NS4 protease inhibitor in rats.

http://informahealthcare.com/xen ISSN: 0049-8254 (print), 1366-5928 (electronic) Xenobiotica, Early Online: 1–12 ! 2014 Informa UK Ltd. DOI: 10.3109/00498254.2014.920116

RESEARCH ARTICLE

Biotransformation and mass balance of faldaprevir, a hepatitis C NS3/NS4 protease inhibitor in rats Linzhi Chen1, Roger St. George1, Stephen H. Norris1, Yanping Mao1, Elsy Philip1, Li-Quan Wang2, Diana Wu2, and Michael J. Potchoiba3 Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT, USA, 2XenoBiotic Laboratories, Inc., Plainsboro, NJ, USA and 3Covance Laboratories, Inc., Madison, WI, USA

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Abstract

Keywords

1. The metabolism, pharmacokinetics, excretion and tissue distribution of a hepatitis C NS3/NS4 protease inhibitor, faldaprevir, were studied in rats following a single 2 mg/kg intravenous or 10 mg/kg oral administration of [14C]-faldaprevir. 2. Following intravenous dosing, the terminal elimination t1/2 of plasma radioactivity was 1.75 h (males) and 1.74 h (females). Corresponding AUC0–1, CL and Vss were 1920 and 1900 ngEq h/mL, 18.3 and 17.7 mL/min/kg and 2.32 and 2.12 mL/kg for males and females, respectively. 3. After oral dosing, t1/2 and AUC0–1 for plasma radioactivity were 1.67 and 1.77 h and 11 300 and 17 900 ngEq h/mL for males and females, respectively. 4. In intact rats, 90.17% dose was recovered in feces and only 1.08% dose was recovered in urine for both iv and oral doses. In bile cannulated rats, 54.95, 34.32 and 0.27% dose was recovered in feces, bile and urine, respectively. 5. Glucuronidation plays a major role in the metabolism of faldaprevir with minimal Phase I metabolism. 6. Radioactivity was rapidly distributed into tissues after the oral dose with peak concentrations of radioactivity in most tissues at 6 h post-dose. The highest levels of radioactivity were observed in liver, lung, kidney, small intestine and adrenal gland.

LC/MS, metabolite identification, pharmacokinetics, QWBA, radiolabeled

Introduction It is estimated that 3–4 million people are infected with hepatitis C (HCV) every year, with about 150 million people being chronically infected worldwide. More than 250 000 people die every year from HCV-related liver disease (World Health Organization, 2012). There are at least 7 genotypes of HCV (Nakano et al., 2011), with genotype 1 being the most prevalent in the United States (75%; Alter et al., 1999) and the most difficult to treat (Fried et al., 2002; Manns et al., 2001). The mainstay of therapy has been combination treatment with interferon and ribavirin. Recently, the addition of direct acting antiviral therapies (HCV NS3/NS4 protease inhibitors), such as boceprevir and telaprevir to this combination treatment, has been shown to significantly improve the sustained viral response (SVR) in both treatment-naı¨ve and treatmentexperienced patients with HCV genotype 1 (Bacon et al., 2011; Jacobson et al., 2011; Poordad et al., 2011; Zeuzem et al., 2011). Faldaprevir is a novel, selective peptide-mimetic inhibitor of the HCV NS3/NS4 protease (Llina`s-Brunet et al.,

History Received 18 March 2014 Revised 25 April 2014 Accepted 28 April 2014 Published online 15 May 2014

2010; White et al., 2010; Figure 1). Faldaprevir is being developed for combination therapy and has achieved high SVR rates in treatment-naı¨ve and treatment-experienced patients with chronic genotype 1 infection, when used in combination with pegylated interferon alfa-2 a and ribavirin (Sulkowski et al., 2013a,b). Faldaprevir has an advantage over the first-generation protease inhibitors in that it is administered once daily, in contrast to the twice or thrice daily regimens required for boceprevir and telaprevir. This study was conducted primarily to study the absorption, distribution, metabolism and excretion (ADME) of faldaprevir in rats. The pharmacokinetics was determined after oral and intravenous (iv) doses of [14C]-faldaprevir and the excretion and metabolite profiles were characterized. The metabolites were identified using HPLC coupled with radiodetection and tandem mass spectrometry (MS/MS). Tissue distribution in Long–Evans and Sprague–Dawley (SD) male rats was determined by quantitative whole-body autoradiography (QWBA).

Material and methods Address for correspondence: Linzhi Chen, Ph.D., Drug Metabolism and Pharmacokinetics US, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road/P.O. Box 368, Ridgefield, CT 06877, USA. Tel: +1 203-778-7870. Fax: +1 203-791-6003. E-mail: Lin_zhi.chen@ boehringer-ingelheim.com

Chemicals and solvents Carbon-14 liquid scintillation cocktail was obtained from R.J. Harvey Instrument Corporation (Hillsdale, NJ). Ready SafeTM

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Figure 1. Structure of [14C]-faldaprevir. The asterisk denotes the 14 C-label.

and Ready ValueTM liquid scintillation cocktails were purchased from Beckman Instruments, Inc. (Fullerton, CA). PEG 400 was obtained from Sigma-Aldrich, Inc. (St. Louis, MO). Meglumine was supplied by Boehringer Ingelheim Pharmaceuticals, Inc. (Ridgefield, CT). All other chemicals and solvents were HPLC or analytical grade and obtained from reliable commercial sources. Radiolabeled drug and reference standards Faldaprevir and [14C]-faldaprevir were synthesized at Boehringer Ingelheim Pharmaceuticals, Inc. (Ridgefield, CT). Chemical identities of these compounds were established by HPLC, mass spectrometry and NMR. The specific activity of [14C]-faldaprevir material was 52.13 mCi/mmol. [14C]-faldaprevir radiochemical purity was determined to be 98.9% by LC/radiochromatography. The structure of [14C]faldaprevir is shown in Figure 1. Animals For the mass balance studies, SD rats [Crl:CD(SD)IGS BR] were obtained from Charles River Laboratories (Portage, MI). The rats were approximately 12 weeks old and weighed 236–287 g (intact), 287–299 g (bile duct-cannulated or BDC) and 234–290 g (jugular vein cannulated or JVC) at the time of dosing. Intact and JVC rats were quarantined for at least 5 days. BDC rats were quarantined for at least 1 day. For the QWBA study, Long–Evans and SD rats were obtained from Harlan Laboratories Inc. (Indianapolis, IN). The animals were acclimated for 4 days before dose administration. At dosing, the animals were 7 weeks of age and weighed 158–167 g. Four SD rats/route/gender (Hilltop Lab Animals, Inc., Scottdale, PA) were used in this pharmacokinetic study. Study protocols were approved by the local Institutional Animal Care and Use Committee. Mass balance studies For the rat [14C]-mass balance studies, a total of 28 SD intact rats and 4 BDC SD rats were used. The animals were assigned to different groups as follows: 16 animals were used for the

