Brief Article pubs.acs.org/molecularpharmaceutics
Biopharmaceutics Permeability Classification of Lorcaserin, a Selective 5‑Hydroxytryptamine 2C Agonist: Method Suitability and Permeability Class Membership Chuan Chen,* Michael G. Ma, Cody L. Fullenwider,† Weichao G. Chen,‡ and Abu J. M. Sadeque Department of Drug Metabolism and Pharmacokinetics, Arena Pharmaceuticals, Inc., 6154 Nancy Ridge Drive, San Diego, California 92121, United States S Supporting Information *
ABSTRACT: The objectives of the study were (1) to demonstrate that a Caco-2 cell-based permeability assay, developed in our laboratory, is suitable to identify the permeability classification according to the US Food and Drug Administration Biopharmaceutics Classification System guidance, and (2) to use the validated Caco-2 method to determine permeability class membership of lorcaserin. Lorcaserin, marketed in United States as Belviq, is a selective human 5hydroxytryptamine 2C agonist used for weight management. First, the permeability of twenty commercially available drugs was determined in the apical-to-basolateral direction at a final concentration of 10 μM, with the pH of transporter buffer in the apical and basolateral compartments being 6.8 and 7.4, respectively. A rank-order relationship between in vitro permeability results and the extent of human intestinal absorption for the drugs tested was observed. Second, the apparent permeability coefficient values of lorcaserin at 2, 20, and 200 μM and apical pH values of 6.8 and 7.4 in the apical-to-basolateral direction were determined using the validated method and found to be comparable to those of the high-permeability internal standard metoprolol. Lorcaserin permeability across Caco-2 cell monolayers was not dependent on the variation of apical pH. Furthermore, lorcaserin was not a substrate for efflux transporters such as P-glycoprotein. In conclusion, using the validated Caco-2 permeability assay, it was shown that lorcaserin is a highly permeable compound. KEYWORDS: BCS, Belviq, Biopharmaceutics Classification System, Caco-2, lorcaserin, oral absorption, P-glycoprotein, permeability
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formulations exhibit rapid dissolution.3−8 Since its introduction, this cost- and time-saving drug development approach has been increasingly applied to support new drug applications (NDAs) and abbreviated new drug applications (ANDAs).3,4,8−11 The BCS biowaiver guidance also outlines methods and acceptance criteria for the BCS classification of drug substances. The guidance allows the use of human pharmacokinetics studies (absolute bioavailability or mass balance study) and various intestinal permeability methods (e.g., using in vitro excised human or animal intestinal tissue, in vitro cultured epithelial cell monolayers such as Caco-2 cells) to determine permeability classification.2 According to the guidance, a drug substance is considered highly permeable if its extent of intestinal absorption in human is 90% or more of an administered dose. The guidance also recommends that, in order to utilize any in vitro permeability method for BCS permeability classification, the suitability of a given method should be established by demonstrating a rank-order relationship between the test permeability values and the extent of drug
INTRODUCTION The US Food and Drug Administration (FDA) Biopharmaceutics Classification System (BCS) is a scientific framework to classify drug substances based on their aqueous solubility and intestinal permeability. Drug substances are classified into four groups: class I (high solubility and high permeability), class II (low solubility and high permeability), class III (high solubility and low permeability), and class IV (low solubility and low permeability).1 In August 2000, the FDA issued a guidance for industry entitled “Waiver of In Vivo Bioavailability and Bioequivalence Studies for Immediate-Release Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System”, also referred to as BCS “biowaiver” guidance.2 According to this guidance, waivers of in vivo bioavailability and bioequivalence studies (“biowaiver”) may be requested for BCS class I drug substances because the rate and extent of absorption of drug substances in this class, when administered orally, is primarily determined by gastric emptying and independent of drug dissolution and intestinal transit time. Therefore, a drug product composed of a BCS class I active pharmaceutical ingredient can essentially be considered equivalent to an aqueous solution of the drug substance. Furthermore, there is continued scientific support for biowaivers of some class III drug substances whose © 2013 American Chemical Society
Received: Revised: Accepted: Published: 4739
August 7, 2013 October 1, 2013 October 8, 2013 October 8, 2013 dx.doi.org/10.1021/mp400468c | Mol. Pharmaceutics 2013, 10, 4739−4745
Molecular Pharmaceutics
Brief Article
