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Analysis of Nifedipine Absorption from Soft Gelatin Capsules Using PBPK Modeling and Biorelevant Dissolution Testing
Analysis of Nifedipine Absorption from Soft Gelatin Capsules Using PBPK Modeling and Biorelevant Dissolution Testing ¨ RG LIPPERT,2 STEFAN WILLMANN2 KIRSTIN THELEN,1 EKARAT JANTRATID,1 JENNIFER B. DRESSMAN,1 JO 1
Institute of Pharmaceutical Technology, Johann Wolfgang Goethe University, 60438 Frankfurt am Main, Germany
2
Competence Center Systems Biology and Computational Solutions, Bayer Technology Services GmbH, 51368 Leverkusen, Germany Received 6 May 2009; revised 30 September 2009; accepted 25 October 2009 Published online 14 December 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.22026 ABSTRACT: Delayed absorption of nifedipine when administered as a 20 mg immediate release soft gelatin capsule to fasted volunteers has been reported. Physiologically based pharmacokinetic (PBPK) modeling and in vitro dissolution data were used to explore our hypothesis that at high doses of nifedipine it precipitates in the stomach. Plasma concentration–time profiles following different doses of nifedipine were simulated using commercial PBPK software and compared to in vivo data. In vitro dissolution tests were performed with Adalat1 10 mg capsules in different volumes of fasted state simulated gastric fluid (FaSSGF). The discrepancy in plasma concentration–time profiles between the different nifedipine doses could be well simulated, assuming protracted dissolution for the 20 mg dose. Nifedipine release from one Adalat1 10 capsule in 250 or 500 mL FaSSGF was completed within 15 min whereas when release from two capsules, corresponding to 20 mg nifedipine, was studied in 250 mL FaSSGF, a maximum of about 75% drug dissolved was observed after 15 min followed by a decline in the % dissolved to a final value of approximately 40%. Based on the in silico and in vitro results it can be concluded that the observed prolongation in nifedipine absorption following the 20 mg dose was likely caused by nifedipine precipitation in human stomach. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:2899–2904, 2010
Keywords: absorption; biorelevant media; computational ADME; dissolution; nifedipine; PBPK modeling; pharmacokinetics; PK-Sim; precipitation; solubility
INTRODUCTION Absorption of orally administered drugs from the gastrointestinal (GI) tract represents a complex process that can be influenced by numerous factors. Physiologically based pharmacokinetic (PBPK) modeling has been established as a useful tool for understanding the impact of the various factors on drug absorption and can therefore contribute to investigating the reasons for poor absorption. Using known physiological parameters and physicochemical properties of the drug, PBPK modeling allows for the prediction of absorption, distribution, metabolism, and elimination (ADME) properties of drugs in the human body.1–4 In vitro dissolution tests provide useful information about the release characteristics of drug formulations and of the drug itself. In 1998 biorelevant media for Correspondence to: Stefan Willmann (Telephone: þ49-214-3036568; Fax: þ49-214-30-50698; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 99, 2899–2904 (2010) ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
simulating the composition of the proximal GI tract were proposed by Dressman et al.5 More recently, fasted state simulated gastric fluid (FaSSGF) was developed by Vertzoni et al.6 This medium appears to be appropriate for simulating fasting conditions in the human stomach. The calcium channel blocker nifedipine is widely used in the treatment of several cardiovascular diseases.7–9 Nifedipine is available in various formulations worldwide with the liquid-filled capsule representing the first formulation available for clinical use.10,11 According to the Biopharmaceutics Classification Systems (BCS) nifedipine is a typical Class II compound.12,13 As a highly permeable drug, orally administered nifedipine is rapidly and completely absorbed over the whole length of the intestinal tract but due to its substantial metabolism in the liver and the small intestine, the mean bioavailability from capsules is only 40–70%.7,14–18 In 1983, Raemsch and Sommer18 demonstrated that the areas under the plasma concentration–time curves (AUC) following oral administration of nifedipine capsules as single doses of 5, 10, and 20 mg showed a linear correlation between the AUC and the
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dose. By contrast, the dose-normalized mean maximum plasma concentration (Cmax) after administration of the highest dose was lower than after administration of a 5 or 10 mg capsule, although concentrations persisted over a longer time frame. However, no explanation was proposed by the authors. On the basis of its low solubility we hypothesized that the observed change in the nifedipine absorption profile might be caused by precipitation of nifedipine from the liquid-filled capsules in the human stomach in the preprandial state. The objective of this study was to test the hypothesis of nifedipine precipitation with the aid of PBPK modeling. The in silico results were subsequently confirmed by experimental assessment of the in vitro dissolution behavior of Adalat1 10 mg capsules in different volumes of FaSSGF.
