Exploring a Bioequivalence Failure for Silodosin Products Due to Disintegrant Excipients

Abstract

Some years ago, excipients were considered inert substances irrelevant in the absorption process. However, years of study have demonstrated that this belief is not always true. In this study, the reasons for a bioequivalence failure between two formulations of silodosin are investigated. Silodosin is a class III drug according to the Biopharmaceutics Classification System, which has been experimentally proven by means of solubility and permeability experiments. Dissolution tests have been performed to identify conditions concordant with the non-bioequivalent result obtained from the human bioequivalence study and it has been observed that paddles at 50 rpm are able to detect inconsistent differences between formulations at pH 4.5 and pH 6.8 (which baskets at 100 rpm are not able to do), whereas the GIS detects differences at the acidic pH of the stomach. It has also been observed that the differences in excipients between products did not affect the disintegration process, but disintegrants did alter the permeability of silodosin through the gastrointestinal barrier. Crospovidone and povidone, both derivatives of PVP, are used as disintegrants in the test product, instead of the pregelatinized corn starch used in the reference product. Permeability experiments show that PVP increases the absorption of silodosin—an increase that would explain the greater Cmax observed for the test product in the bioequivalence study.

Full text

Citation: González-Álvarez, I.;
Sánchez-Dengra, B.; Rodriguez-Galvez, R.;
Ruiz-Picazo,A.; González-Álvarez,M.;
García-Arieta, A.; Bermejo, M.
Exploring a Bioequivalence Failure
for Silodosin Products Due to
Disintegrant Excipients.
Pharmaceutics 2022,14, 2565.
https://doi.org/10.3390/
pharmaceutics14122565
Academic Editor: Patrick J. Sinko
Received: 18 October 2022
Accepted: 15 November 2022
Published: 23 November 2022
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pharmaceutics
Article
Exploring a Bioequivalence Failure for Silodosin Products Due
to Disintegrant Excipients
Isabel González-Álvarez 1, Bárbara Sánchez-Dengra 1, Raquel Rodriguez-Galvez 1, Alejandro Ruiz-Picazo 1,
Marta González-Álvarez 1, *, Alfredo García-Arieta 2, † and Marival Bermejo 1
1Engineering: Pharmacokinetics and Pharmaceutical Technology Area, Miguel Hernandez University,
03550 San Juan de Alicante, Spain
2Service of Pharmacokinetics and Generic Medicines, Division of Pharmacology and Clinical Evaluation,
Department of Human Use Medicines, Spanish Agency for Medicines and Health Care Products,
28022 Madrid, Spain
*Correspondence: marta.gonzalez@umh.es; Tel.: +34-965-919217
†
This manuscript represents the personal opinion of the authors and does not necessarily represent the views
or policy of their corresponding Regulatory Authorities.
Abstract:
Some years ago, excipients were considered inert substances irrelevant in the absorption
process. However, years of study have demonstrated that this belief is not always true. In this
study, the reasons for a bioequivalence failure between two formulations of silodosin are investigated.
Silodosin is a class III drug according to the Biopharmaceutics Classification System, which has
been experimentally proven by means of solubility and permeability experiments. Dissolution tests
have been performed to identify conditions concordant with the non-bioequivalent result obtained
from the human bioequivalence study and it has been observed that paddles at 50 rpm are able to
detect inconsistent differences between formulations at pH 4.5 and pH 6.8 (which baskets at 100 rpm
are not able to do), whereas the GIS detects differences at the acidic pH of the stomach. It has also
been observed that the differences in excipients between products did not affect the disintegration
process, but disintegrants did alter the permeability of silodosin through the gastrointestinal barrier.
Crospovidone and povidone, both derivatives of PVP, are used as disintegrants in the test product,
instead of the pregelatinized corn starch used in the reference product. Permeability experiments
show that PVP increases the absorption of silodosin—an increase that would explain the greater C
max
observed for the test product in the bioequivalence study.
Keywords:
Biopharmaceutics Classification System (BCS); pharmacokinetics (PK);
in vitro dissolution
;
permeability; bioequivalence
1. Introduction
A generic product is a medicine that is developed to be equivalent and interchangeable
with a reference medicinal product that has been previously authorized. In all cases, generic
products must meet the following conditions: to contain the same active ingredient, with
the same strength and the same dosage form for the same route of administration. In
addition, generic products are authorized only after the exclusivity period of the reference
product, which is at least 10 years in the European Union, and only after the patent and its
complementary certificate have expired. Finally, generic products must demonstrate to be
bioequivalent to the reference product [1,2].
Generic products meet the same quality standards as any other medicinal product, and
support their safety and efficacy profile by demonstrating bioequivalence to the reference
product. Generic products are commercialized with a lower price than the original product
because their developmental process is much shorter and cheaper, which contributes to a
more rational use of economic resources in the health system [3].
Pharmaceutics 2022,14, 2565. https://doi.org/10.3390/pharmaceutics14122565 https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2022,14, 2565 2 of 12
Normally, bioequivalence is evaluated in humans employing a randomized, two-
period, two-sequence, single-dose crossover design. In these studies, plasma levels of the
parent drug are measured in different participants and the rate and extent of absorption of
the drug present in each product is compared by means of the primary pharmacokinetic
parameters C
max
(maximum plasma concentration or peak exposure) and AUC (the area
under the concentration time curve), which reflect the absorption rate and the extent of
exposure, respectively [
4
]. Two formulations will be considered bioequivalent, and thus,
with a comparable
in vivo
performance, if the 90% confidence intervals of the ratio of the
geometric means of test and reference for both Cmax and AUC are between 0.80 and 1.25,
which ensures a difference equal to or lower than 20% between test and reference and is
considered an irrelevant difference that does not require an adjustment of the dose [5,6].
According to Gordon Amidon et al., the main processes that affect absorption are the
aqueous solubility of the active substance and its permeability through the intestinal barrier.
Taking these processes into account, they defined the Biopharmaceutics Classification
System (BCS) and divided drugs into four different groups: (I) high permeability and
high solubility, (II) high permeability and low solubility, (III) low permeability and high
solubility, and (IV) low permeability and low solubility [7,8].
The main purpose of the BCS is to reduce the number of generic drugs that must
demonstrate their bioequivalence by means of studies in humans and, therefore, reduce
ethical and economic difficulties in the development of these types of products [
9
]. In
line with this idea, governmental agencies unified criteria in July 2020 and defined a drug
as highly permeable when its oral fraction absorbed is equal to or greater than 85%, and
highly soluble when its highest single therapeutic dose is completely soluble in 250 mL of
aqueous media over the pH range of 1.2–6.8 at 37
±
1
◦
C. (10) Highly soluble drugs (class
I and class III) whose transport through the intestinal barrier follows a passive route can
be candidates for a biowaiver and, thus, demonstrate similarity between formulations by
means of in vitro dissolution tests [10].
Silodosin is a drug whose commercialization was first approved in 2008 by the U.S.
Food and Drug Administration (FDA) and in 2010 by the European Medicines Agency
(EMA). It is indicated for the treatment of benign prostatic hyperplasia as it allows a
better urine flow. In terms of physicochemical properties, silodosin is a weak base with
a pKa equal to 9.66 and a logP of 3.05. It is slightly soluble in water and, according to
the literature, it is classified as class III [
11
–
13
]. Nevertheless, it could not be a candidate
for a BCS biowaiver as studies in Caco-2 cells have demonstrated it is a substrate of
P-glycoprotein (Pgp) and of active absorption processes through MRP3 [14,15].