penicillin, 100 μg/mL streptomycin, and 1% nonessential amino acids at 37 °C in 5% CO2. Caco-2 cells (passage number: 62−67) were seeded at a density of approximately 5 × 104 cells per Transwell insert. Cell culture medium was changed every 2 days. Cells for the transport experiments were used between 22 and 24 days postseeding. BCS Permeability Assay. Before the start of the experiment, cell culture medium in each compartment of the Transwell units was removed. The Caco-2 cell monolayers were preincubated at 37 °C in HBSS supplemented with 20 mM glucose and 10 mM HEPES (pH 7.4) for 60 min. After the preincubation, transepithelial electrical resistance (TEER) for each monolayer was measured using an ohmmeter (model EVOMX; World Precision Instruments; Sarasota, FL). Monolayers registering a resistance of 250 Ω·cm2 or greater were used in the permeability experiments. The stock solutions of antipyrine, atenolol, carbamazepine, cimetidine, furosemide, hydrochlorothiazide, ketoprofen, labetalol, α-methyldopa, metoprolol, minoxidil, nadolol, pindolol, propranolol, theophylline, and verapamil were prepared in DMSO. The stock solutions of caffeine and naproxen were prepared in HPLC-grade water. The stock solution of amoxicillin was prepared in 20 mM ammonium acetate (pH 8.67). The concentration of all stock solutions was 10 mM. The final concentration of organic solvent in the buffer solutions used in all experiments was less than 0.5% (v/v). The permeability of 20 commercially available drugs (i.e., amoxicillin, antipyrine, atenolol, caffeine, carbamazepine, cimetidine, furosemide, hydrochlorothiazide, ketoprofen, labetalol, mannitol, α-methyldopa, metoprolol, minoxidil, nadolol, naproxen, pindolol, propranolol, theophylline, and verapamil) was determined in the apical (A) to basolateral (B) direction at a final concentration of 10 μM for all compounds except for 3 H-mannitol, which had a final concentration of ∼1.0 μCi/mL. The pH value of the transporter buffer used in the apical and basolateral compartments was 6.8 and 7.4, respectively. MES buffer (pH 6.8, 1 M) and HEPES buffer (pH 7.4, 1 M) were used to adjust the pH to 6.8 and 7.4, respectively. The inserts of the Transwell units were transferred to a new 12-well plate after measuring the TEER values. Transport buffer (1.5 mL; HBSS supplemented with 20 mM glucose and 10 mM HEPES [pH 7.4]) was added to each well. The permeability experiment was initiated by adding 0.5 mL of transport buffer (HBSS supplemented with 20 mM glucose and 10 mM MES [pH 6.8]) containing the test compound to the inserts (A). The Transwell units were incubated for up to 90 min on an orbital shaking platform (50 oscillations per min) placed in a 37 °C humidified incubator. The inserts were carefully transferred at 30 min intervals to new 12-well plates with 1.5 mL of transport buffer (HBSS supplemented with 20 mM glucose and 10 mM HEPES [pH 7.4]) in each corresponding well. At the end of the experiment, 0.25 mL aliquots of the solutions in the wells (B), as well as of the solutions in the inserts (A), were collected for liquid scintillation counting or stored at −20 °C until liquid chromatography−tandem mass spectrometry (LC− MS/MS) analysis. The same inserts used above, except those used for 3Hmannitol, were subsequently used to determine the A-to-B permeability of 3H-mannitol. 3H-Mannitol was used as a marker for membrane integrity.20 3H-Mannitol solution (0.25 mL; ∼2.0 μCi/mL), prepared in transport buffer (HBSS supplemented with 20 mM glucose and 10 mM MES [pH 6.8]), was added to the corresponding inserts (A) with 0.25 mL solution
absorption data in human subjects using a sufficient number of model drugs. Following the recommendations in the BCS biowaiver guidance, Kim et al. (2006) demonstrated using a series of 20 drugs that a rat in situ single-pass intestinal perfusion model is suitable for assessing permeability for BCS classification.12 Other studies demonstrated the use of epithelial cell monolayers such as Caco-2 cells and MDCKII-MDR1 for BCS permeability classification.13−18 Caco-2 cells derived from human colorectal adenocarcinoma have characteristics that resemble those of intestinal epithelial cells such as the ability to form a polarized monolayer, the existence of a well-defined brush border on the apical surface, and the presence of intercellular tight junctions. Caco-2 cells also express several membrane-associated efflux transporters, including P-glycoprotein (P-gp).19,20 Thus, measuring the permeability of a test compound across Caco-2 cell monolayers provides insight into its ability to cross the gut wall in the human.19,21 Lorcaserin [(1R)-(+)-8-chloro-2,3,4,5-tetrahydro-1-methyl1H-3 benzazepine], a selective 5-hydroxytryptamine (5-HT) 2C agonist, is commercially available in the United States as Belviq for weight management.22,23 Herein, we demonstrate that a Caco-2 cell-based permeability assay, developed in our laboratory, is suitable to identify the permeability classification of a given compound according to the FDA BCS guidance and apply the validated Caco-2 method to determine permeability class membership of lorcaserin.
