Investigating the Discriminatory Power of BCS-Biowaiver in Vitro

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Investigating the discriminatory power of BCS-biowaiver in vitro methodology to detect bioavailability differences between immediate release products containing a class I drug. Sarin Colon Useche, Isabel Gonzalez Alvarez, Victor Mangas Sanjuan, Marta Gonzalez Alvarez, Pilar Pastoriza, Irene Molina Martinez, Marival Bermejo, and Alfredo Garcia-Arieta Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00076 • Publication Date (Web): 19 Aug 2015 Downloaded from http://pubs.acs.org on August 20, 2015

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Molecular Pharmaceutics

TITLE: Investigating the discriminatory power of BCS-biowaiver in vitro methodology to detect bioavailability differences between immediate release products containing a class I drug. AUTHORS: Sarin Colón-Useche†§∥, Isabel González-Álvarez†, Victor Mangas-Sanjuan†, Marta González-Álvarez†, Pilar Pastoriza§, Irene Molina-Martínez§, Marival Bermejo†*, Alfredo García-Arietaƚ. AFFILIATION: †

Engineering: Pharmacokinetics and Pharmaceutical Technology Area. Miguel Hernandez

University. Spain. §

Pharmacokinetics and Pharmaceutical Technology. Complutense University of Madrid. Spain.

∥ Analysis

ƚ

and Control Department. University of Los Andes. Venezuela.

Service on Pharmacokinetics and Generic Medicines, Division of Pharmacology and Clinical

Evaluation, Department of Human Use Medicines, Spanish Agency for Medicines and Health Care Products, Madrid, Spain.

CORRESPONDING AUTHOR FOOTNOTE *Address for correspondence: Marival Bermejo, [email protected] Edificio Muhammad Al-Shafra, Facultad de Farmacia, UMH, Carretera Alicante Valencia km 87, 03550 San Juan de Alicante, Alicante, Spain. Phone +34 965 919217 Fax +34 963544911

ACS Paragon Plus Environment


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TABLE OF CONTENT GRAPHICS

ABSTRACT The purpose of this work is to investigate the discriminatory power of the BCSbiowaiver in vitro methodology, i.e., to investigate if a BCS-biowaiver approach would have detected the Cmax differences observed between two zolpidem tablets and to identify the cause of the in vivo difference. Several dissolution conditions were tested with three zolpidem formulations: the reference (Stilnox), a bioequivalent formulation (BE), and a non-bioequivalent formulation (N-BE). Zolpidem is highly soluble at pH 1.2, 4.5 and 6.8. Its permeability in Caco-2 cells is higher than that of metoprolol and its transport mechanism is passive diffusion. None of the excipients (alone or in combination) showed any effect on permeability. All formulations dissolved more than 85% in 15 minutes in the paddle apparatus at 50 rpm in all dissolution media. However, at 30 rpm the non-bioequivalent formulation exhibited a slower dissolution rate. A slower gastric emptying rate was also observed in rats for the nonbioequivalent formulation. A slower disintegration and dissolution or a delay in gastric emptying might explain the Cmax infra-bioavailability for a highly permeable drug with short half-life. The BCS biowaiver approach would have declared bioequivalence, although the in vivo study was not conclusive but detected a 14% mean difference in Cmax that precluded the bioequivalence demonstration. Nonetheless, these findings suggest that a slower dissolution rate is more discriminatory and rotation speeds higher than 50 rpm should not be used in BCS biowaivers, even if a coning effect occurs. KEYWORDS Bioequivalence, biopharmaceutics classification system, in vitro, dissolution test, gastric emptying, zolpidem.


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ABBREVIATIONS Bioequivalent (BE), Non Bioequivalent (N-BE), Biopharmaceutics Classification System (BCS), high (H), low (L).