The objectives of this work were to confirm the BCS classification of silodosin and to
explain the reasons for the different bioavailability, which caused a bioequivalence failure,
between a new generic product of silodosin and its reference product. With this aim,
solubility, disintegration, dissolution and permeability were studied.
2. Materials and Methods
2.1. Drugs and Products
Reference product (Silodyx
®
) was acquired from a local pharmacy. Test product and
pure silodosin were kindly supplied by a pharmaceutical company. Reference and test
products were capsules containing 4 mg of silodosin and their different excipients as shown
in Table 1.
Table 1. Qualitative different composition of excipients of the reference and the test products.
Excipients Reference Test
Yellow Iron Oxide
Pharmaceutics 2022, 14, x FOR PEER REVIEW 3 of 13
Table 1. Qualitative different composition of excipients of the reference and the test products.
Excipients Reference Test
Yellow Iron Oxide ✓
Pregelatinized Corn Starch ✓
Crospovidone ✓
Povidone ✓
Sodium chloride, sodium acetate, potassium dihydrogen phosphate, hydrochloric
acid, acetic acid, phosphoric acid, methanol, trifluoroacetic acid (TFA), rhodamine, and
polyvinylpyrrolidone (PVP) were purchased from Sigma® (Barcelona, Spain).
2.2. In Vivo Study
The in vivo study (EudraCT no.: 2017-001854-32) was an open label, balanced, ran-
domized, two-period crossover BE study in 48 healthy subjects. The volunteers received
one immediate release (IR) dose of the test product and one dose of the reference product
in a sequence determined by randomization. Maximum plasma concentration (Cmax) and
the area under the concentration time curve (AUC0-t) were the primary pharmacokinetic
parameters to determine the bioequivalence between the products.
The results of the BE study are described in Table 2, where the confidence intervals
for Cmax and AUC0-t showed the BE failure due to the Cmax value, whose 90% confidence
interval upper boundary was too high. More importantly, as the confidence interval does
not include the 100% value, a statistically significant difference at the α level of confidence
interval was detected between the test and the reference products. This unexpected dif-
ference caused the marginal bioequivalence failure.
Table 2. In vivo results of the failed bioequivalence study between the test and the reference prod-
ucts.
Ratio CI (%)
Cmax 1.12 100.15–126.08
AUC 1.05 95.23–115.09
CI: Confidence interval.
2.3. Solubility Assays: Saturation Shake-Flask Procedure
Silodosin solubility was determined at pH 1.2, 4.5 and 6.8 using an orbital shaker at
100 rpm and 37 °C. First, a preliminary test was performed to determine the time needed
to reach an equilibrium concentration, for which 33 mg of API and 2 mL of buffer were
used. Once the equilibrium time was known, a final test with 1 mg of API and 5 mL of
buffer was performed in which equilibrium solubility at pH 1.2, 4.5 and 6.8 was measured
by HPLC. Standard buffers were prepared according to European Pharmacopeia: pH 1.2
(Sodium chloride 50 mM), pH 4.5 (Sodium acetate 36.5 mM), and pH 6.8 (Potassium di-
hydrogen phosphate 50 mM).
2.4. Disintegration Assays
Disintegration assays were carried out in a PTZ-S disintegration tester (Pharma Test®,
Hainburg, Germany) with 900 mL of buffer at pH 1.2 or 900 mL of water. Temperature
was fixed to 37.3 °C, and the oscillations per minute of the basket to 30 times per minute.
The times at which the formulations began to disintegrate were noted, as well as the times
at which they were completely disintegrated. Statistical differences were evaluated with
software SPSS (V 21.0) assuming a statistical level of 0.05.
Pregelatinized Corn Starch
Pharmaceutics 2022, 14, x FOR PEER REVIEW 3 of 13
Table 1. Qualitative different composition of excipients of the reference and the test products.
Excipients Reference Test
Yellow Iron Oxide ✓
Pregelatinized Corn Starch ✓
Crospovidone ✓
Povidone ✓
Sodium chloride, sodium acetate, potassium dihydrogen phosphate, hydrochloric
acid, acetic acid, phosphoric acid, methanol, trifluoroacetic acid (TFA), rhodamine, and
polyvinylpyrrolidone (PVP) were purchased from Sigma® (Barcelona, Spain).
2.2. In Vivo Study
The in vivo study (EudraCT no.: 2017-001854-32) was an open label, balanced, ran-
domized, two-period crossover BE study in 48 healthy subjects. The volunteers received
one immediate release (IR) dose of the test product and one dose of the reference product
in a sequence determined by randomization. Maximum plasma concentration (Cmax) and
the area under the concentration time curve (AUC0-t) were the primary pharmacokinetic
parameters to determine the bioequivalence between the products.
The results of the BE study are described in Table 2, where the confidence intervals
for Cmax and AUC0-t showed the BE failure due to the Cmax value, whose 90% confidence
interval upper boundary was too high. More importantly, as the confidence interval does
not include the 100% value, a statistically significant difference at the α level of confidence
interval was detected between the test and the reference products. This unexpected dif-
ference caused the marginal bioequivalence failure.
Table 2. In vivo results of the failed bioequivalence study between the test and the reference prod-
ucts.
Ratio CI (%)
Cmax 1.12 100.15–126.08
AUC 1.05 95.23–115.09
CI: Confidence interval.
2.3. Solubility Assays: Saturation Shake-Flask Procedure
Silodosin solubility was determined at pH 1.2, 4.5 and 6.8 using an orbital shaker at
100 rpm and 37 °C. First, a preliminary test was performed to determine the time needed
to reach an equilibrium concentration, for which 33 mg of API and 2 mL of buffer were
used. Once the equilibrium time was known, a final test with 1 mg of API and 5 mL of
buffer was performed in which equilibrium solubility at pH 1.2, 4.5 and 6.8 was measured
by HPLC. Standard buffers were prepared according to European Pharmacopeia: pH 1.2
(Sodium chloride 50 mM), pH 4.5 (Sodium acetate 36.5 mM), and pH 6.8 (Potassium di-
hydrogen phosphate 50 mM).
2.4. Disintegration Assays
Disintegration assays were carried out in a PTZ-S disintegration tester (Pharma Test®,
Hainburg, Germany) with 900 mL of buffer at pH 1.2 or 900 mL of water. Temperature
was fixed to 37.3 °C, and the oscillations per minute of the basket to 30 times per minute.
The times at which the formulations began to disintegrate were noted, as well as the times
at which they were completely disintegrated. Statistical differences were evaluated with
software SPSS (V 21.0) assuming a statistical level of 0.05.
Crospovidone
Pharmaceutics 2022, 14, x FOR PEER REVIEW 3 of 13
Table 1. Qualitative different composition of excipients of the reference and the test products.
Excipients Reference Test
Yellow Iron Oxide ✓
Pregelatinized Corn Starch ✓
Crospovidone ✓
Povidone ✓
Sodium chloride, sodium acetate, potassium dihydrogen phosphate, hydrochloric
acid, acetic acid, phosphoric acid, methanol, trifluoroacetic acid (TFA), rhodamine, and
polyvinylpyrrolidone (PVP) were purchased from Sigma® (Barcelona, Spain).