MATERIALS AND METHODS In Vitro Biorelevant Dissolution Studies Materials Nifedipine substance (purity not <98%) was purchased from Sigma–Aldrich Chemie GmbH (Steinheim, Germany). Nifedipine immediate release (IR) liquidfilled soft gelatin capsules (Adalat1 10 mg) were manufactured by Bayer HealthCare AG (Leverkusen, Germany). The composition of the capsules comprises glycerol, purified water, saccharin sodium, peppermint oil, and macrogol 400. Methanol (gradient grade) and analytical reagent grade dibasic sodium phosphate (Na2HPO4) were obtained from Merck KGaA (Darmstadt, Germany). Hydrochloric acid (37%) and pepsin (Ph. Eur., 0.51 U/mg, lot 1241256) were purchased from Fluka Chemie AG (Buchs, Switzerland). Sodium taurocholate (97% pure, lot 2007100274) was used as received from Prodotti Chimici e Alimentari SpA (Basaluzzo, Italy). Ultrapurified water used for the HPLC mobile phase was Milli-Q grade (Millipore GmbH, Schwalbach, Germany). Media Preparation A dissolution medium representing the preprandial gastric conditions in the stomach, FaSSGF (pH 1.6), was used for the in vitro dissolution tests. The detailed composition and preparation method have been described previously.6 Dissolution Test Conditions The dissolution characteristics of the soft gelatin capsules were investigated by using USP Apparatus 2 (paddle method) and the appropriate mini-paddle (DT700, Erweka, Heusenstamm, Germany). The JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 6, JUNE 2010
mini-paddle assembly is based on the conventional USP paddle setup but scaled down exactly one-third with respect to the dimensions.19,20 In the fasted state, the resting volume of the stomach has been estimated to be about 20– 30 mL.5,21,22 However, when a capsule or tablet is orally administered, a typical fluid volume of 200– 250 mL is coadministered13 resulting in a total volume of 220–280 mL. To exclude discrepancies between the different doses of nifedipine that could affect the in vitro results, all dissolution tests were accomplished using Adalat1 10 mg capsules by adjusting the volume of the dissolution medium and employing appropriate test apparatus to achieve the designated hydrodynamics and nifedipine concentrations according to the different dosing conditions in vivo: (i) one Adalat1 10 in 500 mL FaSSGF (corresponds to a 5 mg dose in 250 mL gastric contents) using the conventional paddle method, (ii) one Adalat1 10 in 250 mL FaSSGF (corresponds to a 10 mg dose in 250 mL gastric contents) using the mini-paddle method, and (iii) two Adalat1 10 in 250 mL FaSSGF (corresponds to a 20 mg dose in 250 mL gastric contents) using the mini-paddle method. Experiments were run in six replicates. The paddle revolution speed was set at 50 rpm and the temperature of the medium within each vessel maintained at 37 0.58C. The sampling times were 5, 10, 15, 20, 30, 45, and 60 min. Approximately 5 mL was withdrawn at each sampling time point with volume replacement. The samples were filtered through a 0.45 mm PTFE filter and then analyzed by HPLC. Nifedipine dissolution tests and analyses were performed in the dark to prevent degradation of nifedipine. Chromatographic Conditions An isocratic reversed-phase HPLC system equipped with a UV detector was used for the drug quantification. The HPLC system consisted of a Merck Hitachi L-7100 LaChrome Pump, a Merck Hitachi L-7200 Autosampler, a Merck Hitachi L-7400 UV Detector (Hitachi Ltd, Tokyo, Japan), and a Zorbax RX-C8 analytical column (4.6 mm 250 mm ID; 5 mm) connected with a Zorbax RX-C8 guard column (Agilent Technologies Deutschland GmbH, Bo¨blingen, Germany). The assays were operated at ambient temperature. The chromatograms were evaluated with EZChrom EliteTM Version 2.8 software (Biochrom Ltd., Cambridge, UK). The mobile phase consisted of 45% 0.01 M Na2HPO4 buffer (pH 6.1) and 55% methanol. The injection volume was 25 mL and the flow rate was set at 1.0 mL/min resulting in a DOI 10.1002/jps
ANALYSIS OF NIFEDIPINE ABSORPTION FROM SOFT GELATIN CAPSULES
run time of 10 min per sample. The detection wavelength was set at 237 nm. Physiologically Based Pharmacokinetic Studies Software Used The PBPK model for nifedipine was built using the commercial software tool PK-Sim1 Version 4.0 (Bayer Technology Services GmbH, Leverkusen, Germany), a whole-body PBPK model. The GI absorption model as well as the substructure of the organs and tissues have been described in detail elsewhere.2,3,23 Physicochemical Data Required for the PBPK Model Physicochemical parameters of nifedipine were taken from the literature and in-house data (Tab. 1). Anthropometric Data The plasma concentration–time profiles obtained from the literature originated from an in vivo study in fasted volunteers.18 Since no detailed information about the study population was given in the publication, a virtual individual was constructed using mean values for the body weight (73 kg) and height (176 cm) of a European male subject at the age of 30 years, with data provided by the software tool.27 Construction of the PBPK Model Physicochemical data and anthropometric data were integrated into the PBPK model. Prior to the simulations of oral nifedipine, to ensure that the distribution and hepatic clearance are described properly by the PBPK model, intravenous (IV) nifedipine administration of 0.015 mg/kg was simulated and compared to the experimental data obtained from the same study.18 Then oral administration of 5, 10, and 20 mg was simulated and compared with in vivo data. Virtually no unchanged nifedipine is excreted renally,7 and CYP3A-mediated metabolism in the liver and gut wall is responsible for the first step of nifedipine biotransformation into pharmacologically Table 1. Physicochemical Parameters Used as Input Parameters for PK-Sim1 Physicochemical Parameter
Value
Log P Intestinal permeability Plasma fraction unbound Molecular weight pKa Solubility in FaSSGF, 378C
2.225 2.079 105 cm/s3 0.037,26 346.3 g/mol3,25 a
10.5 mg/L (in-house)
a Nifedipine was assumed to exist in its nonionized form at all pH values in the GI tract.24
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inactive metabolites.16,17 Consequently, Michaelis– Menten processes were introduced in the gut wall and liver. A Km value of 10.1 mM28 was used for both intestinal and hepatic metabolization, because median Km values have been reported to be similar for different intestinal regions and the liver.29 The hepatic Vmax value was fitted to match the experimental plasma concentration–time profiles following IV administration of nifedipine. Since Vmax values are directly influenced by enzyme expression they vary significantly between tissues. Therefore, the intestinal Vmax values had to be adjusted using oral data. To account for regional activity differences along the GI tract, reported CYP3A catalytic activity ratios toward the probe substrate midazolam were used.29 To adjust the Vmax values toward nifedipine, only one intestinal Vmax value was fitted by means of the lowest oral dose and integrated into the model with the reported proportions in duodenum, jejunum, and ileum. The same Vmax values in the liver and intestine, respectively, were used for all simulations.
