pharmaceutics

Article
On Absorption Modeling and Food Effect Prediction of
Rivaroxaban, a BCS II Drug Orally Administered as an
Immediate-Release Tablet
Varun Kushwah 1 , Sumit Arora 1,2 , Miklós Tamás Katona 3 , Dattatray Modhave 1,4 , Eleonore Fröhlich 1,5
and Amrit Paudel 1,6, *

1 Research Center Pharmaceutical Engineering (RCPE) GmbH, Inffeldgasse 13, 8010 Graz, Austria;
varun.kushwah@rcpe.at (V.K.); sumit0607@gmail.com (S.A.); datta.niper@gmail.com (D.M.);
eleonore.froehlich@medunigraz.at (E.F.)
2 Simcyp Division, Certara UK Limited, Level 2-Acero, Sheffield S1 2BJ, UK
3 Department of Pharmaceutical Chemistry, Semmelweis University, Hőgyes Endre u. 9.,
H-1092 Budapest, Hungary; Katona.Miklos@egis.hu
4 Galapagos, Analytical Development CMC, Generaal De Wittelaan L11 A3, 2800 Mechelen, Belgium
5 Center for Medical Research, Medical University of Graz, Stiftingtalstr. 24, 8010 Graz, Austria
6 Institute for Process and Particle Engineering, Graz University of Technology, Inffeldgasse 13,
8010 Graz, Austria
* Correspondence: amrit.paudel@tugraz.at; Tel.: +43-316-873-30912

Abstract: The present work evaluates the food effect on the absorption of rivaroxaban (Riva), a



BCS II drug, from the orally administered commercial immediate-release tablet (Xarelto IR) using


physiologically based pharmacokinetic (PBPK) and conventional in vitro–in vivo correlation (IVIVC)
Citation: Kushwah, V.; Arora, S.; models. The bioavailability of Riva upon oral administration of Xarelto IR tablet is reported to
Tamás Katona, M.; Modhave, D.; exhibit a positive food effect. The PBPK model for Riva was developed and verified using the
Fröhlich, E.; Paudel, A. On previously reported in vivo data for oral solution (5 and 10 mg) and Xarelto IR tablet (5 and 10 mg
Absorption Modeling and Food Effect
dose strength). Once the PBPK model was established, the in vivo performance of the tablet formu-
Prediction of Rivaroxaban, a BCS II
lation with the higher dose strength (Xarelto IR tablet 20 mg in fasted and fed state) was predicted
Drug Orally Administered as an
using the experimentally obtained data of in vitro permeability, biorelevant solubility and in vitro
Immediate-Release Tablet.
dynamic dissolution data using United States Pharmacopeia (USP) IV flow-through cell appara-
Pharmaceutics 2021, 13, 283. https://
doi.org/10.3390/pharmaceutics
tus. In addition, the mathematical IVIVC model was developed using the in vitro dissolution and
13020283 in vivo profile of 20 mg strength Xarelto IR tablet in fasted condition. Using the developed IVIVC
model, the pharmacokinetic (PK) profile of the Xarelto IR tablet in fed condition was predicted and
Academic Editor: Luisa Di Marzio compared with the PK parameters obtained via the PBPK model. A virtual in vivo PK study was
Received: 23 January 2021 designed using a single-dose, 3-treatment cross-over trial in 50 subjects to predict the PK profile of
Accepted: 12 February 2021 the Xarelto®IR tablet in the fed state. Overall, the results obtained from the IVIVC model were found
Published: 20 February 2021 to be comparable with those from the PBPK model. The outcome from both models pointed to the
positive food effect on the in vivo profile of the Riva. The developed models thus can be effectively
Publisher’s Note: MDPI stays neutral extended to establish bioequivalence for the marketed and novel complex formulations of Riva such
with regard to jurisdictional claims in
as amorphous solid dispersions.
published maps and institutional affil-
iations.
Keywords: in vitro–in vivo correlation; physiologically based pharmacokinetic model; BCS Class II;
Rivaroxaban; Xarelto; food effect; population kinetics

Copyright: © 2021 by the authors.

Licensee MDPI, Basel, Switzerland. 1. Introduction
This article is an open access article
Developing and deploying approaches that enable predicting the in vivo efficacy
distributed under the terms and
conditions of the Creative Commons
and safety profile of pharmaceutical drug products enormously expedite the product and
Attribution (CC BY) license (https://
process development effort as well as reduce the need for expensive clinical studies. For
creativecommons.org/licenses/by/ solid oral dosages, a thorough biopharmaceutical characterization at the in vitro level, such
4.0/). as solubility and dissolution testing using biorelevant media, study of food–formulation

Pharmaceutics 2021, 13, 283. https://doi.org/10.3390/pharmaceutics13020283 https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2021, 13, 283 2 of 22

interaction, in vitro membrane permeability and drug transport studies etc., provides the
input data for in vivo absorption prediction. In recent years, prominent progress has been
made in the in vitro biopharmaceutics profiling as well as in silico modeling for solid drug
products [1]. A set of biorelevant and clinically relevant in silico models are expected to
account for the critical formulation and physiological factors to facilitate the correlation
between in vitro drug dissolution and in vivo pharmacokinetic profiles.
A widely accepted approach to assess the correlation between in vitro dissolution
and in vivo bioavailability of an immediate-release (IR) drug product is based on the
Biopharmaceutics Classification System (BCS). The BCS categorizes drug substances into
one of four classes based on their solubility and permeability. In general, BCS Class I (highly
soluble and highly permeable) drugs are well-absorbed. The rate-limiting step to absorption
is dissolution or gastric emptying. In vitro–in vivo correlation (IVIVC) is expected if
dissolution rate is slower than gastric emptying rate. In the case of IR drug products
containing BCS Class II drug substance (dissolution as a rate-limiting step for absorption),
conventional IVIVC can be used to establish the (cor)relation between the in vitro drug
release and in vivo plasma concentration. Dissolution media and methods that reflect the
in vivo controlling process are particularly important in this case if good in vitro–in vivo
correlations are to be obtained. For BCS Class III drugs (for which permeability is the
rate-limiting step for absorption), limited or no IVIV correlation is expected with the
dissolution rate. Drug products containing BCS Class IV drug substance (low solubility
and low permeability) has to be evaluated case by case; however, these drugs exhibit poor
and variable bioavailability [2,3].
IVIVC is a predictive mathematical model describing the relationship between an
in vitro property and a relevant in vivo response. In the case of immediate release dosage
forms, the main objective of an IVIVC is to reduce the number of BE studies via supporting
the optimization of the drug formulation. A Level A IVIVC is usually established by a
two-stage procedure: in vivo absorption is estimated using an appropriate deconvolution
technique (e.g., Wagner-Nelson, Loo-Riegelman, numerical deconvolution) followed by
the comparison of drug absorbed to the fraction of drug dissolved. Even though the
deconvolution method is often applied for regulatory submission, the method is limited to
linear pharmacokinetics (PK) regimen [4].
Alternatively, the mechanistic deconvolution using the physiologically based pharma-
cokinetic (PBPK) modeling popularly known as physiologically based IVIVC (PB-IVIVC) is
nowadays extensively utilized for biopharmaceutics modeling [5,6]. Besides its applicabil-
ity to the nonlinear PK, the PBPK model also considers the different factors governing the
drug release and absorption such as particle size of the API, food effect, pH-dependent sol-
ubility profile, precipitation, gastric emptying time, drug degradation, drug solubilization
in the presence of excess bile acids and permeation across the intestinal membranes [7–10].
In the present work, we established both PB-IVIVC and conventional (numerical)
IVIVC models for the immediate release oral tablet (Xarelto) formulations containing Ri-
varoxaban (Riva), a BCS II anti-coagulant drug. Riva is reported to exhibit dose-dependent
food effects. More precisely, while the lower dose (10 mg) can be taken with or without
food, the highest dose strength tablet (20 mg) should be taken with food to attain the
positive food effect for oral absorption and systemic availability [6]. As per the regu-
latory requirement, clinical trials are required to establish bioequivalence between the
innovator and generic drug products containing the BCS II drug, and especially exhibiting
a food effect. As the critical formulation and drug product information of Riva is still
covered by patent protection and no biowaiver exists, the design of a generic formulation
containing this drug can be a challenge, especially considering the food effect displayed
by the highest dose strength [11,12]. Here, we first developed an in silico PBPK model
using the fasted conditions and low-dose formulations. On the basis of developed models,
the PK profile of Xarelto formulations of the highest dose strength was predicted in fed
conditions. The in vitro permeability of Riva as the pure API alone, and in the reference
formulations (Xarelto® IR tablet) was determined using Caco-2 cell lines. Using combined
Pharmaceutics 2021, 13, 283 3 of 22