2.2. In Vivo Study
The in vivo study (EudraCT no.: 2017-001854-32) was an open label, balanced, ran-
domized, two-period crossover BE study in 48 healthy subjects. The volunteers received
one immediate release (IR) dose of the test product and one dose of the reference product
in a sequence determined by randomization. Maximum plasma concentration (Cmax) and
the area under the concentration time curve (AUC0-t) were the primary pharmacokinetic
parameters to determine the bioequivalence between the products.
The results of the BE study are described in Table 2, where the confidence intervals
for Cmax and AUC0-t showed the BE failure due to the Cmax value, whose 90% confidence
interval upper boundary was too high. More importantly, as the confidence interval does
not include the 100% value, a statistically significant difference at the α level of confidence
interval was detected between the test and the reference products. This unexpected dif-
ference caused the marginal bioequivalence failure.
Table 2. In vivo results of the failed bioequivalence study between the test and the reference prod-
ucts.
Ratio CI (%)
Cmax 1.12 100.15–126.08
AUC 1.05 95.23–115.09
CI: Confidence interval.
2.3. Solubility Assays: Saturation Shake-Flask Procedure
Silodosin solubility was determined at pH 1.2, 4.5 and 6.8 using an orbital shaker at
100 rpm and 37 °C. First, a preliminary test was performed to determine the time needed
to reach an equilibrium concentration, for which 33 mg of API and 2 mL of buffer were
used. Once the equilibrium time was known, a final test with 1 mg of API and 5 mL of
buffer was performed in which equilibrium solubility at pH 1.2, 4.5 and 6.8 was measured
by HPLC. Standard buffers were prepared according to European Pharmacopeia: pH 1.2
(Sodium chloride 50 mM), pH 4.5 (Sodium acetate 36.5 mM), and pH 6.8 (Potassium di-
hydrogen phosphate 50 mM).
2.4. Disintegration Assays
Disintegration assays were carried out in a PTZ-S disintegration tester (Pharma Test®,
Hainburg, Germany) with 900 mL of buffer at pH 1.2 or 900 mL of water. Temperature
was fixed to 37.3 °C, and the oscillations per minute of the basket to 30 times per minute.
The times at which the formulations began to disintegrate were noted, as well as the times
at which they were completely disintegrated. Statistical differences were evaluated with
software SPSS (V 21.0) assuming a statistical level of 0.05.
Povidone
Pharmaceutics 2022, 14, x FOR PEER REVIEW 3 of 13
Table 1. Qualitative different composition of excipients of the reference and the test products.
Excipients Reference Test
Yellow Iron Oxide ✓
Pregelatinized Corn Starch ✓
Crospovidone ✓
Povidone ✓
Sodium chloride, sodium acetate, potassium dihydrogen phosphate, hydrochloric
acid, acetic acid, phosphoric acid, methanol, trifluoroacetic acid (TFA), rhodamine, and
polyvinylpyrrolidone (PVP) were purchased from Sigma® (Barcelona, Spain).
2.2. In Vivo Study
The in vivo study (EudraCT no.: 2017-001854-32) was an open label, balanced, ran-
domized, two-period crossover BE study in 48 healthy subjects. The volunteers received
one immediate release (IR) dose of the test product and one dose of the reference product
in a sequence determined by randomization. Maximum plasma concentration (Cmax) and
the area under the concentration time curve (AUC0-t) were the primary pharmacokinetic
parameters to determine the bioequivalence between the products.
The results of the BE study are described in Table 2, where the confidence intervals
for Cmax and AUC0-t showed the BE failure due to the Cmax value, whose 90% confidence
interval upper boundary was too high. More importantly, as the confidence interval does
not include the 100% value, a statistically significant difference at the α level of confidence
interval was detected between the test and the reference products. This unexpected dif-
ference caused the marginal bioequivalence failure.
Table 2. In vivo results of the failed bioequivalence study between the test and the reference prod-
ucts.
Ratio CI (%)
Cmax 1.12 100.15–126.08
AUC 1.05 95.23–115.09
CI: Confidence interval.
2.3. Solubility Assays: Saturation Shake-Flask Procedure
Silodosin solubility was determined at pH 1.2, 4.5 and 6.8 using an orbital shaker at
100 rpm and 37 °C. First, a preliminary test was performed to determine the time needed
to reach an equilibrium concentration, for which 33 mg of API and 2 mL of buffer were
used. Once the equilibrium time was known, a final test with 1 mg of API and 5 mL of
buffer was performed in which equilibrium solubility at pH 1.2, 4.5 and 6.8 was measured
by HPLC. Standard buffers were prepared according to European Pharmacopeia: pH 1.2
(Sodium chloride 50 mM), pH 4.5 (Sodium acetate 36.5 mM), and pH 6.8 (Potassium di-
hydrogen phosphate 50 mM).
2.4. Disintegration Assays
Disintegration assays were carried out in a PTZ-S disintegration tester (Pharma Test®,
Hainburg, Germany) with 900 mL of buffer at pH 1.2 or 900 mL of water. Temperature
was fixed to 37.3 °C, and the oscillations per minute of the basket to 30 times per minute.
The times at which the formulations began to disintegrate were noted, as well as the times
at which they were completely disintegrated. Statistical differences were evaluated with
software SPSS (V 21.0) assuming a statistical level of 0.05.

Pharmaceutics 2022,14, 2565 3 of 12
Sodium chloride, sodium acetate, potassium dihydrogen phosphate, hydrochloric
acid, acetic acid, phosphoric acid, methanol, trifluoroacetic acid (TFA), rhodamine, and
polyvinylpyrrolidone (PVP) were purchased from Sigma®(Barcelona, Spain).
2.2. In Vivo Study
The
in vivo
study (EudraCT no.: 2017-001854-32) was an open label, balanced, ran-
domized, two-period crossover BE study in 48 healthy subjects. The volunteers received
one immediate release (IR) dose of the test product and one dose of the reference product
in a sequence determined by randomization. Maximum plasma concentration (C
max
) and
the area under the concentration time curve (AUC
0-t
) were the primary pharmacokinetic
parameters to determine the bioequivalence between the products.
The results of the BE study are described in Table 2, where the confidence intervals
for C
max
and AUC
0-t
showed the BE failure due to the C
max
value, whose 90% confidence
interval upper boundary was too high. More importantly, as the confidence interval does
not include the 100% value, a statistically significant difference at the
α
level of confi-
dence interval was detected between the test and the reference products. This unexpected
difference caused the marginal bioequivalence failure.
Table 2. In vivo
results of the failed bioequivalence study between the test and the reference products.
Ratio CI (%)
Cmax 1.12 100.15–126.08
AUC 1.05 95.23–115.09
CI: Confidence interval.
2.3. Solubility Assays: Saturation Shake-Flask Procedure
Silodosin solubility was determined at pH 1.2, 4.5 and 6.8 using an orbital shaker at
100 rpm and 37
◦
C. First, a preliminary test was performed to determine the time needed to
reach an equilibrium concentration, for which 33 mg of API and 2 mL of buffer were used.
Once the equilibrium time was known, a final test with 1 mg of API and 5 mL of buffer was
performed in which equilibrium solubility at pH 1.2, 4.5 and 6.8 was measured by HPLC.
Standard buffers were prepared according to European Pharmacopeia: pH 1.2 (Sodium
chloride 50 mM), pH 4.5 (Sodium acetate 36.5 mM), and pH 6.8 (Potassium dihydrogen
phosphate 50 mM).
2.4. Disintegration Assays
Disintegration assays were carried out in a PTZ-S disintegration tester (Pharma Test
®
,
Hainburg, Germany) with 900 mL of buffer at pH 1.2 or 900 mL of water. Temperature
was fixed to 37.3
◦
C, and the oscillations per minute of the basket to 30 times per minute.