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INTRODUCTION Some regulatory guidelines allow for waivers of the requirement to conduct in vivo bioequivalence (BE) studies for immediate release solid oral dosage forms containing class I drugs based on the Biopharmaceutics Classification System (BCS)

1-3

i.e., replacement of in

vivo BE studies with in-vitro BE dissolution tests, if dissolution profiles are rapid and similar to those of the reference product. The relationship between the BCS class of a drug and the probability of success / failure to show BE between products containing that drug has been investigated recently.4-6 Unfortunately, the reports on these studies did not include the dissolution profiles at pH 1.2, 4.5 and 6.8. Consequently, it is not possible to know if a failure was caused by differences in dissolution profiles, which would have been detected if a BCS biowaiver had been applied, or if on the contrary, a BCS biowaiver had been granted despite the existence of bioavailability differences because the current in vitro dissolution methodology is not sufficiently discriminative. At least these investigations confirmed that for class II drugs, the current pharmacopeial methods lack predictive power. Importantly, however, Ramirez et al.5 reported the drugs that failed to show BE, which allows us the opportunity to investigate these failures based on the information available in the Spanish Agency for Medicines and Health Care Products. Of the failures reported by Ramirez et al., isoniazid (Class I/III), codeine (class I) and zolpidem are the only class I drugs whose failure was due to the existence of differences in a sufficiently powered BE study. In the isoniazid case, the formulation contained mannitol as diluent. Therefore, the excipient composition can explain the failure and a BCS biowaiver should not have been granted if it had been applied. In the case of codeine, it was part of a fixed dose combination with ibuprofen. Interestingly, the AUC of ibuprofen in the study also failed and it is very unusual that an ibuprofen AUC comparison would fail. Therefore, this dual failure seems to suggest that there may have been an issue with the dosing of this liquid formulation. Consequently, based on Ramirez et al. database the only failure that needs to be explained in order to ensure that current in vitro dissolution methodology is predictive is the case of zolpidem tablets. The purpose of this work is to investigate if the BCS biowaiver approach would have detected the zolpidem Cmax difference observed in the BE study and to identify the cause of the BE failure.


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EXPERIMENTAL SECTION 1. Compounds Zolpidem was kindly provided by a pharmaceutical company. Metoprolol, n-octanol, acetonitrile and methanol were purchased from Sigma (Barcelona, Spain). 2. Zolpidem Formulations Stilnox (Sanofi-Aventis) was used as reference product. Its excipients are: lactose monohydrate, microcrystalline cellulose, hypromellose, sodium starch glycollate and magnesium stearate in the tablet core and hypromellose, titanium dioxide and macrogol 400 in the film coating. Zolpidem bioequivalent (BE) and non-bioequivalent (N-BE) formulations were kindly provided by a pharmaceutical company. Both test product are on the market on a jurisdiction where wider acceptance limits for the IC90% of Cmax were applied. The excipients in the N-BE formulation are: lactose monohydrate, microcrystalline cellulose, hypromellose, sodium starch glycollate and magnesium stearate in the tablet core and hypromellose, titanium dioxide, macrogol 6000 and talc in the film coating: In the BE formulation the excipients are: lactose monohydrate, cellulose, sodium croscarmellose, colloidal silica, and magnesium stearate in the tablet core and hypromellose, titanium dioxide, macrogol and polisorbate 80 in the film coating. These products have not been changed in any way that may affect their bioavailability after their marketing approval. The results obtained in the corresponding 2x2 cross-over BE studies for these products are reported in Table 1. The non-bioequivalent formulation failed to show bioequivalence in Cmax. Further, the 90% confidence interval of Cmax did not include the 100% value indicating that there was a statistically significant difference in Cmax at the significance level employed to construct the confidence interval. 3. Experimental techniques Solubility assays: Saturation Shake-Flask Procedure To estimate zolpidem solubility, an excess of solid drug was added in a standard buffer solution at 37ºC (pH 1.2, 4.5 and 6.8) and samples were taken until the concentration remained unchanged indicating saturated conditions. Flasks were shaken for 4, 8, 24 and 48 h. Sample concentration was determined using HPLC with UV detection. Permeability assays: Cell culture and transport studies Caco-2 cells were grown in Dubelcco’s Modified Eagle’s Media containing Lglutamine, fetal bovine serum and penicillin/streptomycin. Cells monolayers were prepared by seeding 250,000 cells/cm2 on polycarbonate membranes with surface area of 4.2 cm2. They were maintained at 37ºC temperature, 90% relative humidity and 5% CO2 until confluence (1921 days). Afterward, the integrity of each cell monolayer was evaluated by measuring the transepithelial electrical resistance (TEER). SOPs were described and validated previously in our laboratory.7-11 Hank’s balanced salt solution (HBSS) supplemented with HEPES was used to fill