Xenobiotica, Early Online: 1–12

excretion mass balance study after oral (group 1) and iv (group 2) dosing (4 animals/sex/group); four BDC males (group 3) were used for the biliary excretion study after oral dosing; three males (group 4) were used for expired air study after oral dosing; eight animals (group 5) were used for the plasma metabolite profiling/identification study after oral dosing (4 animals/sex); one male (group 6) was used as control to collect control urine, feces and plasma. During the test period, animals were housed as appropriate for sample collection: animals designated for collection of blood samples were housed in individual cages; animals designated for collection of excreta and expired air were housed in glass metabolism cages designed for the separation and collection of urine and feces and collection of expired air; animals designated for collection of bile and excreta were housed in individual Nalgene cages designed for the separation and collection of urine and feces and collection of bile. The rats (except for group 3 rats) were fasted overnight before dosing and for 4 h after dosing. Otherwise, Certified Rodent Diet #5002 M (PMI Feeds, Inc., St. Louis, MO) and water were provided ad libitum. The BDC rats in group 3 received electrolyte-supplemented water (NaCl:KCl: glucose, 0.925:0.05:5.06%, w/v). The oral dose formulations for groups 1, 3, 4 and 5 were prepared by dissolving [14C]-faldaprevir and faldaprevir sodium salt in PEG400. TRIS and meglumine in water were then added dropwise to the PEG400 mixture. The dose formulations were solutions with nominal concentrations of 2 mg/32 mCi/mL in 80% PEG400, 18% water, 1% TRIS and 1% meglumine. The iv dose formulation for Group 2 was prepared by dissolving [14C]-faldaprevir in PEG400. NANOPureÕ water was then added dropwise to the PEG400 mixture. The dose formulation was a solution with a nominal concentration of 2 mg/20 mCi/mL. Each treated rat received a single dose by oral gavage or bolus tail vein injection at a nominal dose based upon individual body weights taken prior to dosing. The actual amount of dose formulation administered to each animal was determined by weighing the loaded dose syringe before dose administration and the emptied syringe after dose administration. The overall mean doses administered to rats ranged from 9.87 mg/kg (154.18 mCi/kg) to 10.13 mg/kg (158.68 mCi/kg) for the oral groups and 1.86 mg/kg (110.96 mCi/kg) for the iv group. Urine from the rats in groups 1 and 2 from 0 to 168 h postdose, and from the rats in group 3 from 0 to 48 h post-dose was collected into tared sample cups and freeze-trapped using dry ice. Feces were collected at the same time periods as the urine at room temperature except group 3, which was collected in sample cups surrounded by dry ice. Cages were rinsed with NANOPureÕ water at 24-h intervals post-dose and thoroughly washed with isopropanol/water (1:1) at 168 h postdose for groups 1 and 2, and at 48 h post-dose for group 3. Bile was collected over dry ice from the rats in group 3 at predose and 0–2, 2–4, 4–6, 6–8, 8–24 and 24–48 h post-dose. Expired air from group 4 was trapped using 100 mL of monoethanolamine:CellosolveÕ (1:1), which had been validated for good trapping efficiency (data not shown). The traps were recharged with fresh solutions at 4, 8 and 24 h post-dose. For plasma metabolite profiling, blood was collected in

DOI: 10.3109/00498254.2014.920116

EDTA-containing tubes on wet ice from the jugular vein cannula (or cardiac puncture for the 8-h time point) of the rats in group 5 at 2, 4 and 8 h post-dose. The blood was centrifuged at 4 C and 2500 rpm (approximately 1400 g) or 10 000 rpm (approximately 9800 g) for 10 min to separate plasma. Animals were euthanized with an overdose of halothane CO2 at the end of the study. Before and after analysis, plasma samples were stored at 70 C, CO2-trapping solutions were refrigerated and all other samples were stored at approximately 20 C.


Pharmacokinetic studies Pharmacokinetic studies were conducted in male and female SD rats (n ¼ 4/sex/route) after oral and iv doses of [14C]faldaprevir. Rats were fasted the evening before dosing and food was returned after the 4 h blood sample was taken. Water was provided ad libitum. The oral and iv doses were prepared in the same manner as the mass balance study. Rats were dosed intravenously by slow bolus injection at a dosage of 2 mg/kg, or orally by gavage at a dosage of 10 mg/kg (free form equivalent). Whole blood was collected in tubes containing K3EDTA as the anticoagulant via a jugular vein cannula at 0.083 (iv only), 0.33, 1, 2 (oral only), 4, 8, 24 and 48 h post-dose. Plasma samples were prepared by centrifugation of the whole blood. The plasma samples were stored at approximately 70 C before analyses. Radioactivity of [14C]-faldaprevir in plasma was determined by liquid scintillation counting (LSC) using an external standard to measure the efficiency of counting and an instrument-stored quench curve generated from sealed quenched standards to obtain disintegrations per min (dpm). For whole blood, samples were thawed and then solubilized in 1 mL of a solution of SolueneÕ -350 tissue solubilizer:isopropyl alcohol (1:1, v:v) for 1.16–3.25 h in an oven at 52 C and the mixture was decolorized by addition of 500 mL of 30% hydrogen peroxide, followed by incubation at room temperature for 15 min and then at 52 C for 30 min before the addition of 15 mL scintillation fluid. Blood and plasma concentrations versus time data for faldaprevir were analyzed by standard non-compartmental methods using Kinetica Version 4.4.1 (Thermo Scientific, Philadelphia, PA). Pharmacokinetic parameters calculated included, but were not limited to, maximum concentration (Cmax), time to reach maximum concentration (tmax), half-life (t1/2) and area under the concentration–time curve from 0 to infinity (AUC0–1). QWBA study The pharmacokinetics and tissue distribution of [14C]faldaprevir-derived radioactivity were determined in nine male Long–Evans rats and two male SD rats, each receiving either a single oral or iv administration of [14C]-faldaprevir sodium salt at a nominal dose of 10 mg/kg (100 mCi/kg) or 2 mg/kg free form equivalent (100 mCi/kg), respectively. One Long–Evans rat/time point was sacrificed at 1, 6, 12, 24, 48, 72, 336 and 672 h following a single oral dose. One Long– Evans rat was sacrificed at 0.083 h after a single iv dose. In addition, one SD rat/time point was sacrificed at 24 and 672 h following a single oral dose. Blood was collected at the time