The times at which the formulations began to disintegrate were noted, as well as the times
at which they were completely disintegrated. Statistical differences were evaluated with
software SPSS (V 21.0) assuming a statistical level of 0.05.
2.5. Dissolution Assays: USP I and USP II
Several dissolution tests were performed at pH 1.2, 4.5 and 6.8 with a PT-DT70 dis-
solution instrument (Pharma Test
®
). In all cases, the temperature was fixed to 37
◦
C, the
volume of buffer employed to 900 mL, and the samples times to 5, 10, 15, 20, 30, 45, and
60 min. The apparatus and the revolutions per minute employed were:
•Paddle apparatus with sinkers to prevent capsules from floating: 50 rpm.
•
Basket apparatus: 100 rpm. The assays with baskets at 100 rpm were performed by
the pharmaceutical company and the sampling times were 7.5, 15, 20, and 30 min.
Dissolution profiles were compared using the similarity factor f
2
(Equation (1)), in
which n is the number of points, R(t) and T(t) are the mean percent of reference or test
product dissolved at time t. Two profiles are considered similar when their f
2
value is
≥
50

Pharmaceutics 2022,14, 2565 4 of 12
or when, in both products, the fraction dissolved is
≥
85% in 15 min, in which case it is not
necessary to calculate f2[10].
f2=50 ×log 100
s1+∑n
t=1[Rt−Tt]2
n (1)
2.6. Permeability Assay: Doluisio Experiment
The product permeabilities were evaluated in rats using the in situ closed loop per-
fusion experiment (Doluisio technique) in three different segments of the intestinal tract
(duodenum, jejunum, and ileum) [
16
,
17
]. The animal experiments, performed in
250–300 g
male Wistar rats, were designed according to the document approved by the Spanish
Government with code A1330354541263. With this aim, three loops were made in each rat
and capsules were opened and resuspended in 250 mL of buffer at pH 6.8. Then, 2 mL
were administered in the duodenum segment, 4 mL in the jejunum one and 4 mL in the
ileum one. Samples were taken at 5, 10, 15, 20, 25, and 30 min and, after being centrifuged,
were analyzed by HPLC.
Once the samples were analyzed, the apparent absorption rate (k
app
) and the apparent
permeability (P
app
) were calculated with Equations (2) and (3), in which C is the luminal
concentration of silodosin at sampling times after correcting it by water reabsorption, C
0
is
the extrapolated concentration of silodosin at time 0, and R is the radius of the intestinal
segment [
18
]. Statistical differences were evaluated with software SPSS (V 21.0) assuming a
statistical significance level of 0.05.
C=C0·e−kapp·t(2)
Papp =kapp·R
2(3)
The Doluisio studies were approved by the Scientific Committee of the Faculty of
Pharmacy, Miguel Hernandez University, and followed the guidelines described in the EC
Directive 86/609, the Council of the Europe Convention ETS 123, and Spanish national
laws governing the use of animals in research.
2.7. In Vitro Permeability Tests
The permeability of the API and the API with crospovidone, povidone, or pregela-
tinized corn starch was evaluated in MDCK-MDR1 monolayers, a cell line which simulates
the intestinal barrier and expresses the P-gp [19].
First, cells were seeded in inserts in 6-well plates and maintained over 7 days as
performed in Sanchez-Dengra et al. [20].
After defrosting the cells, the passage numbers between 30 and 40 were used to
perform the experiments. These cells were maintained in media to grow the cells in
the wells.
The medium used to maintain the growth of the cells was DMEM (Dulbecco’s Modified
Eagle’s Medium with 4500 mg/L glucose, l-glutamine sodium bicarbonate, without sodium
pyruvate from Sigma D5796) (89%) and it was combined with MEM Non-Essential Amino
Acids Solution from Gibco 11140–035 (1%), Fetal Bovine Serum F7524 from Sigma (10%),
and HEPES 1 M 15630-056 by Gibco.
The media were changed three times each week. On the day of the experiment,
the apical side of the wells were filled with the drug solutions so that the Apical-to-
Basolateral effective permeability—P
eff
A-B could be determined. Samples were taken from
the basolateral side at 15, 30, 60, and 90 min. The cell culture is considered acceptable for
transport experiments if all the following criteria are met:

Pharmaceutics 2022,14, 2565 5 of 12
(a) TEER values at the beginning and at the end of the permeability experiments should be
adequate (100 units of difference with blank insert and no more than a 10% difference
between initial values and values at the end of the experiment).
(b) In checking the mass balance after determining the amount of compound in the insert
membranes and inside the cells, the percentage of compound retained in the cell
compartment should typically be less than 5%.
Later on, the samples were analyzed with an HPLC and the apparent permeability
values were calculated using the Modified Non-Sink equation [21].
2.8. Dissolution Experiments: Gastrointestinal Simulator (GIS)
The gastrointestinal simulator is a new device which comprises three dissolution com-
partments: (i) a gastric chamber (GIS Stomach), (ii) a duodenal chamber (GIS Duodenum)
and (iii) a jejunal chamber (GIS Jejunum); it is controlled by a computer. The design of the
GIS is depicted in Figure 1.
Pharmaceutics 2022, 14, x FOR PEER REVIEW 5 of 13
The medium used to maintain the growth of the cells was DMEM (Dulbecco’s Mod-
ified Eagle’s Medium with 4500 mg/L glucose, l-glutamine sodium bicarbonate, without
sodium pyruvate from Sigma D5796) (89%) and it was combined with MEM Non-Essen-
tial Amino Acids Solution from Gibco 11140–035 (1%), Fetal Bovine Serum F7524 from
Sigma (10%), and HEPES 1 M 15630-056 by Gibco.
The media were changed three times each week. On the day of the experiment, the
apical side of the wells were filled with the drug solutions so that the Apical-to-Basolateral
effective permeability—P
eff
A-B could be determined. Samples were taken from the baso-
lateral side at 15, 30, 60, and 90 min. The cell culture is considered acceptable for transport
experiments if all the following criteria are met:
(a) TEER values at the beginning and at the end of the permeability experiments should
be adequate (100 units of difference with blank insert and no more than a 10% differ-
ence between initial values and values at the end of the experiment).
(b) In checking the mass balance after determining the amount of compound in the insert
membranes and inside the cells, the percentage of compound retained in the cell com-
partment should typically be less than 5%.
Later on, the samples were analyzed with an HPLC and the apparent permeability
values were calculated using the Modified Non-Sink equation [21].
2.8. Dissolution Experiments: Gastrointestinal Simulator (GIS)
The gastrointestinal simulator is a new device which comprises three dissolution
compartments: (i) a gastric chamber (GIS Stomach), (ii) a duodenal chamber (GIS Duode-
num) and (iii) a jejunal chamber (GIS Jejunum); it is controlled by a computer. The design
of the GIS is depicted in Figure 1.
Figure 1. Setup and design of the GIS that was applied to test the products in fasted-state conditions.
A tablet of each product was added to the stomach compartment at the start of the
study. The dissolution media, initial volumes, and secretion rates are described in Table
3.
Table 3. Experimental conditions in the GIS for testing the different drug products of the drug.