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the receiver chamber and to prepare the drug solution that was placed in the donor chamber (pH=7 in both chambers). Transport studies were conducted in an orbital environmental shaker at constant temperature (37ºC) and at an agitation rate of 50 rpm. Four samples of 200 µL each were taken, and replaced each time with fresh buffer, from the receiver side at 15, 30, 45 and 90 minutes. Samples of the donor side were taken at the beginning and the end of the experiment. Moreover, the amount of compound in cell membranes and inside the cells was determined at the end of experiments in order to check the mass balance and the percentage of compound retained in the cell compartment that was always less than 5%. Zolpidem transport was studied in solution at four concentrations (5.2, 12, 52 and 120

µM) and in the presence of the formulation excipients at a drug concentration equivalent to 52 µM (equivalent to 1 tablet in 250 mL). To perform the transport studies in presence of the formulation excipients a tablet of each formulation was crushed and dissolved in 250 mL of transport buffer and drug. Transport studies were performed in apical-to-basal (A-to-B) direction and volume of donor and receiver compartments were 2 and 3 mL, respectively. The apparent permeability coefficient was calculated according the following equation:

, =

+ , · −

(

(

$%% ',( ·)·* ,·∆ + + "·#

(1) where Creceiver,t is the drug concentration in the receiver chamber at time t, Qtotal is the total amount of drug in both chambers, Vreceiver and Vdonor are the volumes of each chamber, Creceiver,t−1 is the drug concentration in receiver chamber at previous time, f is the sample replacement dilution factor, S is the surface area of the monolayer, ∆t is the time interval and Peff is the permeability coefficient as were described by Mangas-Sanjuan et al 9. Gastric emptying and intestinal transit Evaluation of gastrointestinal motility was performed in rats with the help of a test meal consisting of a suspension of 7.5 g barium sulphate in 10 mL salt-free water given orally by gavage in a volume of 6 mL/kg body weight. The test meal was administered immediately after oral administration of 3.5 mL/kg of the sample dispersions: Zolpidem tablets (reference and test products) were crushed. An adequate amount of solid powder was weighted to render a Zolpidem concentration of 52 µM in saline. As some of the excipients were not water soluble, dispersions were obtained. Thirty minutes after the administration of the test meal 12, the animals were euthanized with an overdose of isoflurane (above 7%). The stomach and the intestine were exposed by laparatomy and removed. For the evaluation of gastric emptying rate, the removed stomach was weighed, then excised, the contents removed, and the stomach weighed again. The gastric content was


