pharmaceutics
ArticleBioequivalence of Oral Drug Products in the Healthy andSpecial Populations: Assessment and Prediction Using a
Newly Developed In Vitro System “BE Checker”
Takato Masada 1 , Toshihide Takagi 1 , Keiko Minami 1 , Makoto Kataoka 1 , Ken-ichi Izutsu 2 , Kazuki Matsui 3
and Shinji Yamashita 1, *
1

2
3

*

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Citation: Masada, T.; Takagi, T.;
Minami, K.; Kataoka, M.; Izutsu, K.-i.;
Matsui, K.; Yamashita, S.
Bioequivalence of Oral Drug Productsin the Healthy and Special
Populations: Assessment andPrediction Using a Newly Developed
In Vitro System “BE Checker”.
Pharmaceutics 2021, 13, 1136.
https://doi.org/10.3390/
pharmaceutics13081136
Academic Editors:
Isabel Gonzalez-Alvarez andPeter Langguth
Received: 30 June 2021
Accepted: 23 July 2021
Published: 26 July 2021

Faculty of Pharmaceutical Sciences, Setsunan University, Osaka 573-0101, Japan;
18d402mt@edu.setsunan.ac.jp (T.M.); toshihide.takagi@setsunan.ac.jp (T.T.);
keiko.minami@pharm.setsunan.ac.jp (K.M.); makoto@pharm.setsunan.ac.jp (M.K.)
National Institute of Health Sciences, Kanagawa 210-9501, Japan; izutsu@nihs.go.jp
Sawai Pharmaceutical Co. Ltd., Osaka 532-0003, Japan; kazuki.matsui@sawai.co.jp
Correspondence: shinji@pharm.setsunan.ac.jp; Tel.: +81-72-866-3125

Abstract: In order to assess and predict the bioequivalence (BE) of oral drug products, a newin vitro system “BE checker” was developed, which reproduced the environmental changes in thegastrointestinal (GI) tract by changing the pH, composition, and volume of the medium in a single
chamber. The dissolution and membrane permeation profiles of drugs from marketed products wereobserved in the BE checker under various conditions reflecting the inter-patient variations of the GI
physiology. As variable factors, initial gastric pH, gastric emptying time, and GI agitation strength
were varied in vitro. Dipyridamole, a basic drug, showed rapid and supersaturated dissolution
when the paddle speed in the donor chamber was 200 rpm, which corresponds to the high agitation
strength in the stomach. In contrast, supersaturated dissolution disappeared, and the permeatedamount decreased under the conditions with a slow paddle speed (100 and 50 rpm) and short gastric
emptying time (10 min). In those conditions, disintegration of the formulation was delayed, andthe subsequent dissolution of dipyridamole was not completed before the fluid pH was changed toneutral. Similar results were obtained when the initial gastric pH was increased to 3.0, 5.0, and 6.5.
To investigate that those factors also affect the BE of oral drug products, dissolution and permeationof naftopidil from its ordinary and orally disintegrating (OD) tablets were observed in the BE checker.
Both products showed the similar dissolution profiles when the paddle speed and gastric emptying
time were set to 100 rpm and 10 or 20 min, respectively. However, at a low paddle speed (50 rpm),
the dissolution of naftopidil from ordinary tablets was slower than that from the OD tablets, and thepermeation profiles became dissimilar. These results indicated the possibility of the bioinequivalenceof some oral formulations in special patients whose GI physiologies are different from those in thehealthy subjects. The BE checker can be a highly capable in vitro tool to assess the BE of oral drug
products in various populations.

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Keywords: oral drug product; bioequivalence; special population; gastrointestinal physiology;
dissolution; permeation; gastric emptying

iations.

1. Introduction
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

The bioequivalence (BE) study of oral dosage forms is performed to assure therapeutic
equivalence of two drug products containing the same active pharmaceutical ingredient(API), by comparing the rate and extent of drug absorption after oral administration [1–3].
After in vitro tests for drug release/dissolution, human BE study is usually performed
using healthy adult volunteers. Subjects having undesirable diseases or who are taking
concomitant medicines are excluded from these studies according to the exclusion criteria.
This is to avoid the risk of adverse events caused by the test or reference products, andPharmaceutics 2021, 13, 1136. https://doi.org/10.3390/pharmaceutics13081136

https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2021, 13, 1136

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the study design with healthy subjects is rational. However, once the drug products areapproved and put on the market, various patients having diverse chronic disorders, healthconditions, or lifestyles may take them. Such patients often use generic drug products as aswitch from original products. Switched products will show the same efficacy and safety
as the original ones for most patients because they are proved to be BE. However, that may
not be always the case for certain populations. As a typical example, poorly soluble basic
drugs are known to be dissolved in the stomach at a low pH and are then transferred to thesmall intestine as a solution to be absorbed rapidly. In patients with achlorhydria or withconcomitant use of proton pump inhibitors (PPIs), the dissolution of basic drugs in thestomach will be inhibited, and the absorption from the small intestine will be decreased. In
those patients, the rate and extent of drug absorption becomes strongly dependent on theformulation design, and some BE products may show different profiles of absorption that
could not be detected in a BE study with healthy subjects. Other factors of GI physiology
such as gastric emptying rate, agitation strength, GI fluid compositions (including bileconcentrations), and volumes can cause the inequivalent performance of BE products inthe special population. Although the concept of “BE in the special population” is widely
recognized as a critical issue when using a generic drug product, clear rules are not yet
defined because of the lack of clinical evidence and the methodology to detect it.
Due to the less biorelevant conditions that are currently used in in vitro drug dissolution tests defined by the Pharmacopeia in each country (typically a paddle or a basket
in 900 mL vessels), various kinds of in vivo predictive in vitro methods for investigating
drug dissolution in the GI tract have been developed. Several types of multiple compartmental GI transfer systems have been developed so far, in which two or three dissolution
vessels are connected by peristatic pumps for analyzing the drug dissolution in each partof the GI tract [4–11]. As one of those systems, a multicompartmental in vitro dissolution
apparatus, the gastrointestinal simulator (GIS), was shown to be able to detect the effect ofgastric pH on the dissolution profile of dipyridamole, a BCS class II weak base drug, whichcorresponded well to the clinically observed drug–drug interaction with famotidine [12].
An additional function of drug partitioning to the octanol phase was also introduced to
the GIS to observe the process of membrane permeation [13]. Further, as a more complex
system, the TNO gastro-intestinal model is designed to realistically simulate conditionsin the GI tract. Although such systems are successful for simulating the performanceof drug formulations in the GI tract, applications to the test these drugs in various GI
conditions representing those in particular patients are limited because of their complex
and large-scale systems, thus low throughput and difficulty in setting up the system forvariable GI conditions.
In a previous study [14], we have developed an in vitro system (named the Stomach-toIntestine Fluid Changing (SIFC) system) which can reproduce the temporal changes in thepH, composition, and volume of the GI fluid in a single vessel equipped with one syringe
pump. In the SIFC system, the fasted state gastric simulated fluid (FaSSGF) was used as
an initial fluid in the vessel to which drugs could be applied as a powder or formulation.
The concentrated fasted state simulated intestinal fluid (pre-FaSSIF) was then infused to
the vessel at a constant speed to change the entirety of the fluid to FaSSIF (pH 6.5). For
the applied drug or drug formulation, this process corresponded to the transit from thestomach to the small intestine after oral administration. One of the most advantageous
points of this system is that it is possible to respond to a variety of GI conditions by simply
changing the fluid pH or composition, and infusion rate of the GI system. Using this system,
the effects of the initial gastric pH and dose strength in the formulation of telmisartan
were observed to simulate dose-dependent intra-subject variability in the plasma drugconcentration in human BE studies.
In this study, based on the previously developed SIFC system, a more biorelevant
in vitro system (in vitro bioequivalence checking system (BE checker)) was newly designed
to predict the BE of oral drug products in healthy and specific populations. Not only the
dissolution but also the membrane permeation of drugs from marketed products were ob-Pharmaceutics 2021, 13, 1136

