiting determinant of clearance
A major issue not fully recognized in the work of invivo (Cross and Roberts 2006) and secondly,
Cordero and Davis (Cordero etal. 2001; Davis carriage of topical non-steroidal anti-inflammatory
2008) is that the fraction of active bound in the drugs (NSAIDS) to deeper tissues below that
invitro system used to determine potency may application site is also dependent on blood flow
differ from that present in skin penetration studies for highly plasma protein-bound drugs (Dancik
and invivo. In order to simplify the present etal. 2013a, b). Accordingly, we have, for illustra-
description and be consistent with the work tive purposes, assumed both constant binding and
reported by Cordero and Davis, this important clearance and expressed efficacy simply as a ratio
correction will not be included in the following of flux divided by the invitro minimum inhibitory
discussion. Hence, if a cutaneous concentration concentration (MIC), as originally proposed by
for a desired effect is defined as Ceff and that Mertin and Lippold (Mertin and Lippold 1997).
observed after topical application as Cve, the effi- Thus, a greater efficacy is provided by an active
cacy is given by the ratio Cve/Ceff. Cve is defined by with either a higher flux or a greater potency (a
23 Efficacy, Safety andTargets inTopical andTransdermal Active andExcipient Delivery 373
Fig. 23.3(a) Saturated skin flux, (b) invitro anti- 23.1.2 Systemic Concentration
inflammatory activity (MIC) and (c) likely clinical efficacy ofSolutes andTheir Effect
of various non-steroidal anti-inflammatory drugs, expressed onActive Performance
as an efficacy coefficient, calculated as saturated flux/MIC
potency for a range of non-steroidal anti-inflammatory drugs
with varying values for logarithm octanolwater partition The systemic efficacy of many actives, such as
coefficient (log P). Flux and MIC data from Cordero etal. anticonvulsants, anti-infective and cardiac drugs,
(2001) (closed symbols) (Adapted from Jepps etal. (2013)) can be defined by plasma concentrations after
dosing by a particular route of administration. As
lower dose for the same effect). These concepts are shown in Fig.23.5, in transdermal delivery occur-
illustrated in Fig.23.3, where data from Cordero ring from a constant rate transdermal passive
etal. and others for saturated skin flux (Fig.23.3a), delivery patch, there is a lag prior to reaching
374 Y.H. Mohammed et al.
Cv
J s = J sat = J sat f v (23.4)
Sv
Fig. 23.4 Comparison of topical efficacy and systemic As is evident from Eq. (23.4), one way to
safety indices for topical immunosuppressive agents, cyclo- maximize skin penetration is to use a fractional
sporin A, tacrolimus and pimecrolimus treatment in atopic
dermatitis. The corresponding margins of safety are 2400, solubility approaching unity. Poulsen led a range
46 and 457, respectively (Adapted from Davis (2008)) of studies that showed maximal skin penetration
will occur when an active is at saturation in a for-
maximal levels and in returning to baseline on mulation (Poulsen etal. 1978).
patch removal (Roberts and Walters 2007). As discussed in depth by Davis (2008), if an
Further, after reaching a maximum, the serum active is used at a concentration below its solu-
levels slowly decline with time, consistent with a bility in the vehicle, the product will normally
reduction in flux due to a gradual depletion in the have a lower efficacy than if the product was
amount of active in the patch. used at saturation. Consequently, products may
Topical products, especially transdermal be formulated to contain more active than will
patches, are often required to provide constant ther- actually be needed for an adequate effect, as is
apeutically effective plasma concentrations, Css. the case with transdermal fentanyl patches,
The penetration flux Js ideally needed to reach such which have led to numerous deaths. The danger
concentrations is given by Eq. (23.3), where Clbody inherent to fentanyl is its potency (greater than
is the body clearance and Fs is the availability 50100 times that of morphine) and rapidity of
action (Jumbelic 2010). An optimal dosing
Css Clbody strategy demands that the rate of active penetra-
Js = (23.3)
Fs tion into the skin will achieve and sustain bio-
Table 23.1 shows some estimated transdermal logically active free levels at the target site
fluxes required for the topical administration of a within the specified limits. Also, ideally, these
number of pharmaceuticals used in transdermal conditions should be independent of skin site
systems, by substituting desired plasma concen- and skin condition. This is a considerable chal-
trations and body clearances into Eq. (23.3). lenge, given that there is wide variability in skin
permeation, depending on the anatomical site
(Rougier etal. 1988) and the condition of the
23.1.3 Enhancing Efficacy skin, such as in particular disease states (Elias
byIncreasing Fractional and Feingold 1992) or levels of hydration
Solubility ofanActive (Roberts and Walker 1993).
intheFormulation As discussed in other chapters in this book,
another strategy to increase efficacy is to use a
A key goal in formulating an active in a transder- saturated active solution in the vehicle and to fur-
mal product is to obtain a desired steady state ther increase either the stratum corneum solubility
skin penetration flux (amount penetrated over a Ssc and/or its diffusivity Dsc using co-solvents and
23 Efficacy, Safety andTargets inTopical andTransdermal Active andExcipient Delivery 375
enhancers, in accordance with Eq. (23.5), where mulations across silicone membranes.
hsc is the stratum corneum thickness: Supersaturated formulations containing either
propylene glycol (PG)/water or PG/ethanol were
J sat = Ssc Dsc / hsc (23.5)
prepared with varying degrees of saturation (DS)
However, care has to be applied in representing of fentanyl. Both PG/water and PG/ethanol for-
such saturated fluxes as maximum fluxes because mulations showed good correlation between the
increasingly, formulations are being designed to flux and DS.The enhancement observed for PG/
include a volatile component that may result in ethanol formulations confirmed that enhanced
enhanced skin penetration due to the generation active thermodynamic activity was induced due
of a transient supersaturated state. Many research- to ethanol evaporation. In further studies, tape-
ers have now promoted the use of supersaturated stripping was used to show that supersaturation
solutions (Coldman etal. 1969), with several of the active is maintained in the outer layers.
showing that these can lead to even higher fluxes However, if the active lacks potency, a pene-
than those achieved with saturated solutions trating active will still prove to be inefficacious.
(Lippold 1992; Morgan etal. 1998; Iervolino Obviously, an improved evaluation of product
etal. 2001; Timothy etal. 2002; Santos etal. efficacy and safety will contribute to better thera-
2012). More recently, there is an increasing ten- peutic outcomes. Whereas the determination of
dency to either stabilize supersaturated products bioequivalence for two solid oral dosage forms is
by using polymers (Raghavan etal. 2003) or to based on a comparison of active and/or its metab-
generate supersaturation on application to the olite concentrations in blood or urine after dosing
skin as a consequence of the evaporation of the from a generic product or that of an innovator in
volatile components in the product (Hadgraft and healthy volunteers, such considerations are really
Lane 2011). Supersaturated systems may be only appropriate for transdermal products meant
obtained either by design or via solvent evapora- for systemic effect. For most topically acting
tion or by mixing of co-solvents. The stability of actives, such an approach is not appropriate as
supersaturated solutions is an important issue, the site of action is local, not systemic. Hence,
and it would be useful to develop supersaturated generic dermatological products are only required
systems that are stable throughout the product to demonstrate bioequivalence in clinical trials,
shelf life. Santos etal. (2011) investigated the including the vasoconstrictor test used to assess
permeation of fentanyl from supersaturated for- the efficacy of topical corticosteroids (Shah etal.