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calculated from the weight difference between the filled and empty stomach and normalized to 100 g of body weight. Thus, an increase in weight difference (i.e., higher stomach content) indicated inhibition of gastric emptying, whereas a decrease in weight difference indicated enhanced gastric emptying.12 The gastric emptying assay was performed using a parallel design with 6 animals per group (formulation). This sample size was estimated to detect a 25% difference with 80% statistical power considering a model variability of 17%. This sample size is close to the recommended in similar protocols (5 to 10 rats per group)13. Average gastric emptying rate (ml/min) during a meal was found to vary inversely with the nutrient density of the test meal14 thus Barium sulphate as gastric emptying marker could have a longer emptying half-life compared with other substances as Phenol Red. Nevertheless the same marker was used to study the excipient effect by comparing gastric emptying in both groups. Dissolution assays Drug release experiments were performed with Apparatus 2 (Paddle method) (PharmaTest PT-DT70) with 900 mL of different media (Table 2) at 37±0.5 ºC and 50 or 30 rpm agitation. Samples (5 mL) were taken using the Pharma-Test PTFC-2 fraction collector and peristaltic pump (Ismatec IPC). Each sample was filtered on line through a 10 µM (Pharma test) filter. After that samples were centrifuged at 10000 rpm for 1 min (12000 g) and the supernatant was transferred to HPLC vials. To keep the test volume constant throughout the entire test, the sample volume was replaced by fresh preheated medium. In order to compare the dissolution profiles, f2 (similarity factor) was calculated 1, 15. When more than 85% of the drug is dissolved within 15 minutes, dissolution profiles may be accepted as similar without further mathematical evaluation. Disintegration test The disintegration times of six tablets per formulation were determined in distilled water at 37 ±0.50 ºC using the disintegration apparatus (Pharma Test Model PTZ-S, Germany) following European Pharmacopoeia 2.9.1. Test A standard. 4. Analysis of the samples Samples of all studies were analyzed by HPLC (Waters 2695) using a Nova-Pak C18 column (4 µM, 3.9 x 150 mm) at 30oC. For zolpidem the mobile phase was a mixture of 6.5 mM trifluoroacetic acid solution and acetonitrile (60:40, v/v) pumped at a flow-rate of 1.0 mL/min. Column effluent was monitored by ultraviolet absorbance at wavelengths of 245 nm (Waters 2487). The limit of quantification (LLOD) for zolpidem was 0.006 µM. To assay metoprolol the mobile phase consisted of a mixture of methanol, acetonitrile and 6.5 mM trifluoroacetic acid solution (20:20:60, v/v) at flow-rate of 1.0 mL/min. Detection was by fluorescence, with excitation at 231 nm and emission at 307 nm. The method was validated previously in our laboratory 8, 16.



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5. Statistical analysis Results are shown as mean ± standard deviation. Mean values of two groups were compared with two-tailed Student t-tests. Mean values of more than two groups were compared with analysis of variance (ANOVA) and Scheffé post hoc test. A significance level of 0.05 was used. The statistical analyses were conducted with the statistical package SPSS. (SPSS version 22 (IBM United States) licensed to Universidad Miguel Hernández)


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RESULTS The solubility of zolpidem at pH 1.2, 4.5 and 6.8 was 48.24, 23.23 and 6.60 mg/mL, respectively. The lowest solubility was obtained at the highest pH, which is expected for a weak ionizable base with pKa at 5.65 17. Given that, 1.52 mL are necessary to solubilize the highest strength / dose of zolpidem (10 mg), leading to a Dose number (Do) less than 1 (≤ 6.32·10-3), which confirms zolpidem as a highly soluble drug. The permeability of zolpidem was studied at different concentrations and it was compared with metoprolol permeability to verify its BCS classification (Figure 1). As its permeability was larger than that of metoprolol, it can be considered highly permeable, which is consistent with an oral bioavailability of 70% and a first pass hepatic effect of 35%,18 and hence classifies zolpidem as a class I drug. The analysis of variance (ANOVA) performed on the permeability values did not detect differences among the different zolpidem concentrations (p=0.08). Figure 2 shows the permeability of zolpidem in the different formulations. The ANOVA was not able to detect statistically significant differences (p=0.13) and therefore, it can be assumed zolpidem permeability is not affected by these excipients. Figure 3 shows the results of the gastrointestinal motility experiment. An increase in weight difference suggests inhibition of gastric emptying. The ANOVA detected statistically significant differences between formulations (p=0.002). The post-hoc test showed statistically significant differences (p=0.01 and p=0.002) between the non-bioequivalent formulation and the other two formulations. The dissolution profile comparisons of these three formulations showed complete dissolution (>85%) in 15 minutes in the paddle apparatus at 50 rpm in buffered media at pH 1.2, 2.0, 4.5, 6.0, 6.5 and 6.8 with normal buffer capacity (50 mM) or a lower buffer capacity (5 or 10 mM) (See Figures 4 and 5). Therefore, they can be considered similar without any further mathematical calculation. However, at 30 rpm with the paddle apparatus the dissolution profiles were able to detect differences (discriminative), showing a slower dissolution for the nonbioequivalent formulation (Figure 6). The disintegration times of all the assayed formulations are represented in Figure 7. The non-BE formulation showed a longer disintegration time, statistically different from the reference and bioequivalent formulation.



