Abstract
The anti-hepatitis C virus nucleotide prodrug GS-6620 employs a double-prodrug approach, with l-alanine-isopropyl ester and phenol moieties attached to the 5′-phosphate that release the nucleoside monophosphate in hepatocytes and a 3′-isobutyryl ester added to improve permeability and oral bioavailability. Consistent with the stability found in intestinal homogenates, following oral administration, intact prodrug levels in blood plasma were the highest in dogs, followed by monkeys, and then were the lowest in hamsters. In contrast, liver levels of the triphosphate metabolite at the equivalent surface area-adjusted doses were highest in hamsters, followed by in dogs and monkeys. Studies in isolated primary hepatocytes suggest that relatively poor oral absorption in hamsters and monkeys was compensated for by relatively efficient hepatocyte activation. As intestinal absorption was found to be critical to the effectiveness of GS-6620 in nonclinical species, stomach pH, formulation, and food effect studies were completed in dogs. Consistent with in vitro absorption studies in Caco-2 cells, the absorption of GS-6620 was found to be complex and highly dependent on concentration. Higher rates of metabolism were observed at lower concentrations that were unable to saturate intestinal efflux transporters. In first-in-human clinical trials, the oral administration of GS-6620 resulted in poor plasma exposure relative to that observed in dogs and in large pharmacokinetic and pharmacodynamic variabilities. While a double-prodrug approach, including a 3′-isobutyryl ester, provided higher intrinsic intestinal permeability, this substitution appeared to be a metabolic liability, resulting in extensive intestinal metabolism and relatively poor oral absorption in humans.
INTRODUCTION
Approximately 170 million people worldwide are infected by the hepatitis C virus (HCV), and nearly 2% of the U.S. population is estimated to have HCV (1). HCV is a major health issue since approximately 15 to 35% of those who are chronically infected will develop cirrhosis and 1 to 3% will progress to hepatocellular carcinoma over 30 years (2). Currently, the recently approved protease inhibitors (boceprevir and telaprevir) are used in combination with pegylated interferon alpha plus ribavirin (PEG-RBV) for the treatment of chronic HCV infection. While these treatments show improved response rates and the potential for shorter duration of treatment over PEG-RBV alone, they lack efficacy against genotypes other than genotype 1, require thrice-daily dosing, and cause additional side effects from the new direct-acting antivirals on top of the already-challenging tolerability profile of PEG-RBV. Therefore, there is a need for more potent anti-HCV compounds with improved clinical efficacy and greater tolerability in order to more broadly address the unmet medical needs of those with chronic HCV infection. Combinations of antiviral agents targeting viral proteins essential to HCV replication have the potential to achieve increased clinical efficacy across HCV genotypes, have fewer adverse effects, and shorten treatment duration compared to the current standard of care.
GS-6620 is a prodrug of 1′-cyano-2′-C-methyl-4 aza-7,9-dideaza adenosine-5′-monophosphate, which is a potent and selective pangenotype inhibitor of HCV replication (3; see also reference 4 [an accompanying paper]). GS-6620 requires metabolic activation to its triphosphate form, GS-441326, to inhibit the HCV nonstructural protein 5B (NS5B) RNA polymerase. GS-441326 competes with endogenous ATP for incorporation, and once incorporated, results in nonobligate chain termination of viral transcripts (4). When administered by itself, the nucleoside analog lacks antiviral activity due to the inability of cellular nucleoside kinases to phosphorylate it to the monophosphate form. Therefore, a nucleotide prodrug was pursued containing amino acid ester and aryl moieties attached to the 5′-phosphate of the nucleoside analog in order to deliver the monophosphate analog to the cells. While strong antiviral activity was achieved in vitro with this nucleotide prodrug approach, poor oral absorption was observed. Notably, the addition of an ester to the 3′ position of the ribose ring was found to increase the intrinsic permeability. Optimization resulted in the selection of l-alanine-isopropyl ester and phenol moieties for the 5′-phosphate and a 3′-isobutyryl ester (3).
Here, we report the in vitro metabolic and animal pharmacokinetic profiles of GS-6620. The studies we conducted to characterize the metabolic profile of GS-6620 include those regarding: (i) metabolism in primary hepatocytes isolated from hamsters, dogs, monkeys, and humans, (ii) the molecular mechanism of intracellular activation, (iii) stability in plasma and intestinal and hepatic subcellular fractions, (iv) blood plasma and liver pharmacokinetics in golden Syrian hamsters, beagle dogs, and cynomolgus monkeys, and (v) the characterization of factors affecting intestinal absorption. These nonclinical results are used to help interpret findings from the recently reported first-in-human study of GS-6620 in treatment-naive subjects chronically infected with HCV genotype 1 (5).
MATERIALS AND METHODS
Materials.
GS-6620, GS-465124, GS-566650 (metabolite X), GS-441285, GS-558272, and GS-441326 were synthesized at Gilead Sciences, Inc. (Foster City, CA). Metabolite A was enzymatically prepared using GS-6620 and cathepsin A (CatA). The analytical internal standards 5-iodotubercidin and chloro-ATP and all other reagents were of the highest grade available from Sigma-Aldrich. CatA was purified from peripheral blood mononuclear cells (PBMCs), according to a previously published procedure (6). Carboxylesterases 1 and 2 (CES1 and CES2, respectively) were purchased from R&D Systems (Minneapolis, MN), and histidine triad nucleotide binding protein 1 (HINT1) was kindly provided by C. Wagner (University of Minnesota, Minneapolis, MN). Hamster, rat, dog, monkey, and human blood plasma samples were obtained from Bioreclamation (Liverpool, NY). Intestinal and hepatic S9 fractions from hamsters, rats, dogs, monkeys, and humans were obtained from Celsis In vitro Technologies (Baltimore, MD). Primary hepatocytes from humans, hamsters, dogs, and monkeys were purchased from Celsis In vitro Technologies in 12-well plates seeded at confluence (0.88 × 106 cells/well).
Enzyme assays.