RESULTS Physiologically Based Modeling Intravenous Administration of Nifedipine Following IV infusion of 0.015 mg/kg of nifedipine the plasma concentration–time profiles were well described by the PBPK model using a hepatic Vmax value of 0.13 mmol/min/g liver tissues (Fig. 1).
Oral Administration of Different Doses of Nifedipine The established PBPK model was transferred to oral nifedipine administration with additional consideration of nifedipine release from the soft gelatin capsules and gut wall metabolism. The initial setting used for an adequate description of nifedipine release from liquid-filled capsules was a dissolution function of the Weibull-type with a 50% dissolution time of 12 min and a slope of b ¼ 1 at 50% dissolved. Using these parameters and, additionally accounting for gut wall metabolism, the plasma concentration–time profiles following 5 and 10 mg nifedipine soft gelatin capsules were very well described by the model (Fig. 2). As expected, using the identical model for the prediction of plasma concentration–time profiles following oral administration of a 20 mg nifedipine capsule, the observed Cmax was clearly overestimated by the model (Fig. 2, gray line). This could not be attributed to alterations in nifedipine metabolism, because the bioavailability of 20 mg oral nifedipine calculated by the software was in excellent agreement with the in vivo bioavailability reported by the authors (69% in silico vs. 68.2% in vivo). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 6, JUNE 2010
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dissolution time of 36 min and the slope of b ¼ 1 at 50% dissolved, the observed plasma concentration– time profile following oral administration of 20 mg nifedipine could be very well described by the PBPK software. Adalat1 Dissolution
Figure 1. Simulated (solid line) and observed () plasma concentration–time profiles following IV administration of 0.015 mg/kg nifedipine over 3 min to healthy volunteers. Experimental data were taken from Raemsch and Sommer.18 The inset graph presents the same plot on a semilogarithmic scale.
The dissolution profiles of the Adalat1 10 mg capsules in FaSSGF are presented in Figure 3. The release of nifedipine from a single nifedipine 10 mg capsule in 500 mL FaSSGF was very rapid with virtually 100% released and consequently dissolved within 15 min. Likewise, the release from one Adalat1 10 mg capsule in 250 mL FaSSGF was complete within 15 min. By contrast, the dissolution profile of 20 mg nifedipine in 250 mL FaSSGF exhibited a maximum dissolution with about 75% dissolved after 15 min and then declined to reach a final value of approximately 40% of drug dissolved after 60 min.
DISCUSSION To reflect the hypothesis that dissolution in the stomach might be limiting to absorption, the preset model description of nifedipine release was changed with the objective of achieving the best fit of the observed plasma concentration–time curve. Using a dissolution function of the Weibull-type with a 50%
Figure 2. Simulated (solid lines) and observed plasma concentration–time profiles following oral administration of nifedipine capsules in single doses of 5 mg (*), 10 mg (~), and 20 mg () to fasted healthy volunteers. Experimental data were taken from Raemsch and Sommer.18 The gray line indicates the simulated plasma concentration–time profile disregarding the effect of nifedipine precipitation. The inset graph presents the same plots on a semilogarithmic scale. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 6, JUNE 2010
Nifedipine represents a typical BCS Class II compound, which is well absorbed throughout the entire intestine when administered orally.14 Consequently, nifedipine PK should be largely dependent on the release characteristics of the orally administered dosage form. Several years ago, the results of a PK study suggested a linear correlation between the AUCs and the doses following oral administrations of
Figure 3. Dissolution of one Adalat1 10 mg soft gelatin capsule in 500 mL FaSSGF (&), one Adalat1 10 mg soft gelatin capsule in 250 mL FaSSGF () and two Adalat1 10 mg capsules in 250 mL FaSSGF (5). Results are expressed as mean % (SD) dissolved at the given sampling time. DOI 10.1002/jps
ANALYSIS OF NIFEDIPINE ABSORPTION FROM SOFT GELATIN CAPSULES
nifedipine capsules as single doses of 5, 10, and 20 mg. By contrast, mean Cmax after the highest dose (20 mg) was much lower than expected, whereas at later times in the plasma profile concentrations were higher than expected.18 In our study, nifedipine plasma concentration–time profiles following oral administrations of liquid-filled capsules with different doses were simulated using a generic PBPK software tool and compared to the experimental data. Oral administrations of 5 and 10 mg nifedipine capsules could be accurately simulated, whereas, as expected, the Cmax following a single 20 mg dose was clearly overestimated by the model. Furthermore, the shape of the profile was only poorly described by the model. The observed differences in PK following increasing nifedipine doses cannot be attributed to a change in presystemic metabolism since the supposed saturation of the intestinal cytochrome P450 system when given at a higher dose30 would have just the opposite effect on nifedipine disposition and lead to an increase in nifedipine bioavailability and consequently to an underestimation of the observed plasma concentrations by the PBPK model. Furthermore, PK studies suggest a linear correlation between the dose and the AUC and, in addition, a linear correlation between the dose and the Cmax for the tablet formulation of nifedipine.18,31,32 Accordingly, we proposed that the delay in nifedipine absorption could be due to precipitation of nifedipine from the liquid-filled capsules when liberated in the fasted stomach. Following transport along the GI tract, the precipitated nifedipine would redissolve in the small intestine and then be absorbed in a lower part of the small intestine, leading to a delay in the time to reach Cmax (tmax) and the decelerated elimination phase. It was observed that the in vivo plasma concentration–time profiles at a dose of 20 mg could be better described by the PBPK model after integration of a protracted nifedipine release in the model. The best fit in terms of the Cmax, tmax, AUC, and the shape of the profile was achieved assuming a dissolution function of the Weibull type with a 50% dissolution time of 36 min, compared to the presetting of a 50% dissolution time within 12 min. To confirm the results obtained by PBPK modeling, the in vitro dissolution profiles of the three different nifedipine doses were obtained under biorelevant conditions. Since nifedipine precipitation was assumed to occur in human stomach due to the low liquid volume available in the fasted state, we adjusted the number of capsules and the volume of FaSSGF to correspond to a total gastric liquid volume of 250 mL for each dose. Complete and fast drug release of dissolved nifedipine was observed for the 5 and 10 mg doses, DOI 10.1002/jps
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whereas the dissolution profile of 20 mg nifedipine revealed incomplete dissolution. Based on the form of the dissolution profile at the 20 mg dose it can be concluded that nifedipine did precipitate in the vessels during the dissolution test. The observed decrease in nifedipine concentration could not be attributed to nifedipine degradation, because all tests and analyses were performed in the conditions that were protected from light. It should be noted that in all cases the theoretically attainable nifedipine concentration in the vessels was over the equilibrium solubility (10.5 mg/L). Obviously, the formulation can push the drug substance into solution initially. However, this failed to prevent precipitation at the highest dose. The in vitro dissolution results of course cannot entirely reflect the in vivo situation, since the drug substance precipitating in the stomach will subsequently enter the small intestine and will be redissolved—the drug concentration in the intestinal fluid will be kept low by absorption (‘‘extraction’’ across the gut wall). Likewise, the in silico dissolution profile used for the best fit can only approximate the in vivo situation, because nifedipine drug substance is already dissolved in the soft gelatin capsule and then precipitates whereas the implemented dissolution profile assumes a continuous increase in nifedipine dissolution. Nevertheless, from the in silico and in vitro results it can be concluded that the prolongation in nifedipine absorption following the highest dose is likely caused by nifedipine precipitation in the stomach. The results attest to the suitability of PBPK modeling to interpret PK data and to the importance of combining in silico and in vitro approaches to achieve successful predictions of the in vivo performance of drug products.