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DISCUSSION This investigation confirms that zolpidem can be classified as a class I drug according to the BCS, and that the existing in vitro methodology currently employed to perform BCS biowaivers, i.e., paddle apparatus at 50 rpm in pH 1.2, 4.5 and 6.8 media, is not able to detect the existing difference in Cmax that resulted in a failure to show BE in a sufficiently powered BE study, which, on the other hand, could be attributed to the beta error. It is important to highlight that zolpidem is extremely permeable because its permeability is 2-fold higher than that of metoprolol (Figure 1) and its half-life is short (2.4 h), which makes Cmax very sensitive to differences in rate of absorption. No differences in permeability values were observed with different zolpidem concentrations or in the presence of sodium azide, which is used as a metabolic inhibitor to nullify the contribution of any transporters.19 Consequently it is confirmed that zolpidem’s transport mechanism is passive diffusion. Similarly, the permeability values of the three zolpidem formulations (Figure 2) did not show any significant difference, which confirms that excipients are not affecting permeability. Then, the in vivo differences in Cmax are not related to differences of permeability due to excipients, which is expected because the excipients in these formulations are not considered critical, i.e., these excipients are not known to affect bioavailability. Two independent hypotheses were tested separately. First, whether any of the excipients in the tested formulations could alter gastric emptying and affect the absorption rate and Cmax. Second, disintegration time differences could be the origin of the absorption rate differences (reflected on Cmax). The study of gastric emptying in rats is an important endpoint in preclinical drug development. While assessment of gastric emptying is not mandatory for approval of a drug candidate, this test has long been proposed as a supplemental safety pharmacology study according to ICH S7A safety pharmacology guidelines.20 The rat remains the most widely used rodent species in gastrointestinal safety evaluation including gastrointestinal motility assessment.21 The use of rats as animal models of human gastric emptying and intestinal motility has a long history for healthy22 or pathological conditions23 and is routinely used for screening purposes24 or mechanism investigation.25, 26 Similar patterns of nutrients and xenobiotics effects on gastric emptying have been observed between human a rat.25, 27, 28 Based on this background a rat experiment of gastric emptying measurement based on a published protocol12 was selected to study the potential formulation effect on stomach motility. Interestingly, the gastrointestinal motility investigation in rats (Figure 3) showed that the N-BE formulation had a significantly slower gastric emptying rate, which could explain the lower Cmax in the failed BE study. For a high permeability drug the actual limiting step for


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absorption is gastric emptying if the drug is emptied from the stomach as a solution. Then, if a formulation delays slightly the gastric emptying, Cmax will be lower, especially for short halflife drugs. These results in rats offer a feasible hypothesis that should be checked in human volunteers. Kelly et al. demonstrated that a faster disintegration time and gastric emptying of a paracetamol formulation causes a faster absorption and significantly higher Cmax values.29 Paracetamol, like zolpidem, is a very highly permeable drug. However, for the N-BE formulation the excipient composition in the tablet core is qualitatively identical to that of the reference and the expected quantitative differences plus the excipients in the coating are not considered to be able to affect gastric emptying, in contrast to bicarbonate that was used in the paracetamol formulation. Therefore, the different gastric emptying might be related to a different disintegration and dissolution rather to an excipient effect in this case. Another hypothesis could be that the drug is not emptied as a solution from the stomach, if in vivo dissolution were slower than in vitro dissolution in the paddle apparatus at 50 rpm in 900 mL, and the actual in vivo dissolution of the N-BE formulation is slower. At 50 rpm the paddle apparatus dissolved all three products completely, i.e., >85% in 15 minutes, in all the tested media. Therefore, these three products can be considered similar without any mathematical calculation (e.g., f2 similarity factor), according to current BCS biowaiver criteria. However, the N-BE formulation exhibited a slower dissolution rate (f2= 36.8 and 40.7 at pH 1.2 and 6.8, respectively) in the paddle apparatus at 30 rpm (Figure 6). In contrast, the BE formulation showed a similar rate at pH 1.2 (>85% in 15min) and pH 6.8 (f2=72.5). Importantly, this observation is supported by the in vitro in vivo correlation that was developed for the above mentioned paracetamol product when 30 rpm were used30. In accordance with these results the N-BE formulation showed a longer disintegration time while reference and BE formulation did not show any difference. A limitation of our study is that the N-BE formulation has been tested in vivo only once and the observed Cmax result could be a false negative outcome, i.e., a bioequivalent product is considered non-bioequivalent due to the producer risk or beta, which is usually predefined at 10 or 20% in BE studies. Furthermore, it could be argued that a 14% mean difference in Cmax is still within the BE limits and another study with a much larger sample size (over-powered) could be able to conclude equivalence within the BE limits (80-125%). Although this is correct, these findings show that the current in vitro methodology at 50 rpm is not able to detect any difference. Then, the in vitro methodology in this case seems to be less discriminative than the in vivo studies, in contrast to other authors’ results31. It can be speculated that even a larger difference in Cmax might also be undetected, but as this has not been demonstrated, it is not possible to claim that the present BCS biowaiver criteria are incorrect. To this end, it would be necessary to find a bioinequivalent product, i.e., a product with the entire 90% confidence



