WFL Publisher

Science and Technology

Meri-Rastilantie 3 B, FI-00980 Journal of Food, Agriculture & Environment Vol.8 (3&4): 90-95. 2010 www.world-food.net
Helsinki, Finland
e-mail: info@world-food.net

Percutaneous absorption of piroxicam: In vitro release and percutaneous absorption

from different concentration gels
Rym Ben Mustapha 1*, Christine Lafforgue 1 and Nadia Fenina 2
1
Laboratoire de Dermopharmacologie and Cosmétologie, Faculté de Pharmacie. 5, rue Jean Baptiste Clément, 92296
Chatenay-Malabry Cedex, France. 2 Laboratoire de Pharmacologie et Laboratoire de Biologie Végétale, Faculté de
Pharmacie de Monastir, Rue Avicenne-Monastir, Tunsie. *e-mail: rymbenmustapha@gmail.com, zohra-marzouk@voila.fr

Received 5 July 2010, accepted 29 October 2010.

Abstract
The study of molecules skin penetration is very important in the field of the pharmaceutical and cosmetic industry and in toxicology. Various studies have
considered the impact of different physicochemical drug characteristics, skin thickness, or formulation on the transition from the skin surface to
underlying tissues or to the systemic circulation, however, the influence of the drug concentration on the permeation flux of molecules was rarely
raised. Our research aims at finding out the influence of the drug concentration in a formulation. For this purpose, three same base gels were used at
1%, 3% and 5% of piroxicam to evaluate the effect of concentration on in vitro release through synthetic membrane and ex vivo permeation through
human skin of piroxicam using diffusion FranzTM cells. It is immediately apparent the diffusion through epidermal tissue is significantly slower than
through the synthetic membrane which recorded an increase of the flux jointly to the increasing concentration on piroxicam. It was seen, that contrary
to the results of in vitro studies, the percutaneous absorption of piroxicam revealed to be not concentration-dependent. Among all the different
concentrations of piroxicam gel examined, gel 1% applied at 5 mg/cm2 showed the highest permeability coefficient and also the best flux characteristics
across human skin.

Key words: Absorption rate, diffusion coefficient, Franz TM diffusion cells, percutaneous absorption, permeation flow.

Introduction
Piroxicam(4-hydroxy-2-methyl-N-2-2-pyridinyl-2H-1,
2-benzo-thiazine-3-carboxamide-1,1-dioxyde) is one of the most
potent non-steroidal anti-inflammatory drugs (NSAIDs). The
NSAIDs has prominent anti-inflammatory, analgesic and
antipyretic properties 1. Many adverse effects such as upper
abdominal pain and ulceration of the gastro-intestinal mucosa
restrict the oral use of the drug 2. Delivering NSAIDs through
skin is an effective strategy for evading NSAIDs’adverse effects
in the gastro-intestinal tract as well as improving patient
compliance 3. However, the skin provides a barrier, which limits
the number of drug molecules suitable for dermal and transdermal
delivery and limits the efficacy of topical formulations 4. For
this purpose, several studies were conducted to better understand
the barrier function of skin and then, strategies to increase the
skin bioavailability of drugs were set up. Thereby, Goosen et
al. 5 established correlation between physicochemical
characteristics and pharmacokinetics properties of some
NSAIDs and piroxicam showed the best bioavailability. Cheong
and Choi investigated effects of enhancers on the percutaneous
absorption of piroxicam to develop a convenient delivery system
for piroxicam 6.
Few studies have investigated the correlation of piroxicam
concentration and its pharmacokinetic parameters. That’s why,
the main objective of this study has been to formulate gels of
piroxicam at different concentrations and to evaluate the effects
of drug concentration on the in vitro release through a synthetic
membrane of cellulose and on ex vivo percutaneous absorption
of piroxicam. The change in percutaneous penetration from gels
as a result of dose application was also examined.
Materials and Methods
Materials: Carbomer: Carbopol® 940 (carboxy-polymethylene);
oleic acid; piroxicam (Table 1); propylene glycol; triethanolamine
(T.E.A) and distilled water have been used for gel formulation.
Other reactive agents have been used for HPLC analysis. The
gels composition is detailed in Table 2.
Preparation of gels: The composition of the piroxicam gels used
is as per the Table 2. It is gel of carbopol containing 40% of
propylene glycol used as co-solvent (solubilisation agent) in order
to have a vehicle in which the piroxicam is entirely dissolved 7.
The oleic acid has almost proven its efficiency as absorption
promoter. The gels are prepared by dispersing Carbopol® 940 in a
quick agitated mixture of water, propylene glycol and piroxicam.
The oleic acid is then added to the mixture and the pH of the
carbopol dispersions was adjusted to 7.4 with triethanolamine.
Study of the piroxicam solubility in the gels: The solubility of
the drug is an important characteristic for a successful
development of medicaments since it is a key factor which rules