Fasted-State Test Condi-
tions GISStomach GISDuodenum GISJejunum
Dissolution Media Simulated Gastric Fluid (SGF), pH 2.0,
0.01 M HCl + 34.2 mM NaCL
Phosphate Buffer 50 mM
pH 6.5 /
Initial Volumes 50 mL SGF + 250 mL of water 50 mL /
Secretions 1 mL/min of SGF 1 mL/min of Phosphate Buffer
100 mM pH 6.5 /
Figure 1.
Setup and design of the GIS that was applied to test the products in fasted-state conditions.
A tablet of each product was added to the stomach compartment at the start of the
study. The dissolution media, initial volumes, and secretion rates are described in Table 3.
Table 3. Experimental conditions in the GIS for testing the different drug products of the drug.
Fasted-State Test Conditions GISStomach GISDuodenum GISJejunum
Dissolution Media
Simulated Gastric Fluid (SGF), pH
2.0, 0.01 M HCl + 34.2 mM NaCL
Phosphate Buffer 50 mM
pH 6.5 /
Initial Volumes 50 mL SGF + 250 mL of water 50 mL /
Secretions 1 mL/min of SGF 1 mL/min of Phosphate
Buffer 100 mM pH 6.5 /
Gastric emptying was programmed as a first-order kinetic process with a gastric
half-life of 13 min, to mimic the human values reported in the literature [
22
]. Duodenal
volume must remain constant, therefore, the pump which connects the duodenum and the
jejunal compartment should be modulated accordingly in order to achieve this requirement.
The volume in duodenum was 50 mL. The volume in the jejunal compartment was 0 at
the beginning of the experiment and this compartment was an accumulation of all of the
experiments. All compartments were stirred with a paddle system (Muscle Corp., Osaka,
Japan) which allowed for a stir rate of 20 rotations per minute (RPM).
The fluids were transferred from the stomach to the duodenum and from the duode-
num to the jejunum by peristaltic pumps (Ismatec REGLO pump; IDEX Health and Science,
Glattbrugg, Switzerland).
Samples were collected at different times and they were centrifuged and diluted with
methanol. After that, samples were analyzed by HPLC.

Pharmaceutics 2022,14, 2565 6 of 12
2.9. HPLC Analysis
Samples from the solubility, dissolution, and permeability assays were analyzed by
HPLC employing a UV detector (Waters
®
2487, Milford, MA, USA), an X-Bridge
®
C18
column (3.5
µ
m, 4.6
×
100 mm), and a mobile phase of 50% methanol and 50% acid water
(0.05% v/vTFA in water). The wavelength was set to 225 nm, the flow of the mobile phase
to 1 mL/min, and the temperature to 30
◦
C. Under these conditions, the retention time
for silodosin was determined to be 5 min. The lowest limit of detection of silodosin was
0.189 µg/mL and the lowest limit of quantification of silodosin was 0.631 µg/mL.
3. Results
3.1. Solubility Experiments: Saturation Shake-Flask Procedure
Figure 2shows the experimental solubility of silodosin obtained at pH 1.2, 4.5, and
6.8. It can be observed that solubility decreases with pH; however, as the maximum
therapeutic dose of silodosin is 8 mg (8/250 = 0.032 mg/mL), it can be classified as a
high-solubility drug.
Pharmaceutics 2022, 14, x FOR PEER REVIEW 6 of 13
Gastric emptying was programmed as a first-order kinetic process with a gastric half-
life of 13 min, to mimic the human values reported in the literature [22]. Duodenal volume
must remain constant, therefore, the pump which connects the duodenum and the jejunal
compartment should be modulated accordingly in order to achieve this requirement. The
volume in duodenum was 50 mL. The volume in the jejunal compartment was 0 at the
beginning of the experiment and this compartment was an accumulation of all of the ex-
periments. All compartments were stirred with a paddle system (Muscle Corp., Osaka,
Japan) which allowed for a stir rate of 20 rotations per minute (RPM).
The fluids were transferred from the stomach to the duodenum and from the duode-
num to the jejunum by peristaltic pumps (Ismatec REGLO pump; IDEX Health and Sci-
ence, Glattbrugg, Switzerland).
Samples were collected at different times and they were centrifuged and diluted with
methanol. After that, samples were analyzed by HPLC.
2.9. HPLC Analysis
Samples from the solubility, dissolution, and permeability assays were analyzed by
HPLC employing a UV detector (Waters® 2487, Milford, MA, USA), an X-Bridge® C18 col-
umn (3.5 μm, 4.6 × 100 mm), and a mobile phase of 50% methanol and 50% acid water
(0.05% v/v TFA in water). The wavelength was set to 225 nm, the flow of the mobile phase
to 1 mL/min, and the temperature to 30 °C. Under these conditions, the retention time for
silodosin was determined to be 5 min. The lowest limit of detection of silodosin was 0.189
μg/mL and the lowest limit of quantification of silodosin was 0.631 μg/mL.
3. Results
3.1. Solubility Experiments: Saturation Shake-Flask Procedure
Figure 2 shows the experimental solubility of silodosin obtained at pH 1.2, 4.5, and
6.8. It can be observed that solubility decreases with pH; however, as the maximum ther-
apeutic dose of silodosin is 8 mg (8/250 = 0.032 mg/mL), it can be classified as a high-
solubility drug.
Figure 2. Equilibrium concentrations of silodosin at pHs 1.2, 4.5, and 6.8 after 2 h.
Figure 2. Equilibrium concentrations of silodosin at pHs 1.2, 4.5, and 6.8 after 2 h.
3.2. Disintegration Tests
The disintegration times for the reference product and the test product were not statis-
tically different as shown in Figure 3. At pH 1.2, both the beginning of the disintegration
and the completed disintegration process were faster than with water, but in all cases, the
disintegration rate for both formulations was the same.
3.3. Dissolution Tests: USP I and USP II apparatuses
Figure 4shows the results for the different dissolution tests that were carried out
with baskets and paddles, and Table 4shows the similarity results after comparing the
different profiles with the similarity factor f
2
. Only the experiments with paddles at 50 rpm
(
pH 4.5 and 6.8
) are able to detect the dissimilarity between products in accordance with
the in vivo bioequivalence study.

Pharmaceutics 2022,14, 2565 7 of 12
Pharmaceutics 2022, 14, x FOR PEER REVIEW 7 of 13
3.2. Disintegration Tests
The disintegration times for the reference product and the test product were not sta-
tistically different as shown in Figure 3. At pH 1.2, both the beginning of the disintegration
and the completed disintegration process were faster than with water, but in all cases, the
disintegration rate for both formulations was the same.
Figure 3. Disintegration times for reference product and test product in water and buffer at pH 1.2.
3.3. Dissolution Tests: USP I and USP II apparatuses
Figure 4 shows the results for the different dissolution tests that were carried out
with baskets and paddles, and Table 4 shows the similarity results after comparing the
different profiles with the similarity factor f2. Only the experiments with paddles at 50
rpm (pH 4.5 and 6.8) are able to detect the dissimilarity between products in accordance
with the in vivo bioequivalence study.
Table 4. Similarity results obtained from the different dissolution experiments for the reference
product and the test product.
USP RPM pH Similarity
Baskets 100 rpm
1.2 Similar
4.5 Similar
6.8 Similar
Paddles 50 rpm
1.2 Similar
4.5 No similar (f2 = 25.5)
6.8 No similar (f2 = 22.8)
Figure 3. Disintegration times for reference product and test product in water and buffer at pH 1.2.
Pharmaceutics 2022, 14, x FOR PEER REVIEW 8 of 13
Figure 4. Dissolution profiles for test and reference products at pH 1.2, 4.5, and 6.8. Baskets at 100
rpm (A–C) and paddles at 50 rpm (D–F), respectively.