Table 23.1 Transdermal delivery
376
Fig. 23.6 Target organs and sites for some common topically and transdermally delivered substances, highlighting the
skin structure and various points of entry
1998). Franz etal. (2009) suggested that a substi- on active delivery choices. Testing for safety of
tute for clinical bioequivalence testing is to define cosmetic preparations will be discussed in the
the penetration fluxes of an active through excised following sections.
human skin. Their evaluation, based on glucocor- A chemists role in overcoming an actives
ticosteroids and generic tretinoin gels, indicated physicochemical properties to make it suitable for
that the invitro model had greater sensitivity than topical and transdermal delivery is a complicated
the clinical method in detecting small differences task. Formulators have made use of physicochem-
between two products (Franz etal. 2009). It is ical characteristics like molecular weight to stop
unclear, however, how this approach can over- an active from penetrating through the skin. One
come the well-known wide variability in excised such example is a high molecular weight
human skin permeability. More recently, Lehman (>500Da) UVA and UVB absorber Tinosorb M
and Franz have assessed the bioequivalence of (bisoctrizole, INCI: methylene bis-benzotriazolyl
topical retinoid products using pharmacody- tetramethylbutylphenol (and) aqua (and) decyl
namic assays (Lehman and Franz 2012). glucoside (and) propylene glycol (and) xanthan
gum) developed by Ciba Chemicals (now part of
BASF, Ludwigshafen, Germany). Similarly
23.2 R
ole ofIntended Targets
LOrals sunscreen with Uvinul N539 (2-ethyl-
onTopical andTransdermal
hexyl 2-cyano-3,3-diphenyl-2-propenoate, INCI:
Delivery
octocrylene), Parsol 1789 (avobenzone; INCI:
butyl methoxydibenzoylmethane) and Mexoryl
There is a range of skin and systemic sites often
SX (MW-563; ecamsule, INCI: terephthalylidene
targeted by topical and transdermal delivery,
dicamphor sulfon) was formulated to reduce
summarized in Fig.23.6. In this section, we illus-
solar-UV-induced skin damage (Seit etal. 2000).
trate some of the actives and their formulations
Sunscreens containing ecamsule (Mexoryl SX)
used to achieve appropriate targeted delivery by
are exclusive to LOral and its brands.
topical or transdermal delivery.
Chlorhexidine is another example of a mole-
cule which binds extensively to the skin surface
23.2.1 Topical Delivery due to its cationic nature (Judd etal. 2013). This
ensures that it does not penetrate into the skin and
The skin is the site of application of numerous rather provides for a sustained antimicrobial action
cosmetics and cosmeceutical preparations. In this when used topically (Karpanen etal. 2008). Insect
section, we will discuss the importance of targets repellents are another good example of actives that
378 Y.H. Mohammed et al.
are not intended to penetrate. Penetration retar- cial in treating long-term photoaged and photo-
dants and modifiers have been used for this pur- damaged skin (Cho etal. 2005). The stratum
pose (Kaushik etal. 2010). On the other hand, corneum is a major barrier to the penetration of
terbinafine, a highly lipophilic but equally potent lipophilic Vitamin E (Cassano etal. 2009), and
antifungal active, with a log P of 5.5, needs a dif- hydrophilic Vitamin C is prone to excessive oxi-
ferent approach. When formulated as a micro- dation. Oxidation of Vitamin C is triggered by
emulsion to enhance its stratum corneum solubility its ionization in aqueous solution (Stamford
using a mixture of surfactant, co-surfactant and 2012). Another example where erythema and
oil, terbinafine exhibited enhanced permeation papular oedema lesions are seen is Herpes labi-
parameters. Microbiological studies of the micro- alis. In a recent study, iontophoretic application
emulsion showed better antifungal activity against of 5% acyclovir cream was tested in a placebo-
Candida albicans and Aspergillus flavus compared controlled trial for treatment of herpes, among
to marketed products (Baboota etal. 2007). 200 patients with an incipient cold sore outbreak
Retinol, with a high molecular weight and lipophi- at the erythema or papular oedema lesion stage.
licity, is another active that is used for topical treat- A 20min iontophoretic treatment cycle short-
ment of acne. These unfavourable properties were ened the median classic lesion healing time by
overcome by delivering Retinol using solid lipid 35 h in the active group when compared to the
nanoparticles (SLN). The suitability of the SLNs control (Morrel etal. 2006). Steroids have also
was compared to a nanoemulsion of the same size, been popularly used in the treatment of ery-
with respect to the ability to influence active pen- thema and other inflammatory skin conditions
etration and distribution within the skin. In both (Perry and Trafeli 2009; Paller 2012). Numerous
formulations, 500g retinol was applied to por- studies have examined various factors control-
cine skin. Following the SLN dispersion for 6h, a ling percutaneous absorption rates and dermal
high retinol concentration was found in the stra- clearances of steroids (Siddiqui etal. 1989). The
tum corneum and upper epidermis (approximately lipophilicity of steroids has been associated
3400ng or 0.68% in the top layer). The nanoemul- with their tissue accumulation and toxic effects.
sion, however, only transported 2500ng (0.5%) However, Magnusson etal. showed that the tis-
into the upper skin strata (Jenning etal. 2000). sue accumulation of steroids was not linearly
For specific disease, conditions where the related to their lipophilicity, but the binding of
surface of the skin itself is the target of therapy, steroids to tissue components led to their accu-
formulation and delivery strategy are of greatest mulation (Magnusson etal. 2006). Long-term
importance. Creams, lotions, emulsions and corticosteroid usage has been associated with
other topical delivery vehicles have been used skin deterioration, abnormal skin aging and
effectively for this. The challenge here is to cutaneous mast cell depletion (Lavker and
modify the formulation according to the active Schechter 1985; Gonzalez and Sethi 2012).
characteristics and skin condition. Being the
largest organ, skin is exposed to the damaging
effects of UVA and UVB radiation, often lead- 23.2.2 Targeting ofKeratinocytes
ing to erythema. Antioxidants such as Vitamin
A, Vitamin E and Vitamin C have shown benefi- One of the most widely explored aims of cellular
cial effects in reducing oxidative damage to targeting has been in the viable epidermis itself.
critical cellular components (Trevithick etal. Inflammatory conditions broadly known as der-
1993; Stamford 2012). While the photoprotec- matitis or eczema are largely reflected in epider-
tive effect of topical antioxidants applied before mal involvement (Elias etal. 1999), including
UV exposure is well recognized, the beneficial spongiosis, acantosis and parakeratosis, as well
effect of these compounds when administered as some dermal involvement (Freeman-Anderson
after irradiation is less obvious (Wu etal. 2012). etal. 2008). The classical treatment for such con-
Only Vitamin A has been proven to be benefi- ditions is the use of topical corticosteroids, which
23 Efficacy, Safety andTargets inTopical andTransdermal Active andExcipient Delivery 379
can be classified on a four-point scale of potency role of these peptides may provide new strategies
(IIV, mild, moderate, potent, very potent), based for the treatment of diseases such as psoriasis
on their vasoconstrictor effects in humans. The (Harder and Schrder 2002).