3 of 17served. As they are the GI physiological factors that influence the drug release/dissolution
profile, effects of the initial gastric pH, gastric emptying rate, and the agitation strength in
the GI tract were investigated using the BE checker to demonstrate the risk of bioinequivalence in special populations.
2. Materials and Methods
2.1. Materials
Hank’s balanced salt solution (HBSS) was obtained from Gibco Laboratories (Lenexa, KS,
USA). Egg-phosphatidylcholine (lecithin) was obtained from FUJIFILM Wako Pure Chemical(Osaka, Japan). Sodium taurocholate (NaTC) was obtained from Nacalai Tesque (Kyoto,
Japan). Seloken® tablet 20 mg and Persantin® tablets 25 mg were obtained from AstraZeneca(Osaka, Japan) and Nippon Boehringer Ingelheim (Tokyo, Japan), respectively. Flivas® tablets
25 mg and Flivas® orally disintegrating (OD) tablets 25 mg were obtained from Asahi Kasei
Pharma (Tokyo, Japan). All other reagents used were of the highest purity.
2.2. Design of In Vitro Bioequivalence Checking System for Oral Drug Product (the BE Checker)
The BE checker developed in this study is illustrated in Figure 1. A hydrophilic filter
with a pore size of 0.22 µm (Durapore membrane filter, MERCK Millipore, Burlington, MA,
USA) was mounted between the donor and receiver chambers. The effective surface areaof the filter was 6.60 cm2 . Both chambers were made of acrylic plastic. The volume ofthe donor chamber was 40 mL in the bottom, which corresponds to the stomach, and themodel had a total volume of 100 mL. The volume of the receiver chamber was set to 30 mL.

Figure 1. Schematic illustration of the BE checker.

The compositions of FaSSGF and FaSSIF used in this study are summarized in Table 1.
At the beginning of the experiment, the bottom of the donor chamber was filled with 40 mL
of FaSSGF. After administration of the tablet, 1.67-times concentrated FaSSIF (pre-FaSSIF)
was infused into the donor chamber to shift the donor fluid to FaSSIF. Since HBSS contains
19.45 mM D-glucose, the final concentration of the D-glucose in the FaSSIF was 25 mM.

Pharmaceutics 2021, 13, 1136

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Table 1. Compositions of the donor fluid used for the BE checker.
FaSSGF(mM)

FaSSIF(mM)

NaTC
Lecithin
NaCl
Na2 HPO4
Citric acid
NaHCO3D -glucose
HEPES

0.08
0.02
34.2
20
10
-

3
0.75
13.7
20
10
4.17
5.55
10

dissolved in

Purified water

HBSS

The pH of the FaSSGF was adjusted to 1.6, 3.0, 5.0, or 6.5 using the diluted HCl and/or
NaOH solution. Pre-FaSSIF was prepared to finally construct 100 mL of FaSSIF (pH 6.5)
after its addition to 40 mL of FaSSGF.
The receiver chamber was empty at the beginning, and octanol was then infused into
the receiver side at a rate to keep the height of the fluid surface the same as the donor side.
2.3. Simultaneous Evaluation of Disintegration/Dissolution and Permeation of the Drugs from theTablets Administered to the BE Checker
Experimental conditions designed to represent the GI physiology in the fasted state
are shown in Figure 2. A tablet was administered to 40 mL FaSSGF in the bottom of
the donor chamber. After agitation for one minute as a disintegration/dissolution phase,
60 mL pre-FaSSIF was infused into the donor chamber at a constant rate for 10 or 20 min
using a syringe pump as gastric emptying phase. At the same time, water-saturated octanol
was infused into the receiver side. After infusion, dissolution and permeation of the drugin the small intestine were evaluated. Samples were taken from each side of the chamber at
the specified time. Donor samples were immediately filtered through a membrane filter(Millex®-LH, pore size: 0.45 µm, Millipore, MERCK Millipore, Burlington, MA, USA) toremove any undissolved and precipitated drug. The volume of each side was maintainedby adding fresh fluid after sampling. Samples were stored at −80 ◦ C until analysis.

Figure 2. Experimental conditions represent the GI physiology in the fasted state in the BE checker.