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MATERIALS AND METHODS Chemicals. Amoxicillin, antipyrine, atenolol, carbamazepine, cimetidine, dextrorphan, furosemide, hydrochlorothiazide, ketoprofen, labetalol, α-methyldopa, metoprolol, minoxidil, nadolol, naproxen, pindolol, propranolol, theophylline, verapamil, 2-(N-morpholino) ethanesulfonic acid (MES), dimethyl sulfoxide (DMSO) and cyclosporin A were purchased from Sigma-Aldrich (St. Louis, MO). Caffeine was obtained from VWR (West Chester, PA). 3H-Mannitol (1 mCi/mL; specific activity: 20 Ci/mmol) was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). 3H-Digoxin ([3H-G], 1 mCi/mL; specific activity: 40 Ci/mmol) was purchased from PerkinElmer (Wellesley, MA). Lorcaserin was synthesized by Cilag AG (Schaffhausen, Switzerland) for Arena Pharmaceuticals, Inc. Internal standards, lorcaserin-d6 and 1-(3-(4-bromo1-methyl-1H-pyrazol-5-yl)phenyl)-3-(4-fluorophenyl)urea (also referred to as AR116082) were synthesized at Arena Pharmaceuticals, Inc. Hanks balanced salt solution (HBSS), penicillin, streptomycin, nonessential amino acids and 4-(2hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) solution (pH 7.4, 1 M) were purchased from Life Technologies (Carlsbad, CA). Minimum essential medium and fetal bovine serum were purchased from Hyclone (Logan, UT). HPLCgrade acetonitrile and water were purchased from Burdick and Jackson (Muskegon, MI). Transwell Units. Twelve-well Transwell units with an array of 12 individual inserts were purchased from Corning Life Sciences (Acton, MA). In each insert, cells were grown on a porous (0.4 μm) polycarbonate membrane (12 mm in diameter). Each Transwell unit has two compartments: a top (or apical) compartment (A) and a bottom (or basolateral) compartment (B). Caco-2 Cell Culture. The Caco-2 cell line was purchased from the American Type Culture Collection (ATCC; Manassas, VA) and grown with minimum essential medium supplemented with 10% fetal bovine serum, 100 units/mL 4740
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Table 1. Apical to Basolateral Permeability and Recovery for the Twenty Validation Drugs in Caco-2 Cell Monolayers compounda f
1. antipyrine 2. caffeinef 3. carbamazepinef 4. ketoprofenf 5. labetalolg 6. metoprololf 7. minoxidilg 8. naproxenf 9. pindolol 10. propranololf 11. theophyllinef 12. verapamilf 13. amoxicillinf 14. atenololf 15. cimetidine 16. furosemidef 17. hydrochlorothiazidef 18. 3H-mannitolf 19. α-methyldopaf 20. nadolol
Nb
Papp(AtoB) (×10−6 cm/s)c
mass balance (%)d
human intestinal absorption (%)e
permeability class
3 4 4 3 3 4 3 3 4 3 4 3 4 3 3 3 3 3 3 3
41.4 ± 1.16 37.6 ± 1.44 36.3 ± 0.942 26.9 ± 2.31 12.8 ± 0.600 33.3 ± 1.71 7.11 ± 0.238 25.3 ± 1.13 24.2 ± 2.30 26.0 ± 2.77 23.0 ± 1.18 30.3 ± 2.67 0.133 ± 0.00707 0.313 ± 0.220 0.645 ± 0.0244 0.0368 ± 0.0142 0.206 ± 0.00404 0.485 ± 0.0121 not calculatedg 0.0649 ± 0.00815
114 ± 1.47 91.5 ± 2.49 91.0 ± 0.572 106 ± 4.04 82.7 ± 1.04 90.7 ± 1.46 96.1 ± 1.51 90.9 ± 2.06 90.2 ± 2.52 61.7 ± 1.50 92.1 ± 2.49 69.0 ± 6.95 92.4 ± 8.76 86.9 ± 5.72 97.0 ± 5.02 90.7 ± 2.44 78.4 ± 3.96 91.2 ± 1.33 102 ± 5.37 47.6 ± 0.586
100 100 100 100 95.0 95.0 95.0 99.0 89.0 96.0 100 100 45.0 50.0 60.0 61.0 67.0 16.0 45.0 35.0
high high high high high high high high high high high high low low low low low low low low
Apical (A) to basolateral (B) permeability was determined at a final concentration of 10 μM for all drugs except 3H-mannitol, which had a final concentration of ∼1.0 μCi/mL. bNumber of monolayers included. cPermeability result for each drug is presented as mean ± standard deviation. d Mass balance is presented as percent of the initial amount of drug recovered from apical (A) and basolateral (B) compartments of the Transwell units (mean ± standard deviation). eValues of human intestinal absorption (%) for the 20 tested drugs were obtained from the literature.12−14,19 Based on the FDA “biowaiver” guidance, the extent of intestinal absorption in human for drugs belonging to the high permeability class should exceed 90% of the administered dose. fModel drugs suggested for use in establishing suitability of permeability method in the FDA BCS “biowaiver” guidance. gConcentration of α-methyldopa in the receiver side was below the lower limit of quantification (0.625 nM). a