body site to be treated determines the potency of
the steroid to be used. For example, the face
should be treated with mild corticosteroids only, 23.2.3 Targeting ofMelanocytes
such as hydrocortisone and hydrocortisone ace-
tate. Care needs to be taken with the more potent The synthesis and distribution of melanin contrib-
steroids and in the treatment of chronic condi- ute to skin and hair colour. However, melanin and
tions. Intermittent dosing can be effective in these pigmentation-associated skin cancers are a major
circumstances. problem, while there is also interest in cosmetic
Salicylic acid has long been used for condi- skin whitening treatments where increased epi-
tions such as psoriasis and ichthyosis that require dermal melanin synthesis causes unwanted dark-
keratolytic treatment. At lower concentrations, ening of the skin. Accurate determination of
salicylic acid is known to have keratoplastic cutaneous melanin in normal skin and in disease
effects; however at higher concentrations, it is conditions requires the collection of skin biopsies.
found to be keratolytic (Gloor and Beier 1984; However, our recent work has used multiphoton
Schwarb etal. 1999). Coal tar, retinoids and tomography and fluorescence lifetime imaging to
anthralin, as well as corticosteroids are some non-invasively measure levels of epidermal mela-
other commonly used treatments for psoriasis nin, which can be related to the visible skin colour
(Mitra and Wu 2010). Psoriatic skin is one such (Dancik etal. 2013a, b). The use of these tech-
example where the rigidization of skin, niques has implications for lesion diagnosis and
ascribed to an increase in the levels of cholesterol assessment of therapeutic progress.
and fall in the levels of ceramides and the lack of Proteins and peptides play an important role in
hydration, makes the topical route a big chal- skin health as structural components of the skin as
lenge (Wertz etal. 1989). The intricacies of topi- well as activators, regulators or inhibitors of cer-
cal delivery into psoriatic skin have lately been tain biochemical processes occurring in the skin
addressed by lipoidal carrier systems, such as (Heidl 2009). Peptides as cosmeceuticals are a
liposomes. Recent work by Ward etal. describes fast-growing segment of the personal care indus-
the delivery of novel immunotherapy using a try with an increasing trend towards the use of
liposomal platform. Liposomes synthesized to these agents in skin care regimens for protection
incorporate clodronate were injected intrader- from radiation and oxidant damage (Brandt etal.
mally into KC-Tie2 mice. An elimination of the 2011). Peptides also play an important role in
CD11c+, F4 80+ and CD11b+cells was seen in melanogenesis. Melanocyte-inhibiting factor
the skin, and the CD8+ T-cell numbers returned (also known as Pro-Leu-Gly-NH2, Melanostatin,
to levels seen in control mice. Further, there was MSH release-inhibiting hormone or MIF-1) is an
also a marked reduction in the levels of interleu- endogenous peptide fragment derived from oxy-
kin (IL)-1a, IL-6, IL-23 and tumour necrosis fac- tocin (Taleisnik 1971). Melanostatin is shown to
tor (TNF) (Ward etal. 2011). Peptides and inhibit melanin formation in Streptomyces bikini-
proteins such as elafin and psoriasin are known to ensis NRRLB-1049 and B16 melanoma cells. A
be highly upregulated in psoriatic skin (Madsen patent filed in 2010 describes a one-step pro-
etal. 1991; Nonomura etal. 1994; Namjoshi cess for production of esters and acetylated forms
etal. 2008). Peptide T analogue, DAPTA, was of peptides, describing melanostatin (MIF-1).
shown in clinical trials to clear psoriasis lesions This chemical modification approach improved
and significantly inhibit the monocyte and lym- metabolic properties leading to increased effi-
phocyte chemotactic properties of RANTES (a ciency for therapeutic and cosmetic purposes
beta chemokine found in increased amounts in including transdermal and topical administration
psoriatic lesions). A better understanding of the (Skinner 2007). Melanocyte-stimulating hormone
380 Y.H. Mohammed et al.
analogues that function as melanocortin 1 recep- There are many physical approaches, both old
tor (MC1-R) agonists have been investigated as and recent for targeting LCs; we will try to men-
potential topical agents to prevent skin photo- tion them in brief. The most well-known tech-
carcinogenesis. In cultured human melanocytes, nique was: (a) Needle and Syringe: This is an
tetrapeptide -MSH analogues, Ac-His-D-Phe- inefficient and indirect way of targeting the den-
Arg-Trp-NH2, N-pentadecanoyl and 4-phenylbu- dritic cells with DNA.It has resulted in modest
tyryl-His-d-Phe-Arg-Trp-NH2 were more potent immune responses (Mumper and Ledebur 2001).
than -MSH in stimulating the activity of mela- Other disadvantages include risks due to needle
nogenesis, reducing apoptosis and release of stick injuries (Gill etal. 2009; Sullivan etal.
hydrogen peroxide and enhancing repair of 2010). (b) Liquid Jet Injector: This is a needle-
deoxyribonucleic acid (DNA) photoproducts in free approach using high-speed liquid jet injec-
melanocytes exposed to UV radiation (Abdel tors (Furth etal. 1995) (Mark 2008). This
Malek 2006). Melanostatin-5 (Aqua- Dextran- technique has seen a recent resurgence, with liq-
Nonapeptide-1) is a new skin lightening biomi- uid delivered around the LCs in gene transfer and
metic peptide. This peptide acts as a -MSH DNA vaccination experiments (Furth etal. 1995).
antagonist thus preventing and lightening hyper- Current liquid jet injectors typically disrupt the
pigmentation (Dhatt 2006). Melanostatin-5 pro- skin in the epidermal and dermal layers to target
duces noticeable skin lightening by at least 33% LCs. Liquid jet injectors however have been
when formulated into a lightening product, and reported to cause pain to the patients. (c)
showed continued improvement over time. Microneedle Arrays: Unlike current liquid jet
injectors, microneedles can accurately target the
viable epidermis, are simple to use when built up
23.2.4 Targeting ofLangerhans Cells as patches and are relatively pain free. McAllister
etal. (2003) have shown that they can be made
Skin has evolved a highly competent immuno- from a range of materials, including silicon,
logical function, in Langerhans cells (LCs esti- metal and other biodegradable polymers. This
mated population 5001000 cells mm2) (Berman makes microneedles a cost-effective method for
etal. 1983; Chen etal. 1985), which often serves delivering oligonucleotides to epidermal cells for
as the first line of defence against many patho- DNA vaccination (Kendall 2010). (d) Biolistic
gens (Babiuk etal. 2000). The viable epidermis microparticle delivery: In this needle-free tech-
lacks the blood vessels and sensory nerve end- nique, pharmaceutical or immunomodulatory
ings found in the dermis, which makes it an ideal agents, formulated as particles, are accelerated in
site for pain-free delivery with minimal damage. a supersonic gas jet to a sufficient momentum to
There are many approaches for non-invasive tar- penetrate the skin (or mucosal) layer and achieve
geting of viable epidermal cells and DNA vacci- a pharmacological effect. Sanford and Klein
nation against major diseases. There have been (Sanford etal. 1987) pioneered this innovation
attempts at displaying foreign peptides and pro- with systems designed to deliver DNA-coated
teins on virus particles and insertion of mam- metal particles (1m diameter) into plant cells
malian cell-active expression cassettes in for genetic modification.
baculoviruses to express genes efficiently into Follicular targeting has been approached as
many different mammalian cell types (Paul etal. another way of targeting the LCs (Patzelt and
2013). Pearton etal. showed morphological Lademann 2013). Vogt etal. (2006) found a high
changes in human LCs stimulated with influenza density of LCs around hair follicles that were
virus-like particulate vaccines delivered via intra- very susceptible to targeting with the use of
dermal injection. The LCs were more dispersed nanoparticles. Nanoparticles of 40nm diameter,
throughout the epidermis, often in close proximity but not larger particles in the micron size range,
to the basement membrane, and appeared verti- were delivered via the follicles and were able to
cally elongated, which provides essential evidence diffuse through the follicular infundibulum to be
of host response (Pearton etal. 2010). taken up by the adjacent Langerhans cells.