Pharmaceutics 2021, 13, 1136

5 of 172.4. Analytical Methods
Drug concentrations in the samples were determined using an ultra-performance liquid
chromatography (UPLC) system (ACQUITY® UPLC, Waters, MA, USA) equipped with atandem mass spectrometer (ACQUITY TQD, Waters, MA, USA). In metoprolol and dipyridamole, the donor and receiver samples were diluted with 0.1% formic acid/acetonitrile(50:50) and water/acetonitrile (20:80), respectively. In the case of naftopidil, the donor andreceiver samples were diluted with 0.1% formic acid/methanol (50:50) and water/methanol(20:80), respectively. A reversed-phase Waters Acquity UPLC BEH C18 analytical column (50mm × 2.1 mm, 1.7 µm particle size) was used with a mobile phase consisting of 0.1% (v/v)
formic acid (solvent A) and acetonitrile containing 0.1% (v/v) formic acid (solvent B) with agradient time period. In the case of naftopidil, 0.1% (v/v) formic acid was used as solvent A,
and methanol was used as solvent B. The initial mobile phase was 98% solvent A and 2%
solvent B pumped at a flow rate of 0.3 mL/minutes. Between 0 and 1.0 min, the percentage ofsolvent B increased linearly to 95%, which was then maintained for 1.0 min. Between 2.01 and2.5 min, the percentage of solvent B decreased linearly to 2%. This condition was maintained
until 3 min had pased, at which time the next sample was injected into the UPLC system. All
samples were injected at a volume of 5 µL into the UPLC system. The ionization conditions
for the analysis of metoprolol were as follows: electrospray ionization condition, positivemode; source temperature, 150 ◦ C; desolvation temperature, 400 ◦ C; cone voltage, 10 V; andcollision energy, 50 V. Precursor and production ion (m/z) were 267.67 and 77.05, respectively.
The ionization conditions for the analysis of dipyridamole were as follows: electrosprayionization condition, positive mode; source temperature, 150 ◦ C; desolvation temperature,
400 ◦ C; cone voltage, 30 V; and collision energy, 48 V. The precursor and production ion (m/z)
were 505.33 and 385.48, respectively. The ionization conditions for the analysis of naftopidil
were as follows: electrospray ionization condition, positive mode; source temperature, 150 ◦ C;
desolvation temperature, 400 ◦ C; cone voltage, 40 V; and collision energy, 30 V. Precursor andproduction ion (m/z) were 393.39 and 190.09, respectively.
3. Results
3.1. pH-Shift Profiles in the Donor Chamber of the BE Checker
A total of four types of pH-shift profiles from the donor side reproduced in this study are
shown in Figure 3. The pH of the initial FaSSGF was set to 1.6, 3.0, or 5.0 to mimic the gastric
pH of healthy or achlorhydric patients. In all protocols, the fluid pH increased gradually with
the infusion of pre-FaSSIF, and the profiles of the pH shift were smooth in all pH ranges. Since
all fluids contained Na2 HPO4 and citric acid as buffering agents, this profile corresponded
to that of the gradual pH change protocol in our previous report [14]. The final pH after
completing the pre-FaSSIF infusion was close to 6.5 in all experimental conditions.

Figure 3. Time profile of the fluid pH in the donor chamber of the BE checker.

harmaceutics 2021, 13, x

6 of 18

Pharmaceutics 2021, 13, 1136

6 of 173.2. Effect of Paddle Speed on the Dissolution and Permeation of Metoprolol from Seloken®
Tablets
Paddleset toSpeed
50, 100,
or 200rpm in and
the BE
checkeroftoMetoprolol
observe theeffectof ® Tablets
3.2.speed
Effectwasof Paddle
on theDissolution
Permeationfrom
Seloken®
agitation strength
on thedissolution
from
its inmarketed
product.
Selokenthe
Paddlespeed
was set of
to metoprolol
50, 100, or 200rpm
the BE checker
toAobserve
effect oftablet 20 mg
was usedin thissince it wasdemonstrated
that
dissolvesagitation
strength
onstudy
the dissolutionof metoprolol
fromits metoprolol
marketed product.
A Seloken®
completely
in thethe itJapanesePharmacopeia
and its dissolves
tablet20 inmgvitrowas dissolution
used in thisstudy
studyofsince
was demonstrated
that(JP),
metoprololdissolutionisnotinfluenced
by
the
fluid
pH
(paddleapparatus
with
pH
1.2,
4.0,
and
completely in the in vitro dissolution study of the Japanese Pharmacopeia 6.8(JP), and its
medium) [15].
Initial gastricpH and pre-FaSSIF
infusiontime were
fixed towith
pH 1.6dissolution
is not influenced
by the fluid
pH (paddle
apparatus
pHand
1.2,104.0, and 6.8
min, respectively.
medium) [15]. Initial gastric pH and pre-FaSSIF infusion time were fixed to pH 1.6 andAs shown
in respectively.
Figure 4, the dissolution of metoprolol from the Seloken® tablet became
10 min,® tablet became
faster as the paddle
speedinincreased
200 rpm, andmore thanfrom
80% theof themetoprolol
As shown
Figure 4,and
the atdissolutionof metoprolol
Selokenwas dissolved
in
the
donor
chamberwithin30
min.
This
profileofmetoprololdissolution
faster as the paddle speed increased and at 200 rpm, and more than 80% of the metoprolol
is similar wasto thatof the one
fromthe chamberdissolution
test 30
in min.
the JPThis(paddlerotation
speed =dissolution
50
dissolved
in thedonor
withinprofileof metoprololisrpm, 20 mg
in 900was slowedand50 rpmand reachedsimilartomL)
that [15].
of theTheonedissolution
from the dissolution
testatin 100the JP(paddlerotation
speed = 50 rpm,
to 80% within
45inmin
100 [15].
rpm andmin at 50wasrpm,slowed
respectively.
Since50the
shape
20 mg
900atmL)The 60dissolution
at 100 andrpm
andofreached toapparatus80%
and within
the dose/fluid
ratioandwere
betweenthe dissolution
testthein shape of
45 min volume
at 100 rpm
60different
min at 50
rpm, respectively.
Since
the JP andapparatus
the BE checker,
it
is
not
possibletodirectlycomparethecorrelation
betweenand the dose/fluid volume ratio were different between the dissolution test in
the paddlethe
speed
and
dissolution
profilesin bothstudies.
However,these experiJP andthedrug
BE checker,
it is notpossibleto directlycomparethe correlation
between themental conditions,
50 toand200drugrpm dissolutionof paddle speed
in intheboth
BE checker,
were consideredtopaddle speed
profilesstudies. However,these experimental
cover a reasonable
range
of200
agitation
the human
GIchecker,
tract for were
testing
the disso-to cover a
conditions,
50 torpm ofstrength
paddle inspeed
in the BE
consideredreasonable
rangeitofisagitation
the humanfor testing
dissolution oflution of drugs,
althoughdifficult strength
to defineinwhich
speedGIis tract
averagein the thehealthy
drugs, although it is difficult to define which speed is average in the healthy population.
population.

