Journal of Pharmaceutical Sciences 107 (2018) 307-316

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Journal of Pharmaceutical Sciences

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Pharmaceutics, Drug Delivery and Pharmaceutical Technology

The Combination of GIS and Biphasic to Better Predict In Vivo

Dissolution of BCS Class IIb Drugs, Ketoconazole and Raloxifene
Yasuhiro Tsume 1, Naoto Igawa 2, Adam J. Drelich 1, Gregory E. Amidon 1,
Gordon L. Amidon 1, *
1
College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109-1065
2
Analytical Chemistry Department, Analytical 2 Group, Sawai Pharmaceutical Co., Ltd., Osaka, 532-0003, Japan

a r t i c l e i n f o a b s t r a c t

Article history: The formulation developments and the in vivo assessment of Biopharmaceutical Classification System
Received 3 August 2017 (BCS) class II drugs are challenging due to their low solubility and high permeability in the human
Revised 1 September 2017 gastrointestinal (GI) tract. Since the GI environment influences the drug dissolution of BCS class II drugs,
Accepted 7 September 2017
the human GI characteristics should be incorporated into the in vitro dissolution system to predict
Available online 15 September 2017
bioperformance of BCS class II drugs. An absorptive compartment may be important in dissolution
apparatus for BCS class II drugs, especially for bases (BCS IIb) because of high permeability, precipitation,
Keywords:
ASD and supersaturation. Thus, the in vitro dissolution system with an absorptive compartment may help
GIS predicting the in vivo phenomena of BCS class II drugs better than compendial dissolution apparatuses. In
ketoconazole this study, an absorptive compartment (a biphasic device) was introduced to a gastrointestinal simulator.
raloxifene
This addition was evaluated if this in vitro system could improve the prediction of in vivo dissolution for
BCS class II drug
simulation BCS class IIb drugs, ketoconazole and raloxifene, and subsequent absorption. The gastrointestinal
in vivo performance simulator is a practical in vivo predictive tool and exhibited an improved in vivo prediction utilizing the
biphasic format and thus a better tool for evaluating the bioperformance of BCS class IIb drugs than
compendial apparatuses.
© 2018 Published by Elsevier Inc. on behalf of the American Pharmacists Association.

Introduction
In vitro dissolution studies have often been conducted to assess
in vivo gastrointestinal dissolution of oral formulations rather than
performing a quality control type dissolution test with compendial
dissolution systems.1-17 Accordingly, numerous dissolution meth-
odologies have been utilized.1,3,4,9,12,13,18-21 The physicochemical
properties such as pKa, log P, solubility, and solid state of active
pharmaceutical ingredients (API) are important factors for their
dissolution and absorption. The physiological parameters in the
gastrointestinal (GI) tract like pH, buffer capacity, and the volume of
GI contents also significantly affect the dissolution and absorption of
oral dosage forms, especially for Biopharmaceutical Classification
System (BCS) class IIa and IIb drugs. Their drug dissolution depends
on the pH of the local environment at the different GI sites and those
display completely different dissolution profiles.22,23 BCS class IIa
* Correspondence to: Gordon L. Amidon (Telephone: 734-764-2226; Fax: 734-763-
6423).
E-mail address: glamidon@umich.edu (G.L. Amidon).
drugs like ibuprofen have a favorable environment for their solu-
bility at the small intestine, whereas BCS class IIb drugs have an
unfavorable environment for their solubility.24,25 To improve the
dissolution rate and solubility for BCS class IIb drugs, the reduction of
drug particle size, the modification of drug forms/processes such as
solid dispersion, and the addition of surfactant and polymer into oral
dosage forms have been embraced to enhance the oral absorption of
BCS class IIb drugs.26-29 Unlike compendial apparatuses, supersat-
uration and precipitation have been observed by in vitro dissolution
transfer models such as artificial stomach and duodenum (ASD) and
gastrointestinal simulator (GIS).3,4,7,8,12,13,30 However, the observed
precipitation time and rate from in vitro studies tends to be faster
than the precipitation time in vivo or the observed dissolution rate
and dissolved amount tends to be slower than in vivo because most
of in vitro dissolution studies do not have an absorptive phase to
account for the disappearance of drug molecules from the aqueous
lumen.19,31,32 Organic solvents have been introduced into the in vitro
dissolution methodology as an absorptive phase to evaluate in vivo
drug dissolution and to understand it mechanistically, and this
2-phase dissolution platform has provided meaningful in vivo pre-
diction of test drugs.9,11 The oral dosage forms of poorly soluble

https://doi.org/10.1016/j.xphs.2017.09.002
0022-3549/© 2018 Published by Elsevier Inc. on behalf of the American Pharmacists Association.
308 Y. Tsume et al. / Journal of Pharmaceutical Sciences 107 (2018) 307-316