Compound hydrolysis by CatA was measured in a reaction buffer containing 25 mM morpholineethanesulfonic acid (MES) (pH 6.5), 100 mM NaCl, 1 mM dithiothreitol (DTT), 0.1% NP-40, and 1 μg/ml enzyme at 37°C. CES1 and CES2 (5 μg/ml) were assayed in 50 mM HEPES buffer (pH 7.2). The hydrolytic reactions with HINT1 (250 nM) were performed in 20 mM HEPES buffer (pH 7.2) containing 1 mM DTT, 1 mM MgCl2, and 200 ng/ml bovine serum albumin (BSA). The reactions were initiated by adding substrates to the reaction buffers to a final concentration of 30 μM. At various time points, 100-μl aliquots were collected from the reaction mixtures and 200 μl of ice-cold methanol was added to stop the reactions. The samples were incubated at −20°C for 30 min and spun at 13,000 rpm for 30 min at 4°C to remove the denatured proteins. The supernatants were evaporated, resuspended in 100 μl buffer A (25 mM potassium phosphate [pH 6.0], 5 mM tetrabutylammonium bromide) and injected onto a C18 reverse-phase column (5 μm, 2.1 by 100 mm, octadecyl silica 2 [ODS-2]; Phenomenex, Torrance, CA) equilibrated with buffer A. The substrates and reaction products were eluted using a linear gradient of acetonitrile (0% to 65%, 10 min, 0.25 ml/min) in buffer A.
Overexpression of CatA, CES1, CES2, HINT2, and HINT3 in 293T cells.
For transient transfection, human embryonic kidney 293T cells (ATCC, Manassas, VA) were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and seeded into 24-well plates coated with poly-d-lysine (BD Biosciences, San Jose, CA) at 1.5 × 105 cells/well. Transfection was performed the next day with 0.5 μg pcDNA3.1 or pcDNA3.1-CatA, pcDNA3.1-Ces1, pcDNA3.1-Ces2, pcDNA3.1-Hint2, or pcDNA3.1-Hint3 (OriGene Technologies, Rockville, MD) expression plasmids using 1 μl/well TransIT-293 transfection reagent (Mirus, Madison, WI). The expression levels of CatA, CES1, CES2, HINT2, and HINT3 were verified by Western blotting using a goat anti-CatA (R&D Systems, Minneapolis, MN), rabbit anti-HINT2 and anti-HINT3 (Sigma-Aldrich, St. Louis, MO), and custom rabbit anti-CES1 and anti-CES2 polyclonal antibodies (Life Sciences, Grand Island, NY).
Metabolism of GS-6620 in transiently transfected 293T cells.
Metabolic assays were performed 48 h after transfection. The cells were incubated with 10 μM [3H]-GS-6620 (0.5 μCi/ml) for 45 and 90 min in DMEM with 10% FBS. Following incubation, the medium was collected and precipitated with four volumes of ice-cold 100% methanol. The cells were washed with phosphate-buffered saline (PBS) and detached by incubation with trypsin-EDTA in PBS. The trypsin was neutralized with medium, and the cells were harvested by centrifugation for 5 min at 1,500 rpm (Beckman GPR, 4°C). The cell pellets were washed with 4°C PBS, and the GS-6620 metabolites were extracted by adding 400 μl of 80% methanol, which was stored in −20°C until use. The cell extracts and precipitated medium were centrifuged at 13,000 × g for 30 min at 4°C, and the supernatants were evaporated under a vacuum and resuspended in 100 μl of buffer A (25 mM phosphate buffer [pH 6] and 5 mM tetrabutylammonium bromide). Aliquots of the cell extracts were analyzed for their 3H-radioactivity content following the addition of 5.0 ml Ready Safe scintillation fluid (Beckman Instruments, Fullerton, CA).
The column chromatography was performed using a Prodigy column (5 μm, octadecyl silica 3 [ODS-3], 150 by 4 mm; Phenomenex) on a Waters high-performance liquid chromatography (HPLC) system connected to a Radiomatic Flo-One/beta liquid scintillation detector (Packard series A-500). A gradient elution from buffer A (flow rate, 1.0 ml/min at 40°C) to 52% acetonitrile in buffer A for 30 min was followed by a 5-min column wash with 65% acetonitrile in buffer A and used to separate the GS-6620 metabolites. The amount of metabolites was calculated from the calibration curve for GS-6620. The retention times for the metabolites were: GS-441285, 10 min; metabolite X, 17.4 min; GS-441326, 21.3 min; metabolite A, 22.8 min; GS-465124, 27 min; and GS-6620, 32.2 min.
Stability in blood plasma and S9 fractions.
For blood plasma stability, GS-6620 or GS-465124 was incubated at 2 μM in hamster, rat, dog, monkey or human plasma samples for up to 4 h at 37°C. At the desired time points, an aliquot from the incubation was quenched by adding 9 volumes of 100% acetonitrile supplemented with internal standard. Following the last collection, the samples were centrifuged at 3,000 × g for 30 min and the supernatants were transferred to a new plate containing an equal volume of water for analysis by liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS).
For S9 stability, GS-6620 or GS-465124 was incubated at 2 μM in hamster, rat, dog, monkey, or human intestinal and hepatic S9 fractions for up to 90 min at 37°C in the presence of NADPH and uridine 5′-diphosphoglucuronic acid trisodium salt (UDPGA) (Sigma-Aldrich). At the desired time points following compound addition, the samples were quenched with 9 volumes of an aqueous solution containing internal standard, 50% acetonitrile, and 25% methanol. The sample plates were centrifuged at 3,000 × g for 30 min, and 10 μl of the resulting solution was analyzed by LC-MS/MS.
The data (analyte-to-internal standard peak area ratio) were plotted on a semi-log scale and fitted using an exponential fit. The half-life (t1/2) was determined assuming first-order kinetics.
Bidirectional permeability assay.