90 Journal of Food, Agriculture & Environment, Vol.8 (3&4), July-October 2010

Table 1. The physicochemical properties of piroxicam.
Structure Physicochemical Molecular Solubility Log P
formula mass

C5H13N3O4S 331.40 23 mg.l-1 at 22 °C 1.8

Table 2. Composition (% w/w) of piroxicam gels.
Constituents Gel code
Gel A Gel B Gel C
Piroxicam (Sigma) 1 3 5
Carbopol® 940(BF Goodrich) 1 1 1
Propylene glycol (BASF) 40 40 40
Oleic acid (Cooper) 5 5 5 specific device, the samples of skin were mounted into the
T.E.A (Cooper) 1.2 1.2 1.2
Distilled water Qsp 100 Qsp 100 Qsp 100
the access of the drugs through the biologic membranes 8. An
incorrectly estimated solubility could lead to wrong conclusions
on the results obtained from in vitro tests 9. In order to set the
solubility, and thus the availability of the amount of piroxicam in
the gels, we proceeded to extractions of the drug we have prepared.
An amount of 200 mg of each gel was introduced in 5 ml of
acetonitrile. The mixture was vortexed during 2 min and left to
stand for 2 hours. Two ml of the floating liquid was taken and a
high performance liquid chromatography (HPLC) analysis was
performed.
Study of the piroxicam release: The piroxicam release rate from
the gels was measured through synthetic membranes by means
of diffusion cells, Franz cells TM.
The diffusion membranes which were used are membranes made
of nitrate of cellulose impregnated during one night in a mixture of
Ethomeen 15%, isopropyl myristate 10.
The diffusion cells used are cells made of glass, static type
containing 3 separate compartments, one as supplier, containing
the drug. The surface of application is 3.14 cm2. The receiver
compartment has a volume of 11 ml. The receiving liquid is
constituted of isotonic phosphate buffer (PBS) pH = 7.4. The
diffusion membrane is fixed between the two compartments.
An amount of 200 mg of each gel were deposited (6 cells per
gel) on the membranes and samples of the receiving content were
taken each 2 hours until the 8th hour. For each time the survival
liquid was completely renewed.
The determination of the piroxicam concentration was done by
HPLC.
Study of the cutaneous penetration through human skin: The
method applied is in compliance with the recommendation of
the A. A. P. S (American Association of Pharmaceutical
Scientists) and the F. D. A. (Food and Drug Administration).
The ex vivo cutaneous penetration was studied on biopsies of
human skin mounted on Franz TM described previously. The
biopsies worked out are from human origin, obtained during
abdominal chirurgical plastics.
The skin was defrosted the day before of its use. The following
day it was cleaned from the subcutaneous fat by the means of a
scalpel. Then the entire skin was dermatomed with a more or less
constant thickness between 390 and 400 µm using a Brown TM
dermatome (Emergence, 94 573 Rungis). Once dermatomed, the
skin was cut into pieces of 4 cm² each.
Quality control of thickness was made for each biopsy using a
diffusion cells without any other treatment. Once the skin was
deposited, the cells were stabilized into a water bath at 37°C during
the night.Skin temperature was verified by using a mini
thermometer TESTO 0900.0519, it was 32°C ± 0.5.
Gel was filled in the upper compartment (on the epidermis surface
of the cutaneous biopsy) using a syringe string pulling. The
quantities of gel filled were weighed individually: 1, 2 and 5 mg/
cm2 for each gel at 1, 3 and 5% of piroxicam.
The samples of receiving liquid were taken each 2, 4, 6, 8, 10 and
24 hours and analysed by HPLC. Eight to ten cells were made for
each gel and for each applied amount.
Quantitative analysis of piroxicam: Piroxicam was analyzed
using a HPLC system (Interface, Merck Hitachi D-7000; UV-
Detector, Merck Hitachi L-7400; Autosampler, Merck Hitachi
L-7200; Pump, Merck Hitachi L-7100). A reverse phase column
was used (Merck Lichrospher® 100RP 18, 125×4 mm, 5 µm).
The mobile phase consisting of acetonitrile: acetic acid: water
(65:4:31). The elution parameters were a flow rate of 1 ml/min
and an injection volume of 20 µl. The UV detection was at 365
nm. Two types of range of graduation were set, a piroxicam in the
acetonirile (R2= 0.9992) and one piroxicam in the phosphate buffer
pH -7.4 (R2= 0.9995).
Data treatment: Piroxicam release rates were calculated using
the Higuchi equation 11:
Q/A = 2C0 ν(Dt / Π) (1)
where Q is the amount of the drug release, D is the apparent
diffusion coefficient and denotes the diffusivity of the drug in
the vehicle, t is the time, A is the area of the diffusion membrane,
C0 is the initial concentration of the drug in the vehicle and Π a
constant.
Eq. 1 may be simplified to:
Q /A= k t ½ (2)
where k is the release rate constant determined from the slope of
the amount of the drug released per unit area versus square root
of time.
The apparent diffusion coefficient of the drug in the vehicle
was estimated from the release rate constant value.