Biotransformation and mass balance of faldaprevir

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of sacrifice from all animals. The carcasses were immediately frozen in a hexane/dry ice bath for 5 min. Each carcass was drained, blotted dry and placed on dry ice or stored at approximately 70 C for at least 2 h. Each carcass was then stored at approximately 20 C. The frozen carcasses were embedded in chilled carboxymethylcellulose and frozen into blocks. Prior to section collection, standards fortified with [14C]-glucose in 1% CMC were placed into the frozen block containing the carcass and were used for monitoring the uniformity of section thickness. Appropriate sections were collected on adhesive tape at 40 mm thickness, in a Leica CM 3600 cryomicrotome (Leica, Wetzlar, Germany). Sections were collected at five levels of interest in the sagittal plane. All major tissues, organs and biological fluids were represented. Collected sections were dried at approximately 20 C. The sections were mounted and exposed on phosphor imaging screens along with plastic-embedded ARC autoradiographic standards (American Radiolabeled Chemicals, Inc., St. Louis, MO) for subsequent calibration of the image analysis software. Screens were exposed for 4 days. The autoradiographic standard image data were sampled using Imaging Research Inc. AIS software (InterFocus Imaging LTD, Linton, UK) to create a calibrated standard curve. Specified tissues, organs and fluids were analyzed. Tissue concentrations were interpolated from each standard curve as nanocuries/g and then converted to ng equivalents/g on the basis of the specific activity of the test article. Sample preparation and analysis Mass balance Levels of radioactivity in plasma, urine, bile, cage rinse/wash and CO2-trap samples were assayed directly in a LSC. Feces were homogenized with approximately 3–5 times (w/v) mixture of isopropanol:NANOPureÕ water (1:1), and aliquots of the homogenates were then combusted in a Harvey Biological Sample Oxidizer (Models OX-300 and OX-500; R.J. Harvey Instrument Corporation, Hillsdale, NJ). The evolved [14C]-CO2 from the oxidizer was trapped in 15 mL of Harvey Scintillation Carbon-14 Cocktail. All samples were analyzed for radioactivity using LSC (Models LS 6000LL, LS 6000TA, LS 6000IC and LS 5000, Beckman Instruments, Inc., Fullerton, CA) for 10 min or until the 2 sigma error was less than or equal to 2%, whichever came first. Quench correction was performed using an external standard method. Oxidizer efficiencies were determined by combusting a known amount of [14C]-mannitol. Feces were assayed in triplicate while all other samples were analyzed in duplicate. Scintillation counting data expressed in counts/min (cpm) were automatically corrected for counting efficiency using the external standardization technique and an instrument-stored quench curve generated from a series of sealed quenched standards. Metabolite profiling Before thawing of the individual samples, an appropriate volume of formic acid (ca. 1% in each sample, v/v) was added to individual completely thawed plasma, bile and urine samples, and mixed thoroughly. Rat plasma samples collected


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at 2, 4, and 8 h post-dose were pooled according to gender and time point. Three volumes of acetonitrile were added to one volume of pooled plasma and the mixture was vortexed for 2 min. After storage in a refrigerator for ca. 0.5 h, the mixture was centrifuged at ca. 10 000 g and 4 C for 10 min (Hettich Centrifuge, Model Rotina 46 R, Integrated Services TCR Inc, Palisades Park, NJ). The pellets were rinsed twice with acetonitrile. The acetonitrile extract and rinsates were combined and evaporated to dryness under a nitrogen stream by N-Evap Nitrogen Evaporator (Model 111 and 112, Organomation Associates, Inc., Berlin, MA). The residues were reconstituted with an appropriate volume of acetonitrile-water (2:1 v/v) for radio-profiling. The overall extraction recovery from plasma was greater than 95%. The urine samples were pooled according to gender. Only 0–24 and 24–48 h urine samples were used to make the pools, as the majority of urine radioactivity was found in 0–48 h samples and the remaining radioactivity was spread in large volumes of urine across a 120-h period. An equal percentage by volume of individual urine samples from group 1 were combined according to gender and time interval to give four pooled urine samples for metabolite radio-profiling: a 0–24 h and a 24–48 h pool each for males and females. A portion of each pooled urine sample was centrifuged at ca. 10 000 g and 4 C for 10 min. After the supernatant was separated, the residue was extracted with acetonitrile. The extract was combined the supernatant for radio-profiling. The overall extraction efficiency of radioactivity from urine was greater than 88%. The 0–48 h fecal samples contained more than 98% of the total radioactivity excreted in feces and were used for metabolite profiling. Similar to urine, feces samples were combined according to gender and time intervals to make fecal pools: a 0–24 h and a 24–48 h pool each for males and females. For extraction, three volumes of acetonitrile (v/w) were added to a portion (ca. 5 g) of the pooled fecal homogenate. The mixture was shaken for 30 min using a Wrist Action Shaker (Model 75, Burrell Corporation, Pittsburgh, PA), followed by centrifugation at ca. 1800 g for 10 min to separate the supernatant. The residue was extracted twice more with acetonitrile. The radioactivity extraction efficiency from the feces was greater than 96% for 0–24 h pools and greater than 83% for the 24–48 h pools. All the supernatants were combined and evaporated to dryness at room temperature under a nitrogen stream by N-Evap. The resulting radioactive residue was reconstituted with a small volume of acetonitrile/H2O (5:1) for radio-HPLC analysis. The 0–24 and 24–48 h male rat bile samples were combined to make a 0–24 and 24–48 h pool for metabolite profiling. The pooled rat bile was centrifuged to remove sediment and the sediment was further extracted with acetonitrile. The radioactivity extraction efficiency from the bile was greater than 93%. Metabolite profiles in the plasma, urine, feces and bile extracts were determined by HPLC radio-chromatography using a Waters 2695 HPLC system (Waters, Milford, MA) or a Shimadzu HPLC System (LC 10AD VP pump and SIL-HTC injector, Shimadzu Inc., Columbia, MD) coupled with a fraction collector (Model ISCO Foxy 200Õ , ISCO, Inc.,