23 Efficacy, Safety andTargets inTopical andTransdermal Active andExcipient Delivery 381
23.3 R
ole ofSafety andActive response is mounted following re-exposure to the
Toxicity inTransdermal allergen which manifests as ACD (Alenius etal.
Formulation 2008). Cutaneous photosensitivity can be evoked
by both systemic and topical exogenous photo-
Cosmetics and pharmaceuticals must be formu- sensitizers leading to active photosensitivity and
lated not only for efficacy, but also for patient or photo contact dermatitis, respectively.
consumer acceptance. This is influenced by a Photoallergenicity is a well-organized immuno-
perception of safety or lack of toxicity. logical reaction. The current understanding of
Regarding safety and toxicity, it must be under- ordinary contact dermatitis and active hypersensi-
stood that for a topically applied active to have tivity is based on the hapten hypothesis: mole-
either therapeutic or toxic effects, it must first be cules bind covalently to proteins, and the resulting
absorbed through the skin. The toxic effects conjugates can be recognized as immunogenic
caused if the chemicals are absorbed can include determinants. Likewise, photosensitive chemicals
acute irritation, corrosion and skin sensitization have a haptenic moiety.
(Grice etal. 2010). Chemicals applied to the Although the terms structureactivity relation-
skin can also have direct effects on the stratum ship (SAR) and quantitative structureactivity
corneum, resulting in an alteration in its perme- relationship (QSAR) are widely used, for skin
ability. This concept has been effectively applied sensitization, it is more meaningful to describe
in the development of penetration enhancers. the relationship as being between chemistry and
However, the harmful effects of excipients on activity. Skin sensitization induction is a multi-
the skin have been evaluated under increasing stage process (Jowsey etal. 2006): Stage 1,
scrutiny by regulatory authorities. Regulatory Translocation of the sensitizer from the skin sur-
considerations will be discussed later in this face to the epidermal site of action. This depends
chapter. on the dose given and duration of exposure; Stage
Topically applied actives causing adverse 2, Covalent reaction of the sensitizer with skin
effects can be classified as either allergens or irri- protein. This is strongly dependent on the chemi-
tants (Zweiman 1990). These allergens and irri- cal nature of the sensitizer, in particular electro-
tants not only affect the skin but can also affect philic reactivity and hydrophobicity. The nature
other body sites. Cutaneous active reactions can of the skin proteins involved in this process is not
be classified into three types of contact dermati- established. Possibilities range from any protein
tis: (a) Allergic contact dermatitis, (b) irritant encountered in the skin to highly nucleophilic
contact dermatitis and (c) photo contact dermati- proteins, purpose-designed by evolution, asso-
tis. The role and mechanism of irritation caused ciated with epidermal Langerhans cell mem-
by various chemical penetration enhancers has branes; Stage 3, Response of epidermal
been studied extensively. Langerhans cells to the modified protein, result-
ing in migration to the lymph node and presenta-
tion of antigen, leading to antigen recognition by
23.3.1 Mechanisms ofContact nave T-lymphocytes resulting in clonal
andPhoto Allergies expansion. Regulators classify chemicals as
andIrritations being hazardous after an acute dermal contact if
they lead to mortality (owing to absorption),
Allergic contact dermatitis (ACD) results from an cause acute irritation or corrosion and cause skin
adaptive immune response to the penetration of sensitization on multiple contacts. In the EU,
allergens into the skin. ACD progresses in two evaluation of skin irritation/corrosion is manda-
phases: (a) in the initial sensitization phase, the tory for all products likely to be associated with
host is immunized to the allergen, and (b) in the skin exposure. The section below discusses test-
elicitation phase, a rapid secondary immune ing protocols and requirements in the EU.
23 Efficacy, Safety andTargets inTopical andTransdermal Active andExcipient Delivery 383
23.3.2 Testing ofDermatologics, Table 23.2 SCCP requirements for testing of chemical
Cosmetics andTheir Safety and physical properties of cosmetic substances, and the
associated toxicity studies (SCCP 2012)
Evaluation
Chemical and physical
specifications Relevant toxicity studies
The European Commission Scientific Committee
Chemical identity Acute toxicity
on Consumer Safety (SCCP) adopted its most
Physical form Corrosivity and irritation
recent guidelines The SCCSs notes of guidance
Molecular weight Skin sensitization
for the testing of cosmetic substances and their
Characterization and Dermal/percutaneous
safety evaluation, 8th Revision in December purity of the chemical absorption
2012 (SCCP 2012). These set out procedures for Characterization of the Repeated dose toxicity
the safety evaluation of cosmetic substances (sin- impurities or
gle ingredients or defined mixtures) and finished accompanying
contaminants
cosmetic products. In the guidelines, a cosmetic
Solubility Reproductive toxicity
ingredient is defined as any chemical substance
Partition coefficient Mutagenicity, genotoxicity
or mixture of synthetic or natural origin, used in (log Pow)
the formulation of cosmetic products. A cos- Additional relevant Carcinogenicity
metic ingredient may be: physical and chemical
specifications
1. A chemically well-defined single substance Homogeneity and Toxicokinetic studies
stability
with a molecular and structural formula
UVVIS absorption Photo-induced toxicity
2. A complex mixture, requiring a clear defini-
spectrum
tion and often corresponding to a mixture of
Isomer composition Human data
substances of unknown or variable composi- Functions and uses
tion and biological nature
3. A mixture of 1 and 2, used in the formulation
of a finished cosmetic product SCCP recognizes the increasing use of syn-
thetic and semisynthetic ingredients, in addition
A finished cosmetic product is defined as Any to the ingredients derived from natural products
substance or mixture intended to be placed in con- that have been traditionally used in cosmetics. In
tact with the external parts of the human body (epi- addition, their requirements for evaluating the
dermis, hair system, nails, lips and external genital safety requirements for cosmetic products are
organs) or with the teeth and the mucous mem- increasingly being brought into line with those
branes of the oral cavity with a view exclusively or for therapeutic products.