Caco-2 cells were maintained in DMEM with sodium pyruvate, GlutaMax supplemented with 1% penicillin-streptomycin, 1% nonessential amino acids, and 10% FBS in an incubator set at 37°C, 90% humidity, and 5% CO2. Caco-2 cells between passage 43 and 71 were grown to confluence over ≥21 days on 12- or 24-well polyethylene terephthalate plates (BD Biosciences). Experiments were conducted using Hanks' balanced salt solution (HBSS) buffer containing 10 mM HEPES and 15 mM glucose (Invitrogen). The donor buffers had their pH levels adjusted to pH 6.5. The receiver well used HBSS buffer supplemented with 1% BSA. The receiver buffers had their pH levels adjusted to pH 7.4. After an initial equilibration with transport buffer, transepithelial electrical resistance (TEER) values were read to test membrane integrity. The experiment was started by adding buffers containing test compounds, and 100 μl of solution was taken at 1 and 2 h from the receiver compartment. The removed buffer was replaced with fresh buffer and a correction applied to all calculations for the removed material. The compound was tested in 2 separate replicate wells for each condition. All samples were immediately collected into 400 μl 100% acetonitrile to precipitate protein and stabilize test compounds. The cells were dosed on the apical (A) or basolateral (B) side to determine forward (A to B) and reverse (B to A) permeability. Permeability through a cell-free transwell was also determined as a measure of cellular permeability through the membrane. To test for nonspecific binding and compound instability, the total amount of drug was quantified at the end of the experiment and compared to the material present in the original dosing solution as the percent recovery. The samples were analyzed by LC-MS/MS. For experiments assessing the effects of efflux transporter inhibitors, the cells were preincubated with 30 μM cobicistat or 10 μM cyclosporine for 30 min prior to the addition of 10 μM GS-6620.
Kinetic solubility assay.
A 10-mM test compound stock solution in dimethyl sulfoxide (DMSO) was diluted to 100 μM with PBS (pH 7.0) or 0.1 N HCl (pH 1.0). The samples were incubated at room temperature or at 37°C for 1 h, followed by centrifugation at 2,800 relative centrifugal force (RCF) for 30 min at room temperature. The concentration of the compound in the supernatant was determined by a Waters Alliance 2795 HPLC system equipped with Waters photodiode array detector 2996 using an ODS-AQ, 5 μl, 120 Å, 2.0 by 50 mm C18 column (YMC America, Inc., Allentown, PA).
Animal pharmacokinetics.
Golden Syrian hamster studies were conducted at NoAb BioDiscoveries (Mississauga, Ontario, Canada). The animals were housed and handled in accordance with the principles of the Canadian Council on Animal Care (CCAC), and the protocols were reviewed and approved by NoAb BioDiscoveries. Animals weighing 100 to 120 g were used for the in vivo portion of the studies. Each compound was dosed orally by gavage to male golden Syrian hamsters (Charles River Laboratories, St. Constant, Quebec, Canada) in 5% ethanol, 55% polyethylene glycol (PEG) 400, and 40% 50 mM citrate buffer. The oral doses ranged from 10 to 11 mg/kg of body weight. Blood plasma samples were collected at 0.5, 1, 4, 8, and 24 h postadministration, and liver samples were collected at 1, 4, 8, and 24 h postadministration.
Beagle dog studies were conducted at MPI Research, Inc. (Mattawan, MI). The animals were housed and handled in accordance with the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources. The protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of MPI Research, Inc. before the initiation of treatment. Animals weighing 10 to 12 kg were used for the in vivo portion of the studies. The compound was dosed orally at 5 mg/kg of body weight as in aqueous solution and suspension form, or at a 100-mg fixed dose in tablet form by gavage to male beagle dogs (Covance Research Products, Kalamazoo, MI). The oral solution formulation was made up in 20% polyethylene glycol, 19.5% propylene glycol, 5% ethanol, and 0.5% polysorbate 80 in 50 mM citrate buffer. The oral tablet formulation contained 20% GS-6620, 41.5% lactose monohydrate, 30.25% microcrystalline cellulose, 5% sodium croscarmellose, 2% hydroxypropyl cellulose, 0.5% polysorbate 80, and 0.75% magnesium stearate. The oral capsule contained 30% GS-6620, 35% octanoic acid, and 35% decanoic acid (wt/wt). The animals were pretreated with pentagastrin for ∼20 min or famotidine for 1 h prior to the dosing. Plasma samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h postadministration, except in the animals dosed with the solution formulation, in which plasma samples were collected at 0.25, 0.5, 1, 2, and 4 h postadministration. At each terminal collection, the animal was anesthetized under isoflurane and a section of liver (approximately 1 to 2 g in size) was harvested. Liver samples were collected at 4, 8, and 24 h postadministration from the animals dosed with tablets, and liver samples were collected at 4 h postadministration from the animals dosed with the solution formulation. A diastereomeric mixture containing GS-6620 (5 mg/kg) was dosed with the solution formulation to the animals without any pretreatments.
Cynomolgus monkey studies were conducted at MPI Research, Inc. (Mattawan, MI) and Charles River Laboratories (Senneville, QC, Canada). The animals were housed and handled in accordance with the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources. The protocols were reviewed and approved by IACUC of the contract labs. Animals weighing 4 to 6 kg were used for the in vivo portion of the studies. GS-6620 was dosed orally as a suspension in 0.5% hydroxypropyl methylcellulose (HPMC), 0.2% polysorbate 80, 0.9% benzyl alcohol, and 98.4% water at 3.86 mg/kg of body weight by gavage to male cynomolgus monkeys. Plasma samples were collected at 1, 2, 4, 6, 8, 12, and 24 h postadministration, and liver samples (approximately 1 to 2 g in size) were collected at 1, 2, 4, 8, 12, and 24 h postadministration.
The plasma samples from pharmacokinetic studies were subject to protein precipitation by the addition of acetonitrile to final concentrations of 75% containing 5-iodotubercidin as internal standards. The analytes in the plasma samples were separated on a 4-μm 150 by 2 mm Synergi Max-RP column (Phenomenex, Torrance, CA) using mobile phase containing 0.2% formic acid and a linear gradient from 2% to 100% acetonitrile at a flow rate of 250 μl/min over 7 min. Eight-point standard curves prepared in blank plasma samples covered concentrations from 5.1 to 5,000 nM and showed linearity in an R2 value of >0.99. Separately prepared quality-control samples of 120 and 3,000 nM in plasma were analyzed at the beginning and end of each sample set to ensure accuracy and precision within 20%.
The nucleoside triphosphate quantification used an ion-pairing nucleotide detection LC-MS/MS method. The analytes were separated by a 2.5-μm 2.0 by 50 mm Luna C18 column (Phenomenex, Torrance, CA) using an ion-pairing buffer containing 3 mM ammonium phosphate (pH 5) with 10 mM dimethylhexylamine (DMH) and a multistage linear gradient from 10% to 50% acetonitrile at a flow rate of 160 μl/min over 11 min. Seven-point standard curves prepared in blank matrices covered concentrations from 24.0 to 17,500 nM and showed linearity in an R2 value of >0.99.