in vitro solubility, dissolution, and permeation data with literature data as the input in the
developed PBPK model, the extent of food effect in vivo on the oral absorption of the drug
was predicted by mimicking the fed state condition using biorelevant media. In addition, a
conventional IVIVC model was established using the two-stage deconvolution method.
The dissolution of Xarelto 20 mg IR tablet in USP IV apparatus was determined both in
fasted and fed biorelevant media. The correlation was built based on the experimental
in vitro dissolution data in fasted media and the mean in vivo plasma concentration profile
in the fasted state obtained from the literature. Thereafter, the correlation was used to
predict the in vivo profile in the fed state based on the in vitro dissolution in fed biorel-
evant media. The predicted results of the IVIVC model were compared with the PBPK
model. Lastly, a virtual bioequivalence trial was performed to assess the performance of
formulation in the fed state taking into consideration the population variability.

2. Materials and Methods

2.1. Chemical and Reagents
The reference formulations (XareltoTM of 20 mg strength IR tablet) were acquired
from the market. Caco-2 cell line was purchased from American Type Culture Collection
(ATCC) (Rockville, MD, USA). Minimal Essential Medium (MEM), fetal bovine serum
(FBS), L-glutamine, trypsin (0.25%)-EDTA (1 mM), and penicillin-streptomycin mixture
were purchased from Sigma (Vienna, Austria). Simulated fluid powders were purchased
from Biorelevant.com Ltd, the UK, for the cell culture studies. Whereas for the solubility
and dissolution studies, the simulated media were prepared using sodium taurocholate,
lecithin and pepsin purchased from Sigma. The milk used for the solubility studies were
purchased from the local market with a natural fat content of 3.5% fat. All other chemicals
were of analytical reagent grade.

2.2. Software
Simulations were performed using the advanced compartmental absorption and tran-
sit model (ACAT) model implemented in the GastroPlusTM (version 9.0., Simulation Plus,
Inc., Lancaster, CA, USA). The ACAT model serves as a bridge between the formulation
performance and PK parameters of the drug products and hence provides a valuable tool
to guide formulation development to achieve the desired quality target product profile.
In addition, the simulations from the ACAT models were verified using the conventional
IVIVC model developed using the IVIVC Toolkit of Phoenix WinNonlin, Certara, NJ, USA.

2.3. Chromatographic Quantitative Analysis

Ultra-High-Performance Liquid Chromatography (UHPLC) was used to quantify Riva
in the samples obtained during in vitro dissolution, solubility, and permeability. A Waters
Acquity H-Class instrument (Milford, CT, USA) equipped with a PDA detector (operating
at a wavelength value of 248 nm) was used. The column used was Acquity UPLC BEH
C18, 1.7 µm, 2.1 mm × 50 mm (Waters), and the mobile phase was 3:7 (v/v) mixture of
acetonitrile and 0.1M Ammonium-acetate buffer. The analysis was performed applying
isocratic elution at the flow rate of 0.5 ml min−1 . The injection volume was 2 µL, and the
total run time was 5 min. The dissolved amount of Riva was determined based on the area
under the appropriate peak and using external standard calibration.

2.4. Biopharmaceutical Properties of Riva

2.4.1. Biorelevant Solubility Determination
Equilibrium solubility of Riva was determined in water, fasted state simulated gastric
fluid (FaSSGF), fed state simulated gastric fluid (FeSSGF), fasted state simulated intestinal
fluid (FaSSIF), and fed state simulated intestinal fluid (FeSSIF). All the simulated fluids
(version 01) were prepared fresh on the day of the experiments conducted. In the case of
FaSSIF media, 3 mM and 0.75 mM, and for FeSSIF, 15 mM and 3.75 mM of the sodium
taurocholate and lecithin were used, respectively. The FaSSGF media contain 0.08 mM
Pharmaceutics 2021, 13, 283 4 of 22

and 0.02 mM sodium taurocholate and lecithin, whereas the FeSSGF media were prepared
using an equivalent amount of milk and monobasic sodium phosphate buffer of pH 5. The
concentration of different components of the media in detail is mentioned in Table S1 of
the Supplementary Materials.
For the solubility determination, an excess amount of the pure drug was added to
5 mL of different simulated fluids. Thereafter, the sample was incubated at RT for 24 h with
gentle shaking at 100 rpm using a rotatory shaker. The samples were then filtered using
0.22 µm syringe filter, and the filtrate was analyzed using UHPLC [13,14]. The solubility
results of Riva obtained in different simulated media were incorporated in GastroPlusTM .

2.4.2. In vitro Dynamic Biorelevant Dissolution

Dynamic dissolution of XareltoTM (20 mg) tablets were carried out using USP Ap-
paratus 4 fitted with 12 mm tablet cell using the media mimicking both the fasted and
fed gastrointestinal state. In the in vitro fasted state, the tablet was exposed to FaSSGF
(pH 1.6) for 0.5 h, followed by exposure of the same tablet to FaSSIF (pH 6.5) for 5.5 h. The
dissolution study was evaluated with 12 replicates. During the dissolution test, samples
were withdrawn at predetermined time points and then filtered with 0.7 µm GF/F disc
filter (Whatman, Maidstone, UK). The in vitro fed state experiments were conducted in
a similar manner to that of the fasted state, using FeSSGF and FeSSIF as dissolution me-
dia. The dissolution media (FaSSGF, FeSSGF, FaSSIF, FeSSIF) were prepared based on the
composition available at https://biorelevant.com/shop/ (accessed on 24 February 2021);
however, enzymes were excluded and SIF powder was replaced by surfactants such as
tween 80 and sodium lauryl sulfate.