Figure 4. Effect of paddle speed on the dissolution (A) and permeation (B) time profiles of metoprolol from Seloken® tablets
Figure 4. Effect of paddle speed on the dissolution (A) and permeation (B) time profiles of metoprolol from Seloken®
20 mg in the BE checker. Each data point represents mean ± S.D. (n = 3).
tablets 20 mg in the BE checker. Each data point represents mean ± S.D. (n = 3).

The effect of paddle speed on the permeation profile of metoprolol was not clearly
The effectof paddle
on the permeationof phase-profile
metoprolol was
notdissolution.
clearlyobserved
despitespeed
its pronounced
effect on profile
the earlyof theSinceobserved despite
its
pronounced
effectontheearlyphase-profile
ofthedissolution.
Since
the filter was not soaked with the donor and receiver fluids at the beginning andbecause thethe filter was
soakedwith thedonor
and receiver
fluids at the
beginningand because
fluidnotlevel
graduallyraisedto initiate
the permeationof thedrug, early-phasedifferences
the fluid level
graduallyraisedtoinitiatethepermeation
of
the
drug,
early-phasedifferin the dissolution profile may not be completely reflected in the permeation profile. This
ences in the
profile may
beexperimentscompletely reflected
permeation
profile.
is dissolution
a similar situation
to innotvivo
because inthethedrug
dissolution
profile in theThis is a similar
situation
to
in
vivo
experiments
becausethedrug
dissolution
profilein profile ofstomach just after drug administration may not profoundly affect the absorption
the stomach
just
after
drug
administrationmay
notprofoundly
affecttheabsorption
prothe intestine.
file of the intestine.
3.3. Effect of Paddle Speed, Pre-FaSSIF Infusion Time and Initial Fluid pH on the Dissolution and3.3. Effect ofPaddle Speed,
Pre-FaSSIF from
Infusion
Time ®
and
InitialinFluid
pHChecker
on the Dissolution
Permeationof Dipyridamole
Persantin
Tablets
the BE®
and Permeation Dipyridamoleof Dipyridamole isfrom
Persantin
Tablets
intheBE
Checkera weak basic drug and is highly soluble to water at a low pH
Dipyridamoleis a weak
basic
drug
highlyatsoluble
to water
at a low(>100,000 µg/mL
at pH
< 2.0
andand7000isµg/mLpH 3) butis less solubleat pH
a neutral pH(>100,000 (6µ g/mLat pH< 2.0
andThe7000dipyridamoleµ g/mL at pHdissolution3) but is lesssolubleat aits
neutralpH product,
µg/mL
at pH
6.5).
profilefrommarketed
Persantin® tablets 25 mg, using the paddle apparatus was reported to be strongly affectedby medium pH in the dissolution study. Dipyridamole dissolved almost completely within
10 min at pH 1.2 but less than 20% after 360 min at pH 6.5 [15].

(6 µ g/mL at pH 6.5). The dipyridamole dissolution profile from its marketed product, Persantin® tablets 25 mg, using the paddle apparatus was reported to be strongly affected by
Pharmaceutics 2021, 13, 1136
7 of 17
medium pH in the dissolution study. Dipyridamole dissolved almost completely within
10 min at pH 1.2 but less than 20% after 360 min at pH 6.5 [15].
Figure 5A shows the dissolution profiles of dipyridamole from the Persantin® tablet
in the BE checker
at 5A
the shows
paddlethespeedsof 50, 100,or 200rpm. The initial
gastric
pH and ® tablet
Figuredissolution
profilesof dipyridamolefromthe Persantin
pre-FaSSIF
infusiontime wereto pH
1.6 and
10 100,min,orrespectively.
Atinitial
the 200
rpm pH andin theBE checker
at thefixed
paddlespeedsof 50,
200 rpm. The
gastric
paddle speed,
dipyridamole
was dissolved
within
5 min,and the dissolved
pre-FaSSIF
infusion time
were fixedcompletely
to pH 1.6 and
10 min,respectively.
At the 200 rpm
amount in
the donor
chamberthen decreased
gradually.
Because within
the infusion
pre-FaSpaddle
speed,
dipyridamole
was dissolved
completely
5 min,ofandthe dissolved
amount in thechamberthenchanged
decreased
infusionof pre-FaSSIF
SIF was completed
anddonorthe fluid
pH wastogradually.
6.5 by theBecause
10 min the
point,
this timeand the fluid
pH was achanged
to 6.5 by
the 10 min point,
thisthetime-profile
ofprofile ofwas
thecompleted
dissolved amountrepresented
precipitationof dipyridamolefromsuthe dissolved
amountby
represented
a precipitationof dipyridamole
supersaturated
persaturated
solution caused
the pH shift
from 1.6 to 6.5.
In contrast, atfrom
100 theor 50
rpm
by the pHwas
shiftnotfrom
1.6 to 6.5.and
In contrast,
at 100 amountor 50 rpm
paddle speed,of paddlesolution
speed, caused
supersaturation
observed,
the dissolved
ofofdipyrnot25%
observed,
and theamountof dipyridamole
idamole supersaturation
was kept at lesswas
thanthroughout
thedissolved
experiment.
Permeationof dipyr-was kept
25%chamber
throughout
the experiment.
Permeationof dipyridamole
the receiver
idamole at
to less
the thanreceiverin Figure
5B well reflected
the differencein the totimechamber
inFigure5B
wellreflected
thedifferenceinthetime-profile
ofdissolved
amount.
profile of dissolved amount.