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interval outside of the acceptance range (80-125%), showing similar dissolution profiles. Nonetheless, these findings suggest that a slower dissolution rate is more discriminatory and rotation speeds higher than 50 rpm should not be used in BCS biowaivers, even if a coning effect occurs. CONCLUSION Zolpidem is a class 1 drug with very high permeability and short half-life, which makes its Cmax very sensitive to differences in rate of absorption. The current dissolution test with the paddle apparatus at 50 rpm in pH 1.2, 4.5 and 6.8 media was not able to detect the Cmax differences observed in a BE study. Consequently, a BCS biowaiver would have been granted despite a 14% mean difference in Cmax. The in vivo data is not conclusive as the N-BE formulation failed to show BE, but it was not bioinequivalent. Nevertheless, it is important to be reminded that for a high permeability drug with a short half-life, small changes in gastric emptying caused by excipients, disintegration, and dissolution may be undetected by the current in vitro methodology and cause some differences in Cmax, reducing the probability for BE demonstration.


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ACKNOWLEDGEMENTS The

authors

acknowledge

partial

financial

support

to

projects:

DCI

ALA/19.09.01/10/21526/245-297/ALFA 111(2010)29: Red-Biofarma, funded by European Commission. AGL2011-29857-C03-03, Foodomics evaluation of dietary polyphenols, from Spanish Ministry of Science. FA-426-08-07-C, Comparación de los perfiles de disolución de distintos principios activos from CDCHT-ULA. Victor Mangas-Sanjuan received a grant from Ministry of Education and Science of Spain and Miguel Hernandez University (FPU AP20102372) and Sarin Colón-Useche received a grant from DAP, University of Los Andes, Venezuela, to develop this project.