2.4.3. In Vitro Caco-2 Permeability Determination

Caco-2 cells (ATCC) were cultured in Minimal Essential Medium (MEM), 20% fetal
bovine serum, 2 mM L-glutamine, and 1% penicillin-streptomycin at 37 ◦ C in humid air
atmosphere containing 5% CO2 in 75 cm2 cell culture flasks. Freshly prepared FaSSIF was
used for the study.
For the transport studies, 0.5 × 106 cells were seeded per 12-well transwell insert
(translucent, 0.4 µm pore size, Greiner Bio-one® ). Cells were cultured with 500 µL medium
in the upper compartment and 1500 µL in the lower compartment. The medium was
changed every 2 or 3 days, and transepithelial electrical resistance was measured via
EVOM STX-2-electrode (World Precision Instruments, Florida, USA). The cell monolayers
were used for the experiments, once the resistance reached a transepithelial electrical
resistance (TEER) value of >300 Ω*cm2 (18–21 days) [15,16].
Thereafter, the medium was removed, and the cells were coated with 90 µL gastric
porcine mucin (40 mg/ml in MEM + 10%FBS) for 30 min. A concentration of 10 µM of Riva
and XareltoTM (20 mg) tablets (equivalent to 10 µM of Riva) was used. For the preparation
of XareltoTM (20 mg) samples, the tablets were ground using a mortar, and pestle and
the amount of the formulation containing the desired amount of Riva was dispersed in
FaSSIF and stirred for 30 min at RT. The respective suspensions (510 µL) were applied
to the upper compartment of the transwell and 1500 µL Krebs Ringer buffer added in
the lower compartment. Ten microliters were immediately withdrawn from the apical
compartment to determine the total amount applied. Plates with transwell were incubated
upon agitation for a total of 120 min. One-hundred-microliter samples were taken from the
lower compartment at predetermined time points of 0, 30, 60, 90, and 120 min and replaced
by pre-warmed Krebs-Ringer buffer. In addition, at the end of the experiment, 10 µL of the
upper compartment were collected for the calculation of the recovery rate. TEER values
were measured before and after the transport study to identify potential damage to the
cell layer. The permeability of sodium fluorescein (10 µg/mL) in the Krebs-Ringer buffer
was determined to verify the barrier properties of the Caco-2 monolayer. All samples were
stored at −20 ◦ C until further analysis. On the day of analysis, the samples were thawed at
room temperature, and the content was measured using UHPLC.
Pharmaceutics 2021, 13, 283 5 of 22

For the determination of the apparent permeability coefficient (Papp ), the following
equation was used, where dQ/dt is the flux across the cell monolayer (ng/s), A the surface
of the monolayer (cm2 ), and C the initial concentration in the donor compartment (ng/mL):

dQ
Papp = (1)
dt × A × c

2.4.4. Determination of Systemic Disposition Parameters of Riva

The in vivo PK profile of Riva after intravenous administration was not found in
the literature. Therefore, plasma concentration–time profile after 10 mg oral solution
administration under the fasted state was used in the PKPlusTM to obtain the systemic
clearance, volume of distribution, half-life, and distribution constants between the central
and peripheral compartments [17,18]. Models were fitted empirically in the PKPlusTM
employing 1-, 2-, and 3- compartment separately. The Hooke & Jeeves pattern search
method was used during the fitting and the weighing was equal to 1/Yhat2 . Akaike
information criterion (AIC) and Schwarz criterion (SC) were used to select the best-fitted
compartment model. The obtained PK parameters were then fixed and employed in the
simulation of solid oral dosage forms.

2.5. Physiologically Based Gastrointestinal Absorption Modeling

2.5.1. Model Compound Parameters
Physiochemical properties of Riva such as molecular weight and lipophilicity were
compiled from the literature (Table 1) [19]. Human duodenum effective permeability (Peff )
was estimated from in vitro CaCo-2 permeability data using the in-built relation present
in the GastroPlusTM . The experimentally obtained values of solubility, particle size, and
dissolution parameters were used.

Table 1. Input parameters of Riva for building the PBPK model in GastroPlusTM.

Physiochemical Parameter Values

Molecular Weight (g/mol) 435.89
logP 1.36
pKa strongest acidic: 13.6strongest basic: 1.6
water solubility (pH = 7) = 10 µg/mL
pH 1.2, FaSSGF = 11 µg/mL
Solubility vs. pH
pH 6.5, FaSSIF = 9.9 µg/ml
pH 5.0, FeSSIF =16.8 µg/ml
7.5 µm (Xarelto tablet, 20 mg)
Particle Size (Radius)
d90 = 9.4 µm; d50 =3.8 µm; d10 =0.7 µm
Caco-2 Permeability 2.69 ± 0.72 × 10−6 cm/s (Xarelto)
Dissolution Profiles (USP 4) Xarelto IR tablet (20 mg)

2.5.2. Development of In-Silico Physiology Based Gastrointestinal Absorption Model

The ACAT model implemented in GastroPlusTM was used for all the simulations in
the current study. Input parameters for the ACAT model can be categorized into three
classes, i.e., formulation properties (such as particle size distribution, density, and release
profiles of drug products), physicochemical properties of drug substances (such as diffusion
coefficient, lipophilicity, pKa, solubility, and permeability), and pharmacokinetic parame-
ters (such as clearance, the volume of distribution, and the disposition model). All other
parameters were set at default values in GastroPlusTM . The simulations were performed
using the default “Human Physiological-Fasted” and “Opt LogD Model SA/V6.1”, in
order to simulate the plasma concentration profiles of Riva following oral administration of
tablet dose in fed condition. The Opt. LogD SA/V v6.1 model is one of the ACAT models
in GastroPlus software. In the ACAT model, the option “CR Dispersed” was selected using
the USP4 profile to model the release of the drug. Once the drug is released, solubility and
PSD were used to model dissolution of the drug via the Johnson model.
Pharmaceutics 2021, 13, 283 6 of 22

On the basis of LogD value of the drug molecules, the model can automatically fine-
tune the absorption scale factor for the different compartment of the intestine, resulting in
improved simulation and thus prediction of the regional absorption of the API.

Model Verification
Initially, the predictive power, robustness, and the effect of Absorption Scale Factor
(ASF) optimization on the absorptive phase of Riva were evaluated. To do so, the predicted
pharmacokinetic parameters were compared with the in vivo data from literature. The
pharmacokinetic model was developed using oral solution doses (5 and 10 mg) as well as
for oral IR tablet doses (5 and 10 mg) of Riva under the fasted state and compared with the
published data [17,18]. The percent prediction error value was calculated to evaluate the
accuracy of the model. The calibrated ACAT model for Riva was then applied to simulate
the plasma concentration–time profile of Xarelto tablet (20 mg) in the fed state.

Parameter Sensitivity Analysis (PSA)

PSA was performed for the uncertain and key parameters in formulations such as
mean particle radius, dose volume, particle density, effective permeability, precipitation
time, and diffusion coefficient for the dosage forms investigated under the fasted and
fed tates.

