Preclinical Pharmacokinetics and In Vitro Metabolism of BMS-605339: A Novel HCV NS3 Protease Inhibitor

RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism

Preclinical Pharmacokinetics and In Vitro Metabolism of BMS-605339: A Novel HCV NS3 Protease Inhibitor SUSAN JENKINS,1 PAUL SCOLA,2 FIONA MCPHEE,3 JAY KNIPE,1 CHRISTOPH GESENBERG,4 MICHAEL SINZ,1 VINOD ARORA,5 GARY PILCHER,6 KENNETH SANTONE1 1

Department of Metabolism and Pharmacokinetics, Bristol-Myers Squibb Company, Wallingford, Connecticut Department of Chemistry, Bristol-Myers Squibb Company, Wallingford, Connecticut 3 Department of Virology, Bristol-Myers Squibb Company, Wallingford, Connecticut 4 Department of Pharmaceutical Development, Bristol-Myers Squibb Company, New Brunswick, New Jersey 5 Department of Biotransformation, Bristol-Myers Squibb Company, Wallingford, Connecticut 6 Department of Toxicology, Bristol-Myers Squibb Company, Mt. Vernon, Indiana 2

Received 12 January 2014; revised 4 March 2014; accepted 6 March 2014 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23959 ABSTRACT: BMS-605339 is a potent HCV NS3 protease inhibitor that suppresses hepatitis C virus replication and was under investigation as an oral agent for the treatment of this disease. In vitro and in vivo studies were conducted in mouse, rat, dog, and monkey to characterize the pharmacokinetics and metabolism of this compound. BMS-605339 was predicted to be a moderate clearance compound in the human, based on human microsomal and hepatocyte data. Nearly all metabolism of BMS-605339 was oxidative; CYP3A4 is likely to play a key role in the metabolic clearance of this compound. Moderate to high Caco-2 permeability was observed for this compound, with the potential for P-glycoprotein involvement. The oral bioavailability of BMS-605339 was variable and dose dependent, suggesting low absorption, possibly because of transporter involvement. BMS-605339 possesses low intrinsic aqueous solubility and, in both rat and dog, administration of an aqueous suspension suggested that BMS-605339 absorption is likely solubility limited. Liver exposure of BMS-605339 was consistently higher than plasma exposure in all species tested (mouse, rat, and dog), indicating the potential for active uptake into hepatocytes. The C 2014 Wiley overall preclinical pharmacokinetic profile supported the selection and development of BMS-605339 as a clinical candidate.  Periodicals, Inc. and the American Pharmacists Association J Pharm Sci Keywords: preclinical pharmacokinetics; oral absorption; metabolism; hepatic clearance; in vitro/in vivo correlations (IVIVC); ADME; protease

INTRODUCTION Hepatitis C virus (HCV) is a major human pathogen of the liver, infecting an estimated 3% of the world’s population.1,2 Although primary infection with HCV is often asymptomatic, most HCV infections progress to a chronic state that can persist for decades, causing liver cirrhosis, liver failure, or liver cancer.3,4 BMS-605339 (Fig. 1) was selected from a series of molecules designed to optimize the in vitro and in vivo pharmacokinetic properties by systematically exploring variation of the substituents.5 It is a specific and selective competitive inhibitor of the HCV nonstructural (NS) 3/4A serine protease complex that is essential for HCV polyprotein processing and subsequent viral replication. BMS-605339 exerts its antiviral activity in HCV replicon cell-based systems representing genotypes 1a (EC50 = 8 nM) and 1b (EC50 = 3 nM).5,6 Mechanistic and resistance studies suggest that BMS-605339 specifically targets HCV NS3 proteolytic activity. Importantly, BMS-605339 has been demonstrated to be clinically efficacious, producing a maximum decline in HCV RNA viral titer of −1.8 log10 IU/mL after a single 120-mg oral (p.o.) dose in HCV-infected patients.5

Figure 1. Structure of BMS-605339.

The present work was carried out to thoroughly investigate the nonclinical pharmacokinetic and metabolic properties of BMS-605339 and to project the human pharmacokinetics and efficacious doses to support its clinical development, particularly the first-in-human trials.

MATERIALS AND METHODS Correspondence to: Susan Jenkins (Telephone: +203-677-7086; Fax: +203677-6193; E-mail: [email protected]) Journal of Pharmaceutical Sciences

 C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association

Materials BMS-605339 was discovered and synthesized at Bristol-Myers Squibb (Wallingford, Connecticut). Solvents were HPLC grade Jenkins et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism

from Fisher (Waltham, MA) or J.T. Baker (Phillipsburg, NJ) and other chemicals were ACS reagent grade or better. HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] and polyethylene glycol-400 (PEG-400) used for dosing solutions was from Sigma Chemical Company (St. Louis, Missouri). Caco-2 cells were obtained from the American Type Culture Collection (Rockville, Maryland). Heparinized (pooled) plasma from rat, dog, monkey, and human was purchased from Bioreclamation, Inc., (Hicksville, New York). Hanks balanced salt solution (HBSS) was obtained from Gibco Life Technologies (Grand Island, New York). Human liver microsomes and recombinant singly expressed CYP450 enzymes were obtained from BD Biosciences (Woburn, Massachusetts). Rat and monkey liver microsomes were obtained from XenoTech LLC (Kansas City, Kansas). Dog liver microsomes were obtained from In Vitro Technologies (Baltimore, Maryland). Fresh rat and dog hepatocytes and cryopreserved human hepatocytes were obtained from In Vitro Technologies. Male Balb/c mice were obtained from Harlan Breeding Laboratories (Indianapolis, Indiana) and mdr1a knockout (lacking P-glycoprotein, Pgp) and FVB mice were obtained from Taconic Farms (Cranbury Township, New Jersey). Male Sprague–Dawley rats were obtained from Hilltop Lab Animals, Inc. (Scottdale, Pennsylvania), male beagle dogs were obtained from Marshall Farms USA Inc. (North Rose, New York), and cynomolgus monkeys were obtained from Charles River Biomedical Research Foundation (Houston, Texas). Sample Analysis Samples from in vitro and in vivo studies were analyzed by liquid chromatography tandem mass spectrometry.. Proteincontaining samples (plasma, Caco-2, microsomal, and hepatocyte incubates) were treated with two volumes of ACN containing an internal standard (structural analog of BMS-605339). After the precipitated proteins were removed by centrifugation, the resulting supernatants were transferred to autosampler vials and 5 :L was injected onto an HPLC column for analysis. Bile and urine samples were diluted with water and aliquots were directly injected onto the HPLC column. Tissue samples were weighed, and homogenized in two volumes of a solution containing 20% HBSS/80% ACN and centrifuged. An aliquot of each tissue supernatant was removed and processed as described for plasma samples. The HPLC system consisted of two Shimadzu LC10AD pumps (Columbia, Maryland), a Shimadzu SIL-HTC autosampler, and a Hewlett Packard Series 1100 column compartment (Palo Alto, California). The column was a YMC Pro C18 (2.0 × 50 mm2 , 3 :m particles), maintained at 60◦ C. The mobile phase consisted of solvent A (10 mM ammonium formate and 0.1% formic acid in water) and solvent B (10 mM ammonium formate and 0.1% formic acid in methanol) at a flow rate of 0.3 mL/min. The initial mobile phase composition was 95% A/5% B that was changed to 15% A/85% B over 2 min and held at that composition for an additional 2 min. The mobile phase was then returned to initial conditions and the column re-equilibrated. The total analysis time was 5 min. The HPLC was interfaced to a Micromass Quattro tandem mass spectrometer (Beverly, Massachusetts) equipped with an electrospray ionization (ESI) source. Ions representing the (M+H)+ species for both BMS-605339 and the internal standard were selected in MS1 and collisionally dissociated with argon at a pressure of 2 × 10−3 torr to form specific product Jenkins et al., JOURNAL OF PHARMACEUTICAL SCIENCES