Dose normalization was performed based on body surface area according to FDA guidance (7). Specifically, doses given to hamsters (10.6 mg/kg), monkeys (3.86 mg/kg), and dogs (5 mg/kg) corresponded to human equivalent doses of 1.38 mg/kg, 1.24 mg/kg, and 2.78 mg/kg, respectively. Since the human equivalent dose for dogs was approximately 2-fold higher than that for hamsters or monkeys, the results were dose normalized to 2.5 mg/kg.
Metabolism in primary hepatocytes.
The cells were incubated with GS-6620 at 10 μM for 2 h, followed by 22 h in compound-free medium. Following the removal of extracellular medium at select time points (1, 2, 3, 6, 12, and 24 h), the cells were washed twice with 2 ml of ice-cold 0.9% normal saline and scraped into 0.5 ml ice-cold 70% methanol containing 100 nM 2-chloro-adenosine 5′-triphosphate (Sigma-Aldrich, St. Louis, MO) as an internal standard. The samples were stored overnight at −20°C to facilitate nucleotide extraction, centrifuged at 15,000 × g for 15 min, and then the supernatant was transferred to clean tubes for drying in a miVac Duo concentrator (Genevac, Gardiner, NY). The dried samples were then reconstituted in mobile phase A containing 3 mM ammonium formate (pH 5.0) with 10 mM DMH in water for analysis by LC-MS/MS. The analytes were separated using a 50 by 2 mm by 2.5 μ Luna C18(2) HST column (Phenomenex, Torrance, CA) connected to an LC-20ADXR (Shimadzu, Columbia, MD) ternary pump system and HTS PAL autosampler (LEAP Technologies, Carrboro, NC). A multistage linear gradient from 10% to 50% acetonitrile in mobile phase A at a flow rate of 150 μl/min was used to separate the analytes. The analytes were quantified using a 7-point standard curve ranging in concentration from 0.137 to 100 pmol/million cells prepared in cell extract from untreated primary animal hepatocytes.
LC-MS/MS instrumentation.
Pharmacokinetic samples were analyzed using an HTS PAL autosampler with cooled sample storage stacks set to 10°C (Leap Technologies, Carrboro, NC) and an LC-20ADXR ternary pump system (Shimadzu Scientific Instruments, Columbia, MD). An HPLC system was coupled to a Sciex API 4000 mass spectrometer (Applied Biosystems, Foster City, CA). Mass spectrometry was performed in positive-ion and multiple reaction monitoring mode (MRM). In vitro stability assay samples were analyzed using an HTS PAL autosampler with cooled sample storage stacks set to 6°C (Leap Technologies, Carrboro, NC) and a 1200 series quaternary pump system (Agilent Technologies, Santa Clara, CA). Stability assay LC-MS/MS analyses were done in positive-ion and multiple reaction monitoring modes using a Quattro Premier mass spectrometer (Waters, Milford, MA). The analytes were retained using a 2-μm 20 by 2.1 mm Mercury RP C18 column (Phenomenex, Torrance, CA), maintained at 60°C with a mobile phase flowing at 1.0 ml/min, consisting of 0.2% formic acid and a linear gradient from 0 to 85% acetonitrile over 1 min.
The noncompartmental pharmacokinetic parameters were calculated using WinNonlin 5.01 (Pharsight Corporation, Mountain View, CA).
RESULTS
Intracellular activation of GS-6620 in primary hepatocytes.
In order to inhibit the HCV RNA polymerase in hepatocytes, GS-6620 must be metabolized to its triphosphate form (GS-441326). Therefore, in vitro metabolism studies were conducted in primary hepatocytes from hamsters, dogs, monkeys, and humans to characterize the intracellular activation pathway and assess potential species differences in intracellular activation. Experiments were performed by conducting a continuous incubation with 10 μM GS-6620 for 2 h, followed by the replacement with medium not containing the compound and the monitoring of intracellular concentrations of the metabolites over 24 h. The time-dependent metabolite formation in primary human hepatocytes is shown in Fig. 1A. The structures of metabolite X, GS-558272, GS-639477, and GS-441326 are shown in Fig. 2. The highest concentrations of the key alaninyl phosphate intermediate metabolite, metabolite X, were observed at 1 h, the earliest time point collected, and declined rapidly thereafter. Relatively low levels of the nucleoside analog monophosphate, GS-558272, were formed over the 24-h time course, suggesting rapid phosphorylation by nucleotide kinases. Moderate levels of the nucleoside analog diphosphate, GS-639477, were formed over the 2-h incubation period and persisted for 4 h after the removal of GS-6620 from the medium, followed by a slow decline. The pharmacologically active nucleoside analog triphosphate, GS-441326, was the predominant metabolite in human hepatocytes and reached its maximum concentration at 3 h. As described in Fig. 1B and Table 1, significant species differences were observed in the GS-441326 levels in the primary hepatocytes from hamsters, dogs, monkeys, and humans. The highest GS-441326 concentrations were observed in hamster hepatocytes, with a maximum concentration (Cmax) of 543 pmol/million cells, followed by monkey hepatocytes with 233 pmol/million cells. The GS-441326 levels in dog and human hepatocytes were similar, with 99 and 133 pmol/million cells, respectively (Fig. 1B and Table 1). The half-life of GS-441326 was similar across species, ranging from 4.7 to 5.8 h. In addition, the overall kinetics of formation and decline for other metabolites were similar across species (data not shown).
FIG 1.
Intracellular activation of GS-6620 in primary hepatocytes. (A) Intracellular metabolites, metabolite X (GS-566650) (○), GS-558272 (♢), GS-639477 (□), and GS-441326 (△) formed by GS-6620 in primary human hepatocytes. (B) Intracellular formation of the active triphosphates in primary hepatocytes from hamsters (▲), dogs (◆), monkeys (●), and humans (■). The cells were incubated with 10 μM GS-6620 for 2 h before the compound was removed by replacing the medium. The graphs were generated by averaging each time point from the number of experiments shown in Table 1. The Cmax and t1/2 in panel B may not match exactly with the numbers in Table 1, which are averages of the individually determined values.