2.6. IVIVC Studies

In addition to the PBPK model, the conventional IVIVC was used to predict the PK
profile after oral administration of formulation in the fed state using IVIVC Toolkit 8.0 of
Phoenix WinNonlin 8.2.0.4383 for Windows (Pharsight, Certara, USA Inc, St. Louis, MO,
USA). The IVIVC model was developed and calibrated using the in vitro dissolution and
in vivo profile of 20 mg strength of Xarelto IR tablet in the fasted condition. Thereafter,
using the established IVIVC, the PK profile of the Xarelto IR tablet in fed condition was
predicted and compared with the simulation results obtained from the PBPK model.
IVIVC was based on a two-step deconvolution method. Initially, the Weibull function
was fitted to the in vitro data of the Xarelto IR tablet (20 mg tablet) in the fasted condition.
Moreover, the time course of in vivo absorption was derived using deconvolution. The
in vivo data of the 10 mg oral solution published by Kubitza were used as a reference for
calculating the unit impulse response (UIR) function [17,18]. The in vivo data of the PK
profiles of the Xarelto IR tablet (20 mg tablet) were then deconvolved and compared to the
in vitro dissolution profiles using the fraction absorbed (Fabs ) vs. the fraction dissolved
Fdiss plot.
Thereafter, in the second step, a correlation was built between the in vitro drug
release and in vivo drug absorption. The IVIVC model was established based on in vitro
dissolution profile of the Xarelto IR tablet, and in vivo results were obtained from the
literature. As per the condition, the regression slope line most closely aligned with a value
of 1.0, whereas the elimination phase of Riva was calculated using the PK profile of 10 mg
oral solution.
Thereafter, as the final step, validation of the developed IVIVC model is required, in
order to establish quantitative resilience of the predictive capacity of the model. The vali-
dation of the model was performed using the results of the in vivo fate of the formulation
used to establish the model, known as internal validation, and/or by the application of a
different formulation, known as external validation. In the present model development,
the internal validation of the IVIVC model was performed by comparing the predicted and
observed PK profile of the reference product Xarelto 20 mg tablet in the fasted condition.
Thereafter, the predictability of the model was evaluated using the percentage prediction
error (%PE) from the following equation:

Predicted value − Observed value

 
%PE = 100 × (2)
Observed value
Pharmaceutics 2021, 13, 283 7 of 22

For the development of a robust model, the average absolute %PE of ≤10% for the
maximum plasma concentration (Cmax ) and area under the curve (AUC) establishes the
predictability of the IVIVC. Furthermore, the %PE for each formulation should not exceed
15%. If the %PE conditions are not met for the internal validation, further validation using
the external formulation is required [20,21].

2.7. Food Effect (FE) Studies of Riva in Simulated Healthy Population

Simulations were carried out for 20 mg dose strength using the fed state physiology
of GastroPlusTM to evaluate the quantitative prediction of FE based on the measurements
of in vitro biorelevant solubility and dissolution. The percentage prediction error for the
predicted PK parameters was calculated in comparison with the predicted values Xarelto
IR tablet (20 mg tablet).
A single-dose (20 mg), three-period virtual trial in 50 subjects was carried out. Gastro
PlusTM randomly generates subjects by varying physiological factors such as gastroin-
testinal transit times, pH, fluid volumes, PK parameters as well as compound parameters.
Three populations, namely A, B, and C, with 50 subjects each (in order to gain a thorough
sampling across all the variables) were given the same treatment (20 mg strength of Xarelto
IR tablet).

3. Results and Discussion

3.1. Biopharmaceutical Properties of Riva
3.1.1. Equilibrium solubility in simulated media
Table 2 depicts the solubility of the Riva in different biorelevant media. The solubility
of the Riva was found to be comparable among different media, i.e., water, FaSSGF (pH 1.6),
and FaSSIF (pH 6.5). Whereas in the case of FeSSIF and FeSSGF (pH 5.0), the solubility
of the Riva was found to be markedly higher as compared to other simulated biorelevant
media. The FeSSIF contains 15 mM sodium taurocholate and 3.75 mM lecithin as compared
to 3 mM sodium taurocholate and 0.75 mM lecithin in the case of FaSSIF. Thus, an approx.
2-fold increase in the solubility in the Fed conditions could be attributed to the increase
in the lipidic component of the media with a higher fraction solubilized in taurocholate
and lecithin micelles. The increased solubility of Riva in the Fed state is in accordance with
the literature, demonstrating higher bioavailability of equal of more than 80% when taken
with food (for 20 mg dose of Riva) [19,22].

Table 2. Solubility of Riva in biorelevant media.

Solubility in pH Values (µg/mL)

Unbuffered water 7.0 10.0
FaSSGF 1.6 11.0
FaSSIF 6.5 9.9
FeSSGF 4.5 24.0
FeSSIF 5.0 16.8

3.1.2. In Vitro Release Profile of Riva in Fasted and Fed Conditions

Figure 1 demonstrates the in vitro release profiles of the Xarelto IR tablet (20 mg) in
fasted and fed conditions using the dynamic dissolution method. After 30 min, in vitro
release of Xarelto IR tablet in the fed state and fasted simulated gastric fluids were found
to be 27.7 and 11.0%.
Thereafter, the fed and fasted simulated gastric fluid was replaced via simulated
intestinal fluids without removing the Xarelto IR tablets. The in vitro release, and the time
to 80% drug release was found to be the 360 and 210 min in case of fasted and fed state
conditions, respectively. Furthermore, the f 1 (the difference factor) and f 2 (the similarity
factor) were also calculated and found to be 28 and 38, respectively. For bioequivalent
in vitro release profile, the values of f 1 should be between 0 and 15, whereas the value of
f 2 should be between 50 and 100 [23,24]. Thus, the release profile in the case of the fed
Pharmaceutics 2021, 13, 283 8 of 22

condition was found to be significantly higher as compared to the fasted condition. The
results demonstrated that the lower solubility in the absence of food components could be
the rate-limiting factor for the dose-proportional absorption of Riva from Xarelto IR tablets,
independent of the formulation. The significantly higher dissolution in the presence of a
food-induced increase in bile salt concentration was found to be in accordance with the
solubility study and is the key parameter for the establishment of the PBPK model.

Figure 1. In-vitro release profile of Riva from Xarelto IR tablet (20 mg strength) using USP 4 apparatus.
The tablets were transferred from FaSSGF (pH 1.6) and FeSSGF (pH 4.5) media to FaSSIF (pH 6.5)
and FeSSIF (pH 5.0) media, respectively, after 30 min.