ions that were subsequently monitored by MS2. The selected reaction-monitoring transitions used were 714.7 → 439.1 for BMS-605339 and 485.2 → 381.3 for the internal standard. The LLQ of BMS-605339 was 5 nM in all samples, which measured concentrations of at least two-thirds of the quality control samples with 20% of nominal values.

Metabolite Identification Microsome and hepatocyte samples from various animal species (rat, dog, monkey, and human) were treated with an equal amount of acetonitrile to precipitate proteins. Aliquots of the supernatants were used for analysis. Bile and urine samples were obtained from bile duct cannulated (BDC) rats (n = 2) dosed with BMS-605339 [p.o. and intravenous (i.v.): 20 and 5 mg/kg, respectively]. Plasma and liver homogenate samples were obtained from another group of rats (n = 2) dosed with BMS-605339 (p.o. and i.v.: 20 and 5 mg/kg, respectively). Plasma, bile, urine, and liver homogenate samples were obtained from dogs (n = 2) dosed p.o. with BMS-605339 (14 mg/kg) and were pooled across sample collection times. Plasma samples (300 :L) were precipitated with acetonitrile (900 :L), and centrifuged. The supernatants were removed, and evaporated under a stream of dry nitrogen. Residues were then redissolved in 150 :L of acetonitrile–water (1:1, v/v). Liver homogenate samples were precipitated with acetonitrile to yield acetonitrile/sample ratios of 1:1 (v/v). The resulting mixture was centrifuged, and the supernatants were removed for direct analysis. Bile and urine samples were diluted with acetonitrile to yield acetonitrile/sample ratios of 1:1 (v/v) and analyzed. All samples were analyzed by LC-UV tandem mass spectrometry. The HPLC system consisted of a Waters Alliance 2690 separation module interfaced in series with a Finnigan photo diode array detector and a Finnigan Deca XP ion trap mass spectrometer. Samples were chromatographed on an YMC, pro-pack, C18 column, 2.1 × 150 mm2 , 5 :m particles, and at the flow rate of 0.3 mL/min. The mobile phase consisted of 95:5 (v/v) water– acetonitrile, containing 0.1% formic acid as solvent A and acetonitrile as solvent B. The initial mobile phase composition was 30% solvent B. After injection, the composition was maintained at 30% solvent B for 0.5 min and then linearly ramped to 100% solvent B in 20 min, and then held at 100% solvent B for 5 min. The mobile phase was then returned to the initial conditions in 0.1 min and the column was re-equilibrated for 5 min. Total analysis time was 31 min. Ultraviolet chromatographic profiles were obtained at the wavelength of 240 nm. The DECA XP mass spectrometer was equipped with an ESI source operated in positive and negative ion modes with a capillary temperature of 325◦ C and source voltage of 5.0 kV. Ion fragmentation was achieved with a normalized collision energy of 50% and an isolation width of 3.0 Da. Multiple mass spectrometry (MS) experiments were performed in each mass chromatographic analysis using dynamic exclusion experiments in a data-dependent mode containing four scan events:

r Scan event 1: full-scan MS (m/z 100–m/z 1200). r Scan events 2–4: data-dependent scans; full product–ion scans of most intense ion from preceding scan event. DOI 10.1002/jps.23959

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RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism

Permeability and P-gp Interaction Caco-2 cells were seeded onto a collagen-coated polycarbonate filter membrane at a density of 60,000 cells/cm2 . Bidirectional permeability studies were conducted with the monolayers obtained from P-gp expressing Caco-2 cells cultured for approximately 21 days. Because the expression of P-gp is increased with the number of passages,7 the Caco-2 permeability assay was conducted with cells from passage 21 to 40 and between 50 and 80 for the P-gp substrate and inhibitor assay. The transport medium was modified by Hank’s balanced salt solution (HBSS) containing 10 mM N-2-hydroxyethylpiperazine-N -2ethanesulfonic acid (HEPES) (pH 7.4 for both apical and basolateral sides). BMS-605339 stock solution was prepared at 10 mM in dimethyl sulfoxide (DMSO) (final concentration 1% DMSO) and was diluted to the working concentrations of either 50 :M (bidirectional studies) or 10 :M (P-gp inhibition studies) in HBSS buffer. The bidirectional permeability study for BMS-605339 was initiated by adding an appropriate volume of buffer containing the test compound to either the apical (apical to basolateral transport, or A to B) or basolateral (basolateral to apical transport, or B to A) side of the monolayer. The volumes of the apical and basolateral compartments in these studies were maintained at 2.0 and 0.6 mL, respectively. The monolayers were then placed in an incubator for 2 h at 37◦ C. Samples were taken from both the apical and basolateral compartment at the end of the 2-h period and the concentration of test compound was analyzed by an LC/UV method. Permeability coefficient (Pc ) was calculated according to the following equation: Pc = dA/(dt × S × C0 ), where dA/dt is the flux of the test compound across the monolayer (nmol/s), S is the surface area of the cell monolayer (0.33 cm2 ), and C0 is the initial concentration (50 :M) in the donor compartment. The Pc values are expressed as nm/s. Additionally, studies were performed with the P-gp inhibitor, GW-918 (Elacridar) (synthesized at Syngene (Bangalore, India)), incubated in the cell preparations at 2 :M. Serum Protein Binding and Stability The extent of serum protein binding of BMS-605339 was determined in serum from rat, dog, monkey, and human using equilibrium dialysis (Dianorm Equilibrium Dialyser, Munich, Germany). BMS-605339 (10 :M, n = 3) was added to serum and the samples dialyzed against Krebs–Ringer phosphate buffer (pH 7.4) using Diachrema dialysis membranes (10,000 Da cutoff). Equilibrium was achieved by rotating the cells at 10 rpm in a dry air incubator at 37◦ C. Aliquots of buffer and serum were removed at 4 h and analyzed for BMS-605339. The percentage of protein binding was calculated from the ratio of measured concentration in buffer to that in serum. Parallel 4-h stability studies (BMS-605339 at 10 :M) were conducted in serum from all species. Blood Cell Partitioning The stability and blood cell partitioning of BMS-605339 was determined in fresh whole blood from rat, monkey, dog, and human (pooled after being drawn from multiple donors). BMS605339 was added to blood samples at a concentration of 10 :M and incubated at 37◦ C for 2 h. Two aliquots of blood (0.5 mL each) were removed at 0, 0.5, and 2 h. The first aliquot was centrifuged to obtain plasma, whereas the second was treated with water to hemolyze the red cells. Samples were stored at DOI 10.1002/jps.23959