FIG 2.
Proposed metabolic activation pathway of GS-6620. The enzymes involved in the pathway are based on the results shown in Table 2. The primary intracellular activation pathway was proposed to proceed from GS-6620 to GS-465124, to metabolite X, and to GS-558272. The formation of metabolite B was not observed in our biochemical assays.
TABLE 1.
Intracellular activation of GS-6620 in primary hepatocytes from hamsters, dogs, monkeys, and humans
| Hepatocyte type | Triphosphate metabolite |
n | |
|---|---|---|---|
| Cmax (pmol/million) | t1/2 (h) | ||
| Hamster | 543 ± 134 | 4.7 ± 0.4 | 2 |
| Dog | 99 ± 57 | 5.8 ± 0.2 | 2 |
| Monkey | 233 ± 187 | 5.1 ± 2.2 | 2 |
| Human | 133 ± 63 | 5.3 ± 0.6 | 3 |
Cmax and t1/2 values were determined from each experiment and reported as means ± standard deviations.
Activation pathway.
The proposed activation pathway of GS-6620 is shown in Fig. 2. The first step of GS-6620 activation is either hydrolysis of the isopropyl ester in the 5′-phosphoramidate or the 3′-isobutyryl ester. The hydrolysis of these ester bonds was evaluated using human cathepsin A (CatA), carboxylesterase 1 (CES1), and carboxylesterase 2 (CES2). The results demonstrated that CatA and CES1 but not CES2 were able to catalyze the cleavage of the 5′-ester to form metabolite A. On the other hand, only CES2 was able to hydrolyze the 3′-ester to form the des-3′-isobutyryl metabolite, GS-465124 (Table 2). Interestingly, further metabolism of the des-isopropyl metabolite, metabolite A, was not observed in our biochemical assays, while GS-465124 was hydrolyzed to metabolite X by both CES1 and CatA. Although some other unidentified enzymes may be able to hydrolyze metabolite A to either metabolite X and/or the putative nucleoside analog monophosphate 3′-isobutyryl metabolite, metabolite B, we propose that the major activation pathway involves the hydrolysis of the 3′-ester by CES2, followed by the hydrolysis of the 5′-isopropyl ester by CatA or CES1.
TABLE 2.
Activities of enzymes involved in GS-6620 activationa
| Substrate | Product | Enzyme | Specific activity (nmol · min−1 · mg−1)b |
|---|---|---|---|
| GS-6620 | GS-465124 | CatA | No activity |
| CES1 | No activity | ||
| CES2 | 62 ± 2.4 | ||
| GS-6620 | Metabolite A | CatA | 9,834 ± 1,800 |
| CES1 | 5 ± 1.4 | ||
| CES2 | No activity | ||
| GS-465124 | Metabolite X | CatA | 770 ± 180 |
| CES1 | 19 ± 5.6 | ||
| Metabolite A | Metabolite X | CES2 | No activity |
| Metabolite X | GS-558272 | HINT1 | 129 ± 25 |
| Metabolite A | Metabolite B | HINT1 | No activity |
All reactions done with substrate concentrations of 30 μM.
Results were obtained from at least 3 independent experiments and reported as means ± standard deviations.
The involvement of CatA, CES1, and CES2 in GS-6620 metabolism was further evaluated in 293T cells overexpressing these hydrolases. CES1, CES2, and CatA were overexpressed by approximately 15-, 100-, and 20-fold compared to mock-transfected cells, based on Western blot analysis, respectively (data not shown). Total intracellular metabolites were monitored following the incubation of the cells with GS-6620 for 45 min or 90 min. In CES1- or CatA-overexpressed cells, approximately 2- to 3-fold-more intracellular metabolites were formed relative to the mock-transfected control cells, whereas the overexpression of CES2 did not significantly affect the levels of the total intracellular metabolites. Increased levels of metabolites were found in the medium of the cells overexpressing CatA, CES1, and CES2, likely due to efflux of the metabolites (Fig. 3). These results strongly suggest that these three hydrolases are involved in the metabolism of GS-6620.
FIG 3.
Effect of overexpression of enzymes involved in metabolism of GS-6620. CES1-, CES2-, or CatA-overexpressing cells were incubated with 10 μM [3H] GS-6620 for 45 and 90 min, and GS-6620 metabolites in medium and cells were analyzed by HPLC-LS. The efficiency of protein overexpression was monitored by quantitative Western blots.
Histidine triad nucleotide binding protein 1 is a member of the HINT family and has been reported to hydrolyze various phosphoramidates (8–11). The alaninyl phosphate metabolite, metabolite X, was a substrate for HINT1, with a specific activity of 450 nmol/min · mg. The ability of other HINT proteins to catalyze the reaction was assessed in HINT2- or HINT3-overexpressed 293T cells, and the results indicated that in addition to HINT1, HINT2 may be involved in metabolite X metabolism while HINT3 is not (data not shown). The monophosphate metabolite GS-558272 is then consecutively phosphorylated to the diphosphate, GS-639477, and the triphosphate, GS-441325, forms by host kinases. GS-558272 can be dephosphorylated to the nucleoside GS-441285, which was detected in the following pharmacokinetic studies.
Stability in plasma and S9 fractions.
CES1 is predominantly expressed in the liver and CES2 is highly expressed in the small intestine and liver (12–14). CatA is known to be ubiquitously expressed across different tissues (15, 16). Therefore, evaluating the stabilities of GS-6620 and its metabolites during intestinal absorption, hepatic extraction, and systemic circulation is important in understanding their metabolism and pharmacokinetics in vivo. The stabilities of GS-6620 and GS-465124 were measured in blood plasma, intestinal S9, and hepatic S9 fractions from hamsters, dogs, monkeys, and humans in vitro. GS-6620 and GS-465124 were stable in plasma from all species (t1/2 ≥ 80 min; Table 3). In intestinal S9 fractions, GS-6620 was relatively unstable across all the species tested; however, GS-465124 was significantly more stable than GS-6620, particularly in human and dog intestinal S9 (Table 3). Both GS-6620 and GS-465124 were unstable in hepatic S9, with half-lives of ≤25 min across species, indicating that these compounds are efficiently metabolized in hepatocytes.
TABLE 3.