3.1.3. In Vitro Caco-2 Permeability

Permeation of the marker substance, fluorescein, did not cause any alterations of
transepithelial electrical resistance (TEER) values compared to controls (no permeation
performed). The API powder and, to a greater extent, the formulations caused a decrease in
TEER values (Figure 2A). Despite the decrease in TEER values induced by the formulations,
there was no increased transport of Riva across the monolayers. Apparent permeability
coefficient (Papp ) values (2.69 ± 0.72 × 10−6 cm/s) were not increased in the formulations
compared to standard Riva (3.11 ± 0.24 × 10−6 cm/s, (Figure 2B)). The Papp of fluorescein
was 0.72 ± 0.13 × 10−6 cm/s, indicating a good barrier function of the Caco-2 monolayer
and absence of damage by FaSSIF.
Papp and transport rates were identical for formulated products and standard Riva.
The decrease in TEER values was slightly higher in the formulations than in the unformu-
lated Riva but was not reflected in changes of the Papp values. Excipients in the formulations
more likely to decrease the TEER values. However, taking into consideration that there is
no difference in the Papp of pure Riva in comparison with Xarelto (presence of excipients)
despite the change in TEER values of CaCo-2 cells in Xarelto, it can be suggested that
passage of Riva is mainly transcellular through the CaCo-2 cells. Papp values determined in
the study were lower than the values published by Gnoth et al. (8.0 ± 0.6 × 10−6 cm/s) [25].
The most likely reason for this difference is the lack of mucus production of Caco-2 cells,
which can affect permeation. To reproduce the physiological situation, a mucus layer has
been added to the Caco-2 monolayer in this study. The effect of mucus on the permeation
of active pharmaceutical ingredients (APIs) has been reported controversially [26,27]. The
comparison between Caco-2 cells and mucus-producing HT29-MTX did not show promi-
nent differences for many lipophilic and hydrophilic compounds, suggesting that mucus
does not represent a strong barrier for the permeation [28]. The exclusive assessment of the
role of mucus, however, was not possible because HT29-MTX cells lack P-glycoprotein ex-
pression and the lack of reverse transport will increase the measured Papp values. Although
Pharmaceutics 2021, 13, 283 9 of 22

the same cell types were used, another study reported that the permeability of drugs with
a partition coefficient (logP) > 1 was decreased in the mucus-producing cell lines [28].
The passage of Riva might be hindered by mucus because of its logP value of 1.36 [28].
It can be concluded that Riva formulations reacted very similarly and did not display a
permeation-enhancing effect on the permeability of Riva. The results obtained of various
physicochemical parameters such as those from the solubility study, Caco-2 permeability,
and the published literature were used as the input parameters for the development of the
PBPK model. The values of the input parameters used in the PBPK model are mentioned
in Table 1 [19,29–33].

Figure 2. (A) Differences in transepithelial electrical resistance (TEER) values between the start value and measurement
at the end of the permeation studies. Abbreviations: Control (Co), fluorescein (Flu), rivaroxaban (Riva), Xarelto (Xare).
(B) Papp values of rivaroxaban (Riva) and Xarelto (Xare).

3.1.4. Systemic Disposition Parameters of Riva

The two-compartment model was found to be the best fit model according to the
AIC and SC criteria to describe the Riva pharmacokinetics following the administration
of oral solution dose (10 mg) (Figure 3). Values of clearance, the volume of distribution
(Vc) and, T1/2 were in accordance with the literature values. Other parameters such as
the peripheral volume of distribution (V2 ), distribution constant from the central to the
peripheral compartment (K12 ), and distribution coefficient from the peripheral to central
compartment determined (K21 ) from the two-compartment model fitting were used for
further simulations. Table 3 depicts the mean baseline values used for the simulation of
Riva plasma concentration–time profile following the administration of oral solution and IR
formulation doses. The value of clearance and elimination half-life obtained through fitting
is found to be in accordance with the value reported in the Xarelto product information
after intravenous administration of Riva at a dose of 1 mg [34,35].

3.2. Physiology Based Gastrointestinal Absorption Model of Riva Formulation

Prediction of PK Profiles and Optimization of ACAT Model
Taking into consideration the input parameter as mentioned in Table 1 and the phar-
macokinetic parameters mentioned in Table 3, the PK profile of the oral solution dose
(10 mg) of Riva was simulated using the ACAT model in GastroPlusTM with default fasted
human physiology and ASF values (Table 4). Simulation of 10 mg oral dose with the default
GastroplusTM Human Physiology Fasted largely underestimated the absorption phase
of Riva, resulting in the poor fitting of the extracted plasma concentration–time profile
obtained for 10 mg oral dose (Figure 4A). This observation suggested that the default fasted
Pharmaceutics 2021, 13, 283 10 of 22

human physiology in GastroPlusTM was not able to capture the absorption phase of Riva.
Thereafter, the influence of effective permeability (Peff ) on the Cmax and Tmax predictions
under fasted conditions for oral solution dose (10 mg) of Riva were evaluated. The results
showed that even though the Peff was increased by 10 folds to 3.1, Tmax was overpredicted
by 5 folds and Cmax was underpredicted by 1.78 folds compared to the average observed
values (Table 5) [18].

Figure 3. Pharmacokinetic data fitting 2- compartment of oral solution dose (10 mg) of Riva.

Table 3. PK parameters obtained from two-compartment model fitting of oral solution (10 mg)
of Riva.

Parameter Values
Clearance (L/h) 9.43
Vc (L/kg) 0.47
T1/2 (h) 4.62
K12 (1/h) 0.04
K21 (1/h) 0.21
V2 (L/kg) 0.09

Table 4. ASF values before and after optimization of ACAT model.

Compartment Default (GastroPlus) ASF Optimized ASF

Stomach 0 0
Duodenum 2.673 36.44
Jejunum 1 2.658 36.25
Jejunum 2 2.629 35.85
Ileum 1 2.592 35.35
Ileum 2 2.568 35.02
Ileum 3 2.505 34.16
Caecum 0.535 0.535
Asc Colon 1.038 1.038

Furthermore, it is also important to consider the solubility of Riva in gastric medium

and concentration of Riva attained with 5 mg and 10 mg of oral solutions. Table 2 reports
fasted state gastric solubility of 11 µg/mL (i.e., 0.011 mg/mL) for Riva. Considering 250 mL
of dosing volume with instantaneous saturation translates to a solubilization capacity of
2.75 mg in the medium. At a dose of 10 mg, this can generate ~3.6-fold supersaturation,
Pharmaceutics 2021, 13, 283 11 of 22

which can lead to faster absorption as reflected by high Cmax and lower Tmax in the
observed profile (assuming absence of precipitation from the supersaturated state). As
simulations were conducted with solubility values of ~11 µg/mL, this could have led
to underpredictions.

Figure 4. Riva plasma concentration–time profile from solution oral dose (10 mg) (A) before and
(B) after optimization.

Table 5. PK parameters obtained from simulated PK profiles.

Predicted (Before Predicted (After

Parameter Actual (Reported) a
Optimization) Optimization)
PK parameters obtained from simulated PK profile of solution oral dose (10 mg) of Riva before and after
optimization
Cmax (µg/L) 266/25.1 (187–412) 159.27 212.23
Tmax (h) 0.50 (0.25–1.00) 1.92 0.72
AUC (µg·h/L) 997/25.1 (613–1383) 1056 1058
F% >90% 99.54 99.79
Pharmacokinetic parameters for 5 mg oral (solution) dose of Riva in fasted conditions
Cmax (µg/L) 119/18.5 (97.2–158) 80.13 107.06
Tmax (h) 0.63 (0.5–0.75) 1.92 0.66
AUC (µg·h/L) 461/17.2 (348–587) 528.24 529.37
F% >90% 99.58 99.80
a —taken from literature; data are represented as geometric means/percent geometric coefficient of variation and
range in case of reported data [18].