Lincoln, NE). The HPLC was controlled by Millennium32 Data System (Waters, Milford, MA). Metabolite separation was achieved on a Gemini C18 column (3 mm, 150 4.6 mm; Phenomenex, Torrance, CA) at 45 C. The mobile phase A comprised of 40 mM ammonium acetate in H2O, (adjusted to pH ca. 5.5 with acetic acid), and the mobile phase B was acetonitrile. The gradient conditions were initial holding at 100% A for 5 min, to 85% A over 15 min, to 25% A over 40 min and then increased to 100% A and hold for 5 min at 0.7 mL/min. Fractions of chromatography effluents were collected by time (15 s/fraction) to Deepwell LumaPlateTM96 plates (Perkin Elmer, Waltham, MA). The plates were subsequently dried on a SpeedVacÕ concentrator (Model AES2010, Savant Instrument Inc., Holbrook, NY) and then counted for radioactivity on a TopCountÕ NXTTM Microplate counter (Packard Instrument, Meriden, CT). HPLC radio-chromatograms were reconstructed using ARCÕ Convert and Evaluation software (AIM Research Co., Hockessin, DE). Metabolite identification The prepared plasma, urine, bile and fecal samples were analyzed for metabolite identification. Metabolite identification was conducted using an LC/radio-detection/MS system, which consisted of the same HPLC system used for the metabolite profiling (see above), a b-RAM radioactivity detector (Model 3, IN/US Systems Inc., Tampa, FL) equipped with a Ramona 92 (100 mL) or Star (200 mL) glass flow cell (Raytest Co., Straubenhardt, Germany), and a Sciex 4000 QTrap mass spectrometer (Applied Biosystems, Foster City, CA). The mass spectrometer was equipped with a Turbo Ion Spray ion source and operated in positive mode. The key operating parameters included: spray voltage: 5 kV; gas 1: 50 units; gas 2: 30 units; ion source temperature: 550 C; curtain gas: 20 units; declustering potential: 50 V; and exit potential: 10 V.

Results Pharmacokinetics The blood and plasma concentrations versus time profiles of radioactivity following iv administration of [14C]-faldaprevir at 2 mg/mL is shown in Figure 2. Concentrations of radioactivity in the blood and plasma were similar in males and females. The mean initial maximum concentrations of radioactivity in blood at the first sampling time at 5 min were 927 and 912 ng Eq/mL for male and female SD rats, respectively; corresponding plasma concentrations were 1380 and 1320 ng Eq/mL, respectively. The blood and plasma concentrations versus time profiles of radioactivity following oral administration of [14C]-faldaprevir at 10 mg/mL is shown in Figure 3. Concentrations of radioactivity were slightly higher in females. The mean Cmax of radioactivity in blood was 1503 and 2434 ng Eq/mL, at 2 h for males and females, respectively; corresponding plasma concentrations were 2700 and 4190 ng Eq/mL, respectively. For both iv and oral doses, concentrations of radioactivity in blood and plasma were below the limit of quantitation (BLQ) of the assay after 8 h post-dose (after 4 h in male blood only).


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Figure 2. Mean concentrations of radioactivity in blood and plasma following single iv dose of [14C]-faldaprevir (2 mg/kg) to male and female SD rats.

The pharmacokinetic parameters for radioactivity in plasma after iv and oral administration of [14C]-faldaprevir are presented in Table 1. As there was no significant association with red blood cells (see the ‘‘Discussion’’ section), only pharmacokinetic parameters for plasma are discussed below. Following iv dosing, the terminal elimination half-life (t1/2) values of radioactivity in plasma were 1.75 and 1.74 h for males and females, respectively. The corresponding AUC0–1, CL and Vss values were 1920 and 1900 ngEq h/ mL, 18.3 and 17.7 mL/min/kg and 2.32 and 2.12 L/kg for males and females, respectively. After oral dosing, the t1/2 and AUC0–1 for radioactivity in plasma were 1.67 and 1.77 h and 11 300 and 17 900 ng Eq h/mL for males and females, respectively. The oral absorption of [14C]-faldaprevir was similar in males (117 ± 25.9%) and females (188 ± 42.2%). The percent absorption of [14C]-faldaprevir was greater than 100% in both males and females suggesting the presence of non-linear, saturable processes. While enterohepatic recirculation was possible as 34% dose was recovered in the bile (see mass balance results later), the higher bioavailability due to enterohepatic recycling was unlikely. If recycling were to occur, it would also occur after iv administration, thus increasing the AUC after both iv and oral dosing. Mass balance and excretion After iv administration of [14C]-faldaprevir at 2 mg/kg, mean recoveries (0–168 h) of radioactivity in urine (including cage rinse/wash) and feces were 0.51 and 92.11% for males and 0.50 and 90.17% for females, respectively (Table 2). Total recoveries of dosed radioactivity were 92.74% (males) and 90.82% (females). Most of the radioactivity was excreted