mainly to cleaning them, perfuming them, chang-
ing their appearance, protecting them, keeping
them in good condition or correcting body odours. 23.3.3 In Vitro Skin Irritancy Tests:
Table23.2 illustrates the SCCP requirements ANeed forTriangulation
for testing of chemical and physical properties
and the relevant toxicity studies for cosmetic While dermal toxicity to exogenous materials is
substances. seldom associated with mortality, it may involve
Other requirements for SCCP evaluation of significant morbidity. Ethical concerns have
cosmetic substances are included under the gen- brought about a paradigm shift in the way the
eral headings listed in Table23.3. skin irritancy of a cosmetic ingredient is evalu-
The SCCP guidelines cover the evaluation of ated. Prior to its phasing out and eventual ban by
safety and microbiological quality of finished the European Commission, the scientific commu-
cosmetic products under the headings listed in nity has traditionally used invivo animal testing
Table23.4. methods to evaluate the safety of a topically
384 Y.H. Mohammed et al.
Table 23.3 Safety evaluation procedure of cosmetic sub- skin models, the order of permeation was in line
stances as applied by the SCCP: Further requirements for with that observed in exvivo skin. Enzymatic
cosmetic ingredients (SCCP 2012)
equivalence and gene expression were also com-
Toxicological requirements for inclusion of a pared (Hu etal. 2010; Neis etal. 2010). In micro-
substance in one of the Annexes to Regulation (EC)
No 1223/2009 (to be evaluated by the SCCP)Dir. array analysis, Hu etal. (2010) found that the
76/768/EEC expression of a large percentage of the genes was
Basic requirements for cosmetic substances present in consistent between EpiDerm and human skin
finished cosmetic substances (which are to be indicating the presence of similar metabolic path-
evaluated by individual safety assessors) ways. In a 2005 review, Netzlaff etal. evaluated
General principles for the calculation of the Margin of the morphology of commercially available skin
Safety and lifetime cancer risk for a cosmetic
substance models and compared their suitability for testing
The specific assessment of hair dyes and hair dye phototoxicity, irritancy, corrosivity and active
components transport (Netzlaff etal. 2005). They tabulated
The Threshold of Toxicological Concern (TTC) comparative morphological features and test
Aspects to consider with respect to the risk assessment results for these parameters. They commented
for the inhalation route that currently the models come close to reproduc-
Human biomonitoring ing human skin, but only in certain aspects. These
skin models are useful in toxicity testing, includ-
Table 23.4 SCCP evaluation of finished cosmetic prod- ing phototoxicity testing, and to an extent for
ucts: Safety and microbiological quality (SCCP 2012) drug transport studies. The variability in trans-
Toxicological profile of the substances port can be attributed to the incompletely devel-
Stability and physical and chemical characteristics of oped barrier.
the finished cosmetic product Before the enforcement of the EU chemicals
Evaluation of the safety of the finished product policy REACH (Registration, Evaluation and
Microbiological quality: quantitative and qualitative Authorization of Chemicals) in 2009, Basketter
limits etal. (2007) published report and recommenda-
Challenge testing
tions of European Centre for the Validation of
Good manufacturing practice
Alternative Methods (ECVAMs) Workshop 59a.
This workshop was part of a concerted effort in
applied product. There are many invitro testing recognizing the role of percutaneous/dermal
models that have been developed and commer- absorption process in sensitization. They defined
cialized to provide an alternative to animal test- epidermal disposition as the specific location and
ing. Models such as EpiSkin (Episkin SNC, stressed the quantification of chemicals in the epi-
Lyon, France), SkinEthic (SkinEthic laborato- dermal compartment, which are relevant to local
ries (Nice, France) and EpiDerm (Mat-Tek Inc., toxicity endpoints/skin sensitization. OECD Test
Ashland, USA) have reasonable similarities to Guideline (TG) 428 (OECD 2004), for invitro
human tissue in terms of morphology, lipid com- skin absorption, provides general rules on how to
position and biochemical markers (Netzlaff etal. measure systemic availability and concentrations
2005). Numerous reviews and research articles of chemicals that permeate through all layers of
have been written comparing the different skin the skin and the overall kinetics of skin absorption
models to each other and to human skin (Lotte but does not allow for separate evaluation of epi-
etal. 2002; Netzlaff etal. 2005; Schafer-Korting dermal disposition to be derived for local skin
etal. 2006, 2008; Neis etal. 2010; Gotz etal. toxicity endpoints, such as skin sensitization.
2012; Brinkmann etal. 2013). Lotte etal. (2002) Hence, this report emphasizes the need to relate
studied the permeation of lauric acid, caffeine the invivo dosimetry of sensitizers that penetrate
and mannitol in EpiSkin, SkinEthic and into the viable epidermis via the stratum corneum
EpiDerm and observed that although there was of human skin to the concentrations used in
huge variability in permeation data among the invitro applications. This requires the need to
23 Efficacy, Safety andTargets inTopical andTransdermal Active andExcipient Delivery 385
estimate both the applied concentration of chemi- ied in cell culture for a significant period (Deleo
cal onto the skin surface for given finite dose etal. 1996). There are other approaches that can
exposure conditions and to relate it to what is be used to complement the validated invitro
applied to cells invitro as being representative of methods and satisfy the need for triangulation in
the target dose of chemical in human skin. safety evaluation. Human exvivo skin is often
The models proposed by the SCCP for skin used as a realistic model for invivo human skin
safety tests have been validated against invivo in transdermal active delivery. The study of
Draize tests (SCCP 2012). However, these stand- detailed cellular and sub-cellular processes in the
alone methods rely heavily on quantification of skin was previously not possible using conven-
optical density and colorimetry, which lead to a tional light microscopic methods, such as wide-
one-dimensional analysis. Moreover, since field and confocal microscopy. Although these
results obtained from previously conducted tools work reliably in cell cultures and thin tissue
human tests studies on model compounds are sections, their application in thick biological
necessary as reference values for invitro assays, samples such as skin is greatly limited due to
attention should also be paid to the generation of light-scattering and absorbing properties of bio-
invivo data and standardization of invivo test logical tissues. However, this is being addressed
protocols (Ponec 2002). with the increasing use of multiphoton or two
In the last decade, numerous committees and photon microscopy (MPM/TPM). Understanding
taskforces have been set up to constantly review of the process of epidermal and transdermal
the performance of invitro irritancy and genotox- transports of xenobiotics is important for rational
icity assays (Fentem etal. 1998; Eskes etal. 2012; design of topical active delivery and to avoid
Pfuhler etal. 2014). In vitro assays have shown a exposure to toxic and allergenic compounds.
very high rate of positive genotoxicity results that MPM allows 3D visualization of penetration and
do not correlate with invivo genotoxicity or car- distribution with minimal sample preparation.
cinogenicity (Kirkland etal. 2007). Due to the Sanchez etal. have used cellular autofluores-
inability to follow up the invitro tests with invivo cence to show that exvivo skin viability, includ-
assays, many potential new products have been ing metabolic activity, can be preserved up to 7
de-selected. The Cosmetics Europe (formerly days at 4C (Sanchez etal. 2010). MPM has also
COLIPA) Genotoxicity Task Force has driven and been used to demonstrate the importance of stra-
funded projects to help address the high rate of tum corneum, in the sensitization phase of con-
misleading positives in invitro genotoxicity tests tact allergy (Samuelsson etal. 2009). One of the
(Pfuhler etal. 2014). The ongoing 3D skin present drawbacks of MPM is that it tends to be
model project is now validating the use of human qualitative rather than quantitative; however, the
reconstructed skin (RS) models in combination use of fluorescence lifetime imaging (FLIM)
with the micronucleus (MN) and Comet assays. holds promise for quantitative analysis in tissue
Non-testing methods like [quantitative] structure samples (Benati etal. 2011; Koenig etal. 2011).
activity relationships ([Q]SARs) or in silico meth- Excised skin obtained from elective abdomino-
ods are also a valuable way of gaining more plasties is a convenient resource, which when
toxicological information. Formation of chemical used within its viability provides an excellent
categories facilitates the application of read across model. However, not all research facilities have
between similar chemicals based on the assump- access to freshly excised skin. Skin from elec-
tion that their behaviour will be consistent for tive surgery is donated in goodwill to assist sci-
their class (Adler etal. 2011). entific research, and there are ethical constraints
Even from before the complete ban on animal on its use for commercial testing. The EU pro-
testing, alternative techniques to test dermal irri- hibits financial gain through the use of human
tancy and immunogenicity caused by pharma- tissue (Netzlaff etal. 2005). Progress is ongoing
ceutical and cosmetic products were being in the development of advanced skin equivalents
developed. Responses to irritants have been stud- with immune and inflammatory equivalency.