As a result, the ASF values were optimized using the optimization module in the
GastroPlusTM , which could capture the absorption phase of the plasma concentration–time
profile of oral solution (10 mg) (Figure 4B). ASFs in GastroPlusTM are a multiplier used to
Pharmaceutics 2021, 13, 283 12 of 22

scale the effective permeability to account for variations in surface-to-volume ratio, pH

effects, influx, or efflux transporter differences, and other absorption-rate-determining
effects. On the basis of the GI physiology, ASFs are used to scale the effective permeability
of the API across the different sections of the GI tract.
The ASFs were optimized using the PK data set of single-dose oral solution (10 mg) in
the fasted state changing the C1 and C2 coefficients of Opt logD Model SA/V 6.1, which
determines absorption from the small intestinal compartments. However, the C3 and C4
coefficients which determine absorption from the colon were kept at their default values.
The optimized ASF were nearly 14 folds higher than the default values, leading to the faster
absorption of Riva in the small intestine. In addition, the default value for compartment
volume occupation by water in the colon was reduced from 10% to 2% to better account for
measured free water content in the colon [8,36]. All other parameters were set at default
values in GastroPlusTM ; the default and optimized ASF values are mentioned in Table 4.
Table 5 shows the pharmacokinetic parameters obtained from the simulated PK profile
of solution oral dose (10 mg) of Riva before and after optimization of ASF and compared
with the literature data.
In order to verify that the optimized ASF values for the small intestine can reasonably
capture the absorption phase of Riva, simulations of mean plasma concentration–time
profile of Riva following administration of oral solution dose (5 mg and 10 mg) and IR tablet
formulation (5 mg and 10 mg) under the fasted condition were also carried out. Simulated
mean plasma concentration–time profiles of Riva from solution and IR tablet formulation
and corresponding pharmacokinetic parameters (Cmax . Tmax and AUC0–∞ ) calculated are
demonstrated in Tables 5 and 6. All the predictions of pharmacokinetic parameters for
different formulation and doses of Riva were within two folds of the reported values. This
fosters our confidence in the predictive ability of the developed ACAT model for Riva.

Table 6. Pharmacokinetic parameters for IR Tablet in fasted conditions.

Parameter Actual (Reported) a Predicted (Optimized ASF)

Pharmacokinetic parameters for 5 mg oral (IR Tablet) dose of Riva in fasted conditions
Cmax (µg/L) 72/19.7 (55–96) 76.13
Tmax (h) 1.88 (0.5–4.00) 2.1
AUC (µg·h/L) 466/23.0 (348–677) 524.42
F% 80–100% 98.86
Pharmacokinetic parameters for 10 mg oral (IR Tablet) dose of Riva in fasted conditions
Cmax (µg/L) 141/15.5 (112–184) 149
Tmax (h) 2.00 (0.5–2.50) 2.20
AUC (µg·h/L) 1020/14.9 (797–1217) 1037
F% 80–100% 97.75
a —taken from literature; data are represented as geometric means/percent geometric coefficient of variation and
range in case of reported data [18].

Parameter sensitivity studies were performed investigating the impact of key factors
on the bioavailability of Riva. The mean particle size of the API in the IR tablet formulation
was found to be the most important factor influencing the bioavailability of Riva (Data not
shown) irrespective of fasted and fed state. Other factors such as dose volume, particle
density, precipitation time, diffusion coefficient, and Peff seem to have a relatively minor
influence on the bioavailability of Riva.
Once the ACAT model was optimized with modified ASF values, pharmacokinetic
parameter predictions were carried out for Riva 5 mg oral solution dose (Table 5) and
Xarelto IR tablet for 5 mg and 10 mg (Table 6) dose in the fasted condition. The in vitro
release profile and the physicochemical parameters of the API was found to be biopredictive
and was able to describe the plasma concentration profiles, and the predicted values of
Cmax , AUC, and Tmax were found to be in agreement with those of the literature, which
increased the confidence in the developed ACAT model.
In silico simulation of Xarelto (20 mg dose strength) in fasted and fed state
The developed ACAT model was used to simulate the plasma concentration–time
profile of Xarelto (20 mg dose strength) in the fasted and fed state with the dissolution
Pharmaceutics 2021, 13, 283 13 of 22

profiles using the USP 4 flow-through apparatus and biorelevant dissolution medium. It
was observed that the inclusion of dissolution profiles of Xarelto IR Tablet (20 mg) during
modeling led to an improvement in the simulations with predicted values close to the
observed values, as reported in the literature. Table 7 and Figure 5 represent the simulated
plasma concentration–time profile and key pharmacokinetic parameters predicted from
the simulation of Xarelto (20 mg) IR tablet in the fasted and the fed states.

Table 7. Key pharmacokinetic parameters predicted from the simulation of Xarelto (20 mg) IR tablet in the fasted and fed
states.

Parameters Fasted State Fed State

Actual (Reported) a Predicted (Optimized ASF) Actual(Reported) b Predicted (Optimized ASF)
Cmax (µg/L) 173/35.6 (111–294) 171.15 294.4/15 (225.4–360.6) 236.64
Tmax (h) 1.50 (0.5–4.00) 3 3.00 (0.5–6.00) 2.9
AUC (µg·h/L) 1612/36.1 (859–2193) 1433.8 2294/19 (1464–3227) 1857.3
F% 66% 67.57 80–100% 87.54
a—taken from Reference [18]; b—taken from Reference [17]; data are represented as geometric means/percent geometric coefficient of variation and
range in case of reported data.

Figure 5. Simulated plasma concentration-time profile of Xarelto (20 mg) in fasted and fed states
using GastroPlusTM ACAT model.

The results clearly depict the presence of food effects when Xarelto (20 mg) is admin-
istered in the fasted and fed states. However, the food effect was a bit underestimated
compared to that reported in the literature.
As evident from Figure 6, the increase in bioavailability of Xarelto during the fed
state simulation was found, which could be due to the enhanced dissolution of Riva in the
fed state. The increase in solubility in the fed state resulted in a greater fraction of Riva
to be absorbed from the duodenum and Jejunum 1 as compared to the fasted state. The
simulated results are in accordance with the Xarelto product literature outlining site-specific
absorption of Riva [34,35].
Pharmaceutics 2021, 13, 283 14 of 22

Figure 6. Regional amount absorbed from Xarelto in fasted and fed states in the gastrointestinal tract.