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during the 0 to 24 h interval (83.84% for males and 80.37% for females) with lesser amounts excreted during the 24–48 h interval (8.09% for males and 9.58% for females). The remaining radioactivity excreted was less than 1% and spread out over the 48–168 h collection periods for both males and females. The results for the excretion of radioactivity after oral administration of [14C]-faldaprevir were similar to the iv dose results. Most of the radioactivity was excreted during the 0–24 h interval (87.38% for males and 82.47% for females) with smaller quantities excreted during the 24–48 h interval (7.94% for males and 12.25% for females). The predominant route of excretion after oral administration was also via the feces. Over the 168-h study, a mean total of 95.95% was excreted via feces in males and 91.96% in females. Excretion of radioactivity in urine was a minor route of elimination after oral dosing, and a total of 0.35 and 1.08% of the radioactive dose was excreted by this route in males and females, respectively. After oral administration of [14C]-faldaprevir to bile ductcannulated male rats, a mean value of 34.32% of the radioactive dose was excreted in the bile over the 48-h collection period. Total excretion of radioactivity in feces and urine (including cage wash) of bile duct-cannulated rats accounted for 54.95 and 0.27% of the dose, respectively. Total recovery of radioactivity from the bile duct-cannulated rats was 90.68% over a period of 48 h. Collection of expired air indicated that only a trace amount of radioactivity from [14C]-faldaprevir was metabolized to 14 CO2 after a single oral dose of [14C]-faldaprevir. The total amount of radioactivity recovered in CO2 traps during the first 24 h after dosing was 0.02%. Metabolite profiling Metabolite profiling was conducted in the plasma, bile, urine and feces from the oral dose groups only. The contributions of unchanged faldaprevir and its metabolites to matrix radioactivity and percent of dose are listed in Table 3. Representative radiochromatographic profiles from plasma, feces and bile are shown in Figure 4. In plasma at 2, 4 and 8 h after the oral dosing, faldaprevir was predominant and accounted for 96.5–99.2% of the plasma radioactivity. Two trace metabolites, M3 and M4, were identified, and each represented 0.56% of plasma radioactivity. Two additional trace metabolites were observed in some plasma samples but not further identified. In the male rat bile, unchanged faldaprevir was the most abundant radioactive component, accounting for 15.9% dose. One major metabolite, M4, was identified and accounted for 12.9% dose. Five minor metabolites, M1, M2a, M2b, M3 and M5, were also identified in the bile, and each accounted for 0.04–1.73% of the dose. M6, which accounted for 0.35% of the dose, was also found in the dosing solution, and thus believed to be an impurity from the dose rather than a metabolite. Unchanged faldaprevir was also the predominant radiocomponent in the feces, accounting for 79.5 and 78.1% of the dose for the males and females, respectively. Fourteen metabolites were found in feces and each represented 5.03% of the dose. Among them, four were identified to

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be M2a, M2b, M3 and M4, and each accounted for 0.56– 5.03% dose. The remaining fecal metabolites each accounted for 1.06% of the dose and were not further identified. Unchanged faldaprevir was the major radio-component in both male and female rat urine samples. As described above, the total radioactivity excreted in urine was very low, and faldaprevir accounted for only 0.37% of dose. In addition, three minor metabolites, M2a, M2b and M5, were detected in male rat urine, and five minor metabolites, M2a, M2b, M3, M4 and M5, were detected in female rat urine. Quantitative whole-body autoradiography


Table 4 shows the tissue concentrations of total radioactivity following 10 mg/kg oral or 2 mg/kg iv administration of

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[ C]-faldaprevir as determined by QWBA. Following a single oral administration to male Long–Evans rats, radioactivity derived from [14C]-faldaprevir was well absorbed and widely distributed to most tissues at the first sampling time of 1 h post-dose. Radioactivity was primarily associated with the gastrointestinal (GI) tract, liver, lung, kidney, small intestine and adrenal gland. The concentrations of radioactivity in most tissues were not discernible from background or were BLQ by 12 h post-dose. Concentrations of radioactivity were BLQ in tissues of the central nervous system with a blood brain barrier at all time points. Following a single oral administration to SD male rats, the 24 and 672 h data were similar to data obtained from Long–Evans rats, where concentrations of radioactivity in tissues were BLQ at these time points. In pigmented rats, terminal half-life (t1/2) values for radioactivity could be estimated in the matrices analyzed by QWBA. Terminal t1/2 values ranged from 1.35 h in bile to 7.55 h in large intestine. Tmax was 6 h for most tissues. The highest calculated radiation absorbed doses, excluding gastrointestinal contents, were in kidney, renal medulla, thymus, testis, epididymis and liver, with 0.286, 0.333, 0.340, 1.05, 1.77 and 5.60 mrem, respectively. In the Long–Evans rat after iv administration, radioactivity was widely distributed to most tissues at 0.083 h post-dose. Distribution of radioactivity was similar to pigmented rats receiving oral administration except for the presence of measurable levels of radioactivity in the brain. However, concentrations of radioactivity were low in tissues of the central nervous system indicating that [14C]-faldaprevirderived radioactivity did not readily cross the blood brain barrier after oral administration. Metabolite identification The metabolite identification was based on HPLC retention time, radiochromatography and mass spectral analysis. MS/MS analyses were performed for structure elucidation. Faldaprevir

Figure 3. Mean concentrations of radioactivity in blood and plasma following single oral dose of [14C]-faldaprevir (10 mg/kg) to male and female SD rats.