remaining after sampling as described above. The final concentration of 3H-mannitol was ∼1.0 μCi/mL. The inserts were then transferred to a new 12-well plate. The permeability experiment was initiated by adding 1.5 mL of transport buffer (HBSS supplemented with 20 mM glucose and 10 mM HEPES [pH 7.4]) to the wells (B) on the 12-well plate. The Transwell units were incubated for 60 min on an orbital shaking platform (50 oscillations per min) placed in a 37 °C humidified incubator. At the end of the experiment, 0.25 mL aliquots of the solutions in the wells (B), as well as the solutions in the inserts (A), were collected for liquid scintillation counting. Bidirectional Permeability Assay. To demonstrate the presence of functional efflux transporters such as P-gp in the Caco-2 cell monolayers intended to be used for BCS permeability classification, bidirectional permeability of the known P-gp substrate 3H-digoxin in the absence and presence of the P-gp inhibitor cyclosporin A was determined as part of the method suitability study. Additionally, bidirectional permeability of lorcaserin in the absence or presence of cyclosporin A was determined to evaluate whether lorcaserin is a substrate for P-gp. The final concentration of lorcaserin in the dosing solutions was 10 μM. The final concentration of cyclosporin A was 5 μM. The permeation of 3H-digoxin or lorcaserin in the A-to-B direction with and without cyclosporin A was determined according to similar procedures described above. The pH of the transport buffer added to the apical and basolateral compartments was 7.4. For permeation of 3H-digoxin or lorcaserin in the B-to-A direction in the absence and presence of inhibitor, the inserts of the Transwell units were transferred to a new 12-well plate after measuring the TEER values. Transport buffer (0.5 mL) was added to the inserts. The permeability experiment was initiated
by adding 1.5 mL of transport buffer containing the test compound in the absence or presence of inhibitor to the corresponding wells (B). The Transwell units were incubated for up to 90 min on an orbital shaking platform (50 oscillations per min) placed in a 37 °C humidified incubator. Of the solutions in the inserts (A), 0.25 mL was collected at 30 min intervals for scintillation counting and replaced with an equal volume of transport buffer (37 °C) at the 30 and 60 min time points. At the end of the experiment, 0.25 mL aliquots of the solutions in the wells (B) were also collected. MS/MS Analyses. Samples of the nonlabeled drugs were analyzed using MS/MS. Briefly, after sample processing, aliquots (10 or 20 μL) were injected on a HALO (3 × 30 mm, 2.7 μm) column (Mac Mod Analytical, Chadds Fort, PA) at a flow rate of 0.5 mL/min or 0.60 mL/min. Detailed MS/MS conditions are included in Table SI1 in the Supporting Information. Mass spectrometric detection was achieved with an MDS Sciex API-4000 (Applied Biosystems, Foster City, CA) in positive- or negative-ion mode using multiple-reaction monitoring (MRM). Quantification was performed using a linear or quadratic regression (1/x weighting) analysis generated from the peak area ratio of the analyte over the internal standard (lorcaserin-d6, AR116082, or dextrorphan) for the calibration standards. Liquid Scintillation Counting. A 4 mL Ultima Gold liquid scintillation counting cocktail (PerkinElmer, Waltham, Ma) was added to samples in scintillation vials. After thorough mixing and adjustment for temperature and light, the samples were counted with a Wallac 1414 WinSpectral liquid scintillation counter (PerkinElmer). Radioactivity was reported as disintegrations per minute (dpm). Calculations. The apparent permeability coefficient (Papp) was calculated using the following equation: 4741