3.3. IVIVC Studies

3.3.1. Modelling
In Vitro and In Vivo Raw Data
The in vitro release profile of Xarelto 20 mg tablet in the fasted state was used for the
establishment of the IVIVC model. As evident from the dynamic in vitro dissolution of the
Xarelto (Figure 1), after 30 min of incubation in a simulated gastric medium, the simulated
gastric medium was replaced by a simulated intestinal medium. Upon incubating the
tablets for 5.5 hours, the amount of API release was found to be approx. 80 and 90% in case
of fasted and fed conditions, respectively.
The in vivo data of the Xarelto 20 mg tablet and 10 mg oral solution in the fasted
condition were obtained by literature published by Kubitza and co-workers [17,18]. The
Tmax in the case of solution and tablet was found to be 0.5 and 3 h, respectively. In addition,
the Cmax was reported to be markedly higher in the case of the solution as compared to
the tablet. The observation suggests higher absorption of the Riva in presence of solutions,
which is reported to be due to the faster and higher amount of Riva available to be absorbed.
Thus, suggesting the absorption of Riva to be not limited by permeability.

Dissolution Curve Fitting

The fasted state in vitro release profile of the Xarelto 20 mg tablet was fitted with
the Weibull function. As evident from Figure 7, using the Weibull function, the predicted
in vitro release or, more precisely, the fraction of API dissolved was found to be overlapping
with the observed in vitro release data as a function of time, suggesting that the Weibull
function was suitable to fit the dissolution data.
b
h i
Weibull function, y(t) = Fin f 1 − e−(t/MDT ) (3)

where, y(t) or Fdiss(t) - Fraction of drug dissolved; dependent variable of the function, t:
time (h); independent variable of the function, Finf : Fraction of drug dissolved at infinity
time; parameter of the function, MDT: mean dissolution time; parameter of the function, b:
beta; shape parameter of the function

3.3.2. Calculation of Unit Impulse Response (UIR) Function

To obtain the in vivo data, the pharmacokinetic parameters of 10 mg oral solution
was used. Thus, the UIR function was used to calculate the oral pharmacokinetic data. As
evident from Figure 8, using the UIR function, the observed pharmacokinetic profile was
found to be overlapping with the predicted pharmacokinetic profile and a linear relation
was established suggesting the best fit model. The UIR function obtained were then used to
Pharmaceutics 2021, 13, 283 15 of 22

deconvolute the in vivo pharmacokinetic profile of Xarelto 20 mg tablet in fasted condition,

for the assessment of in vivo absorption profile (Figure 9).

UIR Function, Cp(t) = A1 eα1(t-tlag) + A2 eα2(t-tlag) + A3 eα3(t-tlag) (4)

Where, Cp(t): Concentration of drug in plasma [ng/L]; dependent variable of the func-
tion, t: time [h]; independent variable of the function, A (coefficient) and α (exponential);
parameter values, tlag: lagtime; parameter value.

Figure 7. Observed in vitro profile vs. fitted curves of the Xarelto 20 mg tablet.

Figure 8. Properties of the unit impulse response (UIR) function.

3.3.3. Correlation
The dissolved API fraction (obtained from the fitting of in vitro release profile) was
then related with the absorbed in vivo fractions (obtained from the deconvolution of
plasma concentration). As evident from Figure 10, the in vivo fraction absorbed was
found to be linearly correlated with the fraction of API dissolved in vitro. The observed
relation was found to be aligned with the linear regression analysis. Thus, the slope and
regression values of Fabs vs. Fdiss plot suggest the development of a robust mathematical
IVIVC model.

Correlation equation, Fabs = AbsScalexDiss(Tscale × Tvivo − Tshift ) (5)

where, Fabs: Fraction of drug absorbed; dependent variable of the function, Tvivo: in vivo
time (h); independent variable of the function, AbsScale: Absorption scale factor, Tscale :
Time scaling factor, Tshift : Time scale shift, Diss: Internal function that linearly interpolates
Pharmaceutics 2021, 13, 283 16 of 22

the predicted dissolution data, i.e., the predicted data from the dissolution model fitted to
the in vitro data.

Figure 9. Calculated absorption after deconvolution of Xarelto 20 mg tablet.

Figure 10. Fraction absorbed in vivo vs. fraction dissolved in vitro for the Xarelto 20 mg tablet in the
fasted condition.

The AbsScale, Tscale, and Tshift parameters of the correlation equation were found to
be 0.595, 1.494, and 0.188, respectively.

3.3.4. Internal Validation and Prediction

An IVIVC model was established based on the USP IV fasted state dissolution data
and using published in vivo data as an internal validation of the reference product Xarelto
20 mg tablet (Figure 11). The result of the internal validation was promising (<2% error).
The IVIVC model was then used for the prediction of in vivo performance of the
Xarelto 20 mg tablet in the fed state. As evident from Figure 12 and Table 8, the Cmax and
AUC were found to be distinctly higher, whereas no significant difference in the Tmax was
predicted in the case of the fed state as compared to the fasted condition. The increase
in absorption was found to be in accordance with the increase in solubility of Riva in the
presence of simulated fed media. Thus, the developed IVIVC was able to adapt the effect
of fed media, predicting the in vivo profile in the fed state. The developed IVIVC model
Pharmaceutics 2021, 13, 283 17 of 22

(developed for reference product) can also be used as an effective tool to predict the in vivo
fate of formulations with different strength and release profiles, considering an average
percentage error of less than 10% and tolerance limit in the range of 0.8–1.25.

Figure 11. Internal validation (Xarelto 20 mg tablet Fasted condition) of the in vitro–in vivo correla-
tion (IVIVC) model.

Figure 12. Predicted mean in vivo profiles of Xarelto 20 mg tablet using IVIVC model.

In the present report, both PBPK absorption and IVIVC model were developed to
predict the food effect on the in vivo fate of the Riva released from Xarelto 20 mg tablet.
Both models predicted a higher amount of Riva absorption in case of fed conditions,
which could be due to higher solubility and release profile in simulated fed media [17].
The findings are in accordance with the literature, which reported an increase in Riva
AUC and the mean Cmax by 39% and 76%, respectively, when the 20 mg tablet was orally
administered with food [37]. Interestingly, both 10 and 20 mg strength of the marketed
formulation contain sodium lauryl sulfate (SLS) in order to increase the solubility of the
API in the in vivo conditions. However, as evident from the lower bioavailability, the
solubilization efficiency of SLS in increasing the solubility of Riva in the 20 mg tablet was
found to be less as compared to the Riva in 10 mg tablet [37]. This decrease in solubilization
efficiency in the fasted state could be due to a higher amount of API above the saturation
solubility in the case of 20 mg Riva, as the volume of the in vivo fluid remains the same,
potentially resulting in local precipitation and thus reduced bioavailability. Now in the case
of the fed state, the excess of API higher than the saturation solubility could dissolve in
lipidic components of the food and thus resulted in higher AUC and mean Cmax . On further
Pharmaceutics 2021, 13, 283 18 of 22

increasing the dose to supra-therapeutic levels of 50 mg of Riva, no further increase in the

AUC and mean Cmax values was observed even in the presence of fed conditions [33,37].
The ceiling effect observed in the case of 50 mg of Riva dosing could be due to attainment
of maximum solubility in fed conditions, assuming the volume of food is nearly alike.

Table 8. Observed and predicted values of the internal validation (Xarelto 20 mg tablet fasted condition) and predicted
values of theXarelto 20 mg tablet fed condition.