The HPLC retention time of faldaprevir was approximately 56.0 min. Faldaprevir had a protonated molecular ion MH+ at m/z 869 with an isotope ratio of roughly 1:1 for [M + 2]/M

Table 1. Mean (±SD) pharmacokinetic parameters for radioactivity in blood and plasma following a single intravenous (2 mg/kg) or oral (10 mg/kg) dose of [14C]-faldapravir to SD rats (n ¼ 4). Cmaxa tmax AUC0–t AUC0–1 Route Matrix Gender (ng Eq/mL) (h) (ng Eq h/mL) (ng Eq h/mL) IV

Blood Plasma

Oral

Blood Plasma

a

M F M F M F M F

927 ± 336 912 ± 327 1380 ± 548 1320 ± 497 1503 ± 406 2434 ± 383 2700 ± 940 4190 ± 587

– – – – 2 2 2 2

b

NC NC NC NC 1850 ± 523 1920 ± 497 1820 ± 176 1900 ± 151 NC NC NC NC 10 100 ± 4120 11 300 ± 2480 16 500 ± 2870 17 900 ± 4020

For intravenous dose group the value represents C0. Not calculated. c For oral dose group the value represents Clearance/F. d For oral dose group the value represents Vz/F. b

ke (1/h)

t1/2 (h)

CLc (mL/min/kg)

NC NC 0.404 ± 0.0660 0.407 ± 0.0660 NC NC 0.466 ± 0.148 0.458 ± 0.200

NC NC 1.75 ± 0.319 1.74 ± 0.300 NC NC 1.67 ± 0.781 1.77 ± 0.813

NC NC 18.3 ± 4.99 17.7 ± 1.41 NC NC 15.4 ± 3.81 6.67 ± 2.16

Vssd (L/kg)

F (%)

NC – NC – 2.32 ± 1.26 – 2.12 ± 0.821 – NC NC NC NC NC 117 ± 25.9 NC 188 ± 42.4


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Figure 4. Representative radiochromatograms of male 2 h plasma (top), 0–24 h feces (middle), and 0–24 h bile (bottom panel).

Table 2. Mass balance and excretion of [14C]-faldapravir-derived radioactivity following a single iv (2 mg/kg) and oral (10 mg/kg) dose to SD rats (n ¼ 4). Route IV

Gender Urine (%)

Male Female Oral Male Female Oral (Bile) Male

0.51 ± 0.08 0.50 ± 0.10 0.35 ± 0.03 1.08 ± 0.96 0.27 ± 0.03

Feces (%)

Bile (%)

92.11 ± 2.93 – 90.17 ± 1.26 – 95.95 ± 0.96 – 91.96 ± 4.46 – 54.95 ± 18.73 34.32 ± 18.15

Total (%)a 92.74 ± 2.90 90.82 ± 1.23 96.96 ± 0.74 96.35 ± 2.14 90.68 ± 2.63

a

Total percent of radioactivity recovered includes cage wash and cage wipe.

due to the presence of one bromine atom. The positive MS/MS of faldaprevir produced fragment ions at m/z 448, 422 and 352 (Figure 5). The m/z 422 and 448 fragments were formed via the cleavage of the C–O bond connecting quinoline and pyrolidine, and the m/z 352 ion was a further fragment from the m/z 422 product ion. These characteristic fragmentations were useful in structure elucidation for metabolites as discussed below. M4 The MH+ of M4 eluting at approximately 52.4 min was determined to be at m/z 1045, 176 Da higher than

faldaprevir MH+, which was consistent with a glucuronide (gluc) conjugate (+176 Da) of faldaprevir. The isotope pattern MH+ of M4 was similar to that of the parent, indicating the retention of bromine. Same as faldaprevir, M4 gave product ions at m/z 448, 422 and 352 (Figure 6). In addition, M4 produced fragments at m/z 869 and 624. The m/z 869 ion was formed via the loss of the glucuronic acid (176), while the m/z 624 ion corresponded to the m/z 448 fragment with the addition of the glucuronic acid (448 + gluc). Therefore, the formation of the m/z 624 fragment indicated that the glucuronic acid was retained on the pyrolidinecontaining moiety. Although the exact location of the glucuronidation could not be determined by MS/MS, it was likely at the acyl group. M3 MH+ of M3 at approximately 51.0 min in the pooled 0–24 h rat bile was determined to be at m/z 799 with a similar isotope pattern as the parent but 70 Da lower than the parent, suggesting that a des-isopropylcarbonyl metabolite. M3 produced the same fragment ions as faldaprevir at m/z 448 and 352 (data not shown), consistent with a des-isopropylcarbonyl structure.

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Xenobiotica, Early Online: 1–12 14

Table 3. Radioactivity contributions of faldapravir and metabolites in male and female SD rats after oral dose of [ C]-faldapravir. Male SD ratsa

Female SD rats

Plasma (group 5)

Feces and urine (group 1)

Plasma (group 5)

Feces and urine (group 1)

Bile (group 3) 2h 4h 8h Feces Compounds [%dose] (%[14C]) (%[14C]) (%[14C]) (%dose)

Urine (%dose)

Feces + Urine 2h 4h 8h Feces (%dose) (%[14C]) (%[14C]) (%[14C]) (%dose)

M1 M2a M2b M3 M4 M5 M6 Faldapravir

– 50.01 50.01 – – 50.01 – 0.02

– 1.13 2.71 5.03 0.02 50.01 1.74 79.5

0.04 0.41 0.34 1.73 12.9 1.32 0.35 15.9

– – – 0.28 0.28 – – 99.2

– – – 0.23 0.30 – – 98.9

– – – 0.52 0.42 – – 97.5

– 1.13 2.71 5.03 0.02 – 1.74 79.5

– – – 0.28 0.24 – – 97.2

– – – 0.56 0.19 – – 98.4

– – – 0.56 0.45 – – 96.5

– 1.22 1.49 3.94 0.56 – 1.63 78.1

Urine (%dose)

Feces + Urine (%dose)

– 0.04 0.04 0.09 0.04 0.01 – 0.37

– 1.26 1.53 4.03 0.60 0.01 1.63 78.5

a


The remaining radioactivity in each matrix was contributed by a number of minor metabolites, each 51.75% of plasma sample radioactivity, 50.29% of dose in bile, 51.06% of dose in feces and 50.15% of dose in urine.

Table 4. Tissue concentration of total radioactivity (ng equivalents 14 C-faldapravir to male Long–Evans and SD rats.