Figure 5. Effect of paddle speed on the dissolution (A) and permeation (B) time profiles of dipyridamole from Persantin®
Figure 5. Effect of paddle speed on the dissolution (A) and permeation (B) time profiles of dipyridamole from Persantin®
tablets 25 mg in the BE checker. Each data point represents mean ± S.D. (n = 3).
tablets 25 mg in the BE checker. Each data point represents mean ± S.D. (n = 3).

The pre-FaSSIF infusion time was then prolonged to 20 min, and the same experiments
Thewerepre-FaSSIF
infusiontime wasthen 6,
prolonged
to 20 min, andsame experiperformed.
As shown
in Figureinitial supersaturation
andthesubsequent
precipitation
ments were
performed.
As
shown
in
Figure6,initialsupersaturation
andsubsequent
prewere not only observed at 200 rpm, but also at 100 and 50 rpm of paddle speed. The
cipitationdissolved
were not andonlypermeatedobserved at
200 rpm,werebut also
atthe
100sameand 50of paddle
speed.
amountsabout
at rpm
100 and
200 rpm,
while those
The dissolved
andpermeatedamounts
were
about
the
same
at
100
and
200
rpm,
while
at 50 rpm were lower during the initial 30 min of the experiment. Since thepre-FaSSIF
those at infusion
50 rpm were
lower
duringtheininitial
30 min of theSince
the pre-the drug
time inthe BE
checker
vitro corresponds
to experiment.
the time needed
to transfer
FaSSIF infusionin thetoBE
checkerin vitro corresponds
to the intimeneededto transferfrom thetime
stomachthesmall intestinein vivo, the results
Figures
5 and
6 imply that thethe drug combinationfrom the stomach
the smallintestinein thevivo,
the resultsin Figure
5 and
6 implyof thetoagitation
strengthandgastric
emptying
time ispossibleto cause thethat the combinationof the agitation
strength
the gastric
emptying timeinisvivo.
possible tovariety of dissolution
profilesand theandabsorptionof dipyridamole
cause the variety of dissolution profiles and the absorption of dipyridamole in vivo.

Figure 6. Effect of pre-FaSSIF infusion time on the dissolution (A) and permeation (B) time profiles of dipyridamole from
Persantin® tablets 25 mg in the BE checker. Each data point represents mean ± S.D. (n = 3).

Pharmaceutics 2021, 13, 1136

8 of 17

In Figure 7, as another factor to influence the dissolution profile of dipyridamole,the initial pH of FaSSGF was varied from 1.6 to 6.5. For other experimental conditions,
pre-FaSSIF infusion time and paddle speed were fixed to 20 min and 100 rpm, respectively.

Figure 7. Effect of initial FaSSGF pH on the dissolution (A) and permeation (B) time profiles of dipyridamole from
Persantin® tablets 25 mg in the BE checker. Each data point represents mean ± S.D. (n = 3).

As already shown in Figure 5, a supersaturated dissolution and higher permeation of
dipyridamole were observed when the initial FaSSGF pH was set to 1.6, whereas at thehigher initial pH conditions, this supersaturation profile disappeared, and the dissolved
and permeated amounts of dipyridamole decreased as the initial pH increased. Fromthe pH-solubility profile of dipyridamole, this result seems reasonable and suggests thepossibility of lower absorption in patients with high gastric pH.
3.4. Application of the BE Checker for the Assessment of BE between Flivas® Tablets and Flivas®
OD Tablets
To investigate whether the difference in the experimental conditions in the BE checker
reflect whether the variable GI physiology causes the inequivalent performances of two BE
products, ordinary and OD tablets of naftopidil (Flivas® tablets and Flivas® OD tablets)
were used in the experiment. Flivas® OD tablets have been proved to be BE with Flivas®
tablets in human BE studies and are approved for clinical use. Naftopidil is a weak basedrug with a pKa of 3.7 and 6.7 and shows quite low solubility at a neutral pH (<0.1 µg/mL
at pH > 5.0).
As shown in Figure 8, regardless of the pre-FaSSIF infusion time, the dissolution of
naftopidil from the OD tablets was significantly slower than that from the ordinary tablets,
and a clear peak of supersaturation was not observed. Accordingly, the permeation of
naftopidil from the OD tablets was slower during the initial 30 min. Since the OD tablets
are designed to disintegrate quickly in the oral cavity after administration, even without
a concomitant ingestion of water, the results in Figure 8 were completely different fromwhat we expected. To understand the reason why, JP1 solution (pH 1.2), FaSSGF (pH 1.6)
or distilled water was dropped onto the OD tablet in a Petri dish, and the disintegration
process was observed. Figure 9 shows the pictures at 1 and 3 min after the addition ofeach solution.

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Figure 8. Comparison of dissolution (A,C) and permeation (B,D) of naftopidil from Flivas® tablets and Flivas® OD tabletsin the BE checker. Pre-FaSSIF infusion time was set to 10 min (A,B) and to 20 min (C,D). Initial FaSSGF pH was 1.6, andpaddle speed was 100 rpm. Each data point represents mean ± S.D. (n = 3).

Figure 9. Disintegration of Flivas® OD tablets after addition of JP1, FaSSGF, or distilled water onto
the tablets.

When JP1 or FaSSGF was added, a gel-like layer formed on the surface of the OD
tablet, which disturbed further disintegration, while the addition of distilled water quickly
progressed the disintegration. It was reported that the solubility of naftopidil in JP1 solution (pH 1.2, prepared by HCl solution) is lower than that in phosphate buffer at pH 2.0(0.05 mol/L) because the solubility of naftopidil in JP1 is limited by a solubility equilibrium(product) with chloride ions [16]. The formation of the gel-like layer shown in Figure 9 might
be due to the aggregation of quickly dissolving and precipitating naftopidil (as a complex with