drugs regularly contain surfactants and polymers, which improve

the dissolution rate or maintain the higher concentration for
enhanced oral absorption, and often face difficulty in evaluating the
in vivo drug dissolution in these dissolution methodologies due to
the unfavorable interaction (e.g., aggregation) between organic
phase and excipients in the oral dosage forms.
As the stomach in humans introduces disintegrated oral dosage
forms, drug solution, and undissolved drug particles of immediate
release dosage forms into small intestine (e.g., the main absorptive
site), this dissolution methodology in the GIS was designed to
approximate human GI physiology. The objective of this study was
to evaluate the improvement of in vivo prediction by introducing
the absorptive phase into the jejunal chamber in the GIS (GISjejunum),
using raloxifene (lower oral dose 60 mg) and ketoconazole (higher
oral dose 200 mg). This was accomplished by gradually introducing
dissolved drug and drug particles into the GISjejunum, which contains
2 phases (decanol and aqueous) instead of placing the intact oral
dosage form in the biphasic chamber directly. The study was con-
ducted with simple aqueous buffers (50-mM sodium phosphate
buffer) to evaluate the benefits of an organic phase in the GIS for the
in vivo prediction of BCS class IIb drugs.

Materials and Methods

Materials
Ketoconazole tablets were obtained from Taro Pharmaceuticals
U.S.A., Inc. (Hawthorne, NY) through University of Michigan Hos-
pital. Raloxifene tablets and raloxifene standards were provided by
Sawai Pharmaceuticals (Osaka, Japan). Ketoconazole, 1-decanol,
hydrochloric acid, sodium phosphate dibasic, and sodium chloride
were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO).
Trifluoroacetic acid, formic acid, methanol, and acetonitrile were
obtained from Fisher Scientific Inc. (Pittsburgh, PA). All chemicals
were of analytical grade or HPLC grade.
In Vitro Dissolution of Raloxifene and Ketoconazole in Dissolution
Media in the Presence and Absence of Absorptive/Organic Phase
With United States Pharmacopeia Dissolution Apparatus II (Biphasic
Dissolution)
The dissolution profiles of raloxifene and ketoconazole tablets in
the presence and absence of an absorptive phase were determined
using a Hanson SR6 Dissolution Test Station (Chatsworth, CA)
equipped with United States Pharmacopeia (USP) apparatus II
(paddles). The paddle speed was set to 50 rpm. Dissolution tests
were conducted on single dosage unit (a 60-mg raloxifene tablet or
a 200-mg ketoconazole tablet) at 37 ± 0.5
C in either 300 mL or 500
mL of 50-mM sodium phosphate buffer at pH 6.5 with the presence
and the absence of 100 mL of 1-octanol. Tablets were introduced to
the vessel by adding them directly to the aqueous medium without
contacting the organic solvent. Samples (1 mL) were manually
taken from each chamber at the specific time points and super-
natant was diluted with H2O/methanol (1/1, v/v) after centrifuga-
tion (17,000  g, 1 min) at room temperature. The drug
concentration was determined by HPLC analysis.
Raloxifene and Ketoconazole Dissolution Studies With the
Gastrointestinal Simulator (GIS) in the Presence and Absence of the
Absorptive/Organic Phase
Figure 1. The diagram of gastrointestinal simulator (GIS) with the presence of
absorptive/organic phase.
gastric fluid and 250 mL of water as the dose volume). The
duodenal chamber (GISduodenum) was filled with 50 mL of 50-mM
sodium phosphate buffer (pH 6.5) and the volume was main-
tained constant. The jejunal chamber (GISjejunum) was filled with
100 mL of distilled water as the resting volume and simply collected
the output from the GISduodenum. To avoid any unfavorable inter-
action between excipients in aqueous buffer media and organic
phase, distilled water was adopted as the resting volume in the
GISjejunum. With collecting the output (mainly 50-mM sodium
phosphate buffer) from the GISduodenum at the first-order kinetics,
the alteration of experimental condition was thought to be mini-
mal. The presence and absence of organic phase, 100 mL 1-decanol,
as an absorptive phase was incorporated only in the jejunal
chamber. During the experiment, simulated gastric fluid (0.01-N
HCl) and simulated duodenal fluid (100-mM sodium phosphate
buffer, pH 6.5) were pumped into the GISstomach and the GISduodenum
at the constant rate of 1 mL/min, respectively. The tablet of keto-
conazole (200 mg) and raloxifene (60 mg) was placed into the
GISstomach and the dissolution study was started. The test solution,
simulated gastric fluid with test drug, in the GISstomach entered into
the GISduodenum at a first-order rate, which was set 8 min as the
gastric half-emptying time (t1/2 ¼ 8 min). Sampling (100 mL) was
performed at specific time points: 3, 5, 8, 11, 14, 17, 20, 23, 26, 29, 45,
and 60 min, and no replacement of the buffer solution was made.
Those studies were performed, at least, 3 times in each condition, in
the presence or absence of absorptive phase. The sample from the
aqueous phase was spun at 17,000  g for 1 min at room temper-
ature and the supernatant was mixed with the same volume of
H2O/methanol (1/1, v/v) to prevent further precipitation. The
sample from the organic phase was immediately mixed with the
same volume of H2O/methanol (1/1, v/v). Concentration of raloxi-
fene and ketoconazole was determined by HPLC analysis.