14

C-faldapravir/g) after a single oral (10 mg/kg) or iv (2 mg/kg) dose of

Long–Evans rats

SD rats

10 mg/kg oral Tissues Adrenal gland Bile Blood Bone marrow Cecum contents Cerebellum Cerebrum Esophageal contents Brown Fat Harderian gland Kidney Large intestinal content Large intestine Liver Lung Lymph nodes Pancreas Pituitary gland Preputial gland Prostate Renal cortex Renal medulla Salivary gland Skin Small intestinal content Small intestine Spinal cord Spleen Stomach Stomach contents Testis Thymus Thyroid Urinary bladder Uveal tract

1h

6h

12 h

24 h

48 h

2 mg/kg iv 0.083 h

10 mg/kg oral 24 h

691 ND 2250 360 7490 BLQ BLQ 22 900 480 143 800 BLQ 173 22 200 960 131 472 411 244 155 782 819 497 BLQ 23 000 749 BLQ 304 582 233 000 BLQ 96.9 450 157 BLQ

824 58 500 4930 398 89 500 BLQ BLQ BLQ 667 720 1310 34 200 1150 21 800 376 464 875 444 683 585 907 1590 627 364 255 000 863 BLQ 344 796 15 100 382 580 599 1090 110

BLQ 2710 ND ND 101 000 ND ND 1410 ND 212 94.3 195 000 235 604 BLQ ND BLQ ND 120 BLQ BLQ 111 ND BLQ 10 100 96.8 ND ND BLQ 2710 147 111 ND ND ND

ND ND ND ND 10 600 ND ND ND ND ND BLQ 15 700 179 142 ND ND ND ND ND ND ND ND ND ND BLQ ND ND ND BLQ BLQ ND ND ND ND ND

ND ND ND ND 251 ND ND ND ND ND ND 439 ND BLQ ND ND ND ND ND ND ND ND ND BLQ 156 ND ND ND ND BLQ ND ND ND ND ND

6950 91 100 4040 1730 BLQ 51.2 46.5 88 1250 200 3080 BLQ 539 33 300 2640 345 1130 2240 322 195 3300 2580 1910 106 34 500 117 47.2 1900 269 2390 54.8 188 1110 376 269

ND ND ND ND 2000 ND ND ND ND ND ND 5150 ND BLQ ND ND ND ND ND ND ND ND ND BLQ ND ND ND BLQ BLQ ND ND ND ND ND ND

ND: not detectable; BLQ: below limit of quantitation.

M1 M1 in the pooled 0–24 h rat bile eluted at 46.5 min and corresponded to m/z 975, 176 Da higher than M3, suggesting that it was an acyl glucuronide conjugate of M3. M1 gave the same product ions as M3 at m/z 448

and 352 (data not shown). In addition, M1 gave fragments at m/z 799 via the loss of the glucuronic acid (176) and at m/z 624 which corresponded to the m/z 448 fragment with the addition of the glucuronic acid (448 + gluc). As for M4, the observation of m/z 624 fragment in M1 indicated that the glucuronic acid was retained on the


DOI: 10.3109/00498254.2014.920116

pyrolidine-containing moiety and most likely located at the acyl group.


M2a and M2b These two metabolites eluted at approximately 47.5 and 48.1 min, respectively, and corresponded to MH+ ions with same m/z of 885 with a similar isotope pattern as the parent. The determined molecular weight is 16 Da higher than faldaprevir, indicative of mono-oxidation metabolites. Both M2a and M2b gave quite similar MS/MS spectra showing the faldaprevir-characteristic fragment ions at m/z 422 and 352, respectively (Figure 7; only M2a was shown). In addition, M2a and M2b produced a fragment at m/z 464, which corresponded to respective faldaprevir-characteristic fragment ion at m/z 448 with the addition of 16 Da. The formation of m/z 464 ions thus suggested that the oxidation was on the cyclopetanyl ring as shown in the fragmentation pattern. Figure 5. MS/MS spectrum fragmentation of faldaprevir reference standard.

Figure 6. MS/MS spectrum of M4.

and

9

M5 This metabolite eluted at 53.1 min and corresponded to a MH+ at m/z 1031, 162 Da higher than faldaprevir, indicative of a glycoside (glc) conjugate metabolite (+162). It gave faldaprevir-characteristic fragment ions at m/z 448, 442 and 352 (data not shown). In addition, M5 gave product ions at m/z 869 via the loss of glucoside (162) and m/z 610 which corresponded to the m/z 448 fragment with the addition of glucoside. M6 As discussed above, M6 was found in the dose solution and considered as an impurity rather than a metabolite. It had a MH+ at m/z 857 with a similar isotope pattern as the parent but 12 Da lower. The MS/MS gave only one product at m/z 422 (data not shown) and no structure could be proposed solely based mass data.

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L. Chen et al.


Figure 7. MS/MS fragmentation of M2a.


spectrum

and

Discussion Faldaprevir is an inhibitor and a weak to moderate inactivator of CYP3A4 (Li et al., 2013; Sabo et al., 2012). As faldaprevir is also a substrate of CYP3A4 (Li et al., 2013), it may reduce its own metabolism after multiple dosing. As faldaprevir is planned to be dosed chronically (once daily), a human ADME study was conducted in that the disposition was assessed at steady state with a clinically relevant dose. Given the potential for auto-inhibition, the human ADME study also enabled metabolite profiling that is more relevant to the clinical situation than would be predicted from a single-dose study. This single-dose rat ADME study was performed to assess faldaprevir mass balance and biotransformation in rat and to provide justification for using rat as a toxicology species respective of metabolite exposure. Following a single iv administration of [14C]-faldaprevir, the plasma concentrations of radioactivity declined in a multiphasic manner with a terminal half-life of 1.75 h in male rats and 1.74 h in female rats. This relatively short half-life, together with the fact that plasma concentrations of radioactivity in whole blood and plasma could only be measured out to 8 h after dosing, indicates that there were no long-lived metabolites present in the systemic circulation. The plasma clearance of total radioactivity averaged 18.3 and 17.7 mL/ min/kg in male and female rats, respectively. This represents approximately 60% of the hepatic plasma flow in rats (55.2 mL/min/kg; Davies & Morris, 1993). After oral dosing, [14C]-faldaprevir was rapidly absorbed with a mean Cmax of 2 h. Plasma concentrations of total radioactivity were measurable out to 8 h after dosing in both male and female rats. The t1/2 for radioactivity in plasma (1.67 h for males and 1.77 h for females) was rather shorter compared to that in human plasma (22.3 h) at steady state (Chen et al., 2014). After iv and oral dosing, concentrations of total radioactivity in plasma were slightly higher (1.5-fold) than concentrations of total radioactivity in whole blood. This indicates that partitioning of [14C]-faldaprevir into blood cells was low and no significant radioactivity was associated with red blood cells. After oral administration of [14C]-faldaprevir at 10 mg/kg, feces represented the predominant excretion route in both