Journal of Food, Agriculture & Environment, Vol.8 (3&4), July-October 2010 91

 Piroxicam flux through membranes and skin were calculated Table 4. Permeation parameters through cellulose nitrate
using the Fick’s second law of diffusion. According to this law, membrane.
the total amount of drug (Q) appearing in the receptor solution in Gel code J Kp 102 Cum. amount 8h
time t at the steady state is expressed as follows: (µg/cm2/h) (cm/h) (µg/cm2)
Gel A 26.6 6.76 601.67
Q / A = KLC0(D t /L2 – 1/6) (3) Gel B 74.93 7.07 1140.3
Gel C 178.45 9.17 2043.02

where A is the effective diffusion area, C0 is the initial drug

concentration in the vehicle, D is the diffusion coefficient Table 5. Piroxicam release rates and diffusion coefficient
corresponding to the diffusivity of the drug in the membrane, L is values across cellulose nitrate membranes.
the thickness of the membrane and K is the partition coefficient k D 106
of drug between membrane and vehicle. (µg/cm2/ h-1/2) (cm2h-1)
The flux J, was determined from the slope of steady state portion Gel 1% 173.61 (r2 = 0.98) 236.6
of the amount of the drug permeated divided by A versus time. Gel 3% 405.23 (r2 = 0.99) 143.22
Gel 5% 902.04 (r2 = 0.98) 255.49
The lag time values were determined from the x-intercept of the
slope at steady state.
From Eq. 3 the flux is expressed as:
3. Skin permeation study
J = C0KD / L = C0 Kp (4)
The diffusion flux (J): The diffusion flux J is the quantity of
substance absorbed per unit of area and time. The cumulative
where Kp is the permeability coefficient.
amount of the permeated piroxicam was plotted versus time, and
the flux was calculated from the steady-state part of the curve.
Statistical analysis: The amount of piroxicam in the receiver phase
Diffusion flux obtained with different concentration gels weren’t
was assayed by HPLC. The linearity interval established was
significantly different when same quantity of gel was applied (P >
0.5-2000 µg/ml (r > 0.999).
0.05) (Table 6).
The tests were conducted into six samples for the in vitro release
With the three formulations A, B and C, we obtained a good
study and into eight samples for the ex vivo permeation through
correlation between the quantities of gel applied and the diffusion
skin. Results are expressed as averages ± SD (Standard Deviation).
flow J (Fig. 3). It’s confirmed by the high linearity (r2 ≥ 0.99). The
Results were statistically evaluated by ANOVA and using
diffusion flow is more important, for the same amount of piroxicam
Student’s t-test. P ≤ 0.05 was considered significant.
applied, when the gel concentration decreases (Fig. 4).
Results
The lag time Tlag: Lag time is the time requested from which the
1. Study of the solubility of the piroxicam in the gels: The rise of
diffusion flow becomes stable. During our experiments, it varied
measured piroxicam concentration (Table 3) and so of drug part
from 3 to 8 hours (Table 7). It appears immediately more slowly
dissolved in the vehicle was proportional to the increase of the
than obtained with release study through synthetic membrane.
drug concentration in the gel (R2 ≥ 0.993). That indicates a good
As for the diffusion flux, the lag time showed a little variation
availability of the piroxicam under chosen concentrations
when piroxicam concentration in gel increases. It is recorded a
(1/100 to 5/100: w/w).
reduction of the lag time when the deposited gel amount is
increased.
2. Permeation study through nitrocellulose membrane: The
in vitro release profiles of piroxicam from gels A, B and C during
The permeability coefficient: The permeability coefficient values
8 hours through nitrocellulose membrane are represented in
Kp were calculated by the formula 4 described previously and
Fig. 1.
illustrated in Fig. 5. Kp indicates the penetring power of a substance
The steady state flux (J) and the permeability coefficient (Kp)
through the stratum corneum.
values calculated from the slope of the linear portion of the
For a same applied quantity, the permeability coefficient
permeation curve are summarized in Table 4.
decreases when the piroxicam concentration is raised, while for
The release rate constants (k) were obtained by reporting the
the same gel, Kp increases when the applied quantity is increased.
cumulated amount to the square root of the time. In all cases,
The linearity of the obtained curves (Fig. 5) confirms the good
linear plots and good correlation (r² > 0.983) were obtained (Fig. 2).
correlation between the applied gel amount and the permeability
The release rates and diffusion coefficient values for 1, 3 and
coefficient.
5% of piroxicam gels are listed in Table 5. According to the obtained
results, the release rate of piroxicam raised with the increase of its
concentration.