−20◦ C until analysis. The blood-to-plasma concentration ratio (Cb /Cp ) was calculated by dividing the concentration in the control plasma with the concentration in the plasma isolated from the whole blood. Microsomal and Hepatocyte Metabolic Stability The metabolic stability of BMS-605339 was investigated in pooled liver microsomes from human, mouse, rat, dog, and monkey. The concentrations of CYP P450 protein in these preparations were 0.38, 0.52, 0.89, and 1.4 nmol/mg microsomal protein in human, rat, dog, and monkey, respectively. Incubation mixtures were prepared in triplicate for each species and consisted of BMS-605339 (3 :M) in potassium phosphate buffer (0.1 M, pH 7.4) at 37◦ C, liver microsomes (protein concentration 0.9 mg/mL), magnesium chloride (0.033 mM), and a NADPH-regenerating system (NADPH, 0.43 mg/mL; glucose6-phosphate, 0.52 mg/mL; glucose-6-phosphate dehydrogenase, 0.6 units/mL). The total concentration of ACN in the incubation mixtures was less than 0.5%. After a 2-min preincubation, each reaction was initiated by the addition of cofactors and continued for 10 min. Reactions were terminated by adding an aliquot (100 :L) of the incubation mixture to 200 :L of cold acetonitrile containing an internal standard. Following vortex mixing and centrifugation to remove the precipitated proteins, aliquots (10 :L) of the supernatant fluids were analyzed for BMS-605339 by LC/MS/MS. Metabolism of BMS-605339 by Specific CYP Enzymes Reaction phenotyping studies were performed using Supersomes (recombinant singly expressed CYP P450 enzymes). The preparations contained CYP1A1, CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP2A6. All contained 1000 pmol CYP P450/mL. Vector protein devoid of CYP P450 activity was used to adjust the protein content of each incubation to 1 mg/mL. Incubations of each Supersome were performed at 37◦ C in 0.1 M Tris buffer (pH 7.4) containing 1 mM NADPH, 3.3 mM magnesium chloride, 50 pmol/mL CYP P450 Supersome , required amount of vector protein, and 1 or 10 :M of BMS-605339 in a final incubation volume of 1 mL. The organic solvent concentration in each incubation was 0.09% ACN/0.01% DMSO (v/v). After a 5-min preincubation at 37◦ C, each reaction was initiated by the addition of NADPH. After 0 and 30 min, triplicate 100 :L aliquots were removed and added to 200 :L of cold ACN containing internal standard to terminate the reaction. The samples were vortex mixed, centrifuged to remove the precipitated proteins and aliquots (10 :L) of the supernatant were analyzed for BMS-605339. The role of CYP3A4 in the metabolism of BMS-605339 was performed using HLM preparations in the presence of the specific chemical inhibitor, ketoconazole. Incubations were performed in duplicate at 37◦ C in 0.1 M Tris buffer (pH 7.4) containing 1 mM NADPH, 3.3 mM magnesium chloride, 1.0 mg/mL human liver microsomal protein, 1 or 10 :M of BMS-605339, and 0, 1, or 10 :M of ketoconazole. The organic solvent concentration in each incubation was 0.09% ACN/0.01% DMSO (v/v). After a 5-min preincubation at 37◦ C, the reaction was initiated by the addition of NADPH. After 0 and 60 min, triplicate 100 :L aliquots were removed and added to 200 :L of cold ACN containing an internal standard to terminate the reaction. The samples were vortex mixed, centrifuged to remove the R

R

R

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RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism

precipitated proteins and aliquots (10 :L) of the supernatant fluids were analyzed for BMS-605339.

To assess liver exposure, livers were removed from rats at various times after dosing. The tissues were rinsed, blotted dry, weighed, and stored frozen until analysis.