The GIS dissolution diagram and the mass transport kinetics are HPLC Methodology for Raloxifene and Ketoconazole
shown in Figures 1 and 2, and the function and theory of the GIS
have been previously described.7,8,12,13 The gastric chamber All GIS experimental samples were analyzed with an Agilent
(GISstomach) was filled with 300 mL (50 mL of 0.01-N HCl as the HPLC system (Agilent Technologies, Santa Clara, CA). The HPLC
 Y. Tsume et al. / Journal of Pharmaceutical Sciences 107 (2018) 307-316 309

Figure 2. The mass transport kinetics of gastrointestinal simulator (GIS) with the presence of absorptive/organic phase.

system consisted of Agilent pumps (1100 series), an Agilent auto- and ksec(duodenum), to the GISstomach and GISduodenum are both 1 mL/
sampler (1200 series), and an Agilent UV-Vis detector (1100 series) min. GE is a gastric emptying time (gastric half-emptying time
controlled by Chemstation® 32 software (version B.01.03). Samples (t1/2) ¼ 8 min).
were resolved in Agilent Eclipse XDB-C18 reverse-phase column Mass transit kinetics in the GIS was applied to both drug species,
(3.5 mm, 4.6  150 mm) equipped with a guard column for raloxi- undissolved and dissolved drugs. Those transit rates between
fene and ketoconazole. The mobile phase consisted of 0.1% TFA/ chambers were calculated as a function of fluid transfer rate and
water (solvent A) and 0.1% TFA/acetonitrile (solvent B) with the drug amount (observed drug concentration in the GISstomach and
solvent B gradient changing from 0% to 56% at a rate of 2%/min transferred volume) from the previous chamber. The drug amount
during a 14-min run. Standard curves generated for raloxifene and for undissolved and dissolved drugs in each chamber of the GIS can
ketoconazole were utilized for quantitation of integrated area un- be respectively expressed as:
der peaks. The detection wavelength was 254 nm for raloxifene and


ketoconazole. dXudðstomachduodenumÞ dVstomach
¼  þ ksecðstomachÞ
dt dt


Mass Transport Analysis and Model by In Vitro Dissolution XudðstomachÞ
 (4)
Vstomach
The equations and theories of derivation of the models have
been reported previously.9,33 The mass transport of dissolution

studies with GIS, 3 chamber dissolution apparatus, have been dXdðstomachduodenumÞ dVstomach
¼  þ ksecðstomachÞ
described (Fig. 2).7,8,12,13,33 The movement of fluid volume in each dt dt
chamber can be expressed as:

XdðstomachÞ
 (5)

 Vstomach
lnð2Þ
Vstomach ¼ Vstomach;0  e t (1)
GE

dXudðduodenumjejunumÞ dVstomach
¼  þ ksecðstomachÞ
Vduodenum ¼ Vduodenum;0 (2) dt dt


XudðduodenumÞ
ðlnð2ÞÞt
 þ ksecðduodenumÞ
Vjejunum ¼ 100 þ Vstomach;0  e GE þ ksecðstomachÞ Vduodenum
 (6)
þ ksecðduodenumÞ t (3)


dXdðduodenumjejunumÞ dVstomach
where Vstomach, Vduodenum, and Vjejunum are the fluid volume at time ¼  þ ksecðstomachÞ
dt dt
t in each chamber of the GIS, GISstomach, GISduodenum, and GISjejunum 

and Vstomach,0 and Vduodenum,0 are the initial volumes in the XdðduodenumÞ
þ ksecðduodenumÞ (7)
GISstomach and GISduodenum. The fluid secretion rates, ksec(stomach) Vduodenum
310 Y. Tsume et al. / Journal of Pharmaceutical Sciences 107 (2018) 307-316

 GastroPlus™ Simulation of Ketoconazole Absorption

dXudðjejunumÞ dVstomach
¼  þ ksecðstomachÞ þ ksecðduodenumÞ
dt dt

 Oral drug absorption and pharmacokinetics parameters were
XudðduodenumÞ predicted by GastroPlus (version 9.5; SimulationPlus, Inc., Lancas-