dx.doi.org/10.1021/mp400468c | Mol. Pharmaceutics 2013, 10, 4739−4745
Molecular Pharmaceutics Papp =
Brief Article
ΔQ /Δt AC0
where ΔQ/Δt is the appearance rate of radiolabeled test compound (dpm/s) or nonlabeled compound (μmol/s) on the receiver side during the permeation process, A is the surface area of the cell monolayers (1.12 cm2 for the 12-well Transwell inserts used in this study), and C0 is the initial concentration of radiolabeled test compound (dpm/mL) or nonlabeled compound (μmol/mL) on the donor side. 3 H-Mannitol is a low-permeability compound and is often used as a marker for membrane integrity in BCS permeability experiments.2,12 The Papp value of mannitol is typically on the order of 10−7 cm/s and may vary 2-fold to 3-fold among cells cultured by different individuals and between different batches of cells.20 Based on historic Papp values of 3H-mannitol observed in our laboratory (data not shown), we consider Caco-2 cell monolayers as passing the membrane integrity test only if the Papp value of 3H-mannitol determined in these monolayers is less than 0.7 × 10−6 cm/s. Therefore, only permeability results from the monolayers that had passed the membrane integrity test were used for calculating means and standard deviations.
Figure 1. Papp values across Caco-2 cell monolayers versus the extent of human intestinal absorption for 20 tested validation drugs and lorcaserin. The numbers 1 to 20, next to the data points, correspond to those listed in Table 1 next to the corresponding drug names. The data points representing the two low-permeability compounds (atenolol and 3H-mannitol) and three high-permeability drugs (minoxidil, labetolol and metoprolol) are indicated on the figure. The extent of human intestinal absorption for lorcaserin was estimated to be >90% based on the amount of radioactivity recovered in the urine from human subjects in a mass balance study (unpublished data). The highand low-permeability boundary at 90% human intestinal absorption is indicated.
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RESULTS Evaluation of Method Suitability. The Papp values for the 20 tested validation drugs (final concentration: 10 μM for all nonlabeled drugs and 1.0 μCi/mL for 3 H-mannitol) determined using Caco-2 cell monolayers with an apical and basolateral pH of 6.8 and 7.4, respectively, are listed in Table 1. The Papp values for membrane integrity marker 3H-mannitol in the same Caco-2 cell monolayers used to determine the permeability of the validation drugs are listed in Table SI2 in the Supporting Information. The mean Papp values for the highpermeability drugs were on the order of 10 × 10−6 cm/s, with the lowest being 7.11 ± 0.238 × 10−6 cm/s for minoxidil and the highest being 41.4 ± 1.16 × 10−6 cm/s for antipyrine. The mean Papp values for the low-permeability drugs were less than 1.0 × 10−6 cm/s, with the lowest being 0.0368 ± 0.0142 × 10−6 cm/s for furosemide and the highest being 0.645 ± 0.0244 × 10−6 cm/s for cimetidine. A rank-order relationship between the Papp values obtained herein and the extent of human intestinal absorption reported in the literature for the 20 tested validation drugs was established (Figure 1). Furthermore, the functional expression of the efflux transporter P-gp was characterized by determining bidirectional transport of 3Hdigoxin in the absence and presence of the P-gp inhibitor cyclosporin A (Table SI3 in the Supporting Information). The efflux ratio of 3H-digoxin in the absence and presence of cyclosporin A was 17.0 and 0.967, respectively (Table SI3 in the Supporting Information). Lorcaserin Permeability Classification. At pH 6.8 (A)/ pH 7.4 (B), the Papp values of lorcaserin at three different concentrations (2, 20, and 200 μM) in the A-to-B direction were on the order of 30 × 10−6 cm/s (Table 2). In the same groups of cell monolayers, the Papp values of the highpermeability internal standard metoprolol (final concentration: 10 μM) and the low-permeability internal standard 3Hmannitol (final concentration: ∼1.0 μCi/mL) in the A-to-B direction were on the order of 30 × 10−6 cm/s and less than 1.0 × 10−6 cm/s, respectively (Table 2). Similar Papp values were observed at pH 7.4 (A)/pH 7.4 (B) for lorcaserin at three different concentrations (2, 20, and 200 μM) in the A-to-B direction, and for the high- and low-permeability internal