In Vivo Pharmacokinetic Studies All animal studies were performed under the approval of the Bristol-Myers Squibb Animal Care and Use Committee and in accordance with the American Association for Accreditation of Laboratory Animal Care (AAALAC). Mouse Pharmacokinetic studies were performed in male Balb/c, mdr1a gene-deficient (knockout),8 and FVB mouse (control wild-type strain for mdr1a mouse). Weights of all animals averaged between 19 and 23 g. For i.v. administration, BMS-605339 was dissolved in 90% PEG-400/10% ethanol. After i.v. dosing (tail vein), blood samples (∼0.05 mL from orbital sinus or cardiac puncture) were obtained from three animals at 0.05, 0.5, 1, 3, 6, 8, and 24 h. For p.o. administration to Balb/c mice, BMS-605339 was administered as a solution (90% PEG-400/10% ethanol). A solution was also used for dosing the FVB and mdr1a KO animals. Blood samples (orbital sinus or cardiac puncture) were obtained from three animals at 0.5, 1, 2, 5, 7.5, and 24 h after p.o. dosing. Serum was prepared from all collected blood samples and frozen until analyzed. All p.o. dosed animals were fasted overnight prior to dosing. Rat Male Sprague–Dawley rats (300–350 g) with cannulae implanted in the jugular or portal veins and/or the bile duct (BDC) were used. After dosing, serial blood samples (0.3 mL) were obtained from the appropriate cannula of each rat by collection into ethylenediaminetetraacetic acid-containing tubes (Becton Dickinson, Franklin Lakes, New Jersey), and centrifuged to separate plasma. Plasma was frozen until analysis. Bile samples were obtained by continuous collection and stored frozen until analyzed. For i.v. studies, BMS-605339 was dissolved (2–5 mg/mL) in a vehicle of PEG-400/ethanol (90:10, v/v) and dosed (1–2 mL/kg) as a 10-min constant rate infusion into the jugular vein, with serial blood samples collected before dosing and at 0.17, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, and 24 h after dosing (n = 3 rats/dose group). Intraportal (IPT) infusion studies were conducted in a similar manner with infusion over 30 min into the portal vein. For p.o. solution dosing, BMS-605339 was administered by gastric gavage as an aqueous solution [PEG-400/ethanol (90:10, v/v)] or a suspension (0.75% methocel/0.1% Tween-80 in water) containing either amorphous or crystalline (of various particle sizes) material. Serial blood samples were collected before dosing and at 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, and 24 h after dosing (n = 3 rats/dose group). Prior to all p.o. dosing, the rats were fasted overnight, with free access to water. To evaluate routes of elimination of unchanged BMS-605339, BDC rats supplemented with bile from donor animals were administered i.v. (5 mg/kg; n = 3), IPT (5 mg/kg; n = 3), and p.o. (20 mg/kg; n = 2) with BMS-605339 dissolved in PEG400/ethanol (9:1) at 5 mg/mL. Dosed animals were placed in metabolism cages and bile and urine samples were collected over intervals of 0–2, 2–4, and 4–8 h or 0–3, 3–6, and 6–24 h after dosing. Samples were frozen after collection and processed for analysis in the same manner described for plasma samples. Jenkins et al., JOURNAL OF PHARMACEUTICAL SCIENCES

Monkey Pharmacokinetic studies of BMS-605339 were conducted in a crossover fashion in three male cynomolgus monkeys (9 ± 1 kg) bearing vascular access ports to facilitate blood collection. There was a 2-week washout period between the i.v. and p.o. studies. For i.v. administration, BMS-605339 was infused via the venous port over 5 min at a constant rate of 0.2 mL/min kg. Serial blood samples were collected from the arterial port before dosing and 0.083, 0.17, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, and 24 h after dosing. In the p.o. study, monkeys were fasted overnight prior to dosing and BMS-605339 was administered by oral gavage (3 mg/kg). Serial blood samples were collected before dosing and 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, and 24 h after dosing. The vehicle for both dosing routes was PEG-400–water (85:15), in which BMS-605339 was dissolved at 3 mg/mL. Dog Male beagle dogs (9–12 kg) bearing venous and arterial vascular access ports were administered i.v. or p.o. (solution or suspension) doses of BMS-605339. There was at least a 1-week washout period between studies. For i.v. dosing, BMS-605339 was infused into the venous port at a constant rate over 5 min and serial blood samples (0.3 mL) were collected at 0.083, 0.17, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, and 24 h after dosing. Plasma prepared from the collected blood samples was stored frozen until analysis. The dosing vehicle for i.v. administration to dogs was PEG-400–water (85:15, v/v). Prior to all p.o. dosing, dogs were fasted overnight and BMS605339 was administered by oral gavage as a PEG-400–water (85:15) solution. Additionally, a 3-mg/kg dose was administered as an aqueous suspension (0.75% methylcellulose/0.1% Tween 80 in water) of crystalline (0.40 :m particles) material. After all dosing, serial blood samples were collected at 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, and 24 h. In a study to evaluate the liver exposure of BMS-605339 in dog, three dogs (7–9 kg) were administered 120 mg of BMS605339 contained in three capsules (PEG-900 proprietary formulation). At 2, 8, and 24 h after dosing, dogs were euthanatized and samples of liver, spleen, bile, and urine were obtained. Following removal, tissue samples were rinsed, blotted dry, weighed, and stored frozen until processed for analysis. Blood samples were collected at intervals from all dogs until the time of euthanasia. Plasma prepared from the collected blood samples was stored frozen until analysis. Data Analysis The pharmacokinetic parameters of BMS-605339 were obtained by noncompartmental analysis of plasma concentraTM tion versus time data (KINETICA software; Version 2.4; InnaPhase Corporation, Philadelphia, Pennsylvania). The peak concentration (Cmax ) and time for Cmax (Tmax ) were recorded directly from experimental observations. The area under the curve from time zero to the last sampling time [AUC(0–t) ] and the area under the curve from time zero to infinity [AUC(inf) ] were calculated using a combination of linear and log trapezoidal summations. The whole body plasma clearance (CLp ), DOI 10.1002/jps.23959

RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism

steady-state volume of distribution (Vss ), apparent terminal t1/2 , and mean residence time were estimated following i.v. administration. The absolute oral bioavailability (F) was estimated as the ratio of dose-normalized AUC values following p.o. and i.v. doses. The hepatic intrinsic clearance (CLh,int , mL/min kg) of BMS605339 in various species was estimated from liver microsomes or hepatocytes data.9–11 Assuming linear kinetics, similar protein binding in microsomes (or hepatocytes) and blood, and similar CLh,int of unbound drug in vitro and in vivo, the CLh,int was calculated from the disappearance of the parent drug in liver microsomes or hepatocytes incubations9–11 using 45 mg protein/g liver weight for microsomes and 120 × 106 cells per gram of liver weight for hepatocytes. The liver weight relative to body weight in mouse, rat, monkey, dog, and human are 90, 40, 32, 32, and 21 g/kg, respectively.12,13 The hepatic blood clearance (CLh,b , mL/min kg) was estimated using the wellstirred model, using a hepatic blood flow (HBF) of 90, 70, 44, 35, and 20 mL/min kg in mouse, rat, monkey, dog, and human, respectively.12,13 Assuming the CL of BMS-605339 is primarily hepatic, extraction ratios were calculated from the blood CL and HBF in the respective species.