Vduodenum ter, CA). Our goal in this study is to illustrate the effect of the
(8) presence of the organic phase for oral absorption of ketoconazole
using drug properties and in vitro dissolution results. The phar-

 macokinetics parameters (maximum concentration; Cmax and area
dXdðjejunumÞ dVstomach
¼  þ ksecðstomachÞ þ ksecðduodenumÞ under the plasma concentration time curve) between clinical data
dt dt

 and in silico results were compared.34 The physicochemical and
XdðduodenumÞ biopharmaceutical properties of raloxifene and ketoconazole used

Vduodenum in the GastroPlus prediction are presented in Table 134-43 Single
(9) trial was performed with the release profiles using the GastroPlus
standard physiological condition: Human Physiological-Fasted and
where Xud(stomach), Xud(duodenum), and Xud(jejunum) are the amount of Opt LogD Model SA/V 6.1. With extensive metabolism including
undissolved drug and Xd(stomach), Xd(duodenum), and Xd(jejunum) are first-pass metabolism and the large distribution volume of raloxi-
the amount of dissolved drug in each chamber of the GIS (Fig. 2). fene, the meaningful plasma profile of raloxifene was not pre-
With Fick's First Law and assumptions, the flux can be expressed as: dicted.42,44-46 The dissolution profile with USP apparatus II, which




 was determined in pH 6.5, was used for the in vivo prediction. The
dM dV Dor Daq Kap Cor dissolution profiles of a 200-mg ketoconazole tablet were created
j¼ ¼ C¼ Caq 
dt dt Daq hor þ Dor haq Kap Kap from the range of in vitro dissolution profiles (from the fastest to the
(10) slowest dissolution profiles) in the presence and the absence of
organic phase with the GIS by measuring and combining the drug
Experimentally, a drug product is introduced into the aqueous concentration in the GISduodenum and GISjejunum. The oral absorption
buffer of the diphasic schematic diagram (Fig. 1). Caq is the total of ketoconazole was predicted by GastroPlus with those dissolution
drug concentration in the aqueous, Cor is the drug concentration in profiles and physicochemical properties of ketoconazole. No drug
the organic, Daq is diffusion coefficient in the aqueous, Dor is absorption from the stomach was assumed in this set of predictions
diffusion coefficient in the organic, Kap is drug apparent partition and the drug release from a dosage form was simulated in Gas-
coefficient, haq and hor are diffusion layer thickness in the aqueous troPlus based on the dissolution profiles of ketoconazole obtained
and the organic phases, respectively. The time-dependent drug with the USP apparatus II and the GIS.
concentration in the aqueous in the GISjejunum can be expressed
with several theories explained in the previous report9 as:
Results
dXd;aqðjejunumÞ A
¼ j Ketoconazole Dissolution Study in the USP II
dt Vjejunum;aq;t
 