Pharmaceutics 2021, 13, 1136

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chloride ions) at the surface of the OD tablet. In the in vivo situation for drug administration,
if the OD tablets are directly administered into the stomach, the same phenomenon will occur.
However, since the OD tablets are disintegrated and dispersed quickly in the oral cavity
before reaching to the stomach, the delay in the dissolution and absorption was not observedin the human BE study in vivo [16]. This is even true when the OD tablets were taken withoutwater because the OD tablets can be disintegrated by saliva.
To reflect the situation of in vivo oral drug administration, the process of tablet disintegration in water was introduced to the experimental protocol of the BE checker. In the newprotocol, first, the bottom of the donor chamber was filled with 37.5 mL of distilled water.
After 1-min of incubation with the tablet, 0.25 mL of concentrated FaSSGF was added to
set the donor fluid volume as 40 mL FaSSGF (pH 1.6), assuming the orally administeredtablet will reach the stomach in 1 min.
By introducing this disintegration time to the experimental protocol, dissolutionof naftopidil from the OD tablets became faster and showed similar dissolution andpermeation profiles to those from the ordinary tablets (Figure 10). In this experiment, a
new protocol with 1-min disintegration in water was applied both for the ordinary andOD tablets. The longer infusion time of pre-FaSSIF (20 min) increased the dissolved andpermeated amount of naftopidil, whereas the dissolution and permeation profiles were
almost the same between the ordinary and the OD tablets.

Figure 10. Comparison of dissolution (A,C) and permeation (B,D) of naftopidil from Flivas® tablets and Flivas® OD
tablets in the BE checker. Experiments were performed with a new experimental protocol with 1-min disintegration time.
Pre-FaSSIF infusion time was set to 10 min (A,B) or to 20 min (C,D). Initial FaSSGF pH was 1.6, and paddle speed was 100
rpm. Each data point represents mean ± S.D. (n = 3).

Using the same protocol including 1-min disintegration time, the effects of agitation
strength on the dissolution and permeation of naftopidil were observed by changing thepaddle speed to 50 rpm (Figure 11). As seen in Figure 11, the dissolved and permeatedamounts of naftopidil decreased significantly from both the ordinary and the OD tablets,
compared to those in Figure 10 at 100 rpm of paddle speed.

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Figure 11. Effects of paddle speed (50 rpm) on the dissolution (A,C) and permeation (B,D) of naftopidil from Flivas® tablets
and Flivas® OD tablets in the BE checker. Experiments were performed with a new experimental protocol with 1-min
disintegration time. Pre-FaSSIF infusion time was set to 10 min (A,B) or to 20 min (C,D). Initial FaSSGF pH was 1.6. Eachdata point represents mean ± S.D. (n = 3).

However, since the effect of decreased paddle speed was more pronounced on theordinary tablet, clear differences in dissolution and permeation profiles were observed
between the ordinary and OD tablets, at both pre-FaSSIF infusion times of 10 and 20 min.
To quantitatively assess the similarity level between the ordinary and OD tablets, thef2 value was calculated as a similarity factor for the naftopidil permeation profiles fromtwo products in the BE checker using the following equation


100

(1)
f2 = 50 × log q2∑ni=1 (Ti−Ri)1+nwhere Ti and Ri indicate the permeated amount of naftopidil (% of dose) from the ordinary
tablets and the OD tablets, respectively, and n indicates the number of measurement points
to be compared. In the calculation, f2 was evaluated at four time points, 1/4Ta, 1/2Ta,
3/4Ta, and Ta, where the permeated amount from the ordinary tablet at 120 min was set as
100%, and Ta was the time point of about 85%. Table 2 represented the similarity factor(f2 value) obtained from the results in Figure 8, Figure 10, and Figure 11. If the border of thesimilarity was set to 50, it was could be concluded that both tablets gave similar profiles ofnaftopidil permeation at the 100 rpm paddle speed when the protocol with disintegrationin water was applied, whereas at 50 rpm, a similarity in the permeation profile was not
obtained. These results might imply that the ordinary and OD naftopidil tablets are BE
in subjects with strong (or normal) stomach agitation strength, but there is a risk of being
bioinequivalence in subjects whose agitation strength is weak.

Pharmaceutics 2021, 13, 1136

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Table 2. Assessment of the similarity in drug permeation profiles between Flivas® tablets and Flivas®
OD tablets using similarity factor.
Similarity Factor
Protocols

Without Disintegration Timein WaterWith Disintegration Timein Water

100 rpm, 10 min
100 rpm, 20 min
50 rpm, 10 min
50 rpm, 20 min

31
23
–
–

60
63
34
41

4. Discussion
Although various in vitro systems have been developed and used to assess thebioavailability and/or bioequivalence of oral drug products, starting with a simple dissolution test, it is fair to say that no in vitro systems can perfectly mimic these highly complex
in vivo drug absorption processes. For all systems, there are some pros and cons. With
those as a basic premise, we have designed a new in vitro system, the BE checker, for
testing the BE of oral drug products with the following questions in mind;

X
X
X
X

How is the system biorelevant?
How can the various physiological conditions in the GI tract be reflected?
What kind of data can be obtained from evaluating BE?
What is the throughput and cost-performance?

As a biorelevant system, it is essential to expose drug products to different environments in the GI tract, to those in the stomach first, and then to those in the small intestine.
Multiple compartmental GI transfer systems reproduced these processes by connecting several vessels filled with biorelevant fluids [4,6–10,12,13]. Small particles or dissolved drugs
in the fluids were transferred from one vessel to the next using a peristatic pump to mimica GI transit in vivo. Although such compartmental systems are successful in observing
the disintegration/dissolution of drug formulations in each part of the GI tract, systems
are relatively large and complicated. In addition, the peristatic pump sometimes fails toadequately transfer the drug particles that are not dissolved or yielded by precipitation.
In the BE checker, the GI transit of drug formulations or drugs was embodied bychanging the surrounding environments while the drugs were kept in the same vessel. Thisis the most important and advantageous feature of this system, which was adapted from thepreviously developed SIFC system [14]. This concept is noted as “environmental changes
in the GI tract from the stomach to the small intestine are reproduced in one chamber by
gradually changing the fluid pH, composition and volume without transferring drugs or
drug formulations”. Only one donor chamber equipped with one syringe pump is neededto achieve this process in the BE checker.
Another point to be addressed is that a receiver chamber was added to the system,
which, when equipped with another pump to infuse octanol as a receiver fluid, can observe
the membrane permeation process of the dissolved drug. We have already developeda small-scale, side-by-side chamber system and dissolution/permeation (D/P) system
to investigate the oral absorption of drugs, mainly the fraction/dose absorbed from thesmall intestine (Fa). In the D/P system, a Caco-2 cell monolayer was used as a model
membrane of the human intestine, and dissolved and permeated amounts (during thedefined period) were measured after the addition of the drug to the donor chamber as apowder. A permeated amount in the D/P system was then converted to the Fa found in
humans by using a standard curve obtained in advance using various marketed drugs.
The D/P system is now used to screen the oral absorbability of APIs as well as to checkthe effect of food intake or different formulations on it [17–19]. In the BE checker, insteadof a Caco-2 cell monolayer, a hydrophilic filter was mounted between the chambers toprevent the mixing of donor and receiver fluids. As a receiver fluid, octanol was usedbecause the partition of octanol is known as a good surrogate for drug permeation acrossPharmaceutics 2021, 13, 1136