386 Y.H. Mohammed et al.
Better culture media to maintain the viability of lished and validated to replace invivo animal
exvivo skin and skin biopsies are also under testing that can no longer be used. A triangula-
development. tion approach is suggested to deal with the
Chau etal. (2013) recently described an problem of false positives in the invitro tests.
advanced static 3D skin equivalent which com-
bines human keratinocytes, human dendritic AcknowledgementsThe authors thank the National
cell and fibroblasts combined into a three- Health & Medical Research Council of Australia for sup-
port of this work.
layer CellGrown tissue culture insert com-
prising keratinocyte and fibroblast layers at a
thickness of 100m and 30m, respectively.
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Ethical Considerations inResearch
Involving Dermal andTransdermal 24
Drug Delivery
DusankaKrajnovic andNinaDragicevic
benefit worldwide. Transdermal drug delivery of which are internationally known scandals,
has been accepted as a potential non-invasive such as medical experimentation in Nazi
route of drug administration, because of its many Germany; the Tuskegee Syphilis Study and
advantages, such as avoidance of the first-pass Milgram experiments. A clear appreciation of
metabolism by the liver, possible sustained drug general ethical principles in research and specific
release, minimization of pain, reduction of side ethical issues that each study method and design
effects and better patient compliance and adher- might request is equally important to the
ence. However, as an innovative area of pharma- researcher generating experimental data as to the
ceutical science, drug delivery science is user of this data (e.g. patients, consumers, gov-
sometimes underestimated, when measured by ernmental agencies or community). Ethics of
the number of medicines with topical or transder- research involving human beings are related to a
mal routes of delivery compared with oral ther- variety of scientific disciplines including biotech-
apy (Barry 2001). As human skin inhibits drug nology as well as biomedical, social and human
permeation and limits daily dosage delivery, life sciences.
many approaches have been developed in the
attempt to enhance transdermal drug delivery and
achieve systemic therapeutic concentration 24.2.1 General Ethical Principles
(Barry 2001; El Maghraby et al. 2008). inResearch Involving Human
Techniques used to enhance dermal and transder- Beings
mal drug delivery may include different
approaches to increase drug permeation, such as Depending on the research settings, purpose(s) of
physical enhancers (ultrasound, iontophoresis, the study, research objects (humans, animals, tis-
electroporation, magnetophoresis, micronee- sue samples or models) and using invivo/in vitro
dles), chemical enhancers (sulphoxides, azones, techniques, some generally agreed ethical prin-
glycols, alkanols, terpenes, etc.), particulate sys- ciples for good preclinical (research) and clinical
tems (liposomes, niosomes, transfersomes, inva- practice must be followed. For clinical research
somes, microemulsion, solid lipid nanoparticles) involving human participants, including research
and others (Barry 2001; Dragicevic-Curic et al. using identifiable human materials and existing
2008, 2009a, b, 2010; Ers et al. 2012).. The data, these principles are stated in some interna-
proper analysis of ethical and moral implications tionally accepted ethical normatives such as
of these methods is needed before the researches Declaration of Helsinki (ethical principles for
protocols are approved, because of the diversity medical research involving human subjects, the
of the enhancers and methods themselves. In latest version since 2013, which replaces the
some studies, ethical issues can be extensive and original 1964 versions and all subsequent ver-
could be related to the vulnerable subjects as par- sions) and CIOMS Guidelines (International
ticipants, research protocols in poor-resources Ethical Guidelines for Biomedical Research
countries, cultural difficulties toward skin and Involving Human Subjects, the latest revised ver-
animal testing, bioengineered skin surrogates or sion since 2002). The Declaration of Helsinki
difficulties to obtain human skin samples (Barry incepted in 1964 elaborated and clarified 10 prin-
2001; Hikima et al. 2012). ciples stated in Nrnberg Code and provided the
ethical foundation for Proposed International
Guidelines endorsed by Council for International
24.2 General Ethical Organizations of Medical Sciences in collabora-
Considerations inResearch tion with World Health Organisation in 1982,
focusing on research in developing countries.
Although highly unethical research is not wide- Both documents evolved to their current forms
spread, the history of biomedical research and are still considered to be the core of interna-
includes many cases of unethical research, some tional normatives encouraging cultural sensitivity
24 Ethical Considerations inResearch Involving Dermal andTransdermal Drug Delivery 395
in implementing same standards for research in protecting their own interests. More formally,
developing and resource-rich countries. Although they may have insufficient power, intelligence,
internationally accepted as ethical guidelines, education, resources, strength or other needed
these documents could not provide universal attributes to protect their own interests.
answers to all ethical issues, but clearly could Individuals whose willingness to volunteer in a
guide the researchers to confront the challenging research study may be unduly influenced by the
task to design, conduct, monitor, control and expectation, whether justified or not, being of
oversee human research project. Later in 1996 benefits associated with participation, or of a
these documents influenced the development of retaliatory response from senior members of a
the international guidelines proposed by hierarchy in the case of refusal to participate
International Conference of Harmonization may also be considered vulnerable or explored
(ICH): Good Clinical Practice Guidelines (ICH subjects. Examples are members of a group with
GCP/E6) as well as regional EU legal documents a hierarchical structure, such as medical, phar-
such as the European Clinical Trial Directive macy, dental, and nursing students, subordinate
(2001/20/EC), the new Clinical Trials Regulation hospital and laboratory personnel, employees of
(CTR) EU No 536/2014 and Good Clinical the pharmaceutical industry, members of the
Practice Directive (2005/28/EC). Subsequently, armed forces and persons kept in detention.
many ethical guidelines and recommendations Other vulnerable persons include patients with
for human studies as well as legal rules for human incurable diseases, people in nursing homes,
studies are developed based on national basis in unemployed or impoverished people, patients in
resource-poor and resource-rich settings (see emergency situations, ethnic minority groups,
Table 24.1.) Table24.1 summarizes some of homeless people, nomads, refugees, minors and
national guidelines and recommendations for those incapable of giving consent. This list may
ethical conduct in research, selected in a conve- not be exhaustive as there may be circumstances
nient way to include some of the BRIC and ICH in which other groups are considered vulnera-
countries along with some transition countries ble, women for example, in an orthodox patriar-
and developing countries. All the listed guide- chical society or adult young men having to ask
lines could be found in extensor, from the sources a chieftain (Marshall 2007; Benatar 2002, 2004;
included in the reference list. Nuffield 2002; CIOMS 2012; WMA 2013; ICH
These normatives established universally 1996).