standards (Table 2). The percent of the initial amount of lorcaserin, metoprolol, and 3H-mannitol recovered from the apical and basolateral compartments of the Transwell units under each condition of the permeability study is reported in Table SI4 in the Supporting Information. The Papp values of lorcaserin (final concentration: 10 μM) in the A-to-B and B-to-A directions in the absence and presence of cyclosporin A (5 μM) are listed in Table 3. The efflux ratios (Papp(BtoA)/Papp(AtoB)) of lorcaserin in the absence and presence of cyclosporin A were 1.19 and 1.09, respectively (Table 3). The bidirectional permeability of the known P-gp substrate 3Hdigoxin across Caco-2 cell monolayers was determined along with lorcaserin (Table 3). The efflux ratios of 3H-digoxin in the absence and presence of cyclosporin A were 24.5 and 2.08, respectively (Table 3).
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DISCUSSION In this study, the permeability of 20 commercially available drugs across Caco-2 cell monolayers was determined to demonstrate that a Caco-2 cell-based permeability assay developed in our laboratory is suitable for BCS permeability classification according to the criteria described in the BCS “biowaiver” guidance. Among the 20 drugs tested, 15 are recommended by the BCS “biowaiver” guidance for use in such method suitability studies.2 Polyethylene glycol 400, 1000, and 4000 recommended in the FDA “biowaiver” guidance for BCS permeability method validation are oligomers of polyethylene glycol with nominal molecular weight of 400, 1000, and 4000. Each of these oligomers sold commercially is a mixture containing a rather broad distribution of sizes. The oligomers may form either singly or multiply charged ions with cations such as Na+ in an electrospray ionization source commonly used in LC−MS/MS methodology.27 Overall, the sensitivity of the LC−MS/MS method developed for these compounds is low. For examples, Bhaskar et al. (2013) reported that the lower limit of quantitation for polyethylene glycol 400 using the LC−MS/MS method was only 1.01 μg/mL or ∼2.5 μM.28 Therefore, accurate and robust quantitation of these oligomers in BCS permeability assay with LC−MS/MS conditions 4742
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Table 2. Permeability of Lorcaserin, Metoprolol, and 3H-Mannitol across Caco-2 Cell Monolayers in the Apical (A) to Basolateral (B) Direction Papp(AtoB) (×10−6 cm/s)c group lorcaserin lorcaserin lorcaserin lorcaserin lorcaserin lorcaserin
(2 μM) (20 μM) (200 μM) (2 μM) (20 μM) (200 μM)
pH (A/B)
a
6.8/7.4 6.8/7.4 6.8/7.4 7.4/7.4 7.4/7.4 7.4/7.4
b
N
lorcaserin
4 4 5 4 3 4
31.9 32.8 37.0 30.4 31.4 35.4
± ± ± ± ± ±
3.57 0.145 3.05 0.810 0.834 1.64
metoprolold 33.8 31.5 31.6 32.4 32.3 29.7
± ± ± ± ± ±
2.20 3.25 1.12 1.71 4.53 1.17
H-mannitold
3
0.602 0.451 0.491 0.507 0.519 0.562
± ± ± ± ± ±
0.0824 0.0560 0.0723 0.0691 0.0307 0.0598
a
A: apical compartment of the Transwell unit. B: basolateral compartment of the Transwell unit. bNumber of monolayers included. cPermeability result for each condition (A-to-B direction) is presented as mean ± standard deviation. dThe high-permeability internal standard metoprolol was included in the donor fluid along with lorcaserin. The same monolayers used to determine the permeability of lorcaserin and metoprolol were subsequently used to evaluate the permeability of the low-permeability internal standard 3H-mannitol.