In vitro stability of GS-6620 and GS-465124 in plasma and S9 fractions from hamsters, dogs, monkeys, and humans
| Biological matrix | Compounda | Stability (t1/2 [min])b |
|||
|---|---|---|---|---|---|
| Hamsters | Dogs | Monkeys | Humans | ||
| Plasma | GS-6620 | 124 ± 4 | 429 ± 86 | 80 ± 9 | 371 ± 4 |
| GS-465124 | 345 ± 160 | 662 ± 162 | NDc | 360 ± 32 | |
| Intestinal S9 | GS-6620 | <2 | 33 ± 3 | 5.6 ± 0.3 | 17 ± 1 |
| GS-465124 | 2.6 ± 0.5 | 343 ± 39 | ND | 261 ± 5 | |
| Hepatic S9 | GS-6620 | <2 | 12 ± 1 | 5.7 ± 0.1 | 4.0 ± 0.2 |
| GS-465124 | 2.6 ± 0.5 | 25 ± 2 | ND | 5.3 ± 0.2 | |
Compounds were incubated at 2 μM.
Data are means ± standard deviations (n = 2).
ND, not determined.
Bidirectional permeability in Caco-2 cells.
In order to assess the intestinal absorption potential of GS-6620, the concentration-dependent bidirectional permeability of GS-6620 was examined using Caco-2 monolayers. GS-6620 demonstrated dose-dependent forward and reverse permeability across the range of concentrations tested (Fig. 4A). Saturable efflux transport was observed, ranging from an efflux ratio of 11.4 at 1 μM to 1.62 at 100 μM.
FIG 4.
Concentration and efflux transport inhibitor-dependent permeability of GS-6620 through monolayers of Caco-2 cells. (A) Concentration-dependent permeability was determined using 1, 10, and 100 μM GS-6620. (B) Effects of efflux inhibitors, cobicistat (30 μM), or cyclosporine (CsA) (10 μM) on permeability were assessed using 10 μM GS-6620. The white and black bars indicate forward (apical to basolateral) and reverse (basolateral to apical) permeability, respectively. The numbers above the bars indicate the efflux ratios.
The pharmacoenhancer cobicistat is a known inhibitor of intestinal efflux transporters P-glycoprotein (Pgp) and breast cancer-resistant protein (BCRP) (17). The effects of cobicistat and another known transport inhibitor, cyclosporine (CsA), on GS-6620 permeability were tested. In the presence of either 30 μM cobicistat or 10 μM CsA, the efflux ratio was reduced to unity, and the forward permeability of GS-6620 was increased approximately 2-fold (Fig. 4B).
Plasma pharmacokinetics.
Hamsters, dogs, and monkeys were orally dosed at 10.6, 5.0, and 3.86 mg/kg, respectively, and the plasma levels of GS-6620, GS-465124, and the nucleoside metabolite, GS-441285, were determined. Taking the body surface area into account (see Materials and Methods), equivalent doses were given to hamsters and monkeys, and an approximately 2-fold-higher equivalent dose was given to dogs. Therefore, dose-normalized values were reported for dogs (Table 4). The GS-6620 plasma pharmacokinetic (PK) profiles are shown in Fig. 5A. GS-6620 was rapidly absorbed, with time to maximum concentration of drug in serum (Tmax) values within 1.0 h for all species. Exposures to GS-6620 and GS-465124 were transient (<20 min), followed by more persistent exposure to GS-441285. GS-441285 was the predominant metabolite in all species tested, accounting for >70% of the total exposures. There were large differences in the exposures to GS-6620 and its metabolite GS-465124 across species. Dose-normalized Cmax values were 2.5- and 12.5-fold higher in dogs than in monkeys or hamsters, respectively.
TABLE 4.
Pharmacokinetics of GS-6620, GS-465124, and GS-441285 following a single oral dose of GS-6620 to hamsters, monkeys, and dogs
| Species | Dose (mg/kg)a | Parameter | GS-6620 | GS-465124 | GS-441285 |
|---|---|---|---|---|---|
| Hamster | 10.6 | t1/2 (h) | NCb | NC | 16.71 |
| Cmax (μM) | 0.02 | 0.02 | 0.71 | ||
| Tmax (h) | 0.50 | 0.50 | 5.5 | ||
| AUC0–t (μM · h) | NC | NC | 9.87 | ||
| Monkey | 3.86 | t1/2 (h) | NC | NC | 3.36 |
| Cmax (μM) | 0.10 | 0.10 | 0.15 | ||
| Tmax (h) | 1.0 | 1.0 | 4.0 | ||
| AUC0–t (μM · h) | NC | NC | 1.13 | ||
| Dog | 2.5c | t1/2 (h) | 0.29 | 0.31 | 10.22 |
| Cmax (μM) | 0.25 | 0.25 | 0.43 | ||
| Tmax (h) | 0.38 | 0.50 | 3.0 | ||
| AUC0–t (μM · h) | 0.13 | 0.15 | 1.36 |
Human equivalent dose of approximately 1.4 mg/kg.
NC, not calculable due to limited number of quantifiable data.
Dose normalized based on body surface area. The dogs were dosed at 5 mg/kg in the experiment.
FIG 5.
Plasma and liver PK profiles of GS-6620. (A) Plasma PK following a single oral dose of GS-6620 to hamsters, dogs, and monkeys at 10.6, 2.5, and 3.86 mg/kg, respectively. (B) Effects of formulation, stomach pH, or feeding condition on GS-6620 plasma PK following a 100-mg oral dose of GS-6620 to dogs. (C) Liver PK of GS-441326 following a single oral dose of GS-6620 in a solution form to hamsters and dogs, a suspension form to monkeys, or a tablet form to dogs with or without food.
Effect of different formulations, stomach pH, and food on plasma exposure to GS-6620.