Table 3. Determination of extracted piroxicam concentration (µg/ml).

Gel code Gel A Gel B Gel C
Portion of piroxicam in formulae (w/w) 1/100 3/100 5/100
Measured concentration of piroxicam (µg/ml) 1966.00 5296.93 9723.97

92 Journal of Food, Agriculture & Environment, Vol.8 (3&4), July-October 2010

Table 6. Average values of diffusion flux J (µg/cm2/h) for gels applied at
different quantities (N = 8, ± SD).
Quantities of gel Gel 1% Gel 3% Gel 5%
1 mg/cm2 0.0272 (± 0.0008) 0.0274 (± 0.002) 0.0360 (± 0.001)
2 mg/cm2 0.0641 (± 0.002) 0.0590 (± 0.001) 0.0676 (± 0.003)
5 mg/cm2 0.1686 (± 0.001) 0.157 (± 0.003) 0.1768 (± 0.005)

Table 7. Lag times Tlag (h) (n = 8).

Gel 1% Gel 3% Gel 5%
1 mg/cm2 8.03 7.41 5.85
2 mg/cm2 3.56 3.88 3.68
5 mg/cm2 4.42 4.25 3.28

Discussion
Our study was focusing on a comparison of various release and
skin absorption parameters of piroxicam.
Firstly, piroxicam solubility in vehicle (criterion limiting) was
checked. The rise of piroxicam extracted concentration was
proportional to the increase of the drug concentration in the gel
(R2 ≥ 0.993) indicating a good availability of the piroxicam. In fact,
only portion of drug dissolved in vehicle could be released to
skin surface and diffuse.
The effect of piroxicam concentration on drug release across
synthetic nitrocellulose membrane was studied. Drug amount
released was plotted against the square root of time. Results
showed a satisfactory release rate of drug (173.61, 405.23 and
902.04 µg/cm2/h-0.5,respectively, for gel 1, 3 and 5%) and in all
cases, linear plots and good correlation were obtained (r² > 0.983)
showing that the membrane has no substantial effect on the release
determination, and the properties of the formulation controlled
the release of the drug 12.
Cumulative amount of piroxicam per unit area was plotted against
the time. Steady state flux J and permeability coefficient Kp values
were calculated from the slope of the linear portion of the permeation
curve. They increased proportionally to increase of piroxicam
concentration in gels (r > 0.978 for J and r > 0.919 for Kp). Data
revealed that the amount of drug released rose with an increase in
drug concentration. Cumulative amounts after 8 h were 601.67,
1140.3 and 2043.02 µg/cm2, respectively, for gel 1, 3 and 5%.
Data resulted from skin absorption were different from those
obtained with the synthetic membranes. Although the effect of
piroxicam concentration was found to be significant in in vitro
release studies for the three different gels, no significant difference
could be observed in the ex vivo skin permeation of piroxicam
from gels at 1%, 3% and 5% of drug concentration applied at the
same amount. Generally, there is a positive correlation between
drug concentration and release rate and percutaneous absorption
of the drug due to the increase in the thermodynamic activity.
Furthermore, the skin has a limited capacity for the transport of
some drugs 13. Because of these reasons, percutaneous absorption
of piroxicam from 1, 3 and 5% of carbopol gel formulations is
slightly different.
For the same gel (then same piroxicam concentration) the
diffusion flux increased with increasing of applied gel amount