13 of 17

the intestinal membrane. In an in vitro bi-phasic system, octanol is used as an absorptive
compartment [11]. Shi et al. have measured the time-profile of a drug amount partitioned tooctanol from the dissolution medium in a bi-phasic system and reported its high possibilityof assessing the drug absorption profile in vivo [20]. In the BE checker, the height of thefluid surface on both sides is kept always the same by adjusting the octanol infusion speed.
The surface area available for drug permeation gradually increases as the amount of thefluid increases. This may partially reflect the in vivo process of fluid spreading into theintestinal tract to increase the membrane surface area for drug absorption.
As with the case of a D/P system, the amount of the permeated drug (% of dose) in the
BE checker was much smaller than that in vivo absorption (Fa). This is simply due to thelower permeation (absorption) clearance of the filter/octanol system in the BE checker thanthat in the in vivo absorption from the small intestine because of the smaller membranesurface area/volume ratio (A/V ratio) in this system. To evaluate the Fa from the datafrom the BE checker, the same method as that used for a D/P system is applicable inthat the correlation between the permeated amount and Fa was used as a standard curve.
Additionally, it is possible to convert the drug permeation profile in the BE checker to its
plasma concentration profile after oral administration using in silico PK modeling. The
obtained parameters such as AUC and Cmax will then be used to predict the BE of the test
formulation. This is the next project to make the BE checker more useful for BE analysis
using in vitro–in silico–in vivo extrapolation (IVISIVE). The study is currently in progress
and will soon be reported.
In this study, the BE checker was designed as a 1/4 scale model of the human GI tract
in volume. In some cases, drug products containing a 1/4 dose API of maximum dosestrength are commercially available. By using a 1/4 dose strength product, the result in the
BE checker can be regarded as representing the performance of the product with maximal
dose strength in vivo, although the ratio of other excipients might be different. This is to
reduce a cost and increase the throughput of the experiment. If necessary, it is not difficult
to prepare a 1/1 scale system with a similar shape and functions.
The fasted state fluid volume in the stomach immediately after the ingestion of 150 mL ofwater (standard volume used for human BE study in Japan) was reported as 160–200 mL [21];thus, the initial volume of FaSSGF was set to 40 mL in the BE checker. The final volume of thedonor chamber (as FaSSIF) should correspond to the volume of the small intestine. However,in this study, it was defined as 100 mL despite the fact that the fasted state intestinal volume
was reported to be around 100–200 mL, even after the ingestion of 250 mL of water [22].
This is due to the absorption of water by the small intestine; thus, it was assumed that the
total volume of water to which drugs are exposed to in the small intestine is larger than
the reported value. To determine whether the final volume of FaSSIF in the BE checker isadequate or not to assess BE, additional studies should be done to compare the results in the
BE checker with those in human BE studies for various drug products. Those studies are now
on going and will be reported in the near future.
Other factors of the GI physiology relating to drug dissolution/absorption werealso reflected in the experimental protocol of this study by considering the variation
among patients. Among them, agitation strength in the stomach is the most difficult to beparameterized for in vitro study because in vivo strength in the stomach is not uniform,
and the peristaltic movement is too complex to correctly mimic with paddle rotation. In
the results using the Seloken® tablet (Figure 4), a 200-rpm paddle speed in the BE checker
gave a dissolution profile similar to that of metoprolol observed at 50 rpm of the paddle
apparatus (in 900 mL vessel) [15]. Although a 50-rpm paddle speed is often used indissolution tests, it does not necessarily reflect the in vivo situation properly, and in vivo
agitation strength is generally considered to be weaker [23]. In this study, including theinter-subject variability, following experiments were done at three different paddle speeds,
50, 100, and 200 rpm.
Although human gastric pH is generally considered to be less than 2 in fasted condition, it was reported that a higher gastric pH (>3) was detected in about 10% of subjects,