A Xd;orðjejunumÞ;t The dissolution studies with ketoconazole in the USP II were
¼ Xd;aqðjejunumÞ;t 
Vjejunum;aq;t Kap;t conducted with 50-mM sodium phosphate buffer (pH 6.5) with the
presence and the absence of 1-octanol (biphasic study). Figure 3
(11)
exhibits ketoconazole concentration-time profiles and less than
2% of total dissolution in SIF (pH 6.5) of the dose. The ketoconazole
Drug concentration in organic phase concentration in the aqueous phase did not show a difference
Kap ¼ (12)
Drug concentration in aqueous phase between the presence and the absence of 1-octanol but the keto-
conazole concentration in the organic phase did increase to around
where Xd,aq(jejunum) is the drug concentration in the aqueous phase, 12 mg/mL (Fig. 3a). With the presence of organic phase, the aggre-
A is the surface area of the aqueous-organic interface, Vjejunum,aq is gation of ketoconazole oral product was observed in the aqueous
the volume of aqueous at time t. Kap is drug apparent partition phase. Since small amount of 1-octanol can be dissolved in the
coefficient in the aqueous and the organic media. With mass bal-
ance of drug molecules between the aqueous phase and the organic
phase, the drug concentration in the organic phase as a function of Table 1
time can be described as: Chemical/Physiological/Pharmacological Parameters of Raloxifene and Ketoconazole
for GastroPlus Simulation
2 3
Description Raloxifene Ketoconazole
Mt 6 7
61  eVjejunum;aq;t pð1þbÞt 7
A
Xd;orðjejunumÞ;t ¼ 4 5 (13) MW 504.6 531.4
Vor ð1 þ bÞ Dose (mg) 60 200
Dose number 35a 321a
Dose volume (mL) 25035 25035
Solubility (pH 7.8 or 8) (mg/mL) 13.3  103,36 2.5  103,37
Vjejunum;aq log P 1.638 4.339
b¼
Kap Vor pKa 8.9538 2.9/6.540
Human Peff ( 104 cm2/s) 3.141,b 1.37c
Body weight (kg) 70 70
where Xd,or(jejunum),t is the drug concentration in the organic phase
Vc (L/kg) 234842 0.743
at time t, Vor is the volume of the organic phase, P is permeation CL (L/h/kg) 44.142 0.3834
rate across the aqueous and organic diffusion layers, the Mt is
Vc, volume of central compartment.
the mass of dissolved drug at time t, and b is the volume ratio a
Calculated by GastroPlus 9.0.
aqueous to organic media normalized by the apparent partition b
Calculated based on the Caco-2 permeability.
c
coefficient, Kap. Calculated by ADMET predictor.
 Y. Tsume et al. / Journal of Pharmaceutical Sciences 107 (2018) 307-316
Figure 3. The concentration of ketoconazole-time profiles in USP II with the presence
and the absence of absorptive phase (a): black circle, red square, and green triangle
represent the observed drug concentration with the absence of absorptive phase, the
observed drug concentration in the aqueous phase with the presence of absorptive
phase, and the observed concentration in the absorptive phase, respectively. The dis-
solved amount (%) of ketoconazole-time profiles in USP II (b): black circle and red
square represent the observed dissolved drug amount (%) with the absence of
absorptive phase and the total observed dissolved drug amount (%), the combined
amount in the aqueous and organic phases, with the presence of absorptive phase,
respectively. Each data point represents mean ± SD (n ¼ 3).
aqueous phase, 1-octanol in the aqueous phase interacted with
excipients of oral dosage forms to cause the aggregation, which
might prohibit the further drug dissolution.
Raloxifene Dissolution Study in the USP II
The dissolution studies with raloxifene in the USP II were con-
ducted with 50-mM sodium phosphate buffer (pH 6.5) with the
presence and the absence of 1-octanol (biphasic study). Figure 4a
shows raloxifene drug concentration in each phase. The decline
of raloxifene concentration in the aqueous phase was observed
with a corresponding increase of raloxifene concentration in the
organic phase (Fig. 4a). However, the total amount of dissolved
raloxifene was similar regardless of the presence or absence of
organic phase (Fig. 4b). As seen in ketoconazole, the aggregation of
raloxifene oral product was also observed in the aqueous phase
with the presence of organic phase.
Ketoconazole Dissolution Study in the GIS
The dissolution studies with an IR ketoconazole tablet in the GIS
were conducted with 50 mL of 50-mM sodium phosphate buffer
(pH 6.5) in the GISduodenum and 100 mL of distilled water with the
311
Figure 4. The concentration of raloxifene-time profiles in USP II with the presence and
the absence of absorptive phase (a): black circle, red square, and green triangle
represent the observed drug concentration with the absence of absorptive phase, the
observed drug concentration in the aqueous phase with the presence of absorptive
phase, and the observed concentration in the absorptive phase, respectively. The dis-
solved amount (%) of raloxifene-time profiles in USP II (b): Black circle and red square
represent the observed dissolved drug amount (%) with the absence of absorptive
phase and the total observed dissolved drug amount (%), the combined amount in the
aqueous and organic phases, with the presence of absorptive phase, respectively. Each
data point represents mean ± SD (n ¼ 3).
presence and the absence of 100 mL organic solvent, 1-decanol, in
the GISjejunum (biphasic study). Figure 5 shows similar dissolution
profiles of ketoconazole in the aqueous phase in each chamber,
GISstomach, GISduodenum, and GISjejunum, regardless of the presence or
absence of organic phase. Since the saturated drug concentration of
ketoconazole would be around 10 mg/mL in 50-mM sodium phos-
phate buffer (pH 6.5) at 37
C, observed ketoconazole concentration
was tenfold and fourfold higher in the GISduodenum and GISjejunum
than the saturated concentration, suggesting supersaturation
(Figs. 5b and 5c and Table 1). The gradual decline of drug concen-
tration was observed in the GISjejunum, suggesting the precipitation,
even though the supersaturation level was maintained during
60-min experiment (Fig. 5c). In the GIS, the decline of ketoconazole
concentration in the aqueous phase of the GISjejunum was not
observed even though the mass transport of ketoconazole was
observed from the aqueous phase to the organic phase in the
2-phase experiments (Fig. 5c).
Raloxifene Dissolution Study in the GIS
The dissolution studies with raloxifene in the GIS were con-
ducted under the same condition as the ketoconazole experiments.
Figure 6 displays the similar dissolution profiles of raloxifene in the
312 Y. Tsume et al. / Journal of Pharmaceutical Sciences 107 (2018) 307-316
Figure 5. The concentration of ketoconazole-time profiles in the gastric (a), the
duodenal (b), and the jejunal chambers (c): solid line represents the theoretical con-
centration curve and dot line represents the saturated drug concentration in 50-mM
sodium phosphate buffer (pH 6.5) at 37
C. Black circle, red square, and green trian-
gle represent the observed drug concentration with the absence of absorptive phase,
the observed drug concentration with the presence of absorptive phase, and the
observed concentration in the absorptive phase, respectively. Each data point repre-
sents mean ± SD (n ¼ 3).
aqueous phase in each chamber, GISstomach, GISduodenum, and
GISjejunum, regardless of the presence or absence of organic phase.