(Fig. 3). Furthermore, when the same quantity of piroxicam
deposited, the higher flux value J was obtained with the less
concentrated gel. For example, when 157 µg of piroxicam was
applied (as 5 mg/cm2 from gel 1% or 1 mg/cm2 from gel 5%), the
flux is more important from gel 1%. Similarly, as shown in Fig. 4, we
noted that for 200 µg of piroxicam, the flow J from gel 3% (≈ 0.06 µg/
cm2/h) was higher than obtained from gel 5% (≈ 0.04 µg/cm2/h).
It was immediately apparent that the diffusion through epidermal
tissue is significantly slower than through the synthetic membrane.
During skin absorption experiments, lag time, Tlag, varied from 3
to 8 hours. For the same amount of gel applied, Tlag varies little
when the gel concentration increases. Highest values of Tlag
were obtained when gels were applied at 1 mg/cm2. Steady state
was achieved more rapidly with dose of 2 mg/cm2 for the three
gels (Table 7).
The permeability coefficient Kp indicates the penetrating
power of a substance through the stratum corneum. Values of Kp
increased with increasing applied amount of gel (Fig. 5), but for
the same amount of piroxicam, highest values of Kp were obtained
with less concentrated gel (Fig. 6). These results seem to be similar
to those obtained for the flux, and besides, very good correlations
were obtained between diffusion flux and permeability coefficients:
R2 ≥ 1 for gels A, B and C (Fig. 7).
Conclusions
It was seen that, contrary to the results of in vitro studies, the
percutaneous absorption of piroxicam was not concentration-
dependent. On the other hand, the transport through nitrocellulose
membrane impregnated with isopropyl myristate is more rapid than
through epidermis. So, use of synthetic membrane is useful in
assessments of batch-to-batch variation in quality assurance but
gives no indication of how formulation will behave when it is
used on skin. As a result of in vitro studies, it is obvious that the
applied amount of formulation affects the percutaneous absorption
of piroxicam strongly, but increasing drug concentration in vehicle
does not necessarily increase skin permeation.
This kind of studies may be useful in case the active drug is
distributed under various dosages, therefore the key question is
to know whether it is preferable to prescribe the most concentrated
formulation or to adapt a more intensive treatment with a less
concentrated dosage. Among all the different concentrations of
piroxicam gel examined, gel 1% applied at 5 mg/cm2 showed the
highest permeability coefficient and also the best flux
characteristics across human skin.
The present study was conducted on piroxicam while the issue
of how the other physicochemical molecules classes will react
remains to be elucidated.

Journal of Food, Agriculture & Environment, Vol.8 (3&4), July-October 2010 93

94
Gel 1%
Gel 1%
Gel 3% Gel 3%

Flux (µg/cm2/h)
Gel 5% Gel 5%

Amount of piroxicam penetrated (µg/cm2)

Time (hours) Piroxicam amount (µg)
Figure 1. Profile of the in vitro release of piroxicam through a nitrocellulose Figure 4. Diffusion flow J vs. quantity of piroxicam applied (µg) for
membrane (n= 6). each gel.