3.4. Permeability Assay: Doluisio’s Experimental Technique
Permeability values of the products containing silodosin are shown in Figure 5. It can
be observed that, for the reference product, the permeability gets lower while it passes
from the duodenum to the jejunum and then to the ileum, while for the test product, the
permeability keeps constant throughout the intestinal tract.
Figure 4.
Dissolution profiles for test and reference products at pH 1.2, 4.5, and 6.8. Baskets at
100 rpm (A–C) and paddles at 50 rpm (D–F), respectively.

Pharmaceutics 2022,14, 2565 8 of 12
Table 4.
Similarity results obtained from the different dissolution experiments for the reference
product and the test product.
USP RPM pH Similarity
Baskets 100 rpm 1.2 Similar
4.5 Similar
6.8 Similar
Paddles 50 rpm 1.2 Similar
4.5 No similar (f2= 25.5)
6.8 No similar (f2= 22.8)
3.4. Permeability Assay: Doluisio’s Experimental Technique
Permeability values of the products containing silodosin are shown in Figure 5. It can
be observed that, for the reference product, the permeability gets lower while it passes
from the duodenum to the jejunum and then to the ileum, while for the test product, the
permeability keeps constant throughout the intestinal tract.
Pharmaceutics 2022, 14, x FOR PEER REVIEW 9 of 13
Figure 5. Final product permeability in duodenum, jejunum, and ileum.
3.5. In vitro Permeability Tests
In Figure 6, it can be observed how the use of crospovidone as a disintegrant, at the
concentration present in the test formulation, significantly increases the permeability of
silodosin in regard to the API on its own and the API with povidone and pregelatinized
corn starch.
Figure 6. Permeability of silodosin on its own and in presence of excipients.
Figure 5. Final product permeability in duodenum, jejunum, and ileum.
3.5. In Vitro Permeability Tests
In Figure 6, it can be observed how the use of crospovidone as a disintegrant, at the
concentration present in the test formulation, significantly increases the permeability of
silodosin in regard to the API on its own and the API with povidone and pregelatinized
corn starch.

Pharmaceutics 2022,14, 2565 9 of 12
Pharmaceutics 2022, 14, x FOR PEER REVIEW 9 of 13
Figure 5. Final product permeability in duodenum, jejunum, and ileum.
3.5. In vitro Permeability Tests
In Figure 6, it can be observed how the use of crospovidone as a disintegrant, at the
concentration present in the test formulation, significantly increases the permeability of
silodosin in regard to the API on its own and the API with povidone and pregelatinized
corn starch.
Figure 6. Permeability of silodosin on its own and in presence of excipients.
Figure 6. Permeability of silodosin on its own and in presence of excipients.
3.6. Dissolution Experiments: Gastrointestinal Simulator (GIS)
The use of a more complex dissolution apparatus, the GIS, demonstrated the ability
to show the lack of similarity between the test and the reference product in the stomach
compartment, as shown in Figure 7. Nonetheless, differences are not maintained in the
other compartments.
Pharmaceutics 2022, 14, x FOR PEER REVIEW 10 of 13
3.6. Dissolution Experiments: Gastrointestinal Simulator (GIS)
The use of a more complex dissolution apparatus, the GIS, demonstrated the ability
to show the lack of similarity between the test and the reference product in the stomach
compartment, as shown in Figure 7. Nonetheless, differences are not maintained in the
other compartments.
Figure 7. Experimental values of amount dissolved (%) for reference and test silodosin products in
each GIS chamber ((A)—Stomach, (B)—Duodenum, and (C)—Jejunum).
4. Discussion
This study confirms the BCS classification of silodosin and explains that the P-gp
inhibition caused by crospovidone is the most likely reason for the bioequivalence failure
(i.e., non-bioequivalence) of a new generic product of silodosin with respect to its refer-
ence product (Silodyx®), because, as it can be observed from the results of the human bio-
equivalence study (Table 2), the upper boundary of the 90% confidence interval of Cmax
was 126.08%, which is marginally above the upper limit for bioequivalence (1.25).
Solubility assays have confirmed that silodosin can be considered a drug with high
solubility as its maximum therapeutic dose (8 mg) can be freely dissolved in 250 mL of
buffer at pH 1.2, 4.5, and 6.8 (Figure 2). In addition, according to the API permeability
assays (Figure 6), silodosin can be considered a drug with low permeability, as its perme-
ability coefficient in MDCK-MDR1 (2.38 × 10−5 cm/s) is lower than that of metoprolol in
the same cell line (6.77 × 10−5cm/s) [23], an metoprolol is the model drug used to classify
permeability [8,24]. Therefore, it has been confirmed that silodosin is a BCS class III drug.
According to Table 1, the reference and test capsules differ in their excipient compo-
sition: pregelatinized corn starch in the reference was replaced by crospovidone and by
povidone in the test product, as all of them are commonly used as disintegrants [25–28].
Povidone and crospovidone are obtained from PVP and, depending on the degree of
polymerization, they can be classified as soluble PVP (povidone) or insoluble PVP (cro-
spovidone) [26]. In terms of disintegration, Figure 3 shows that the change was not rele-
vant as there are no differences in the disintegration process between the reference prod-
uct and the test product.
In class III drugs, the dissolution process tends to be less important and, in fact, from
the results obtained in the USP I and the USP II, shown in Table 4 and Figure 4, the only
conditions that are able to simulate the non-bioequivalence observed in vivo are the ex-
periments with paddles at 50 rpm (6.8). Importantly, at pH 4.5, the dissolution profiles are
in the reverse order. The reference product dissolves more rapidly, which illustrates that
the results of the paddle apparatus are not consistent and, therefore, unreliable. The M9
Figure 7.
Experimental values of amount dissolved (%) for reference and test silodosin products in
each GIS chamber ((A)—Stomach, (B)—Duodenum, and (C)—Jejunum).
4. Discussion
This study confirms the BCS classification of silodosin and explains that the P-gp
inhibition caused by crospovidone is the most likely reason for the bioequivalence failure
(i.e., non-bioequivalence) of a new generic product of silodosin with respect to its reference
product (Silodyx
®
), because, as it can be observed from the results of the human bioequiv-
alence study (Table 2), the upper boundary of the 90% confidence interval of C
max
was
126.08%, which is marginally above the upper limit for bioequivalence (1.25).
Solubility assays have confirmed that silodosin can be considered a drug with high
solubility as its maximum therapeutic dose (8 mg) can be freely dissolved in 250 mL of
buffer at pH 1.2, 4.5, and 6.8 (Figure 2). In addition, according to the API permeability assays

Pharmaceutics 2022,14, 2565 10 of 12
(Figure 6), silodosin can be considered a drug with low permeability, as its permeability
coefficient in MDCK-MDR1 (2.38
×
10
−5
cm/s) is lower than that of metoprolol in the
same cell line (6.77
×
10
−5
cm/s) [
23
], an metoprolol is the model drug used to classify
permeability [8,24]. Therefore, it has been confirmed that silodosin is a BCS class III drug.
According to Table 1, the reference and test capsules differ in their excipient compo-
sition: pregelatinized corn starch in the reference was replaced by crospovidone and by
povidone in the test product, as all of them are commonly used as
disintegrants [25–28]
.