Because of their relatively similar in vitro stability and metabolism profiles, dogs were chosen as an animal model for predicting human PK and doses. Therefore, the effects of different formulations, stomach pH, and feeding conditions on plasma exposure to GS-6620 were assessed in dogs (Table 5 and Fig. 5B). In order to assess the stomach pH effects, pentagastrin and famotidine were used, which acidify and alkalize gastric fluid, respectively. When GS-6620 was dosed to fasted dogs in a solution form, pentagastrin did not affect the plasma exposure to GS-6620. The 100-mg clinical tablet performed similarly to the solution in pentagastrin pretreated fasted dogs. Poor exposure was observed under fed conditions or when fasted animals were pretreated with famotidine. These results are consistent with our solubility data, in which GS-6620 in its final dosage form showed poor solubility at a neutral pH (5 μg/ml or 10 μM at pH 7.0) and high solubility at an acidic pH (>100 μM at pH 1.0) (data not shown). In contrast to the fasted animals, little or no effect of pentagastrin or famotidine was observed on the low exposures observed in the fed dogs. This large food effect may be related to pH and other parameters, including gastrointestinal emptying time. Since different formulations (solution, tablet, and lipid-filled capsule) were used in these studies, the formulation might have also contributed to some of the differences in the exposures observed across groups.
TABLE 5.
Effect of formulation, pretreatment, and feeding condition on GS-6620 dog pharmacokinetics
| Form (all 100 mg) | Pretreatment | Fasted/fed status | Cmax (μM) | AUC0–t (μM · h) |
|---|---|---|---|---|
| Solutiona | Noneb | Fasted | 0.68 | 0.77 |
| Pentagastrin | Fasted | 0.83 | 0.43 | |
| Clinical tablet | Pentagastrin | Fasted | 0.80 | 0.61 |
| Pentagastrin | Fed | 0.13 | 0.09 | |
| Famotidine | Fed | 0.06 | 0.08 | |
| Capsule | Famotidine | Fasted | 0.14 | 0.10 |
Dose normalized from the 5 mg/kg dose used in the experiment to 100 mg fixed.
Study was done with diastereomeric mixture containing GS-6620.
Liver pharmacokinetics.
Potency best correlates with the levels of the pharmacologically active triphosphate formed in the liver. Therefore, the pharmacokinetics of the triphosphate metabolite, GS-441326, in the liver was determined following oral administration of an equivalent amount of GS-6620 to hamsters, monkeys, and dogs. Further studies assessed the effects of formulation and food on liver levels in dogs (Table 6 and Fig. 5C). Consistent with the similarity in dose-normalized plasma exposures between the solution and 100-mg tablet under fasted conditions, the liver triphosphate levels were similar between these two dose groups. Following the Cmax values, GS-6620 produced relatively high levels of GS-441326, ranging from 3.7 to 9.0 μM in all three species (Table 6). Consistent with the efficient intracellular triphosphate formation, the highest dose-normalized exposure to GS-441326 was observed in hamsters. The liver exposure to GS-441326 in dogs (100-mg tab, fasted) was approximately 2-fold higher than in monkeys (Table 6). However, this may be due to sample collection error at the 4-hour time point for monkeys, which showed the lowest concentration of all the time points collected (Fig. 5C). Therefore, it is likely that the liver triphosphate levels were comparable between dogs and monkeys. The t1/2 values for GS-441326 were similar across species (8.5 to 10.4 h) (Table 6). Consistent with the plasma PK, exposure to GS-441326 in fasted dogs administered the 100-mg tablet was higher (3.5-fold) than in the fed dogs.
TABLE 6.
Liver pharmacokinetics of the active triphosphate metabolite (GS-441326) in hamsters, monkeys, and dogs following oral administration of GS-6620
| Species | Dose (mg/kg) | t1/2 (h) | Cmax (μM) | Tmax (h) | AUC0–t (μM · h) |
|---|---|---|---|---|---|
| Hamster | 10.6 | 8.47 | 8.98 | 4 | 135 |
| Monkey | 3.86 | 10.4 | 3.68 | 2 | 35.8 |
| Dog | 2.5a | NAb | 5.60 | 4 | NA |
| 2.5 (tablet, fed)c | 10.1 | 1.43 | 4 | 18.8 | |
| 2.5 (tablet, fasted)c | 8.95 | 6.17 | 4 | 66.3 |
Dose normalized based on body surface area so that results are comparable to those observed in hamsters and monkeys. The dogs were dosed at 5 mg/kg in the experiment.
NA, not available. Only 4-hour time point was collected for dog liver in the study assessing a solution.
Dose normalized from 100-mg tablet (8.3 mg/kg based on beagle dog body weight of 12 kg) to 2.5 mg/kg.
DISCUSSION
GS-6620 is a novel adenosine nucleotide analog double-prodrug containing the 3′-isobutyryl ester and the 5′-phosphoramidate moiety. The 3′-isobutyryl ester substitution was designed to improve the oral absorption of the diastereomeric mixture containing GS-6620 (3). In order to inhibit HCV NS5B RNA polymerase, GS-6620 requires metabolic activation to its triphosphate form. The first step of the proposed activation pathway of GS-6620 is the cleavage of the 3′-isobutyryl ester by CES2 or hydrolysis of the ester in the 5′-phosphoramidate moiety by CES1 and CatA. It is unknown if the cleavage product of the 5′-ester, metabolite A, can be further metabolized to metabolite X. Our biochemical results indicated that CES2 was unable to hydrolyze the 3′-isobutyryl ester of metabolite A. In a metabolite profiling study following oral administration of GS-6620 to bile duct-cannulated rats, the major metabolite found in bile was metabolite A (data not shown). Therefore, at least in rats, high levels of metabolite A may form in the liver and be excreted in bile as a dead-end metabolite.
Since CES2 is known to be one of the major esterases expressed in intestinal tissues (12–14), it is likely that GS-6620 can be converted to GS-465124 by CES2 during intestinal absorption. The presence of intestinal metabolism was supported by studies in portal vein-cannulated dogs where GS-465124 was observed in the portal vein, although the levels of GS-465124 were substantially less than the levels of GS-6620 (>5-fold; data not shown). Intestinal metabolism resulting in the formation of GS-465124, an efflux substrate with poor intestinal permeability, is suboptimal. CES2 is also highly expressed in the liver, and in addition to causing intestinal degradation, it may play an important role in the productive conversion of GS-6620 to GS-465124 in hepatocytes.