Since the saturated drug concentration of raloxifene would be
around 13.3 mg/mL in 50-mM sodium phosphate buffer (pH 6.5) at
37
C, observed raloxifene concentration in the GISduodenum was
Figure 6. The concentration of raloxifene-time profiles in the gastric (a), the duodenal
(b), and the jejunal chambers (c): solid line represents the theoretical concentration
curve and dot line represents the saturated drug concentration in 50-mM sodium
phosphate buffer (pH 6.5) at 37
C. Black circle, red square, and green triangle represent
the observed drug concentration with the absence of absorptive phase, the observed
drug concentration with the presence of absorptive phase, and the observed concen-
tration in the absorptive phase, respectively. Each data point represents mean ± SD
(n ¼ 3).
about sevenfold higher than the saturated concentration, suggest-
ing supersaturation (Fig. 6b and Table 1). However, observed
raloxifene concentration in the GISjejunum was not as high as the
concentration in the GISduodenum, suggesting the quick precipitation
(Fig. 6c). In the GIS, the decline of raloxifene concentration in the
 Y. Tsume et al. / Journal of Pharmaceutical Sciences 107 (2018) 307-316
aqueous phase of the GISjejunum was observed, unlike ketoconazole
studies, due to the mass transport of raloxifene from the aqueous
phase to the organic phase in 2-phase setting (Fig. 6c). This phe-
nomenon in raloxifene was also observed in the USP II dissolution
studies (Fig. 4a). Since the dose and dose number of raloxifene are
smaller, 60 mg and 35, compared with ketoconazole's, 200 mg and
321, the drug movement of raloxifene from the aqueous phase to
the organic phase, either in 1-octanol or 1-decanol, would affect
their mass balance (Table 1).
Simulation of Pharmacokinetic Data After Oral Administration of
200-mg Ketoconazole
Based on the in vitro dissolution profiles with the GIS and USP II,
the fastest and the slowest release profiles of ketoconazole were
generated for in silico simulation to estimate the range of plasma
concentration-time profiles (Fig. 7). Prediction results with those
release profiles were summarized in Table 2 and Figure 8. Those
simulation results supported the in vitro dissolution results and
captured the improvement with the presence of organic phase in
the GIS for in vivo predictive dissolution. The simulated pharma-
cokinetic parameters with the predicted in vivo dissolution profiles
based on the in vitro dissolution profiles with the GIS are closer to
the clinical results than ones with the predicted in vivo dissolution
profile with the USP apparatus II. The addition of biphasic phase in
the GIS improved the prediction of ketoconazole oral absorption,
Cmax 40.9%-47.8% but did not see any change in area under the
plasma concentration time curve, suggesting the incorporation of
biphasic phase into the GIS would help with predictions of in vivo
oral absorption for BCS class IIb drugs.
Discussion
The information about the level and the duration of supersat-
uration is valuable for BCS class II drugs because the drug
concentration in the GI tract will be the driving force for the oral
absorption. Since highly permeable drugs like BCS class II drugs will
be constantly absorbed in the human small intestine, the apparent
drug concentration in the intestinal lumen will be reduced and the
in vivo precipitation may be slower than observed one by in vitro
experiments.19,30-32,47 To predict in vivo dissolution and absorption,
newer dissolution methodologies such as ASD, GIS and biphasic
models, and different buffer systems along with in silico
Figure 7. The ketoconazole release profiles of a 200-mg ketoconazole (IR) tablet for
GastroPlus simulation. Black solid and dot lines represent the fast release profile and
the slow release profile of ketoconazole with the presence of organic phase in the GIS.
Gray solid and dot lines represent the fast release profile and the slow release profile of
ketoconazole with the absence of organic phase in the GIS. Gray dot dash lines
represent the fast and the slow release profiles of ketoconazole with USP II up to 3 h.
313
Table 2
Summary of Pharmacokinetic Data After the Prediction of 200-mg Dose of Ketoco-
nazole by GastroPlus Simulation
Description Cmax Difference AUC Difference
(mg/mL) (%) (mg-h/mL) (%)
Clinical results34 4.22 e 14.74 e
USP II without Organic 0.02 99.5 0.12 99.2
USP II with Organic 0.04 99.0 0.40 97.4
GIS without Organic 1.78-2.15 49.1-57.8 7.14 51.6
GIS with Organic 2.63-3.03 28.2-37.7 7.14 51.6
technologies have been utilized to determine the insightful pa-
rameters to predict accurate in vivo and develop better oral
formulation.5,7-9,11-14,31-33,37,48-63 ASD, GIS, and TNO gastrointes-
tinal model are dissolution apparatuses incorporating pH changes
and drug movement of the GI tract and can monitor not only the
dissolution rate in different buffers but also the movement of dis-
solved and undissolved drugs to understand in vivo phenomena
like supersaturation and precipitation.3,7,8,12-14 Biphasic dissolution
studies have also been conducted to examine the dissolution-
absorption kinetics of test oral dosage forms to understand and
predict the in vivo situation.9,11,55,63 Raloxifene and ketoconazole
have different doses and their oral bioavailabilities were 2% and
76%, respectively.44,46,64 The low oral bioavailability of raloxifene is
attributed to its high first-pass effect and extensive metabolism
even though raloxifene is orally absorbed well (~60%).44-46 The
observed amounts of dissolved drug of raloxifene and ketoconazole
in compendial dissolution systems are too low to explain their good
oral absorption (Figs. 3 and 4).45,64 In the GIS study, the observed
concentrations of raloxifene and ketoconazole in the duodenal and
jejunal chambers were 2.0- to 14.8-fold higher than the concen-
tration observed in the compendial dissolution systems, respec-
tively, and those higher drug concentrations, supersaturation, in
the GI tract attribute to their better oral absorption.65-68 The better
oral absorption of weak base drugs has been elucidated by alter-
native dissolution methodologies rather than the compendial
dissolution apparatuses.7,8,13,30,52 In the GISjejunum, the raloxifene
and ketoconazole concentrations in the aqueous phase were
maintained at higher concentrations than ones observed in the USP
Figure 8. The predicted ketoconazole plasma concentration-time profile by GastroPlus
and the clinical result following a single oral administration of 200-mg ketoconazole
(IR) tablet. Black circle and minus represent the plasma concentration-time profiles of
ketoconazole with the fastest dissolution profiles and the slowest dissolution profiles
from the GIS with the presence of organic phase. Red square and minus represent the
plasma concentration-time profiles of ketoconazole with the observed fastest disso-
lution profiles and the slowest dissolution profiles from the GIS with the absence of the
organic phase. Green triangle and minus represent the plasma concentration-time
profiles of ketoconazole with the dissolution profiles from the USP II with the pres-
ence and absence of organic phase.
314 Y. Tsume et al. / Journal of Pharmaceutical Sciences 107 (2018) 307-316