male and female rats. A similar excretion pattern was seen after iv administration of [14C]-faldaprevir at 2 mg/kg. Therefore, it appears that the rat gender and route of administration had little effect on the pattern of radioactivity excretion after administration of [14C]-faldaprevir. In both dose routes, very good recoveries of dosed radioactivity were obtained. One can estimate oral absorption by comparing the % dose recovered in urine from the oral dose group with that from the iv dose group. However, in this study, only trace amounts of radioactivity were recovered in urine, which made such a comparison less reliable. This excretion pattern also extended to the bile duct-cannulated rats after the oral dose of [14C]-faldaprevir where 34.3 and 55.0% dose was recovered in bile and feces with only 1.4% being found in urine. Faldaprevir was predominant (>96%) and very few metabolites were found in both the male and female rat plasma. In rat bile, however, faldaprevir glucuronidation was a major metabolic pathway as faldaprevir glucuronide (M4) was the most abundant metabolite and present at a similar level as the parent (Table 3). In the intestine, glucuronide is expected to be hydrolyzed back to the parent, contributing to a high level of faldaprevir in feces (>78% dose). It was expected that biliary excretion was responsible for the radioactivity in feces although intestinal excretion may have contributed as well. Unchanged faldaprevir and its metabolites, once formed in the liver, were transported into bile and then eliminated by biliary excretion. The metabolic pathways for faldaprevir in rats are shown in Figure 8. The major metabolic routes included hydroxylation, amide hydrolysis and conjugation with a glucuronic acid or glucose. Hydroxylation on the cyclopentyl ring resulted in the formation of M2a and M2b. Amide hydrolysis gave rise to metabolite M3, which could be further metabolized to M1. Direct glucose and glucuronic acid conjugation was responsible for the formation of M5 and M4, respectively. M4 underwent amide hydrolysis and produced M1. No gender differences in metabolic pathways were observed. Predominant fecal excretion (98.7% of dose) with minimal radioactivity recovered in urine (0.1% dose) was also seen in the human ADME study in which [14C]-faldaprevir was given

DOI: 10.3109/00498254.2014.920116


11

balance excretion study, where the administered radioactivity was rapidly eliminated within 24 h after an oral dose with approximately 87% of the dose recovered in feces from intact male rats and approximately 34% recovered in bile from BDC male rats though 48 h post-dose.

Conclusions


This study demonstrates that faldaprevir is well absorbed following oral administration and was excreted mainly in feces via bile. Overall elimination of total radioactivity following intravenous and oral administration was similar in male and female rats. Faldaprevir Phase-I metabolism was minimal but glucuronidation plays a major role in faldaprevir metabolism in rats. There were no gender differences in metabolite profiles of faldaprevir.

Acknowledgements

Figure 8. Metabolic pathways of faldaprevir in rats.

following eight daily doses of cold faldaprevir (Chen et al., 2014). The human ADME study revealed few metabolites present in human plasma. However, human feces, which represented the predominant excretion route as in rats, contained much higher levels of M2a (21.6% dose) and M2b (19.4% dose) in addition to unchanged parent drug (49.9% dose). At toxicological doses, both M2a and M2b were adequately represented in monkey feces (unpublished). The extent of glucuronidation in humans was also evaluated using in vitro microsomal and hepatocyte systems and was determined to be a minor pathway (10%; Ramsden et al., 2014). Biodistribution of radioactivity into tissues was widespread at 1 h after a single oral dose of [14C]-faldaprevir to male Long–Evans rats. Peak concentrations of radioactivity in most tissues occurred at 6 h post-dose. Radioactivity was rapidly eliminated from most tissues by 12 h post-dose, at which time the levels of radioactivity were below measurable concentrations. Excluding bile and GI tract contents, the highest levels of radioactivity in tissues were observed in liver, lung, kidney, small intestine and adrenal gland. Biodistribution across the blood–brain barrier did not occur after oral administration in either rat strain with only low levels measured in central nervous system tissues after intravenous administration to a Long–Evans male rat. Bile concentrations of radioactivity were substantially greater than those observed in urine indicating that hepatic clearance and biliary excretion played an important role in the elimination of [14C]-faldaprevir-derived radioactivity whereas urinary excretion was a minor elimination route in male rats. After iv administration to a male Long–Evans rat, the biodistribution was similar to the oral data confirming substantial amounts of radioactivity present in bile as early as 0.083 h post-dose. For the SD male rat at the first sampling time of 24 h post-dose, all tissues were devoid of radioactivity with only measurable amounts present in the cecum and large intestinal contents. These results are consistent with the mass

The authors would like to thank Dr. Bachir Latli for synthesis and purification of [14C]-faldaprevir. We also thank Drs. Jeff Duggan, Donald Tweedie, and Yongmei Li for their valuable input. The in-life part of mass balance study and most metabolite profiling and identification was carried out at XenoBiotic Laboratories, Plainsboro, NJ. The in-life tissue distribution study and QWBA analysis were conducted by Covance Laboratories Inc., Madison, WI.

Declaration of interest LC, RSG, SHN and YM are EP are employees of Boehringer Ingelheim Pharmaceuticals, LQW and DW are employees of XenoBiotic Laboratories, and MJP is an employee of Covance Laboratories. This study was financially supported by Boehringer Ingelheim Pharmaceuticals, Inc. The authors alone are responsible for the content and writing of the paper.

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