GS-465124 is a nucleotide phosphoramidate, and the metabolic activation of this type of prodrug when applied to different nucleotide analogs has been characterized (18–20). Similar to the other phosphoramidate prodrugs targeting HCV, including sofosbuvir (GS-7977) and PSI-353661, GS-465124 was hydrolyzed by CES1 and CatA to form metabolite X, followed by HINT1-mediated removal of l-alanine resulting in the formation of the nucleoside analog monophosphate, GS-558272 (8, 9). Our overexpression experiments indicated that HINT2 but not HINT3 was also able to catalyze the deamination reaction. However, it is likely that HINT1 is the major enzyme for GS-6620 activation, since HINT2 is a mitochondrial enzyme (21, 22). The monophosphate metabolite must be phosphorylated to the active triphosphate form, GS-441326, by cellular nucleotide kinases.
Plasma PK studies demonstrated significant species differences in exposures to GS-6620 and its metabolites. In addition to the species presented in the manuscript, we also studied GS-6620 in rats, in which GS-6620 and GS-465124 are highly unstable due to high plasma levels of esterase activity (data not shown). For the current publication, we chose to focus only on the animal species in which GS-6620 was relatively stable in plasma (half-life >1 h in vitro). Plasma exposures to GS-6620 and GS-465124 in dogs were higher than in monkeys (2.5-fold), while those in hamsters were 5- to 12.5-fold lower than those in dogs or monkeys. This may be explained by intestinal S9 stability of GS-6620 and GS-465124, where GS-6620 was highly unstable in hamster intestinal S9 compared to those in any other species tested. The species differences in intact GS-6620 found in plasma may reflect intestinal CES2, whose substrate specificity and expression levels are known to be highly species dependent (23). Unlike plasma PK, the liver PK demonstrated similar levels of the triphosphate (GS-441326) formation across species. The liver GS-441326 levels are dependent upon two main factors: intestinal absorption and the efficiency of metabolism in hepatocytes. As described above, plasma exposures to GS-6620 and GS-465124 in hamsters or monkeys were significantly lower than those in dogs, suggesting less efficient absorption in these species. However, GS-6620 was more efficiently metabolized to GS-441326 in primary hamster or monkey hepatocytes relative to those in dogs. Therefore, the lower absorption of intact prodrug in hamsters or monkeys relative to those in dogs may have been compensated for by the relatively efficient metabolism in liver cells resulting in the formation of higher liver levels of GS-441326 in hamsters and comparable levels in monkeys relative to those in dogs in vivo. GS-6620 metabolism in primary human hepatocytes was similar to that in dog hepatocytes. Based on the similar in vitro stability and metabolism profiles, dogs were chosen as an animal model for predicting human PK.
Our dog data demonstrated that the plasma exposure to GS-6620 was markedly affected by a combination of different factors, including stomach pH and food. Pentagastrin is known to stimulate gastric acid secretion and lower gastric pH. The solubility results demonstrated that GS-6620 is highly soluble at acidic pH levels, suggesting that pentagastrin treatment increased the solubility of GS-6620 in vivo. In pentagastrin pretreated and fasted dogs dosed in a tablet form, the exposure to GS-6620 was very similar to that with the acidic solution formulation, indicating that the tablet is completely solubilized under these conditions. As expected based on the pH-dependent solubility profile, famotidine pretreated dogs showed significantly reduced exposure. There was a large food effect, with approximately 7-fold-higher exposure in the fasted state relative to the fed state in pentagastrin-treated dogs. The food effect appeared to be pH independent, as the exposure to GS-6620 was relatively low and similar between pentagastrin- and famotidine-pretreated fed dogs. Our in vitro absorption studies in Caco-2 cells suggested that efflux transport might be saturable at the solubility limit, but it is unlikely that the efflux transport is saturated in vivo. Therefore, intestinal absorption appeared to be highly concentration dependent and sensitive to changes in the parameters affected by food, including gastric pH and gastric emptying time.
The safety, tolerability, pharmacokinetics, and anti-HCV activity of GS-6620 were assessed in genotype 1 treatment-naive subjects over 5 days in a first-in-human clinical study (5). GS-6620 demonstrated the potential for potent anti-HCV activity, but substantial intra- and intersubject pharmacokinetic and pharmacodynamic variability were observed. For example, in the 900 mg twice a day (BID) cohort, exposures to GS-6620 and GS-465124 were reported to be 339 ng · h/ml and 916 ng · h/ml, respectively. The coefficient of variation in this cohort was >70% and maximal viral load reductions ranged from 1.14 to >4.37 log. In dogs, exposures to GS-6620 and GS-465124 after a surface area-adjusted dose of 900 mg based on data presented in Table 4 would be 905 ng · h/ml and 930 ng · h/ml, respectively. Comparing dogs and humans, nearly a 3-fold drop in GS-6620 exposure and a 3-fold increase in the ratio of GS-465124 to GS-6620 (from approximately one in dogs to 2.7 in humans) was observed, suggesting less efficient absorption of the prodrug and a higher rate of intestinal metabolism in humans. In light of the results discussed above in dogs showing a large effect of food and stomach pH, an oral solution was administered as part of the clinical trial. While the administration of a solution reduced pharmacokinetic and pharmacodynamic variability and increased GS-6620 plasma exposure by 60% relative to the tablet formulation administered at the same dose, evidence for high levels of intestinal metabolism was still observed by the lack of an effect on the GS-465124-to-GS-6620 ratio. The results with the oral solution suggest that intestinal metabolism, with a lesser contribution from solubility, was the major cause for the relatively poor pharmacokinetics and variable antiviral response observed clinically. The lack of correlation between the oral performance of GS-6620 in dogs and humans is likely related to higher expression levels of CES2 in humans than in dogs (23).
In this study, we assessed species differences in the metabolism and pharmacokinetics of GS-6620. A large species difference was observed in plasma exposures to GS-6620 and its metabolites, which is primarily due to differential intestinal absorption likely mediated by CES2-catalyzed hydrolysis of the 3′-ester of GS-6220. Furthermore, a species difference was seen in the metabolism of GS-6620 in primary hepatocytes from different species. In the three animal models used in this study, these two independent factors, intestinal absorption and intracellular activation, partially offset each other in animals and resulted in highest liver GS-441326 levels in hamsters and similar levels in dogs and monkeys. High levels of intestinal metabolism and the resulting poor intestinal absorption coupled with relatively inefficient activation in human hepatocytes likely explain the requirement for higher doses than anticipated and the observation of high levels of pharmacokinetic and pharmacodynamic variability in the clinic.
Footnotes
Published ahead of print 13 January 2014
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