Pharmaceutics 2021, 13, 1136

14 of 17even among young healthy people [24]. In the elder population, a relatively high percentage of achlorhydric subjects whose gastric pH is almost neutral in all conditions was foundin Japan [25–27]. To reflect these inter-subject variabilities, in this study, the initial gastricpH was varied from 1.6 to 6.5, as shown in Figure 7.
In the study with the BE checker, the infusion time of pre-FaSSIF corresponds to thegastric emptying time in vivo. In our previous report, in which the gastric emptying rateof the ingested fluid was evaluated in humans, the half-life of the fluid emptying was
calculated as 3–5 min [21]. Takeuchi et al. reported that when the gastric half-life was
set to 8 min, multiple compartment systems gave the most relevant dissolution profile ofpropranolol and metoprolol for simulating its plasma concentration profile in humans [4].
Therefore, we have employed 10 and 20 min as the pre-FaSSIF infusion times. In addition,
since longer half times for gastric emptying were also presented in some reports [28,29],
we have checked the dissolution and permeation profiles of dipyridamole by changing the
pre-FaSSIF infusion time to 30 min as well as to 10 and 20 min. A thirty-minute infusion
time gave the similar results to those obtained with a 20-min infusion time (data not shown).
Subsequent studies were then performed with 10 and 20 min of pre-FaSSIF infusion.
Results in Figure 5; Figure 6 clearly indicated the importance of both the agitation
strength in the stomach and the gastric emptying time to evaluate the oral absorption of
dipyridamole, which dissolved rapidly in the stomach under low pH conditions and thenprecipitated in the small intestine. In the patients with low agitation strength and fast gastric
emptying, the absorption of dipyridamole might decrease due to a less supersaturated
dissolution in the stomach. This is also the same for the patients with high gastric pH, asdemonstrated by the experiments with various initial FaSSGF pHs (Figure 7). Clinically,
the absorption of dipyridamole is known to be inhibited by co-medication with PPIs due toelevated gastric pH [27]. The results with the BE checker corresponded well to the in vivo
observations of the decreased absorption of dipyridamole in such patients.
Although the effects of GI physiological factors such as gastric agitation strength, emptying time, and fluid pH on oral drug absorption primarily depend on the physicochemical
properties of APIs, if the effects are also formulation-dependent, it is possible to affectand deviatethe bioequivalency of the products in special populations. The ordinary andOD naftopidil tablets, which have been proved as BE in human BE studies, were used tocheck the possibility of bioinequivalence in special GI situations. As a result, in the firstexperiment, the OD naftopidil tablets showed slow and incomplete dissolution profiles in
the BE checker compared to the ordinary tablets under standard experimental conditions(initial gastric pH was 1.6, paddle speed was 100 rpm, pre-FaSSIF infusion time was 10
or 20 min), even though the OD tablet was designed to disintegrate rapidly in the oralcavity. It was revealed that the solubility of naftopidil in a solution containing a highconcentration of HCl, such as FaSSGF or JP1, is limited by a solubility equilibrium withchloride ions and, in such solutions, a gel-like layer was formed on the surface of the OD
tablets due to the aggregation of the quickly dissolved and precipitated naftopidil as acomplex with chloride ions (Figure 9). The effect of the gel-like layer was not observedin the in vivo BE studies (thus being proved as BE) because the OD tablets were quickly
disintegrated and dispersed in the ingested water (or in the saliva) before arriving in thestomach. To mimic this situation, a 1-min disintegration time in water was added to theexperimental protocol. Since this new protocol is relevant to the process of in vivo oral
drug administration, subsequent studies including those with ordinary tablets were done
according to it.
In the conditions where initial gastric pH was 1.6 and the paddle speed was 100 rpm,
the ordinary and OD tablets showed similar dissolution and permeation profiles regardlessof the pre-FaSSIF infusion time (Figure 10), suggesting the BE of these two products in thehealthy population. However, this might not be true for the special patients whose gastricagitation strength is weaker. As shown in Figure 11, low paddle speed (50 rpm) caused
the difference in the dissolution profile between both naftopidil products, which was lessdissolved in the ordinary tablets than in the OD tablets. In the case of the OD tablets, rapid

Pharmaceutics 2021, 13, 1136

15 of 17disintegration can progress the initial dissolution of naftopidil under low pH conditions,even with a low agitation strength, whereas the dissolution was highly inhibited in theordinary tablets due to the slow disintegration in those conditions. This effect was more
distinct when the pre-FaSSIF infusion time was short, although the difference was stillobserved at 20 min of infusion time.
The similarity level between the two formulations were quantified by calculating f2
values for the time-profile of the permeated drug amount (% of dose). Since an f2 function
is usually applied to the dissolution profile in BE dissolution studies [3], the adequacy orrationality of applying this function to the drug permeation profile in the BE checker has not
yet been confirmed. However, considering the features of this function [30], calculated f2
values can give some indications of the similarity level of the drug permeation time profiles
for both products. If the border of the similarity and dissimilarity was set to 50, the resultin Table 2 implies the risk of bioinequivalence in the patients with weak gastric agitation
strength. It is possible to suggest that the two formulations may become bioinequivalence
inpatients with gastric diseases or gastrectomy and elder or younger patients, although
there is no clinical evidence to prove this at the present.
Currently, the use of in silico models and simulation technologies are accelerated andare expected to make a “virtual BE study” feasible in the near future [31]. However, evenwith a highly sophisticated in silico program, it is still difficult to predict the entire oral
drug absorption process from various formulations under various GI conditions with ahigh reliability. A combination with biorelevant in vitro data is essential for the use of insilico technology in the assessment of the BE of oral drug products. As a next study with
the BE checker, in vitro drug permeation data will be converted to its plasma concentration
time profile in silico to predict pharmacokinetic parameters such as Cmax and AUC in thehealthy and special populations.
5. Conclusions
The BE checker was designed to reproduce the environmental changes in the GI tract
associated with the transit of orally administered drugs from the stomach to the small
intestine by changing the pH, composition, and volume of the fluid of the GI tract with apre-set rate. This in vitro system enabled the assessment of drug dissolution/permeation
profiles from oral formulations under various GI conditions, which reflected those inhealthy and special populations in vivo. For ordinary and OD naftopidil tablets, different
dissolution and permeation profiles were observed when the paddle speed was slowed,
suggesting the possibility of bio-in-equivalent absorption in patients whose gastric agitation
strength are weaker than that of healthy subjects. Although the clinical evidence pertaining
to “BE in special populations” is not enough to confirm the results obtained in this study atpresent, the BE checker is considered to be a highly capable in vitro tool to assess the BE oforal drug products in various populations and to ensure their reliability for clinical use.
Author Contributions: Conceptualization, T.T., M.K., K.I., K.M. (Kazuki Matsui), and S.Y.; methodology, T.M., T.T., and S.Y.; software, T.M. and T.T.; validation, K.M. (Keiko Minami), M.K., K.-i.I., andK.M. (Kazuki Matsui); investigation, T.M., T.T., K.M. (Keiko Minami), M.K., and S.Y.; resources, S.Y.;
writing—original draft preparation, T.M., T.T., and S.Y.; writing—review and editing, K.M. (Keiko
Minami), M.K., K.-i.I., and K.M. (Kazuki Matsui); visualization, T.M., T.T., and S.Y.; supervision,
S.Y.; project administration, S.Y.; funding acquisition, S.Y. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was partly funded by AMED, grant number JP17ak0101074.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All of the data described in this report is included in the publication oravailable in the cited references.

Pharmaceutics 2021, 13, 1136

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Conflicts of Interest: Sawai Pharmaceutical Co. Ltd. had no role in the design of the study; in thecollection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.

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