CONCLUSIONS At the 5 and 10 mg doses the plasma concentration– time profiles of orally administered nifedipine could be well described by the PBPK model. By contrast, the concentration profiles at the 20 mg dose could only be described well under the assumption of precipitation. In vitro studies likewise indicated that precipitation is only likely to occur at the highest dose. It therefore appears that precipitation of nifedipine from the soft gelatin capsule formulation offers a plausible explanation for the subproportional Cmax and persistence of levels in the in vivo plasma curves following the 20 mg dose. It appears that the PBPK software tool combined with the biorelevant dissolution tests is well suited for formulation development and analyses of unexpected in vivo results in the pharmaceutical industry. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 6, JUNE 2010
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ACKNOWLEDGMENTS We would like to thank Dr. Erich Brendel (Bayer Schering Pharma AG, Elberfeld, Germany) for valuable discussions. Kirstin Thelen is supported financially by Bayer Technology Services GmbH and Ekarat Jantratid by F. Hoffmann La Roche (Nutley, NJ). No other sources of funding were used to assist in the preparation of this work.
REFERENCES 1. Schmitt W, Willmann S. 2005. Physiology-based pharmacokinetic modelling: Ready to be used. Drug Discov Today 2:125– 132. 2. Willmann S, Lippert J, Sevestre M, Solodenko J, Fois F, Schmitt W. 2003. PK-Sim: A physiologically based pharmacokinetic ‘whole-body’ model. BIOSILICO 1:121–124. 3. Willmann S, Schmitt W, Keldenich J, Lippert J, Dressman JB. 2004. A physiological model for the estimation of the fraction dose absorbed in humans. J Med Chem 47:4022–4031. 4. Willmann S, Lippert J, Schmitt W. 2005. From physicochemistry to absorption and distribution: Predictive mechanistic modelling and computational tools. Expert Opin Drug Metab Toxicol 1:159–168. 5. Dressman JB, Amidon GL, Reppas C, Shah VP. 1998. Dissolution testing as a prognostic tool for oral drug absorption: Immediate release dosage forms. Pharm Res 15:11–22. 6. Vertzoni M, Dressman JB, Butler J, Hempenstall JM, Reppas C. 2005. Simulation of fasting gastric conditions and its importance for the in vivo dissolution of lipophilic compounds. Eur J Pharm Biopharm 60:413–417. 7. Bayer Vital GmbH. Fachinformation Adalat 5, 10 Kapseln, 2004. Rote Liste Service GmbH, 2006. 8. Bossert F, Vater W. 1971. Dihydropyridines, a new group of strongly effective coronary therapeutic agents. Naturwissenschaften 58:578. 9. Sorkin E, Clissold S, Brogden R. 1985. Nifedipine—A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy, in ischaemic heart disease, hypertension and related cardiovascular disorders. Drugs 30:182–274. 10. Bayer Schering Pharma AG. 2007. Adalat Homepage. www. adalat.com (accessed March 4, 2009). 11. Toal C. 2004. Formulation dependent pharmacokinetics—Does the dosage form matter for nifedipine? J Cardiovasc Pharmacol 44:82–86. 12. Amidon GL, Lennernas H, Shah VP, Crison JR. 1995. A theoretical basis for a biopharmaceutic drug classification: The correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm Res 12:413–420. 13. Food and Drug Administration. 2000. Guidance for Industry: Waiver of In vivo bioavailability and bioequivalence studies for immediate-release solid dosage forms based on a biopharmaceutics classification system. Rockville MD, USA: U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research. 14. Bode H, Brendel E, Ahr G, Fuhr U, Harder S, Staib A. 1996. Investigation of nifedipine absorption in different regions of the human gastrointestinal (GI) tract after simultaneous administration of 13C- and 12C-nifedipine. Eur J Clin Pharmacol 50:447–451.