accepted principles in human research addressing Research ethics committee (REC) is an inde-
privacy, confidentiality, autonomy, beneficence pendent multidisciplinary board of individuals
and non-maleficence, beneficiary, justice, intel- who undertake the review of research protocols
lectual property, information and consent, par- for ethical considerations. It is also known
ticipation and voluntariness and scientific and inlocal, regional and international regulations
ethical review of the research by ethics commit- under different names: ethical review board
tee. Some of them will be discussed shortly in the (ERB), ethical review committee (ERC) are the
following text of this section. The integrity of the most common used in Europe whereas in USA,
principal investigator throughout the protocol this committee is known as human research eth-
development, research process and publication ics committee [HREC], institutional review
process of the study results, as well as clearly board [IRB] (EC 2001; Garrard and Dawson
stated conflict of interest if there is any, are also 2005; CIOMS 2002; WMA 2005, WMA 2013;
ethical principles, generally required in all kinds Council of Europe 2005; ICH 1996). The EU
of research. Directive on Clinical Trials defines a research
Vulnerable (research) participants are not ethics committee as: an independent body in a
included in the research unless it is absolutely Member State, consisting of healthcare profes-
proven necessary. Vulnerable persons are those sionals and nonmedical members, whose
who are relatively (or absolutely) incapable of responsibility it is to protect the rights, safety
396 D. Krajnovic and N. Dragicevic
Table 24.1 Summary of ethical guidelines and recommendations for human studies from 16 selected countries around
the world. Documents are sorted alphabetically by countrys origin regardless of documents title in English and the date
of endorsement
Year of National guideline or recommendation(title in
Country endorsement English) References
Australia 2000, 2006 Note for Guidance on Good Clinical Practice Therapeutic Goods
(CPMP/ICH/135/95) Administration (2000, 2006)
The Australian Clinical Trial Handbook, 2006
Bosnia 2010 Rules on clinical testing and medical devices The Ministry of Civil Affairs of
Bosnia and Herzegovina (2010)
Brazil 2010 Good Clinical Practices: Document of the Pan American Health
Americas. Organization (2010)
Canada 2006 Guidance for records related to clinical trials Health Canada (2006)
Croatia 2010 Rules on clinical trials and good clinical practice Ministry of Health and Welfare
(2010)
France 2006 Good clinical practice for biomedical research on Dcision du 24 novembre (2006)
human subjects
Germany 2012 Good clinical practice - Verordnung (Verordnung ber die Anwendung
der Guten Klinischen Praxis bei
der Durchfhrung von klinischen
Prfungen mit Arzneimitteln zur
Anwendung am Menschen)
(GCP-Verordnung GCP-V)
Great 2005 Practice Guidance on Pharmacy Services for
Britain Clinical Trials
India 2011 Draft Guidance on Approval of Clinical Trials and
New Drugs
UK consolidated version of the Medicines for
Human Use (Clinical Trials) Regulations 2004 (SI
1031)with all amendments (2006, 2008, 2009,
2010)
New 2000 Note for Guidance on Good Clinical Practice Therapeutic Goods
Zealand (CPMP/ICH/135/95) Administration (TGA): Note for
Guidance on Good Clinical
Practice (2000)
Nigeria 2009 Good Clinical Practice Regulations National Agency for Food and
Drug Administration and Control
Nigeria (2009a, b)
Norway 2010 Guidelines on Notification for Clinical
Investigation of Medical Devices in Norway
Serbia 2011 Rules on the content of the request or the Ministry of health Serbia (2011)
documentation for the approval of clinical trials of
medicinal products and medical devices as well as
the way to clinical trials and medical devices
Singapore 2011 Guidelines for Clinical Practice Ministry of health Singapore,2011
South 2006 South African Good Clinical Practice Guidelines Department of Health: Pretoria,
Africa South Africa (2006)
USA 2011 Good Clinical Practice Resource Guide DMID: Good Clinical Practice
Resource Guide (2011)
and wellbeing of human subjects involved in a ties, and on the methods and documents to be
trial and to provide public assurance of that used to inform trial subjects and obtain their
protection, by, among other things, expressing informed consent (EC 2001). Most ECs review
an opinion on the trial protocol, the suitability study protocols for a single institution, such as
of the investigators and the adequacy of facili- hospital, with or without academic affiliation,
24 Ethical Considerations inResearch Involving Dermal andTransdermal Drug Delivery 397
while some are centralized, and review proto- Consent form should be easily understandable
cols from more than one institution/clinic, and in writing, that documents a potential partici-
which is very useful for multi-centred studies. pants consent to be involved in research and
Unfortunately they do not exist in many devel- which describes the rights of an enrolled research
oping and poor-resource countries where much participant. This form, in a clear and respectful
research is conducted nowadays. In some coun- manner, should include the following: title of
tries, protocols for studies are reviewed by hos- research; researchers involved; research time
pital and faculty ethics committees which serve frame; purpose of research; description of
as research ethics committees (investigational research; potential harms and benefits; treatment
boards) as well. alternatives; statement of confidentiality; infor-
Informed consent is a decision to participate in mation and data to be collected; how long the
research, taken by a competent individual who data will be kept, how it will be stored and who
has received the necessary information and has can access it; any conflicts of interest; a statement
adequately understood the information, and who, of the participants right to withdraw from par-
after considering the information, has reached the ticipation at any point without any circumstances;
decision without having been subjected to coer- and declarative statement of understanding that a
cion, undue influence or inducement, or intimida- participant agrees to and signs. The consent form
tion. Involvement of uncompetent subjects are should be in a language that the potential partici-
rarely needed for skin research in general, and pant understands and for those with limited liter-
could be justified for dermal and transdermal acy, the verbal communication of the consent
drug delivery studies, only if acceptable risk- document details should be provided along with
benefit balance is estimated and predictable risks proper documentation of consent, if it will be
and burdens are indentified. In the case of some given (WMA 2013; CIOMS 2002; ICH 1996;
vulnerable subjects not legally competent, DHHS 2012).
informed consent is obtained from the legal Ethics review and evaluation of the research
guardians or parents. In a case of subjects liter- must take into account at least three consider-
acy, when written consent is not possible to ations: ethical challenges, the institutional
obtain, an eye witness oral informed consent is requirements and the legal constraints. Applicable
recommended (WMA 2013; CIOMS 2002; law and legal rules are not always consistent and
NAFDAC 2009a, b; Nuffield 2007). An informed they could differ greatly. Besides, adhering ethical
or valid consent must address to three questions: principles to national regulations and translating
(1) does the potential participant have the capac- and applying them to studies carried out in devel-
ity to consent requiring consideration of such oping countries is sometimes difficult because of
issues as age, maturity, cognitive ability; (2) is social and cultural environments. Marchall elabo-
the consent voluntary (i.e. is the decision made rated several recommendations for researchers in
free from coercion, inducement, or intimidation multinational studies conducted in developing
including pressure from a family member); and countries. They are listed below:
(3) has the potential participant received suffi-
cient information on which to base a decision? 1. Respect the cultural traditions of studied pop-
The CIOMS guidelines also stress that consent is ulations and communities
not an event, but a process in which potential par- 2. Strengthen capacity for developing collabora-
ticipants need to have time to study information, tive partnerships
think about them and ask additional questions 3. Strengthen education in research ethics for
before being asked to make a decision. It is of investigators
absolute importance that information should be 4. Strengthen capacity for independent ethical
available in appropriate languages and written in review of protocols
a style that is understandable by patients, taking 5. Ethical challenges in study design and
into consideration relevant factors, and including informed consent for health research in
cultural and sometimes religious differences. resource-poor settings
398 D. Krajnovic and N. Dragicevic
choice of the specific ethical considerations are Good Clinical Practice, research must confirm to
possible, as well as financial constraints and prob- generally accepted scientific principles meaning
lems with limited availability of models or facili- that it should be conducted in adequate settings
ties, especially in resource-poor settings (Marshall (laboratory equipment and qualified research
2007). As methods for measuring skin penetra- staff) and be based on through knowledge of the
tion and dermal delivery could be divided into two scientific literature and relevant source of infor-
categories: invivo and invitro method, several mation (adequate study design and materials)
different ethical issues have to be taken into (WMA 2013; ICH 1996).