Gel 1%
Gel 1%
Gel 3%

Kp (cm.h-1)
Gel 3%
Gel 5%
Gel 5%

Cumulative amount (µg/cm2)

Square root of time (h-1/2) Amount of gel applied (mg/cm2)
Figure 2. Cumulated quantity of piroxicam (µg/cm²) according to the Figure 5. Correlation between the quantity gel applied and the
square root of the time (h-1/2). permeability coefficient Kp.

Gel 1% R2 = 1
Gel 3% R2 = 0.9999
Gel 1%
Gel 5% R2 = 0.9993
Kp (cm.h-1)

Gel 3%
Linéaire (Gel 1%)
Linéaire (Gel 3%) Gel 5%

Diffusion flow (µg/cm2/h)

Linéaire (Gel 5%)

Piroxicam gel deposited quantity (mg/cm2) Piroxicam amount (µg)

Figure 3. Variation of the diffusion flow J according to the quantity of gel Figure 6. Permeability coefficient Kp vs. quantity of piroxicam applied (µg).
applied (n = 8).

Journal of Food, Agriculture & Environment, Vol.8 (3&4), July-October 2010

 R2 = 1
Kp (cm.h-1)

Gel 1%
R2 = 1 Gel 3%
R2 = 1 Gel 5%

J (µg/cm2/h)
Figure 7. Correlation between the diffusion flux J and the permeability
coefficient Kp.

References
1
Babar, A., Solanki, U. D., Cutie, A. J. and Plakogiannis, F. 1990. Piroxicam
release from dermatological bases: in vitro studies using cellulose
membrane and hairless mouse skin. Drug Dev. Ind. Pharm.
16:523- 540.
2
Santoyo, S., Arellano, A., Ygartua, P. and Martin, C. 1996. In vitro
percutaneous of piroxicam through synthetic membranes and abdominal
rat skin. Pharm. Acta Helv. 71:141-146.
3
Schiantarelli, P., Cadel, S., Acerbi, D. and Pavesi, L. 1982.
Antiinflammatory activity and bioavailability of percutaneous
piroxicam. Arzneim. Forsch. Drug Res. 32:230-235.
4
Doliwa, A., Santoyo, S. and Ygartua, P. 2001. Effect of passive and
iontophoretic skin pretreatments with terpenes on the in vitro skin
transport of piroxicam. Int. J. Pharm. 229:37-44.
5
Goosen, C., Du-Plessis, J., Muller, D. G. and Janse-van-Rensburg, L. F.
2001. Correlation between physicochemical characteristics,
pharmacokinetic properties and transdermal absorption of NSAID’s.
Int. J. Pharm. 163:203-209.
6
Cheong, H. A. and Choi, H. K. 2003. Effect of ethanolamine salts and
enhancers on the percutaneous absorption of piroxicam from a pressure
sensitive adhesive matrix. Eur. J. Pharm. Sci. 18:149-153.
7
Santoyo, S., Arellano, A., Ygartua, P. and Martin, C. 1995. Penetration
enhancers effects on the in vitro percutaneous absorption of piroxicam
through rat skin. Int. J. Pharm. 117:219-224.
8
Barry, B. W. 2001. Novel mechanisms and devices to enable successful
transdermal drug delivery. Eur. J. Pharm. Sci. 14:101-114.
9
Faller, B. and Ertl, P. 2007. Computational approaches to determine
drug solubility. Adv. Drug Deliv. Rev. 59:533-545.
10
Hadgraft, J. and Ridout, G. 1983. Development of model membranes
for percutaneous absorption measurements. I. Isopropyl myristate.
Int. J. Pharm. 39:149-156.
11
Higuchi, W. I. 1962. Analysis of data on the medicament release from
ointments. J. Pharm. Sci. 51:802-804.
12
Guy, R. H. and Hadgraft, J. 1990. On the determination of drug release
rates from topical dosage forms. Int. J. Pharm. Sci. 60:R1-R3.
13
Akhter, S. A. and Barry, B. W. 1985. Absorption through human skin
of ibuprofen and flubiprofen: Effect of dose variation, deposited drug
films, occlusion and the penetration enhancer N-methyl-2-pyrrolidone.
J. Pharm. Pharmacol. 37:27-37.

Journal of Food, Agriculture & Environment, Vol.8 (3&4), July-October 2010 95