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15. Foster T, Hamann S, Richards V, Bryant P, Graves D, McAllister R. 1983. Nifedipine kinetics and bioavailability after single intravenous and oral doses in normal subjects. J Clin Pharmacol 23:161–170. 16. Guengerich F, Martin M, Beaune P, Kremers P, Wolff T, Waxman D. 1986. Characterization of rat and human liver microsomal cytochrome P-450 forms involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug metabolism. J Biol Chem 261:5051–5060. 17. Guengerich F, Brian W, Iwasaki M, Sari M, Ba¨a¨rnhielm C, Berntsson P. 1991. Oxidation of dihydropyridine calcium channel blockers and analogues by human liver cytochrome P-450 IIIA4. J Med Chem 34:1838–1844. 18. Raemsch K, Sommer J. 1983. Pharmacokinetics and metabolism of nifedipine. Hypertension 5:18–24. 19. Klein S, Dressman JB. 2006. The mini paddle apparatus—A useful tool in the early developmental stage? Experiences with immediate-release dosage forms. 33rd Annual Meeting and Exposition of the Controlled Release Society. Vienna, Austria. 20. Klein S, Shah VP. 2008. A standardized mini paddle apparatus as an alternative to the standard paddle. AAPS PharmSciTech 9:1179–1184. 21. Lydon A, Murray C, McGinley J, Plant R, Duggan F, Shorten G. 1999. Cisapride does not alter gastric volume or pH in patients undergoing ambulatory surgery. Can J Anaesth 46:1181– 1184. 22. Maltby JR, Sutherland AD, Sale JP, Shaffer EA. 1986. Preoperative oral fluids: Is a five-hour fast justified prior to elective surgery? Anesth Analg 65:1112–1116. 23. Willmann S, Schmitt W, Keldenich J, Dressman JB. 2003. A physiologic model for simulating gastrointestinal flow and drug absorption in rats. Pharm Res 20:1766–1771. 24. Odou P, Ferrari N, Barthe´le´my C, Brique S, Lhermitte M, Vincent A, Libersa C, Robert H. 2005. Grapefruit juice–nifedipine interaction: Possible involvement of several mechanisms. J Clin Pharm Ther 30:153–158. 25. MedicinesComplete. 2006. Nifedipine: Clarke’s analysis of drugs and poisons, http://www.medicinescomplete.com/mc/ (accessed July 17, 2007). 26. Kelly J, O’Malley K. 1992. Clinical pharmacokinetics of calcium antagonists—An update. Clin Pharmacokinet 22:416–433. 27. Valentin J. 2002. Annals of the ICRP—Basic anatomical and physiological data for use in radiological protection: Reference values. Oxford, UK: Pergamon. 28. Galetin A, Houston J. 2006. Intestinal and hepatic metabolic activity of five cytochrome P450 enzymes: Impact on prediction of first-pass metabolism. J Pharmacol Exp Ther 318:1220– 1229. 29. Paine MF, Khalighi M, Fisher JM, Shen DD, Kunze KL, Marsh CL, Perkins JD, Thummel KE. 1997. Characterization of interintestinal and intraintestinal variations in human CYP3A-dependent metabolism. J Pharmacol Exp Ther 283: 1552–1562. 30. Zhang Q, Kaminsky L, Dunbar D, Zhang J, Ding X. 2007. Role of small intestinal cytochromes P450 in the bioavailability of oral nifedipine. Drug Metab Dispos 35:1617–1623. 31. Banzet O, Colin J, Thibonnier M, Singlas E, Alexandre J, Corvol P. 1983. Acute antihypertensive effect and pharmacokinetics of a tablet preparation of nifedipine. Eur J Clin Pharmacol 24:145–150. 32. Taburet A, Singlas E, Colin J, Banzet O, Thibonnier M, Corvol P. 1983. Pharmacokinetic studies of nifedipine tablet—Correlation with antihypertensive effects. Hypertension 5:29–33.
DOI 10.1002/jps
Institute of Pharmaceutical Technology, Johann Wolfgang Goethe University, 60438 Frankfurt am Main, Germany
2
Competence Center Systems Biology and Computational Solutions, Bayer Technology Services GmbH, 51368 Leverkusen, Germany Received 6 May 2009; revised 30 September 2009; accepted 25 October 2009 Published online 14 December 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.22026 ABSTRACT: Delayed absorption of nifedipine when administered as a 20 mg immediate release soft gelatin capsule to fasted volunteers has been reported. Physiologically based pharmacokinetic (PBPK) modeling and in vitro dissolution data were used to explore our hypothesis that at high doses of nifedipine it precipitates in the stomach. Plasma concentration–time profiles following different doses of nifedipine were simulated using commercial PBPK software and compared to in vivo data. In vitro dissolution tests were performed with Adalat1 10 mg capsules in different volumes of fasted state simulated gastric fluid (FaSSGF). The discrepancy in plasma concentration–time profiles between the different nifedipine doses could be well simulated, assuming protracted dissolution for the 20 mg dose. Nifedipine release from one Adalat1 10 capsule in 250 or 500 mL FaSSGF was completed within 15 min whereas when release from two capsules, corresponding to 20 mg nifedipine, was studied in 250 mL FaSSGF, a maximum of about 75% drug dissolved was observed after 15 min followed by a decline in the % dissolved to a final value of approximately 40%. Based on the in silico and in vitro results it can be concluded that the observed prolongation in nifedipine absorption following the 20 mg dose was likely caused by nifedipine precipitation in human stomach. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:2899–2904, 2010
Keywords: absorption; biorelevant media; computational ADME; dissolution; nifedipine; PBPK modeling; pharmacokinetics; PK-Sim; precipitation; solubility
INTRODUCTION Absorption of orally administered drugs from the gastrointestinal (GI) tract represents a complex process that can be influenced by numerous factors. Physiologically based pharmacokinetic (PBPK) modeling has been established as a useful tool for understanding the impact of the various factors on drug absorption and can therefore contribute to investigating the reasons for poor absorption. Using known physiological parameters and physicochemical properties of the drug, PBPK modeling allows for the prediction of absorption, distribution, metabolism, and elimination (ADME) properties of drugs in the human body.1–4 In vitro dissolution tests provide useful information about the release characteristics of drug formulations and of the drug itself. In 1998 biorelevant media for Correspondence to: Stefan Willmann (Telephone: þ49-214-3036568; Fax: þ49-214-30-50698; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 99, 2899–2904 (2010) ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