Formulation Parameter Predicted Observed a %PE Ratio

Xarelto 20 mg tablet Fasted condition AUClast (µg·h/L) 1381.946 1361.125 1.52966 1.015297
Xarelto 20 mg tablet Fasted condition Cmax (µg/L) 143.567 146.000 −1.66657 0.983334
Tmax
Xarelto 20 mg tablet Fasted condition 3.0 1.5 N/A N/A
(h)
Xarelto 20 mg tablet Fed condition AUClast (µg·h/L) 1543.120 1750.175 13.41790 1.134179
Xarelto 20 mg tablet Fed condition Cmax (µg/L) 174.566 241.000 38.05670 1.380567
Xarelto 20 mg tablet Fed condition Tmax (h) 2.0 3.0 N/A N/A
a —taken from References [17,18].

The Cmax and AUC values predicted by the PBPK and IVIVC model were found to
be comparable. However, the Cmax and AUC values in the case of the PBPK model were
slightly higher compared to the IVIVC model. The increase in Cmax and AUC values in
the case of the PBPK model could be due to the incorporation of different physicochemical
and formulation properties, for the development of the model, whereas the IVIVC model
lacks integration of such parameters. In the present case, as evident from the parameter
sensitivity studies, the particle size of the API was found to be a critical factor affecting
the release profile and thus the bioavailability of the Riva. The impact of particle size
on the bioavailability was found to be in accordance with the product filling, as the
reference formulation was developed using the micronized Riva in order to improve
oral bioavailability via increasing solubility [37]. Thus, the development/optimization of
the PBPK model using the particle size distribution of API could be responsible for the
slight difference in the predicted Cmax and AUC values compared to the IVIVC model.
Thus, the developed model can further be used to develop and optimize the formulation
parameters, mainly in the early stage development phase, reducing preclinical and clinical
time and cost.

3.4. Food Effect (FE) Studies of Riva in Simulated Healthy Subjects

A virtual trial is a stochastic simulation that randomly samples parameters from
predefined distributions. In order to take into account the effect of population variability
on the plasma concentration profile of Riva following administration of Xarelto, a single
dose (20 mg) 3-period virtual study design was carried out in the fed state. Three virtual
populations of 30-year American Male/Female A, B, and C, each of 50 subjects, were
created and subjected to Xarelto IR tablet (20 mg tablet). GastroPlusTM randomly generates
subjects by varying the physiological factors such as gastrointestinal transit times, pHs,
fluid volumes, and pharmacokinetics parameters, as well as compound parameters [38].
Table 9 and Figure 13 provides a summary of in silico investigation of food effect variability
across virtual populations.

Table 9. Summary in vivo pharmacokinetic profile of Xarelto IR tablet (20 mg Strength) for Popula-
tions A, B, and C in fed conditions.

Parameters Population A Population B Population C

Cmax (ng/mL) 238 236 237
AUC0–∞ (ng·h/mL) 1856 1888 1988
Pharmaceutics 2021, 13, 283 19 of 22

Figure 13. Virtual in vivo pharmacokinetic profile of Xarelto IR tablet (20 mg Strength) for Popula-
tions (A–C) in fed conditions.

The predicted population in vivo pharmacokinetic profile of Xarelto IR tablet (20 mg

strength) in the fed condition was found to be comparable in populations A, B, and C in
the conditions, i.e., the average value of population geometric means were found to be
within the range of 80–125% compared to the mean predicted in vivo profile mentioned in
in Table 7, Figure 5 (Cmax and AUC0–∞ value of 236.64 and 1857.3, respectively). However,
Pharmaceutics 2021, 13, 283 20 of 22

marked difference (>80–125%) was observed in case of fasted conditions (Cmax and AUC0–∞
value of 171.15 and 1433.8, respectively), as compared to the predicted in vivo population
pharmacokinetics of Xarelto IR tablet (20 mg strength) in fed conditions.
During the virtual simulations, the physiological and pharmacokinetic parameters of
the same subjects were identical for reference formulations. However, in reality, physio-
logical and pharmacokinetic parameters could fluctuate within the same subjects if they
were given different formulations on different occasions. Thus, by incorporating this
intra-subject variability, it is possible that in vivo profile of Xarelto IR tablet in the fasted
condition might be bioequivalent to the population kinetics of Xarelto IR tablet (Fed) in
population A since its 80% confidence interval for the AUC value (77%) is close to the edge
of the BE limits.

4. Conclusions
In the present manuscript, a mechanistic physiology-based model for the Xarelto IR
tablet was developed considering different physicochemical and pharmacokinetic parame-
ters. In addition, a conventional IVIVC model was also developed in order to verify the
in vivo profile obtained via the PBPK model. The validation results demonstrated the
development of successful models, and the predicted in vivo profiles from both models
were found to be comparable. The results demonstrated a significant food effect increasing
the Cmax of the Riva, which could be due to higher solubility in Fed conditions. The devel-
oped model strategy can be effectively adopted to increase the confidence of the model.
Furthermore, the PBPK model can also lead to the establishment of the biased dissolution
methods crucial for the generic company to establish bioequivalence mainly focusing on
new formulations with a similar drug release mechanism using external validation.
Supplementary Materials: The following are available online at https://www.mdpi.com/1999-492
3/13/2/283/s1, Table S1: Concentration of different components of the simulated media used in the
solubility and dissolution studies, Figure S1: Predicted combined effect of Riva particle size and dose
on the bioavailability.
Author Contributions: Conceptualization, A.P., S.A.; methodology, S.A., D.M., and E.F.; Software,
S.A. and M.T.K.; Formal analysis, V.K., S.A., D.M. and M.T.K.; Data curation, V.K. and S.A.;Writing—
original draft preparation, V.K.; Writing—review and editing, V.K., M.T.K. and A.P.; supervision,
A.P.; project administration, A.P. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by Egis Pharmaceuticals.
Institutional Review Board Statement: Not applicate.
Informed Consent Statement: Not applicate.
Data Availability Statement: The data presented in this study are available in the research article
and supplementary material here.
Acknowledgments: Miklós Katona would like to thank Krisztina Takács-Novák (Department of
Pharmaceutical Chemistry, Semmelweis University) for her useful suggestions.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

Absorption Scale Factors ASF

Active pharmaceutical ingredients APIs
Advanced compartmental absorption and transit ACAT
Akaike information criterion AIC
Apparent permeability coefficient Papp
American Type Culture Collection ATCC
Pharmaceutics 2021, 13, 283 21 of 22

Bioavailability BA
Bioequivalence BE
Biopharmaceutical Classification System BCS
Immediate release IR
In vitro–in vivo correlation IVIVC
Minimal Essential Medium MEM
Fasted state simulated gastric fluid FaSSGF
Fasted state simulated intestinal fluid FaSSIF
Fed state simulated gastric fluid FeSSGF
Fed state simulated intestinal fluid FeSSIF
Fetal bovine serum FBS
Parameter Sensitivity Analysis PSA
Pharmacokinetics PK
Physiologically based pharmacokinetic PBPK
Rivaroxaban Riva
Schwarz criterion SC
Sodium lauryl sulfate SLS
Transepithelial electrical resistance TEER
Ultra-High-Performance Liquid Chromatography UHPLC
Unit impulse response UIR
United States Pharmacopeia USP

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