precipitation rate constant by simple in vitro studies (Fig. 9). Indeed,

simulation results by GastroPlus suggested slight improvement of
in vivo prediction for ketoconazole as the drug concentration was
maintained or slightly increased with the addition of biphasic
format into the GIS and, as previously reported, substantial
improvement of in vivo prediction compared with one with com-
pendial dissolution methodologies (Table 2 and Fig. 8).7-9,11,30,53,55
It is important to note that, in this study, the biphasic GISjejunum
did not define the dissolution-partition relation of a test oral drug
dosage forms like the typical biphasic dissolution study due to the
change of aqueous volume along with mixing condition, and the
loss of sink conditions. Mudie et al.9 have demonstrated the
appropriate conditions of biphasic dissolution for potential test
drugs to determine the parameters for its in vivo prediction. With
Equation 13, the drug concentrations in the organic phase were
predicted and the results were displayed along with the observed
drug concentration in both phases in Figure 9. For both drugs, those
calculations by Equation 13 were under-predicted at early time
points and over-predicted at later time points suggesting the ratio
(A/Vaq) between the surface area of the aqueous-organic interface
and the aqueous volume were not ideal to predict in vivo phe-
nomena as suggested previously (0.16 < A/Vaq < 0.26) suggesting
the importance of the relationship between the surface area of the
aqueous-organic interface (A) and the aqueous volume (Vaq) for the
in vivo prediction (Fig. 10).9 The partitioning of these 2 BCS class IIb
drugs, raloxifene and ketoconazole, into the organic phase occurred

Figure 9. The total dissolved amount (%) of ketoconazole-time profiles in (a) and
raloxifene-time profiles in (b): Black circle represents the dissolved drug amount (%) in
SIF (pH 6.5) with USP II, red square represents the total dissolved drug amount (%) in
GISduodenum and GISjejunum, and green triangle represents the total dissolved drug
amount (%) in GISduodenum, GISjejunum, and organic phase as an absorptive phase in
GISjejunum. Each data point represents mean ± SD (n ¼ 3).