RESULTS BMS-605339 (Fig. 1) has low aqueous solubility of 7 :g/mL at pH 6.5 and contains weakly basic (isoquinoline) (pKa = 2.4) and acidic (sulfonamide) (pKa = 4.8) functionalities. Although BMS-605339 was stable in neutral and basic aqueous solutions at room and elevated temperatures, it slowly degraded at low pH values. Permeability and P-gp Interaction The average Pc of BMS-605339 in Caco-2 cells at pH 7.4 was 103 nm/s. This value is similar to that of compounds that exhibit good (>50%) absorption in human. In subsequent studies using Caco-2 cells overexpressing P-gp, BMS-605339 exhibited an A to B permeability of 13 nm/s and a B to A permeability of 153 nm/s, yielding an apparent efflux ratio of 12. In the presence of the P-gp inhibitor, GW-918 (Elacridar) (10 :M), the efflux ratio was reduced to 1.8. Both studies suggested that BMS605339 is a P-gp substrate. Mouse pharmacokinetic studies (discussed below) further indicated that P-gp may modulate the oral bioavailability of BMS-605339. Serum Protein Binding and Stability BMS-605339 (10 :M) was 99.1% bound to human serum proteins and 98.2%–99.9% bound in the animal species studied Table 1.

5

(rat: 99.9%, monkey: 99.3%, and dog: 98.2%) BMS-605339 was stable in the serum of all species at 37◦ C for up to 4 h. Blood Cell Partitioning The blood-to-plasma concentration ratio of BMS-605339 ranged from 0.67 (rat) to 0.95 (dog), with a ratio of 0.67 in the monkey and 0.77 in the human. These results indicate minimal blood cell uptake of BMS-605339 with rat, human, or monkey blood cells, with a higher association with dog blood cells. Microsomal and Hepatocyte Metabolic Stability Table 1 summarizes the in vitro metabolism of BMS-605339, as measured by the disappearance of BMS-605339, in incubations with liver microsomes and hepatocytes of various species. The rank order of the species, based on the predicted hepatic intrinsic clearance, was monkey > human > mouse > dog > rat. The in vivo clearance estimates made from these in vitro data indicate that, relative to HBF, BMS-605339 would be predicted to be a low-clearance compound in mouse and rat (≤20% HBF), moderate in dog and human (25%–50% HBF) and highest in monkey (∼70% HBF) from microsomal preparations. Hepatocyte incubations were conducted to determine whether there was significant metabolism not addressed with microsomal preparations. Similar results (within twofold clearance prediction) were observed for hepatocyte incubations in comparison to microsomal data. These results indicate that hepatic metabolism is the primary clearance pathway for this compound. Metabolism of BMS-605339 by Specific CYP Enzymes Metabolic turnover of BMS-605339 was observed when incubated with microsomal preparations of individually expressed recombinant human CYP450 proteins. The greatest turnover was observed (30 min incubation) with CYP3A4 (40% and 23% turnover at BMS-605339 concentrations of 1 and 10 :M, respectively). Turnover values for the other CYP isoforms were all less than 10%. Chemical inhibition studies showed significant inhibition (∼90%) of BMS-605339 metabolism by ketoconazole in human liver microsomal preparations (data not shown). These data suggest that CYP3A4 is the primary CYP enzyme responsible for the oxidative metabolism of BMS-605339 in human liver. CYP450 Inhibition Preliminary data for BMS-605339 showed IC50 values of greater than 40 :M against recombinant CYP1A2, CYP2C9, CYP2C19, and CYP2D6. This suggests that BMS-605339 is unlikely to significantly alter the metabolic clearance of drugs metabolized by those isozymes. When evaluated against CYP3A4,

Predicted Versus Observed Systemic Clearance of BMS-605339 in Liver Microsomes and Hepatocytes Microsomes

Hepatocytes

In Vivo

Species

Rate of Metabolism (pmol/minmg Microsomal Protein)

Predicted CL In Vivo (mL/min kg)

Rate of Metabolism (pmol/min 106 Cells)

Predicted CL In Vivo (mL/min kg)

Observed CL (mL/min kg)

Human Rat Mouse Dog Monkey

45 17 22 20 166

7.5 8.6 17 9.5 27

27 10 NDa 16 32

11 15 ND 15 16

4.4 47 18 17

a

ND = Not determined

DOI 10.1002/jps.23959

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Figure 2. Metabolic profile of BMS-605339 in preclinical species.

BMS-605339 showed an IC50 of 32 :M (average of three determinations) when 7-benzyloxy-4-trifluoromethylcoumarin was the substrate and an IC50 of 2.3 :M (average of three determinations) when benzoylresorufin was the substrate. There was no time-dependent change in the IC50 value for CYP3A4 over an incubation period of 5–45 min, indicating no potential for time-dependent inhibition. These preliminary data suggest that BMS-605339 may have the potential to function as a weak/moderate CYP3A4 inhibitor, in vivo. Elucidation of Key Metabolic Pathways Biotransformation studies of BMS-605339 (30 :M) were carried out using microsomes and hepatocytes from rat, monkey, dog, and human as well as plasma, bile urine, and feces from the various species. The major metabolic pathways included monooxygenation, leading to the formation of metabolites M6, M8, M9, M12, and M16, double oxidation resulting in bisoxygenated metabolites M4 and M10, O-demethylation (M5) and its monooxidation (M2) and bisoxidation (M1) products, N-dealkylation (M3), amide hydrolysis (M11 and M14), enzymatic hydration of the terminal olefin (M7), dihydrodiol (M15), and glucuronide conjugates M5 and M13 (Fig. 2). BMS-605339 was found as the only drug-related species present in rat plasma and liver samples. Eight minor metabolites, M3, M5, M7-M9, M11, M12, M15, and M16, were detected in rat bile samples. BMS-605339 Jenkins et al., JOURNAL OF PHARMACEUTICAL SCIENCES

was the only drug-related species present in mouse and dog plasma samples at various time points. Dog livers, however, contained parent molecule as the major component, two major metabolites (M3 and M5) and several minor metabolites detected by MS. Pharmacokinetics in Mouse Pharmacokinetic parameters of BMS-605339 following i.v. and p.o. administration to Balb/c mice are summarized in Figure 3a and Table 2. Following i.v. dosing, the total body clearance (46.6 mL/min kg) was approximately half of the hepatic blood flow13 indicating that BMS-605339 was a moderate clearance compound in mouse. After p.o. solution dosing (10 and 30 mg/kg), both the serum AUC and Cmax increased in a greater than doseproportional manner, with bioavailability estimates increasing from less than 1% (10 mg/kg) to 11% (30 mg/kg). This observation is consistent with the results in Caco-2 cells suggesting that BMS-605339 is a P-gp substrate. To further evaluate the potential role of P-gp in the disposition of BMS-605339, a study was conducted in mdr1a-deficient mice.8 The pharmacokinetic parameters following the i.v. (5 mg/kg) and p.o. (20 mg/kg) administration of BMS-605339 to mdr1a deficient (KO) and FVB control mice are given in Table 3. Differences in the pharmacokinetic parameters after i.v. dosing to the two strains were observed (higher CLp and Vss , shorter t1/2 DOI 10.1002/jps.23959