simulating the composition of the proximal GI tract were proposed by Dressman et al.5 More recently, fasted state simulated gastric fluid (FaSSGF) was developed by Vertzoni et al.6 This medium appears to be appropriate for simulating fasting conditions in the human stomach. The calcium channel blocker nifedipine is widely used in the treatment of several cardiovascular diseases.7–9 Nifedipine is available in various formulations worldwide with the liquid-filled capsule representing the first formulation available for clinical use.10,11 According to the Biopharmaceutics Classification Systems (BCS) nifedipine is a typical Class II compound.12,13 As a highly permeable drug, orally administered nifedipine is rapidly and completely absorbed over the whole length of the intestinal tract but due to its substantial metabolism in the liver and the small intestine, the mean bioavailability from capsules is only 40–70%.7,14–18 In 1983, Raemsch and Sommer18 demonstrated that the areas under the plasma concentration–time curves (AUC) following oral administration of nifedipine capsules as single doses of 5, 10, and 20 mg showed a linear correlation between the AUC and the
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dose. By contrast, the dose-normalized mean maximum plasma concentration (Cmax) after administration of the highest dose was lower than after administration of a 5 or 10 mg capsule, although concentrations persisted over a longer time frame. However, no explanation was proposed by the authors. On the basis of its low solubility we hypothesized that the observed change in the nifedipine absorption profile might be caused by precipitation of nifedipine from the liquid-filled capsules in the human stomach in the preprandial state. The objective of this study was to test the hypothesis of nifedipine precipitation with the aid of PBPK modeling. The in silico results were subsequently confirmed by experimental assessment of the in vitro dissolution behavior of Adalat1 10 mg capsules in different volumes of FaSSGF.
MATERIALS AND METHODS In Vitro Biorelevant Dissolution Studies Materials Nifedipine substance (purity not <98%) was purchased from Sigma–Aldrich Chemie GmbH (Steinheim, Germany). Nifedipine immediate release (IR) liquidfilled soft gelatin capsules (Adalat1 10 mg) were manufactured by Bayer HealthCare AG (Leverkusen, Germany). The composition of the capsules comprises glycerol, purified water, saccharin sodium, peppermint oil, and macrogol 400. Methanol (gradient grade) and analytical reagent grade dibasic sodium phosphate (Na2HPO4) were obtained from Merck KGaA (Darmstadt, Germany). Hydrochloric acid (37%) and pepsin (Ph. Eur., 0.51 U/mg, lot 1241256) were purchased from Fluka Chemie AG (Buchs, Switzerland). Sodium taurocholate (97% pure, lot 2007100274) was used as received from Prodotti Chimici e Alimentari SpA (Basaluzzo, Italy). Ultrapurified water used for the HPLC mobile phase was Milli-Q grade (Millipore GmbH, Schwalbach, Germany). Media Preparation A dissolution medium representing the preprandial gastric conditions in the stomach, FaSSGF (pH 1.6), was used for the in vitro dissolution tests. The detailed composition and preparation method have been described previously.6 Dissolution Test Conditions The dissolution characteristics of the soft gelatin capsules were investigated by using USP Apparatus 2 (paddle method) and the appropriate mini-paddle (DT700, Erweka, Heusenstamm, Germany). The JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 6, JUNE 2010
mini-paddle assembly is based on the conventional USP paddle setup but scaled down exactly one-third with respect to the dimensions.19,20 In the fasted state, the resting volume of the stomach has been estimated to be about 20– 30 mL.5,21,22 However, when a capsule or tablet is orally administered, a typical fluid volume of 200– 250 mL is coadministered13 resulting in a total volume of 220–280 mL. To exclude discrepancies between the different doses of nifedipine that could affect the in vitro results, all dissolution tests were accomplished using Adalat1 10 mg capsules by adjusting the volume of the dissolution medium and employing appropriate test apparatus to achieve the designated hydrodynamics and nifedipine concentrations according to the different dosing conditions in vivo: (i) one Adalat1 10 in 500 mL FaSSGF (corresponds to a 5 mg dose in 250 mL gastric contents) using the conventional paddle method, (ii) one Adalat1 10 in 250 mL FaSSGF (corresponds to a 10 mg dose in 250 mL gastric contents) using the mini-paddle method, and (iii) two Adalat1 10 in 250 mL FaSSGF (corresponds to a 20 mg dose in 250 mL gastric contents) using the mini-paddle method. Experiments were run in six replicates. The paddle revolution speed was set at 50 rpm and the temperature of the medium within each vessel maintained at 37 0.58C. The sampling times were 5, 10, 15, 20, 30, 45, and 60 min. Approximately 5 mL was withdrawn at each sampling time point with volume replacement. The samples were filtered through a 0.45 mm PTFE filter and then analyzed by HPLC. Nifedipine dissolution tests and analyses were performed in the dark to prevent degradation of nifedipine. Chromatographic Conditions An isocratic reversed-phase HPLC system equipped with a UV detector was used for the drug quantification. The HPLC system consisted of a Merck Hitachi L-7100 LaChrome Pump, a Merck Hitachi L-7200 Autosampler, a Merck Hitachi L-7400 UV Detector (Hitachi Ltd, Tokyo, Japan), and a Zorbax RX-C8 analytical column (4.6 mm 250 mm ID; 5 mm) connected with a Zorbax RX-C8 guard column (Agilent Technologies Deutschland GmbH, Bo¨blingen, Germany). The assays were operated at ambient temperature. The chromatograms were evaluated with EZChrom EliteTM Version 2.8 software (Biochrom Ltd., Cambridge, UK). The mobile phase consisted of 45% 0.01 M Na2HPO4 buffer (pH 6.1) and 55% methanol. The injection volume was 25 mL and the flow rate was set at 1.0 mL/min resulting in a DOI 10.1002/jps