II until the end of those studies (Figs. 5c and 6c). The dose number
of raloxifene (35) is lower than ketoconazole (321) and raloxifene
dissolves relatively quicker than ketoconazole in the GISstomach
(Figs. 5a and 6a and Table 1). Since undissolved particles may
accelerate the precipitation by the growth of small particles and the
initiation of nucleation, the quick dissolution in the stomach before
moving into the small intestine would extend the supersaturation
period and slow the precipitation rate.69,70 Also, the reduction of
apparent drug concentration in the small intestinal lumen by
absorption would theoretically slow its drug precipitation. For
investigating the precipitation behavior of oral drug products,
biphasic dissolution formats may be an appropriate approach to
predict in vivo than compendial dissolution formats due to the
dissolution-partitioning phase for test drugs.9,11,63,71,72 In this study,
the addition of organic phase (biphasic format) into the GIS was
evaluated to improve in vivo prediction. Several reports suggest
that in vitro experimental results tend to overestimate the precip-
itation rate due to the lack of absorptive phase in the dissolution Figure 10. The drug concentration in the jejunal chamber of GIS (mg/mL) of
system.32,58,65,68,73 The calculated precipitation rate constants for ketoconazole-time profiles in (a) and raloxifene-time profiles in (b): dot lines represent
ketoconazole with the GIS with the absence of organic phase agree the calculated concentration curves of ketoconazole and raloxifene in the organic
with the previous reports.30,31,74 However, the results of ketoco- phase, respectively. Black circle represents the drug concentration in the aqueous
phase in GISjejunum and red square represents the drug concentration in the organic
nazole displayed slower decline of drug concentration in the small phase in GISjejunum. Arrows represent the ideal range of the ratio between the surface
intestinal chambers with the presence of absorptive phase than area of the aqueous-organic interface and the aqueous volume. Each data point rep-
those without, suggesting the potential overestimation of resents mean ± SD (n ¼ 3).
 Y. Tsume et al. / Journal of Pharmaceutical Sciences 107 (2018) 307-316
more rapidly at the earlier time points when A/Vaq ratio exceeded
the suggested upper limit (0.26), whereas the partitioning drug into
the organic phase occurred more slowly at later time points when
A/Vaq ratio approached the suggested lower limit (0.16). The par-
titioning and the possible reasons of these observations are: (1) the
ratio (A/Vaq) was out of the recommended range; and (2) the stir-
ring was insufficient to maintain consistent hydrodynamic condi-
tions (e.g., consistent diffusional resistance) in the aqueous and
organic phases at the later time points (Fig. 9). Still, the calculations
of the drug concentrations in the organic phase exhibited compa-
rable to the observed drug concentrations (R2 ¼ 0.65 for raloxifene
and R2 ¼ 0.77 for ketoconazole), suggesting acceptable prediction,
and the GIS with biphasic format helps to understand the in vivo
bioperformance of these test drugs.
Poorly soluble weak base drugs, BCS class IIb drugs, often
contain polymer excipients to prolong the duration of supersatu-
ration and/or decelerates precipitation rate.68,75 Those additives,
excipients in oral formulation, often interact with organic solvent
and cause aggregation in simple biphasic dissolution systems.
Unlike a typical biphasic dissolution setting, the GIS gradually
transfers drug solution/suspension into the absorptive site,
biphasic site in the GISjejunum, from the gastric chamber like the
human GI tract instead of dumping the whole oral dosage form into
the biphasic site. This may help better understand the in vivo
dissolution and partition of test oral dosage forms. Altogether, it is
vital to develop the in vitro dissolution systems which not only can
provide the in vivo predictive dissolution profiles including the
observation of supersaturation and precipitation to identify
formulation differences in test formulations but also can analyze
the mass transport of oral test formulations.
Conclusion
The GIS with biphasic platform can provide valuable informa-
tion to predict in vivo drug dissolution profiles including super-
saturation and precipitation as well as mass transport analysis for
BCS class IIb drugs even though these oral dosage forms may
contain unfavorable excipients such as polymers and surfactants
for biphasic dissolution. Due to the physicochemical properties of
test drugs, BCS class IIa, IIc, IVa and IVc drugs might be most
appropriate drug groups to conduct the dissolution studies with
the absorption phase for in vivo prediction than BCS class IIb drugs
where, even today, precipitation is difficult to predict. The GIS can
be a practical dissolution system not only to predict in vivo per-
formance of oral dosage forms but also to potentially predict the
difference in bioavailability and bioequivalence of test formulations
and, hence, be useful for the development of oral formulation and
quality by design.
Acknowledgment
This work was supported by grant# HHSF223201310144C and
HHSF223201510157C.
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