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RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism

Figure 3. Plasma concentration versus time profiles of BMS-605339 following i.v. and p.o. administration to mouse (a), rat (b), monkey (c), and dog (d).

and lower serum, and liver AUC values in the FVB animals compared with the KO animals), but the critical observation from this study was the increase in serum AUC (∼eightfold) following p.o. dosing in the KO animals relative to the FVB strain. The increased plasma exposure after p.o. dosing is consistent with P-gp-mediated modulation of intestinal absorption of BMS-605339. The lower CLp observed after i.v. dosing suggests that P-gp may play a role in the elimination of BMS605339, possibly via modulation of biliary efflux. The results indicated that the in vitro observation that BMS-605339 was

Table 2.

a P-gp substrate in Caco-2 cells was predictive of the in vivo results. Pharmacokinetics in Rat The pharmacokinetic parameters of BMS-605339, administered as a PEG-400/ethanol (9:1) solution, following i.v., IPT, and p.o. administration to rats are summarized in Figure 3b and Table 2. Linear pharmacokinetics of BMS-605339 was observed following i.v. dosing over a fivefold dose range. AUC values increased in approximate proportion to the administered

Pharmacokinetic Parameters from Various Nonclinical Species

Species

Route

Dose (mg/kg)

Cmax (:M)

Tmax (h)

AUC0–t (:M h)

t1/2 (h)

CL (mL/min kg)

Vss (L/kg)

Mouse

i.v. p.o. p.o.

4.4 10 30

46.6

1.37

0.5 0.5

2.31 0.05 1.65

1.61

0.04 1.41

i.v. i.v. IPT p.o.

2 10 10 20

4.7 4.4 4.4

5.3 4.4

0.17 1.4

9.03 53.8 54.0 14.1

Dog

i.v. i.v. p.o.

1 4 3

0.6 5.1

17.6 7.4

0.40 0.33

0.75

1.33 13.9 2.15

Monkey

i.v. i.v. p.o.

1 3 3

1.37 3.91 0.03

0.8 3.7

17.4 17.9

0.26 0.60

Rat

DOI 10.1002/jps.23959

58.6 5.26

1.85

0.04

2

F (%)

1 11 0.4 0.4 100 13

51

<1

Jenkins et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism

Table 3.

Pharmacokinetic Parameters in mdr1a Knockout Mouse (P-gp-Deficient) Compared with FVB (Wild-Type) Mouse

Mouse Strain MDR1A FVB MDR1A FVB

Route

Dose (mg/kg)

i.v. i.v. p.o. p.o.

5 5 20 20

Cmax (:M)

0.86 0.19

Tmax (h)

AUC0–t (:M h)

t1/2 (h)

CL (mL/min kg)

Vss (L/kg)

1.7 1.5

42.8 63.8

2.6 3.7

3 0.5

2.7 1.8 6.36 0.72

dose, whereas the CLp (4.4–5.9 mL/min kg) and Vss (0.40–0.45 L/kg) values were similar with increasing dose. BMS-605339 is a low-clearance compound in the rat and the Vss approximated total body water (0.4 L/kg).13 Following IPT administration, the AUC, CLp , and t1/2 values were equivalent to those following i.v. administration, suggesting that the hepatic uptake of BMS-605339 was comparable after either route of administration. Following p.o. administration, incomplete and variable bioavailability (5%–18%) was observed in the rat. In contrast to the approximately dose-linear pharmacokinetics following i.v. and IPT administration, a greater than dose-proportional increase in both plasma Cmax and AUC values occurred with increasing p.o. dose (both parameters increased ∼35× over a 10-fold dose range). These results are consistent with saturation of an absorption process. The plasma exposure after p.o. suspension dosing (5 mg/kg) was only 25% compared with an equivalent solution dose, suggesting that dissolution-limited absorption may be responsible for the low oral bioavailability. A rat BDC study was conducted to evaluate BMS-605339 elimination routes in the rat. The data from this experiment are summarized in Table 4. After i.v. administration, approximately 35% of the administered dose was eliminated unchanged in bile and less than 0.2% in the urine over 24 h, indicating that biliary excretion of unchanged BMS-605339 was a major elimination pathway in rat. Similar results were also observed following IPT administration. Low recovery of administered dose was obtained following p.o. administration (2.4% in bile) again indicating that low oral bioavailability is because of absorption rather than first-pass elimination. To investigate the hepatic exposure of BMS-605339 in rat, the time course of liver and plasma concentrations was determined over 24 h in rats administered a p.o. (15 mg/kg) dose. Figure 4 illustrates the concentration versus time curve of liver and plasma exposure, whereas Table 5 gives the relevant pharmacokinetic parameters from these data. Liver Tmax occurred 6 h after dosing. Liver exposure remained elevated over plasma exposure throughout the experiment. The plasma and liver elimination t1/2 values were comparable (∼3–6 h), suggesting that a liver–plasma equilibrium had been attained.

Table 4.

Recovery of BMS-605339 in Bile and Urine from BDC Rats

F (%)