ANALYSIS OF NIFEDIPINE ABSORPTION FROM SOFT GELATIN CAPSULES
run time of 10 min per sample. The detection wavelength was set at 237 nm. Physiologically Based Pharmacokinetic Studies Software Used The PBPK model for nifedipine was built using the commercial software tool PK-Sim1 Version 4.0 (Bayer Technology Services GmbH, Leverkusen, Germany), a whole-body PBPK model. The GI absorption model as well as the substructure of the organs and tissues have been described in detail elsewhere.2,3,23 Physicochemical Data Required for the PBPK Model Physicochemical parameters of nifedipine were taken from the literature and in-house data (Tab. 1). Anthropometric Data The plasma concentration–time profiles obtained from the literature originated from an in vivo study in fasted volunteers.18 Since no detailed information about the study population was given in the publication, a virtual individual was constructed using mean values for the body weight (73 kg) and height (176 cm) of a European male subject at the age of 30 years, with data provided by the software tool.27 Construction of the PBPK Model Physicochemical data and anthropometric data were integrated into the PBPK model. Prior to the simulations of oral nifedipine, to ensure that the distribution and hepatic clearance are described properly by the PBPK model, intravenous (IV) nifedipine administration of 0.015 mg/kg was simulated and compared to the experimental data obtained from the same study.18 Then oral administration of 5, 10, and 20 mg was simulated and compared with in vivo data. Virtually no unchanged nifedipine is excreted renally,7 and CYP3A-mediated metabolism in the liver and gut wall is responsible for the first step of nifedipine biotransformation into pharmacologically Table 1. Physicochemical Parameters Used as Input Parameters for PK-Sim1 Physicochemical Parameter
Value
Log P Intestinal permeability Plasma fraction unbound Molecular weight pKa Solubility in FaSSGF, 378C
2.225 2.079 105 cm/s3 0.037,26 346.3 g/mol3,25 a
10.5 mg/L (in-house)
a Nifedipine was assumed to exist in its nonionized form at all pH values in the GI tract.24
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inactive metabolites.16,17 Consequently, Michaelis– Menten processes were introduced in the gut wall and liver. A Km value of 10.1 mM28 was used for both intestinal and hepatic metabolization, because median Km values have been reported to be similar for different intestinal regions and the liver.29 The hepatic Vmax value was fitted to match the experimental plasma concentration–time profiles following IV administration of nifedipine. Since Vmax values are directly influenced by enzyme expression they vary significantly between tissues. Therefore, the intestinal Vmax values had to be adjusted using oral data. To account for regional activity differences along the GI tract, reported CYP3A catalytic activity ratios toward the probe substrate midazolam were used.29 To adjust the Vmax values toward nifedipine, only one intestinal Vmax value was fitted by means of the lowest oral dose and integrated into the model with the reported proportions in duodenum, jejunum, and ileum. The same Vmax values in the liver and intestine, respectively, were used for all simulations.
RESULTS Physiologically Based Modeling Intravenous Administration of Nifedipine Following IV infusion of 0.015 mg/kg of nifedipine the plasma concentration–time profiles were well described by the PBPK model using a hepatic Vmax value of 0.13 mmol/min/g liver tissues (Fig. 1).
Oral Administration of Different Doses of Nifedipine The established PBPK model was transferred to oral nifedipine administration with additional consideration of nifedipine release from the soft gelatin capsules and gut wall metabolism. The initial setting used for an adequate description of nifedipine release from liquid-filled capsules was a dissolution function of the Weibull-type with a 50% dissolution time of 12 min and a slope of b ¼ 1 at 50% dissolved. Using these parameters and, additionally accounting for gut wall metabolism, the plasma concentration–time profiles following 5 and 10 mg nifedipine soft gelatin capsules were very well described by the model (Fig. 2). As expected, using the identical model for the prediction of plasma concentration–time profiles following oral administration of a 20 mg nifedipine capsule, the observed Cmax was clearly overestimated by the model (Fig. 2, gray line). This could not be attributed to alterations in nifedipine metabolism, because the bioavailability of 20 mg oral nifedipine calculated by the software was in excellent agreement with the in vivo bioavailability reported by the authors (69% in silico vs. 68.2% in vivo). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 6, JUNE 2010
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dissolution time of 36 min and the slope of b ¼ 1 at 50% dissolved, the observed plasma concentration– time profile following oral administration of 20 mg nifedipine could be very well described by the PBPK software. Adalat1 Dissolution
Figure 1. Simulated (solid line) and observed () plasma concentration–time profiles following IV administration of 0.015 mg/kg nifedipine over 3 min to healthy volunteers. Experimental data were taken from Raemsch and Sommer.18 The inset graph presents the same plot on a semilogarithmic scale.
The dissolution profiles of the Adalat1 10 mg capsules in FaSSGF are presented in Figure 3. The release of nifedipine from a single nifedipine 10 mg capsule in 500 mL FaSSGF was very rapid with virtually 100% released and consequently dissolved within 15 min. Likewise, the release from one Adalat1 10 mg capsule in 250 mL FaSSGF was complete within 15 min. By contrast, the dissolution profile of 20 mg nifedipine in 250 mL FaSSGF exhibited a maximum dissolution with about 75% dissolved after 15 min and then declined to reach a final value of approximately 40% of drug dissolved after 60 min.