Povidone and crospovidone are obtained from PVP and, depending on the degree of poly-
merization, they can be classified as soluble PVP (povidone) or insoluble PVP (crospovi-
done) [
26
]. In terms of disintegration, Figure 3shows that the change was not relevant as
there are no differences in the disintegration process between the reference product and the
test product.
In class III drugs, the dissolution process tends to be less important and, in fact,
from the results obtained in the USP I and the USP II, shown in Table 4and Figure 4, the
only conditions that are able to simulate the non-bioequivalence observed
in vivo
are the
experiments with paddles at 50 rpm (6.8). Importantly, at pH 4.5, the dissolution profiles
are in the reverse order. The reference product dissolves more rapidly, which illustrates that
the results of the paddle apparatus are not consistent and, therefore, unreliable. The M9
ICH guideline on the BCS biowaivers proposes the use of the paddle apparatus at 50 rpm or
the basket apparatus at 100 rpm, indistinctly, for evaluating the bioequivalence
in vitro
[
10
].
Nevertheless, according to the results presented here, it is not the same to use baskets at
100 rpm as it is to use paddles at 50 rpm. As a matter of fact, the pharmaceutical company
that failed in the development of the generic drug carried out dissolution tests with baskets
at 100 rpm, from which they concluded that both formulations were similar. However,
had they used paddles, they would have observed that the formulations were not similar
and, therefore, could have made some changes before going into the bioequivalence study
in humans. Even if these dissolution tests are not entirely predictive, it would have been
advisable to show similar dissolution profiles before conducting expensive bioequivalence
studies in humans.
The dissolution experiments carried out with the GIS show that a more complex
dissolution method is also able to detect the differences between the reference product and
the test product, as it can also be observed in the stomach compartment (Figure 7). In this
apparatus, in which the stomach compartment has a lower volume compared with the USP
apparatus and the hydrodynamic conditions better mimic the human stomach, it is possible
to observe the difference in disintegration and dissolution. Dissolution is similar in the
next segments, but a faster dissolution in the stomach combined with better permeability in
the intestines may give the test formulation an advantage for its absorption rate. Although,
unless an absorption window in the duodenum exists, the limited permeability of silo-
dosin would make the dissolution difference in the stomach irrelevant. The permeability
differences are considered the more relevant factor for explaining the difference in C
max
for
low-permeability drugs without an absorption window in the duodenum.
Some years ago, excipients were considered inert substances irrelevant in the absorp-
tion process. However, years of study have demonstrated that this belief is not always
true [
29
]. Silodosin transport across the intestinal membrane is a combined transport
of passive diffusion and active efflux transport through Pgp and active influx transport
through MRP3. Thus, an interaction between the transporters and any of the excipients
could alter silodosin permeability [14,15].
Reference and test formulations were tested and their permeability experiments show
that the absorption of the reference product decreases while it advances through the
gastrointestinal tract [
30
]. The test formulation contained crospovidone and povidone and
its permeability values remained constant, which shows that povidone and crospovidone
could inhibit Pgp or activate MRP3 (Figure 5).
This inhibition has been confirmed in the
in vitro
permeability study as shown in
Figure 6. In addition, it has been observed that the disintegrant that is responsible for

Pharmaceutics 2022,14, 2565 11 of 12
the failure in the bioequivalence study is crospovidone seeing as the permeability of the
test drug increases considerably in the presence of crospovidone, whereas its permeability
does not increase significantly with the API alone, the API with povidone, or with the
pregelatinized corn starch.
5. Conclusions
The BCS classification of silodosin (class III) has been experimentally confirmed in this
study. The main reason for the borderline bioequivalence failure, due to a small difference
in C
max
, was the use in the test product of crospovidone as a disintegrant instead of
pregelatinized corn starch as was used in the reference product. It can be concluded that the
selection of the correct excipients is a key step in the development of new generic products.
It is advisable not to change the excipients in the case of the BCS class III drugs, unless it is
mandatory due to patent protection, since knowing how the excipients affect the release
from the product is as important as knowing their potential effects on the gastrointestinal
system and its functions.
Author Contributions:
B.S.-D. and R.R.-G. performed the disintegration and dissolution assays;
B.S.-D., A.R.-P. and I.G.-Á. performed the solubility and permeability tests and the sample and data
analysis; and M.G.-Á., M.B., A.G.-A. and I.G.-Á., designed the study and coordinated it. All the
authors participated in the manuscript drafting and revision. M.B. assisted with funding acquisition.
A.G.-A. assisted with contextualization. All authors have read and agreed to the published version of
the manuscript.
Funding:
Modelos
in vitro
de evaluación biofarmacéutica” SAF2016-78756(AEI/FEDER, EU) funded
by the Agencia Estatal Investigación and the European Union, through FEDER (Fondo Europeo de De-
sarrollo Regional). Bárbara Sánchez-Dengra received a grant from the Ministry of Science, Innovation
and Universities of Spain (FPU17/00530), and the Ministry of Science in Spain
PID2021-123888OB-100.
Acknowledgments:
The authors acknowledge partial financial support to projects: “Modelos
in vitro
de evaluación biofarmacéutica” SAF2016-78756(AEI/FEDER, EU) funded by the Agencia Estatal
Investigación and the European Union, through FEDER (Fondo Europeo de Desarrollo Regional).
Bárbara Sánchez-Dengra received a grant from the Ministry of Science, Innovation and Universities
of Spain (FPU17/00530), and the Ministry of Science in Spain PID2021-123888OB-100.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
FDA Generic Drugs: Overview & Basics. Available online: https://www.fda.gov/drugs/generic-drugs/overview-basics
(accessed on 12 January 2021).
2.
European Medicines Agency Generic Medicine. Available online: https://www.ema.europa.eu/en/glossary/generic-medicine
(accessed on 12 January 2021).
3.
Laosa, O.; Guerra, P.; López-Durán, J.L.; Mosquera, B.; Frías, J. Bioequivalence Studies: Need for the Reability of Generic Drugs.
Rev. Peru. Med. Exp. Salud. Publica. 2009,26, 553–562.
4. EMA Guideline on the Investigation of Bioequivalence; European Medicines Agency: Amsterdam, The Netherlands, 2010.
5.
Obach Vidal, R.; Colom Codina, H.; Domenech Berrozpe, J. Ensayos de Bioequivalencia: Metodología. In Tratado General de
Biofarmacia y Farmacocinética—Volumen I: LADME; Domenech Berrozpe, J., Martinez Lanao, J., Peraire Guitart, C., Eds.; Sintesis:
Madrid, Spain, 2015; pp. 629–660; ISBN 978-84-995895-2-7.
6.
Obach Vidal, R.; Colom Codina, H.; Domenech Berrozpe, J. Ensayos de Bioequivalencia: Realización Del Estudio. In Tratado
General de Biofarmacia y Farmacocinética—Volumen I: LADME; Domenech Berrozpe, J., Martinez Lanao, J., Peraire Guitart, C., Eds.;
Sintesis: Madrid, Spain, 2015; pp. 661–686; ISBN 978-84-995895-2-7.
7.
Baena, Y.; Ponce D
´
Leon, L.F. The Biopharmaceutic Drug Classification, the Theoretical Basis and Its Importance in the Biowaiver
Studies. Rev. Colomb. Cienc. Químico Farm. 2008,37, 18–32.
8.
Amidon, G.L.; Lennernäs, H.; Shah, V.P.; Crison, J.R. A Theoretical Basis for a Biopharmaceutic Drug Classification: The
Correlation of in Vitro Drug Product Dissolution and in Vivo Bioavailability. Pharm. Res.