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REFERENCES 1. (EMA), E. M. A. Committee for Medicinal Products for Human Use (CHMP). Guideline on the Investigation of Bioequivalence 20-1-2010. 2. US FDA; US Departament of Health and Human Services; Center for Drug Evaluation and Research. Guidance for Industry: Bioavailability and Bioequivalence Studies for Orally Administered Drug Products-General Considerations. 2003. 3. WHO. Multisource (generic) pharmaceutical products: guidelines on registration requirements to establish interchangeability. WHO Technical Report Series 2006, 937, 347-390. 4. Lamouche, S.; Leonard, H.; Shink, E.; Tanguay, M. The biopharmaceutical classification system: Can it help predict bioequivalence outcome? A CRO retrospective analysis. Accessed January 10, 2012, at: http://www.aapsj.org/abstracts/AM 2008/AAPS2008– 002992.PDF 2008. 5. Ramirez, E.; Laosa, O.; Guerra, P.; Duque, B.; Mosquera, B.; Borobia, A. M.; Lei, S. H.; Carcas, A. J.; Frias, J. Acceptability and characteristics of 124 human bioequivalence studies with active substances classified according to the Biopharmaceutic Classification System. Br J Clin Pharmacol 2010, 70, (5), 694-702. 6. Cristofoletti, R.; Chiann, C.; Dressman, J. B.; Storpirtis, S. A comparative analysis of biopharmaceutics classification system and biopharmaceutics drug disposition classification system: a cross-sectional survey with 500 bioequivalence studies. J Pharm Sci 2013, 102, (9), 3136-44. 7. Ruiz-Garcia, A.; Lin, H.; Pla-Delfina, J. M.; Hu, M. Kinetic characterization of secretory transport of a new ciprofloxacin derivative (CNV97100) across Caco-2 cell monolayers. J Pharm Sci 2002, 91, (12), 2511-9. 8. Oltra-Noguera, D.; Mangas-Sanjuan, V.; Centelles-Sanguesa, A.; Gonzalez-Garcia, I.; Sanchez-Castano, G.; Gonzalez-Alvarez, M.; Casabo, V.; Merino, V.; Gonzalez-Alvarez, I.; Bermejo, M. Variability of permeability estimation from different protocols of subculture and transport experiments in cell monolayers. J Pharmacol Toxicol Methods 2014, 71C, 21-32. 9. Mangas-Sanjuan, V.; Gonzalez-Alvarez, I.; Gonzalez-Alvarez, M.; Casabo, V. G.; Bermejo, M. Modified nonsink equation for permeability estimation in cell monolayers: comparison with standard methods. Mol Pharm 2014, 11, (5), 1403-14. 10. Gundogdu, E.; Mangas-Sanjuan, V.; Gonzalez-Alvarez, I.; Bermejo, M.; Karasulu, E. In vitro-in situ permeability and dissolution of fexofenadine with kinetic modeling in the presence of sodium dodecyl sulfate. Eur J Drug Metab Pharmacokinet 2012, 37, (1), 65-75. 11. Gonzalez-Alvarez, I.; Fernandez-Teruel, C.; Garrigues, T. M.; Casabo, V. G.; RuizGarcia, A.; Bermejo, M. Kinetic modelling of passive transport and active efflux of a fluoroquinolone across Caco-2 cells using a compartmental approach in NONMEM. Xenobiotica 2005, 35, (12), 1067-88. 12. Pestel, S.; Martin, H. J.; Maier, G. M.; Guth, B. Effect of commonly used vehicles on gastrointestinal, renal, and liver function in rats. J Pharmacol Toxicol Methods 2006, 54, (2), 200-14. 13. Guillaume, P.; Provost, D.; Lacroix, P. Gastrointestinal models: intestinal transit, gastric emptying, and ulcerogenic activity in the rat. Curr Protoc Pharmacol 2008, Chapter 5, Unit5 3. 14. Kalogeris, T. J.; Reidelberger, R. D.; Mendel, V. E. Effect of nutrient density and composition of liquid meals on gastric emptying in feeding rats. Am J Physiol 1983, 244, (6), R865-71.


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FIGURE CAPTIONS

Figure 1. Permeability values of zolpidem obtained in vitro in Caco-2 cell monolayers at four drug concentrations and in the presence of Sodium Azide.

Figure 2. Zolpidem permeability as active substance and combined with the excipients of the different formulations (reference product, non-bioequivalent formulation (N-BE) and bioequivalent formulation (BE).

Figure 3. Effect of zolpidem formulations (reference product, non-bioequivalent formulation (N-BE) and bioequivalent formulation (BE)) on gastric emptying after p.o. administration to rats.

Figure 4. Dissolution profiles of three zolpidem formulations (reference product, nonbioequivalent formulation (N-BE) and bioequivalent formulation (BE)) in the paddle apparatus at 50 rpm in buffered media at pH 1.2, 4.5 and 6.8.

Figure 5. Dissolution profiles of three zolpidem formulations (reference product, nonbioequivalent formulation (N-BE) and bioequivalent formulation (BE)) in the paddle apparatus at 50 rpm in buffered media at pH 2.0, 6.0 and 6.5 (upper); and 1.2, 4.5, 6.0, 6.5 and 6.8 (bottom).

Figure 6. Dissolution profiles of three zolpidem formulations (reference product, nonbioequivalent formulation (N-BE) and bioequivalent formulation (BE)) in the paddle apparatus at 30 rpm in buffered media at pH 1.2 and 6.8.

Figure 7. Disintegration times of zolpidem formulations, reference, BE formulation and N-BE formulation. * denotes statistically significant differences p

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