Table 3. Bidirectional Permeability of Lorcaserin and 3HDigoxin across Caco-2 Cell Monolayers with and without Cyclosporin A compounds lorcaserin (10 μM) lorcaserin (10 μM) + cyclosporin A (5 μM) 3 H-digoxin (25 nM) 3 H-digoxin (25 nM) + cyclosporin A (5 μM)
Papp(AtoB) (×10−6 cm/s)a
Papp(BtoA) (×10−6 cm/s)a
31.3 ± 2.74 31.7 ± 1.43
37.3 ± 3.69 34.5 ± 3.36
0.485 ± 0.248 1.19 ± 0.244
11.9 ± 1.52 2.48 ± 0.285
permeability classification studies, in addition to those drugs recommended by the current BCS “biowaiver” guidance (e.g., metoprolol),2 a recent study from Amidon’s group indicated that labetalol is not an ideal permeability standard for BCS classification because of its concentration-dependent permeability and polarized efflux across Caco-2 cells subjected to inhibition by the P-gp inhibitor verapamil.29 Their findings further suggest that the selection of appropriate high permeability standards should be based on not only their proximity to the low/high-permeability class boundary but also other factors including efflux transport interaction. In addition, compatibility of a particular test drug substance with potential high-permeability internal standards (i.e., lack of any substantial physical, chemical, or permeation interactions) needs to be considered before performing future BCS permeability assays. In this study, the recovery of only four tested validation drugs was less than 80% using the Caco-2 cell assay (Table 1). Two of the drugs were high-permeability drugs (i.e., propranolol and verapamil, with mass balance of 61.7% and 69.0%, respectively), and the other two were low-permeability drugs (i.e., hydrochlorothiazide and nadolol with mass balance of 78.4% and 47.6%, respectively). Thus far, there is no consensus on acceptable minimum mass balance values for in vitro permeability assays. In general, a low recovery of a test compound indicates that the true permeability value may be underestimated. Although it is likely that the true permeability values for the four aforementioned drugs could be higher than the values observed, these drugs were correctly classified into its respective permeability class based on the Papp values observed in this study. As shown in Table 3 and Table SI3 in the Supporting Information, polarized efflux transport of 3H-digoxin, a known substrate for P-gp across the Caco-2 cell monolayers, was observed. Furthermore, cyclosporin A, a known P-gp inhibitor, abolished the P-gp-mediated efflux transporter of 3H-digoxin. These results indicate that the Caco-2 cell monolayers maintained in our laboratory express functional P-gp transporter. Taken together, Table 1, Table 3, Table SI3 in the Supporting Information, and Figure 1 show that (1) a rankorder relationship exists between in vitro permeability results and the percent of administered drugs absorbed in human, and (2) the presence of functional efflux transporter such as P-gp in the Caco-2 cells. Therefore, the Caco-2 cell-based permeability assay developed herein is deemed suitable for BCS permeability classification. Similarly, Pham-The et al. (2013) reported a good correlation between Caco-2 permeability and the extent
efflux ratiob 1.19 1.09 24.5 2.08
Result for each condition is presented as mean ± standard deviation of three separate experiments conducted in triplicate. bEfflux ratio = Papp(BtoA)/Papp(AtoB). a
routinely used in small molecule drug discovery and development settings is challenging, if not impossible. As a result, polyethylene glycol 400, 1000, and 4000 were not used in this study to validate the Caco-2 cell-based BCS permeability method. If appropriate detection methods are available in a particular laboratory or 14C-labeled oligomers are used instead of nonlabeled ones, these polyethylene glycol oligomers can be included to validate in vitro BCS permeability methods. Additionally, ranitidine in the FDA guidance was replaced with another H2-receptor antagonist cimetidine, which belongs to the same permeability class as ranitidine. Four additional drugs (labetalol, minoxidil, pindolol, and nadolol) were selected as validation drugs, because the extent of human intestinal absorption of these drugs is known in the literature.12−14,19 Twelve of the tested validation drugs are considered highly permeable drugs, and eight belong to the low-permeability class (Table 1) based on the definition of a highly permeable drug (i.e., a drug substance of which the extent of absorption in human is determined to be 90% or more of an administered dose).2 The BCS “biowaiver” guidance recommends that a high- and a low-permeability model drug should be included as internal standards to determine the permeability of a given drug substance and that a model drug with permeability near the low-/high-permeability class boundary is preferred as a highpermeability standard.2 In this study, two high-permeability drugs, i.e., labetalol (Papp: 12.8 × 10−6 cm/s) and minoxidil (Papp: 7.11 × 10−6 cm/s), were close to the low-/highpermeability class boundary (Figure 1) and exhibited more than 80% recovery in our permeability assay system. Although these findings support the selection of labetalol and minoxidil as potential high-permeability internal standards in future BCS 4743
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Molecular Pharmaceutics
Brief Article
values for the membrane integrity marker 3H-mannitol in the same Caco-2 cell monolayers used to determine the permeability of the validation drugs. Table SI3 summarizing the bidirectional permeability of 3H-digoxin across Caco-2 cell monolayers with and without cyclosporin A. Table SI4 summarizing the recovery of lorcaserin, metoprolol and 3Hmannitol from the apical and basolateral compartments of the Transwell units in the BCS permeability assay using Caco-2 cell monolayers. This material is available free of charge via the Internet at http://pubs.acs.org.