1995
,12, 413–420. [CrossRef] [PubMed]
9.
Benet, L.Z. The Role of BCS (Biopharmaceutics Classification System) and BDDCS (Biopharmaceutics Drug Disposition Classifica-
tion System) in Drug Development. J. Pharm. Sci. 2013,102, 34–42. [CrossRef] [PubMed]
10.
Bransford, P.; Cook, J.; Gupta, M.; Haertter, S.; He, H.; Ju, R.; Kanodia, J.; Lennernäs, H.; Lindley, D.; Polli, J.E.; et al. ICH M9
Guideline in Development on Biopharmaceutics Classification System-Based Biowaivers: An Industrial Perspective from the IQ
Consortium. Mol. Pharm 2020,17, 361–372. [CrossRef] [PubMed]

Pharmaceutics 2022,14, 2565 12 of 12
11.
Chemicalize Chemicalize—Instant Cheminformatics Solutions. Available online: https://chemicalize.com/welcome (accessed
on 3 May 2020).
12.
National Library of Medicine Silodosin—PubChem. Available online: https://pubchem.ncbi.nlm.nih.gov/compound/5312125#
section=Melting-Point (accessed on 15 January 2021).
13.
Department of Health, Therapeutic Goods Administration. Therapeutic Goods Administration Australian Public Assessment Report for
Silodosin About the Therapeutic Goods Administration (TGA); Department of Health, Therapeutic Goods Administration: Canberra,
Australia, 2017.
14.
Wang, Z.; Xiang, Q.; Cui, Y.; Zhao, X.; Zhou, Y. The Influence of UGT2B7, UGT1A8, MDR1, ALDH, ADH, CYP3A4 and
CYP3A5 Genetic Polymorphisms on the Pharmacokinetics of Silodosin in Healthy Chinese Volunteers. Drug Metab. Pharm.
2013
,
28, 239–243. [CrossRef] [PubMed]
15.
Matsubara, Y.; Kanazawa, T.; Kojima, Y.; Abe, Y.; Kobayashi, K.; Kanbe, H.; Harada, H.; Momose, Y.; Terakado, S.; Adachi, Y.; et al.
Pharmacokinetics and Disposition of Silodosin (KMD-3213). Yakugaku Zasshi 2006,126, 237–245. [CrossRef] [PubMed]
16.
Doluisio, J.T.; Billups, N.F.; Dittert, L.W.; Sugita, E.T.; Swintosky, J.V. Drug Absorption I: An in Situ Rat Gut Technique Yielding
Realistic Absorption Rates. J. Pharm. Sci. 1969,58, 1196–1200. [CrossRef] [PubMed]
17.
Caldeira, T.G.; Ruiz-Picazo, A.; Lozoya-Agullo, I.; Saúde-Guimarães, D.A.; González-Álvarez, M.; de Souza, J.; González-Álvarez,
I.; Bermejo, M. Determination of Intestinal Permeability Using in Situ Perfusion Model in Rats: Challenges and Advantages to
BCS Classification Applied to Digoxin. Int. J. Pharm. 2018,551, 148–157. [CrossRef] [PubMed]
18.
Figueroa-Campos, A.; Sánchez-Dengra, B.; Merino, V.; Dahan, A.; González-Álvarez, I.; García-Arieta, A.; González-Álvarez, M.;
Bermejo, M. Candesartan Cilexetil In Vitro-In Vivo Correlation: Predictive Dissolution as a Development Tool. Pharmaceutics
2020
,
12, 633. [CrossRef] [PubMed]
19.
Tang, F.; Horie, K.; Borchardt, R.T. Are MDCK Cells Transfected with the Human MRP2 Gene a Good Model of the Human
Intestinal Mucosa? Pharm. Res. 2002,19, 773–779. [CrossRef] [PubMed]
20.
Sánchez-Dengra, B.; González-Álvarez, I.; González-Álvarez, M.; Bermejo, M. New In Vitro Methodology for Kinetics Distribution
Prediction in the Brain. An Additional Step towards an Animal-Free Approach. Animals 2021,11, 3521. [CrossRef] [PubMed]
21.
Sánchez-Dengra, B.; González-Álvarez, I.; Sousa, F.; Bermejo, M.; González-Álvarez, M.; Sarmento, B. In Vitro Model for Predicting
the Access and Distribution of Drugs in the Brain Using HCMEC/D3 Cells. Eur. J. Pharm. Biopharm.
2021
,163, 120–126. [CrossRef]
[PubMed]
22.
Hens, B.; Brouwers, J.; Anneveld, B.; Corsetti, M.; Symillides, M.; Vertzoni, M.; Reppas, C.; Turner, D.B.; Augustijns, P.
Gastrointestinal Transfer: In Vivo Evaluation and Implementation in in Vitro and in Silico Predictive Tools. Eur. J. Pharm. Sci.
2014,63, 233–242. [CrossRef] [PubMed]
23.
Cui, Y.; Desevaux, C.; Truebenbach, I.; Sieger, P.; Klinder, K.; Long, A.; Sauer, A. A Bidirectional Permeability Assay for beyond
Rule of 5 Compounds. Pharmaceutics 2021,13, 1146. [CrossRef] [PubMed]
24.
Incecayir, T.; Tsume, Y.; Amidon, G.L. Comparison of the Permeability of Metoprolol and Labetalol in Rat, Mouse, and Caco-2
Cells: Use as a Reference Standard for BCS Classification. Mol. Pharm. 2013,10. [CrossRef] [PubMed]
25.
Villafuerte Robles, L. The Excipients and Their Functionality in Pharmaceutical Solid Products. Rev. Mex. Cienc. Farm.
2011
,
42, 18–36.
26.
Awasthi, R.; Manchanda, S.; Das, P.; Velu, V.; Malipeddi, H.; Pabreja, K.; Pinto, T.D.J.A.; Gupta, G.; Dua, K. Poly(Vinylpyrrolidone).
In Engineering of Biomaterials for Drug Delivery Systems: Beyond Polyethylene Glycol; Elsevier Inc.: Amsterdam, The Netherlands,
2018; pp. 255–272; ISBN 9780081017517.
27.
Brady, J.; Dürig, T.; Lee, P.I.; Li, J.-X. Polymer Properties and Characterization. In Developing Solid Oral Dosage Forms; Academic
Press: Cambridge, MA, USA, 2017; pp. 181–223; ISBN 9780128024478.
28.
Hiremath, P.; Nuguru, K.; Agrahari, V. Material Attributes and Their Impact on Wet Granulation Process Perfor-
mance.
In Handbook of Pharmaceutical Wet Granulation: Theory and Practice in a Quality by Design Paradigm
; Elsevier: Amsterdam,
The Netherlands, 2019; pp. 263–315; ISBN 9780128104606.
29.
Ruiz-Picazo, A.; Lozoya-Agullo, I.; González-Álvarez, I.; Bermejo, M.; González-Álvarez, M. Effect of Excipients on Oral
Absorption Process According to the Different Gastrointestinal Segments. Expert Opin. Drug Deliv. 2020. [CrossRef] [PubMed]
30. Dou, L.; Mai, Y.; Madla, C.M.; Orlu, M.; Basit, A.W. P-Glycoprotein Expression in the Gastrointestinal Tract of Male and Female
Rats Is Influenced Differently by Food. Eur. J. Pharm. Sci. 2018,123, 569–575. [CrossRef] [PubMed]