58 9.8

Pharmacokinetics in Monkey The pharmacokinetic parameters of BMS-605339 following i.v. and p.o. administration to monkeys are summarized in Figure 3c and Table 2. After an i.v. dose at 1 mg/kg, BMS-605339 exhibited a moderate plasma clearance (17 mL/min kg), a low Vss (0.2 L/kg), and short t1/2 (0.8 h). Similar clearance (17 mL/min kg) was observed when the dose was increased to 3 mg/kg, indicating linear clearance processes over this dose range. After a p.o. dose of 3 mg/kg, BMS-605339 had a bioavailability of less than 1%. The poor p.o. bioavailability may be because of the rapid metabolism of BMS-605339 in the monkey. Consistent to this in vivo observation, a high metabolic rate was observed in monkey liver microsomes (Table 1). Pharmacokinetics in Dog Following i.v. dosing at 1 mg/kg, BMS-605339 exhibited a plasma clearance of 17.6 mL/min kg in the dog (Fig. 3d and Table 2), indicating moderate clearance. Vss was moderate (0.40 L/kg) and t1/2 was short (0.6 h). However, when the i.v. dose was increased to 4 mg/kg, a greater than dose-proportional increase in AUC was observed, along with a lower clearance (7.4 mL/min kg) and longer t1/2 (5.1 h), suggesting the potential for saturation of the elimination pathway(s). Following p.o. administration as a PEG-400/water solution to dogs, the oral bioavailability showed a nonlinear increase in exposure with increasing dose, from 23% at 1 mg/kg to 51% at 3 mg/kg. The absorption after all doses was rapid, with Tmax values of 0.75 h. Oral bioavailability following p.o. suspension dosing (3 mg/kg) was considerably lower (∼8%) compared with the equivalent solution dose (51%), similar to what was observed in the rat. The results from a liver uptake study in dog are summarized in Figure 4 and Table 5. In all three dogs, liver levels of BMS-605339 were considerably higher than those of plasma. The liver/plasma ratios (51–64) were comparable over time (2, 8, and 24 h), suggesting a parallel elimination of BMS-605339 from liver and plasma. BMS-605339 concentrations in spleen were lower than those of liver, suggesting a preferential uptake into hepatic tissue. Substantial concentrations of BMS605339 were detected in bile, with significantly less in urine. Even though these were single time point collections, the data strongly suggest that in dog, as in rat, biliary elimination is the major pathway for the elimination of BMS-605339 and/or its metabolites.

Route

Dose (mg/kg) Bile (%) Urine (%)

i.v.

IPT

p.o.

5 34.9 ± 9.1a 0.2

5 42.6 ± 5.3 <0.1

20 2.4 <01

a Values are the mean ± SD of three rats after i.v. and IPT dosing and the average of two rats after p.o. dosing.

Jenkins et al., JOURNAL OF PHARMACEUTICAL SCIENCES

DISCUSSION BMS-605339 is a linear peptide mimetic inhibitor that binds to the active site of the NS3/4A protease complex preventing polyprotein processing and subsequent generation of infectious virus.14–17 As viral replication occurs within hepatocytes, effective uptake into hepatocytes is an important attribute of an efficacious anti-HCV agent. A common characteristic observed DOI 10.1002/jps.23959

RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism

9

Figure 4. Concentration versus time curves of rat and dog plasma compared with liver.

Table 5. Exposure of BMS-605339 in Plasma and Liver of Rats and Dogs Following Oral Administration Species Rat

Dog

Plasma AUC0–24 (:M h) 3.6 Liver AUC0–24 (:M h) 139 Liver–plasma AUC ratio 39

0.85 46.3 55 Sampling Time (h)

2 Plasma (:M) Liver (:M) Liver–plasma ratio

8

24

2

8

24

0.49 0.071 0.005 0.116 0.042 0.006 9.69 6.79 0.74 5.96 2.32 0.38 19.8 95.6 148 51.4 55.1 64.0

during preclinical pharmacokinetic evaluations of the various NS3/4A small molecule inhibitors has been their ability to attain liver concentrations at relatively high multiples of those in plasma (generally ≥30-fold, dependent on preclinical species). As antiviral activity of BMS-605339 and initial pharmacokinetic screening in the rat looked promising, additional preclinical in vitro and pharmacokinetic evaluations were conducted to provide a comprehensive profile of this molecule. Permeability of BMS-605339 in Caco-2 cells (103 nm/s) is comparable to compounds that exhibit moderate-to-good absorption in human. The high efflux ratio in Caco-2 cells overexpressing P-gp indicates the potential to be a P-gp substrate. Because of its low aqueous solubility (7 :g/mL), absorption of BMS-605339 may be limited by efflux from the gastrointestinal tract. BMS-605339 exhibited high protein binding in all DOI 10.1002/jps.23959

species, with 99.1% bound in human and ranging from 98.2% in dog to 99.9% in rat. Preliminary reaction phenotyping studies were carried out to understand the P450 enzymes involved in the oxidative metabolism of BMS-605339. In the presence of cDNA-expressed human CYP proteins, BMS-605339 was metabolized primarily by CYP3A4. In pooled HLM, metabolism of BMS-605339 was inhibited upon coincubation with ketoconazole (90%). These in vitro studies suggest that CYP3A4 might be the key oxidative pathway for metabolic clearance of BMS-605339. In all species tested, oxidation was the key metabolic event. The nature of the metabolites was similar between in vitro (liver microsomes, hepatocytes) and in vivo (rat and dog plasma, bile and tissue homogenates) samples. Glucuronidation of the oxidation products was a minor metabolic pathway, as shown in Figure 2. In vitro data indicated that BMS-605339 is a weak/moderate inhibitor of CYP3A4. This finding, plus the fact that BMS605339 may attain human liver levels in excess of those in plasma, increases the potential for drug–drug interactions with coadministered CYP3A4 substrates. As indicated, BMS-605339 was also identified as a CYP3A4 substrate. Potential interactions with coadministered CYP3A4 inhibitors may significantly raise liver or plasma BMS-605339 concentrations, leading to potentially adverse effects. However, the contribution of CYP3A4 to the overall human clearance of BMS-605339 cannot be determined preclinically. BMS-605339 was characterized as a low or moderate clearance compound in mouse, rat, monkey, and dog in terms of total hepatic blood flow. The rate of in vitro turnover in liver microsomes and hepatocytes was used to predict the in vivo CL in each species using physiologically based scaling factors.12,13 A good correlation (within twofold as established by projection Jenkins et al., JOURNAL OF PHARMACEUTICAL SCIENCES