account, assuming an acceptable risk-benefit bal-
ance, addressing privacy, confidentiality, intellec-
tual property, consistency with local legislative 24.3.1 Ethical Issues Related
and regulatory requirements, appropriate training toInVivo Techniques
and qualifications of the investigations and proper
data management during and after the research. Much attention related to percutaneous penetra-
There are two kinds of invivo measurements of tion studies have been discussed and introduced
skin penetration studies: (1) animal studies and recently, with the intention to assess the penetra-
(2) human studies, and both need a proper analy- tion characteristics of specific substances (test
sis of their ethical implications. Which method to substance). The gold standard, invivo tech-
apply will depend on the research question to be nique when the test substance (drug) is applied
answered, which is probably one of the reasons to the skin of human volunteers and blood and/
why the present OECD guideline for studies on or urine is collected and analyzed, has been used
percutaneous penetration accepts several methods before all other techniques were ever considered
(OECD 2004a, c). In vivo transdermal and dermal and is still used where there is no adverse risk
human studies are carried out to determine the to the human volunteers participating (Benech-
flux, the amount of drug delivered to the skin, as Kieffer et al. 2003; Hueber-Becker et al. 2004,
well as the therapeutic effectiveness. A drugs der- 2007; Lammers et al. 2005; Nohynek et al. 2004,
mal delivery rate is mainly of interest to regula- 2006). The amount of test substance measured
tory agencies concerned with toxicity or chemical in the blood and/or urine gives a good indica-
exposures in the workplace. As human skin has tion of the amount of substance absorbed through
only limited availability, alternatives were neces- the skin into the systemic circulation. In trans-
sary and during the last decade several experi- dermal/dermal drug delivery research combined
mental models (in vitro technique) related with therapy, the human volunteers are not
totransdermal penetration and percutaneous healthy persons but patients, and rarely, even
penetration have been standardized and validated. vulnerable participants. When knowledge of the
In general, the invitro models have the advantage drug (active substance) is limited or incomplete
of avoiding almost all ethical aspects associated and research is intended to disclose different
with invivo skin penetration studies (Holmgaard unknown characteristics, the risk assessment may
and Bo Nielsen 2009). Ethical considerations in not be completely explained, which would give
skin penetration studies include an assessment of rise to ethical concern and would limit the use
predictable risk, burdens and scientific impor- of healthy volunteers or patients. In traditional
tance of the study, but also sensitive privacy con- invivo human technique, many ethical issues are
cerns, content of informed consent and the process to be considered by the investigator and evalu-
of obtaining consent. The recruiting method and ated by the ethics committee before the research
possible advertising for recruitment of partici- is even allowed, in order to protect life, health,
pants are also of relevance and must be thoroughly dignity, integrity, right to self-determination, pri-
addressed by ethics committees evaluation. vacy and confidentiality of those who gave con-
Following the Declaration of Helsinki and ICH sent to voluntarily participate.
400 D. Krajnovic and N. Dragicevic
24.3.2 Ethical Issues ofInVitro mouse, hairless rat, hamster (cheek pouch), snake
Techniques Involving Human (shed skin) and cow udder have been suggested as
Skins alternatives (Haigh and Smith 1994).However, in
comparison to human skin, animal species have dif-
During the last decade research efforts have been ferent permeability of skin, so various enhancement
directed towards standardization and validation of of animal skins permeability was also investigated.
experimental models. Various tissue culture meth- In general, porcine skin, especially the ear which is
ods have been rigorously evaluated and accepted also easily available, is reported to match human
in Europe as total replacements for animal-based skin best, and there are references pointing also to
skin absorption studies (OECD 2004c; PETA the alternative of cow udder skin. Searching for
2013; Holmgaard and Bo Nielsen 2009). These other alternative methods as well as bioengineered
methods use skin from a variety of sources to mea- skin surrogates (artificial human skin) in the future
sure the passage of a test chemical into and across would be additionally fostered with the EU ban of
skin to a fluid reservoir. By using an invitro model, tests on animals for cosmetic products and of the
the ethical topics are minimized since the use of marketing of final cosmetic products/ingredients
human volunteers or animals are excluded and the tested on animals, from September 2009 (Klein
donor skin samples would be sent to destruction 2012; EC 2003, 2009).
anyway, or artificial models are used (Holmgaard
and Bo Nielsen 2009). Other advantages of the Conclusion
invitro model are the possibilities to replicate the Transdermal and dermal drug delivery is one
experiment with samples from the same person. of the most promising routes of drug adminis-
Also different species can be studied under identi- tration. There are invitro as well as invivo
cal laboratory conditions and enable comparisons methods to investigate skin drug delivery
within and between species. The reliability and through skin, and both have their own set of
relevance of invitro skin absorption studies have advantages, limitations and disadvantages.
been thoroughly established through a number of Which method will be used by researchers will
international expert reviews, and these methods depend on the research question(s), available
have been codified and accepted as an official test resources and laboratory conditions, and scien-
guideline of the Organization for Economic tific knowledge. Institutional requirements,
Cooperation and Development (OECD 2004b, c; applicable local and regional laws and regula-
Lehman et al. 2011). tions have to be correctly addressed for the
research to be conducted, and sometimes spe-
cific cultural and moral-defined issues must be
fulfilled as well. Several guiding ethical prin-
24.3.3 Ethical Issues inAnimal
ciples are widely acknowledged including
InVitro Tests
risk-benefit balance, vulnerable and incapable
participants, privacy concerns, informed con-
Animal invitro tests included using different forms
sent, conflict of interest, confidentiality, scien-
of animal skins, but having a number of scientific
tific integrity and intellectual property.
disadvantages over the non-animal tests. Due to
widespread demands in skin absorption studies
Acknowledgement DKs work is down within the frame-
based on regulatory aspects on the one hand, and
work of a project financially supported by the Ministry of
scientific and public empowerment to ending live Science and Education of the Republic of Serbia (Project
animal tests on the other hand, the need to establish No. 41004). The funding agreement ensured the authors
alternative models has been recognized. In addition, independence in designing the research, interpreting the
data, writing and publishing the report.
some researchers have ethical and cultural restraints
DK wishes to thank her colleague Mrs Andrijana
to use human skin (Hikima et al. 2012).Animal skin Miloevi Georgiev for technical assistance with the final
forms from pig (ear, flank, abdomen, or back), preparation of the text.
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Index