DISCUSSION To reflect the hypothesis that dissolution in the stomach might be limiting to absorption, the preset model description of nifedipine release was changed with the objective of achieving the best fit of the observed plasma concentration–time curve. Using a dissolution function of the Weibull-type with a 50%
Figure 2. Simulated (solid lines) and observed plasma concentration–time profiles following oral administration of nifedipine capsules in single doses of 5 mg (*), 10 mg (~), and 20 mg () to fasted healthy volunteers. Experimental data were taken from Raemsch and Sommer.18 The gray line indicates the simulated plasma concentration–time profile disregarding the effect of nifedipine precipitation. The inset graph presents the same plots on a semilogarithmic scale. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 6, JUNE 2010
Nifedipine represents a typical BCS Class II compound, which is well absorbed throughout the entire intestine when administered orally.14 Consequently, nifedipine PK should be largely dependent on the release characteristics of the orally administered dosage form. Several years ago, the results of a PK study suggested a linear correlation between the AUCs and the doses following oral administrations of
Figure 3. Dissolution of one Adalat1 10 mg soft gelatin capsule in 500 mL FaSSGF (&), one Adalat1 10 mg soft gelatin capsule in 250 mL FaSSGF () and two Adalat1 10 mg capsules in 250 mL FaSSGF (5). Results are expressed as mean % (SD) dissolved at the given sampling time. DOI 10.1002/jps
ANALYSIS OF NIFEDIPINE ABSORPTION FROM SOFT GELATIN CAPSULES
nifedipine capsules as single doses of 5, 10, and 20 mg. By contrast, mean Cmax after the highest dose (20 mg) was much lower than expected, whereas at later times in the plasma profile concentrations were higher than expected.18 In our study, nifedipine plasma concentration–time profiles following oral administrations of liquid-filled capsules with different doses were simulated using a generic PBPK software tool and compared to the experimental data. Oral administrations of 5 and 10 mg nifedipine capsules could be accurately simulated, whereas, as expected, the Cmax following a single 20 mg dose was clearly overestimated by the model. Furthermore, the shape of the profile was only poorly described by the model. The observed differences in PK following increasing nifedipine doses cannot be attributed to a change in presystemic metabolism since the supposed saturation of the intestinal cytochrome P450 system when given at a higher dose30 would have just the opposite effect on nifedipine disposition and lead to an increase in nifedipine bioavailability and consequently to an underestimation of the observed plasma concentrations by the PBPK model. Furthermore, PK studies suggest a linear correlation between the dose and the AUC and, in addition, a linear correlation between the dose and the Cmax for the tablet formulation of nifedipine.18,31,32 Accordingly, we proposed that the delay in nifedipine absorption could be due to precipitation of nifedipine from the liquid-filled capsules when liberated in the fasted stomach. Following transport along the GI tract, the precipitated nifedipine would redissolve in the small intestine and then be absorbed in a lower part of the small intestine, leading to a delay in the time to reach Cmax (tmax) and the decelerated elimination phase. It was observed that the in vivo plasma concentration–time profiles at a dose of 20 mg could be better described by the PBPK model after integration of a protracted nifedipine release in the model. The best fit in terms of the Cmax, tmax, AUC, and the shape of the profile was achieved assuming a dissolution function of the Weibull type with a 50% dissolution time of 36 min, compared to the presetting of a 50% dissolution time within 12 min. To confirm the results obtained by PBPK modeling, the in vitro dissolution profiles of the three different nifedipine doses were obtained under biorelevant conditions. Since nifedipine precipitation was assumed to occur in human stomach due to the low liquid volume available in the fasted state, we adjusted the number of capsules and the volume of FaSSGF to correspond to a total gastric liquid volume of 250 mL for each dose. Complete and fast drug release of dissolved nifedipine was observed for the 5 and 10 mg doses, DOI 10.1002/jps
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whereas the dissolution profile of 20 mg nifedipine revealed incomplete dissolution. Based on the form of the dissolution profile at the 20 mg dose it can be concluded that nifedipine did precipitate in the vessels during the dissolution test. The observed decrease in nifedipine concentration could not be attributed to nifedipine degradation, because all tests and analyses were performed in the conditions that were protected from light. It should be noted that in all cases the theoretically attainable nifedipine concentration in the vessels was over the equilibrium solubility (10.5 mg/L). Obviously, the formulation can push the drug substance into solution initially. However, this failed to prevent precipitation at the highest dose. The in vitro dissolution results of course cannot entirely reflect the in vivo situation, since the drug substance precipitating in the stomach will subsequently enter the small intestine and will be redissolved—the drug concentration in the intestinal fluid will be kept low by absorption (‘‘extraction’’ across the gut wall). Likewise, the in silico dissolution profile used for the best fit can only approximate the in vivo situation, because nifedipine drug substance is already dissolved in the soft gelatin capsule and then precipitates whereas the implemented dissolution profile assumes a continuous increase in nifedipine dissolution. Nevertheless, from the in silico and in vitro results it can be concluded that the prolongation in nifedipine absorption following the highest dose is likely caused by nifedipine precipitation in the stomach. The results attest to the suitability of PBPK modeling to interpret PK data and to the importance of combining in silico and in vitro approaches to achieve successful predictions of the in vivo performance of drug products.
CONCLUSIONS At the 5 and 10 mg doses the plasma concentration– time profiles of orally administered nifedipine could be well described by the PBPK model. By contrast, the concentration profiles at the 20 mg dose could only be described well under the assumption of precipitation. In vitro studies likewise indicated that precipitation is only likely to occur at the highest dose. It therefore appears that precipitation of nifedipine from the soft gelatin capsule formulation offers a plausible explanation for the subproportional Cmax and persistence of levels in the in vivo plasma curves following the 20 mg dose. It appears that the PBPK software tool combined with the biorelevant dissolution tests is well suited for formulation development and analyses of unexpected in vivo results in the pharmaceutical industry. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 6, JUNE 2010
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ACKNOWLEDGMENTS We would like to thank Dr. Erich Brendel (Bayer Schering Pharma AG, Elberfeld, Germany) for valuable discussions. Kirstin Thelen is supported financially by Bayer Technology Services GmbH and Ekarat Jantratid by F. Hoffmann La Roche (Nutley, NJ). No other sources of funding were used to assist in the preparation of this work.
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DOI 10.1002/jps