10

RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism

methods18 ) in the predicted and observed CL values was obtained. On the basis of this correlation and the fact that there was no underprediction of systemic CL in any species, human CL was predicted to be moderate. The systemic oral bioavailability of BMS-605339 ranged from 8% in mouse to 51% in dog. Low bioavailability (<1%) in monkey was attributed to relatively high first-pass extraction. As the passive permeability for BMS-605339 (103 nm/s) indicated a good potential for p.o. absorption,19 studies were performed to more fully characterize the causes for the incomplete bioavailability. In rat, the systemic bioavailability after IPT administration were equivalent to those following i.v. dosing (Table 2), suggesting that the relatively lower plasma exposure after p.o. dosing resulted from low absorption at the intestinal level, possibly because of poor dissolution, intestinal wall metabolism, or effects of P-gp-mediated efflux. BMS605339 showed nonlinear pharmacokinetics in the mouse, rat, and dog based on increasing oral bioavailability with increasing dose. Experiments in both Caco-2 cells and pharmacokinetic studies in wild-type (FVBeta) and mdr1a-knockout mouse suggested that BMS-605339 was a P-gp substrate (Table 3). As the interaction of a compound with P-gp at the intestinal level is capable of adversely affecting oral bioavailability by modulating intestinal absorption via the efflux pump function of the enzyme16 and this phenomenon can be pronounced for compounds having low aqueous solubility,17 the data indicate that a component of the low oral bioavailability of BMS-605339 is its interaction with P-gp. It has been shown that P-gp and CYP3A act together in restricting oral bioavailability of compounds such as paclitaxel20 and N-methyl erythromycin.21 Although low bioavailability may be because of intestinal CYP3A4 metabolism in the mouse, it is unlikely that metabolism is the primary source because CYP3A activity in mdr1a-knockout mice is equivalent to control mice.22 The available data suggest that after p.o. administration of relatively low doses, despite acceptable intrinsic permeability, effects of poor aqueous solubility and P-gp-mediated efflux combine to limit the intestinal absorption of BMS-605339. The greater-than-dose proportional increases in plasma exposure likely result from saturation of P-gp-mediated efflux with increasing dose. In both rat and dog, the administration of aqueous suspensions of crystalline BMS-605339 resulted in oral bioavailability values considerably lower than those following equivalent solution doses (Table 2). As BMS-605339 possesses low aqueous solubility (7 :g/mL at pH 6.5), these results suggested that BMS-605339 absorption from an aqueous vehicle is likely solubility limited. As the target organ for HCV is the liver, an important attribute to the selection of an anti-HCV compound has been the demonstration of adequate liver exposure following p.o. administration. Despite its very high plasma protein binding (98%– 99%) and low-to-moderate Vss , BMS-605339 was capable of effectively distributing to the liver in mouse (data not shown), rat, and dog. Although it appears that the relatively low Vss and high liver exposure is incongruous, it has been shown that traditional means of calculating Vss may result in low values for compounds that are active uptake substrates.23 In other studies, BMS-605339 was found to be an active uptake substrate in rat, dog, and human hepatocytes (manuscript in progress). Following p.o. dosing, the liver AUC values were considerably greater than those of plasma, with liver–plasma AUC ratios of 55-fold in both rat and dog and were maintained at least Jenkins et al., JOURNAL OF PHARMACEUTICAL SCIENCES

through 24 h (Fig. 4). The 24-h concentration in rat liver exceeded the replicon genotype1a EC50 (8 nM) by 84-fold and 1b EC50 (3 nM) by 224-fold, whereas the 24-h concentration in dog liver exceeded 1a EC50 by 48-fold and 1b by 128-fold. As limited metabolic turnover of BMS-605339 was observed in rat liver microsomes and biliary excretion of unchanged BMS-605339 is a significant route of elimination in rat (and dog, but available data are more limited), saturation of biliary elimination would have significant effects on clearance. As BMS-605339 was observed to be a P-gp substrate, modulation of bile BMS-605339 elimination by P-gp may contribute to total body clearance and/or the efflux of BMS-605339 from hepatocytes into bile. The differences in the pharmacokinetic properties of BMS-605339 between mdr1a knockout mouse and the wild-type (FVB) strain are consistent with this possibility. Although there are animal models for this disease, including transgenic mouse and chimpanzee,24 there are scientific, economic, and ethical concerns with these models. Therefore, a prediction of human exposure necessary for obtaining efficacy was based on published clinical data for an HCV protease inhibitor (BILN-2061) in clinical trials.14 Although this analysis predicts only efficacious plasma levels, it is the concentration of BMS-605339 in liver that was considered to have primary importance in determining antiviral activity.25,26 Extrapolation of liver levels from plasma data is subject to a high degree of uncertainty. However, if the assumption is made that BMS-605339 and BILN-2061 are capable of producing liver concentrations with a comparable multiple of their plasma values, then the assumption that BMS-605339 will be efficacious when its plasma AUC values fall within those bounded by BILN-2061 may be valid. Limited in-house rat liver uptake data generated using BILN-2061 support this conclusion (data not shown). Without an animal efficacy model, it is difficult to estimate what liver concentrations must be maintained over a given interval (24 h) to produce a sustained antiviral response. On the basis of in vitro potency values, a targeted minimum liver total drug concentration of 80 nM (10× 1a EC50 ) was chosen as a threshold level (Cmin ) that should be maintained over 24 h to ensure activity. On the basis of a liver–plasma 0–24 h AUC ratio of 30, the minimum target concentration to maintain in plasma over 24 h would be 2.7 ng/mL. BMS-605339 was predicted to be a low/intermediate clearance compound in human based on human liver in vitro metabolism data. Allometric scaling of the in vivo clearance estimates from four species (mouse, rat, dog, and monkey) predicted that BMS-605339 will be an intermediate clearance compound in human (predicted total body clearance, 9.7 mL/min kg). Likewise, human Vss was projected to be 0.4 L/kg based on comparable Vss in the preclinical tested species (0.4–0.5 L/kg) and human plasma t1/2 was projected to be 1–1.5 h. As described above, BMS-605339 has been shown to attain liver levels approximately 30 times those of plasma in both rat and dog. Similar liver exposure was expected for the human. On the basis of these predictions, the proposed human efficacious dose was determined to be 50 mg administered on a twice daily or three time daily basis. BMS-605339 was administered to human subjects infected with genotype 1 HCV at doses of 10, 60, and 120 mg. The antiviral response was dose-dependent with a mean 1.8 log10 reduction in viral load observed 12 h after a single 120 mg dose. Although there was a favorable antiviral response in human subjects, clinically important electrocardiographic DOI 10.1002/jps.23959

RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism

changes were noted and further development of this compound was discontinued.5 As a result of these findings, further studies with a close analog of BMS-605339 led to the discovery of asunaprevir (BMS-650032).27 Combination of asunaprevir with daclatasvir, an HCV NS5A inhibitor, has been shown to be highly efficacious in difficult-to-treat patients infected with HCV genotype 1b.28,29

CONCLUSIONS Several in vitro and in vivo experiments were performed to characterize the nonclinical pharmacokinetic properties of BMS-605339. Although the oral bioavailability was dose dependent in the nonclinical species examined, the sustained and elevated liver levels observed after p.o. dosing to the rat and dog provided the confidence to progress BMS-605339 into advanced profiling and toxicology studies. Taken together, these studies demonstrated that BMS-605339 possessed nonclinical pharmacokinetic characteristics sufficient to advance the molecule to the clinical development stage.

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DOI 10.1002/jps.23959