of human intestinal absorption for 282 drugs, among which a rank-order relationship for high permeable drugs was more evident than that of low permeable drugs.30 The other objective of this study was to classify the permeability of lorcaserin according to BCS guidance using the Caco-2 cell assay developed and validated herein. The mean Papp values of lorcaserin at the three concentrations (2, 20, and 200 μM) used were comparable to those of the highpermeability internal standard metoprolol (Table 3). The mean Papp values of the high- and low-permeability internal standards, i.e., metoprolol and 3H-mannitol, respectively (Table 3), were similar to those obtained in the aforementioned method suitability study (Table 1). Different apical pH values (6.8 vs 7.4) appeared to have no major effects on the permeability of lorcaserin (Table 3). These pH values correspond to the pH value of United States Pharmacopeia (USP) simulated intestinal fluid (SIF) without enzymes2,26 and the physiological pH value, respectively. The lorcaserin concentrations (2, 20, and 200 μM) tested were approximately 0.01, 0.1, and 1 times the concentration of highest dose strength (10 mg) dissolved in 250 mL of water. The percent of the initial amount of lorcaserin, metoprolol, and 3H-mannitol recovered from the apical and basolateral compartments of the Transwell units (Table SI4 in the Supporting Information) indicates that lorcaserin is neither highly bound nonspecifically to the assay system (Transwell unit and cells) nor extensively retained within the cells. The bidirectional permeability of lorcaserin across Caco-2 cell monolayers was determined at a final concentration of 10 μM in the absence and presence of cyclosporin A (5 μM) to evaluate whether lorcaserin is transported by efflux transporters such as P-gp. Cyclosporin A is a potent inhibitor of P-gp. It also inhibits other transporters such as multidrug resistanceassociated protein 2 (MRP2, expressed in Caco-2 cells) and organic anion transporting polypeptide 1B1 (predominantly expressed in human liver).24,25 Lorcaserin did not exhibit polarized efflux transport across the Caco-2 cell monolayers, whereas, as expected, transport of 3H-digoxin across the Caco-2 cell monolayers was highly polarized and was inhibited by cyclosporin A (Table 3). These findings suggest that lorcaserin is not a substrate for efflux transporters such as P-gp. Taken together, these results support that lorcaserin can be classified as a highly permeable compound according to the criteria outlined in the BCS “biowaiver” guidance. This conclusion is consistent with the in vivo findings that >90% administered radioactivity was recovered in the urine of human subjects in a mass balance study (unpublished data). In summary, the Caco-2 cell-based permeability assay developed herein is suitable for BCS permeability classification based on the rank-order correlation between the permeability values of the 20 tested validation drugs and their extents of intestinal absorption, as well as the presence of functional efflux transporters such as P-gp in the cells. The permeability of lorcaserin was determined using this validated Caco-2 cell permeability assay, indicating that lorcaserin can be classified as a high-permeability compound according to the scientific framework of BCS.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +1 (858) 453-7200. Fax: +1 (858) 453-7210. E-mail:
[email protected]. Present Addresses †
C.L.F.: Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield CT 06877. ‡ W.G.C.: Vertex Pharmaceuticals, Inc., 11010 Torreyana Road, San Diego, CA 92121. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
The authors greatly appreciate Dr. Ron Christopher for critically reviewing the manuscript and helpful suggestions. The authors also thank Wim D’Haeze for the technical writing support.
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ASSOCIATED CONTENT
S Supporting Information *
Table SI1 summarizing the LC−MS/MS conditions for the validation drugs, lorcaserin, and internal standards used in the accompanying manuscript. Table SI2 summarizing the Papp 4744
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Molecular Pharmaceutics
Brief Article
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