Mechanism for Effective Lymphoid Cell and Tissue Loading Following

Jun 27, 2012 - ... Animal Care and Use Committee at SRI International (Menlo Park, CA). ...... Bhoopathy, Siddhartha; Hidalgo, Ismael J.; Rhodes, Gera...
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Mechanism for Effective Lymphoid Cell and Tissue Loading Following Oral Administration of Nucleotide Prodrug GS-7340 Darius Babusis, Truc K. Phan, William A. Lee, William J. Watkins, and Adrian S. Ray*

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Gilead Sciences, Inc., Foster City, California 94404, United States ABSTRACT: GS-7340 is a prodrug of tenofovir (TFV) that more efficiently delivers TFV into lymphoid cells and tissues than the clinically used prodrug TFV disoproxil fumarate, resulting in higher antiviral potency at greatly reduced doses and lower systemic TFV exposure. First-pass extraction by the intestine and liver represents substantial barriers to the oral delivery of prodrugs designed for rapid intracellular hydrolysis. In order to understand how GS-7340 reduces first-pass clearance to be an effective oral prodrug, its permeability and stability were characterized in vitro and detailed pharmacokinetic studies were completed in dogs. GS-7340 showed concentration-dependent permeability through monolayers of caco-2 cells and dose-dependent oral bioavailability in dogs, increasing from 1.7% at 2 mg/kg to 24.7% at 20 mg/kg, suggesting saturable intestinal efflux transport. Taking into account a 65% hepatic extraction measured in portal vein cannulated dogs, high dose GS-7340 is nearly completely absorbed. Consistent with the proposed role of intestinal efflux transport, coadministration of low dose GS-7340 with a transport inhibitor substantially increased GS-7340 exposure. The result of effective oral absorption and efficient lymphoid cell loading was reflected in the high and persistent levels of the pharmacologically active metabolite, TFV diphosphate, in peripheral blood mononuclear cells following oral administration to dogs. In conclusion, GS-7340 reaches the systemic circulation to effectively load target cells by saturating intestinal efflux transporters, facilitated by its high solubility, and by maintaining sufficient stability in intestinal and hepatic tissue. KEYWORDS: prodrug, tenofovir, intestinal absorption, P-glycoprotein, hepatic extraction, carboxylesterase, cathepsin A

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INTRODUCTION Highly active antiretroviral therapy (HAART) for the treatment of the human immunodeficiency virus (HIV), often containing 2 nucleos(t)ide reverse transcriptase inhibitors (NRTIs) and a third agent from another drug class, has markedly reduced HIVassociated mortality to near levels in the general uninfected population.1 However, the life expectancy of an HIV infected patient is still significantly reduced, often estimated to be approximately 10 years shorter, relative to an uninfected person.2,3 In addition to the effects of low level HIV replication, it has been established that specific components of HAART increase the rate of comorbid conditions generally thought of as non-HIV related, including cardiovascular, renal, and hepatic disease.4,5 The long-term effects of infection and treatment are set against a backdrop of an aging HIV population and comorbidities present irrespective of HIV infection status, including an epidemic of obesity, type II diabetes, and hypertension.6,7 The requirement for treatment over the duration of a normal life span raises new challenges for HIV therapy. In addition to potency and durability, improving the long-term safety profile of components of HAART is of critical importance to further advancing the treatment of HIV patients. Tenofovir (TFV) disoproxil fumarate (TDF), the salt of a lipophilic prodrug of the NRTI TFV, is a component of preferred first-line regimens for HIV infection.8−10 While TFV disoproxil is not observed in plasma at any time following oral © 2012 American Chemical Society

administration, studies in dogs and monkeys have established that the prodrug plays a direct role in loading TFV and its phosphorylated metabolites into lymphoid cells.11,12 GS-7340 is an alternate prodrug of TFV that is more stable in human plasma and more efficiently delivers TFV into lymphoid cells and tissues than TDF.13 Loading of target cells by GS-7340 is facilitated by specific cleavage and activation by lysosomal cathepsin A (CatA), which is highly expressed in cells of lymphoid origin.14 In monotherapy studies in HIV-1 infected patients naive to prior therapy, GS-7340 has demonstrated superior efficacy at markedly lower doses and reduced TFV plasma exposure compared to TDF.15 Monotherapy with 25 mg of GS-7340 resulted in an approximately 0.5 log greater reduction in viral load on day 11, a 7-fold higher intracellular level of the active metabolite TFV-diphosphate (TFV-DP) in circulating lymphocytes, and a 90% lower level of TFV in plasma relative to TDF administered at 300 mg.16 The increased efficiency of target cell loading and reduced off-target exposure resulting from lower systemic TFV suggest that GSSpecial Issue: Prodrug Design and Target Site Activation Received: Revised: Accepted: Published: 459

April 12, 2012 June 20, 2012 June 27, 2012 June 27, 2012 dx.doi.org/10.1021/mp3002045 | Mol. Pharmaceutics 2013, 10, 459−466

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carcinoma cell line caco-2 seeded in 12-well plates as previously reported.20,21 The effects of either concentration (10, 100, or 1,000 μM) or efflux transporter inhibition on the permeation of GS-7340 were studied. Effect of the inhibition of efflux transporters including P-glycoprotein (Pgp) was assessed following a 30 min preincubation of cell monolayers with 10 μM cyclosporin A (CsA) in transport buffer to allow for saturation of transporter binding sites. Following preincubation, fresh assay buffer containing CsA and GS-7340 was added and the assay was started. Each determination was performed in duplicate, and the permeability of control compounds (atenolol, propranolol, and digoxin) was determined to meet acceptance criteria for each batch of assay plates. Animals. Male beagle dogs (Canis lupus familiariz) between the ages of 6 and 18 months were used for the in life portion of this study. The animals were housed in accordance with the standards of the American Association for Accreditation of Laboratory Animal Care and were receiving a standard commercial diet. Animals were handled in strict accordance with the Guide for the Care and Use of Laboratory Animals,22 and the protocol was reviewed by the Institutional Animal Care and Use Committee at SRI International (Menlo Park, CA). Animals were between 7 and 11 kg at dosing. Drug Administration. For intravenous administration, GS7340 was formulated in 5% dextrose in water. For oral administration, GS-7340 was formulated in 50 mM citric acid (pH 5.0) at doses of 2 to 10 mg/kg. For the 20 mg/kg dose, GS-7340 was formulated in 50 mM citric acid (pH 5.5) with 0.1% Polysorbate 20. To test the effect of efflux transport inhibition, dogs were administered 2 mg/kg GS-7340 1 h following pretreatment with a 75 mg capsule of CsA (Novartis, East Hanover, NJ). Plasma and PBMC Sample Collection. Blood (approximately 0.5 mL) was collected at specified time points over 24 h from the jugular vein (intraportal vein infusion and oral pharmacokinetic studies), the jugular and portal veins (oral administration to portal vein cannulated dogs), or the cephalic vein (jugular vein infusion). Plasma was isolated in Vacutainer tubes containing EDTA as an anticoagulant (Becton Dickinson, Franklin Lakes, NJ) by centrifugation. At select time points (1, 4, 8, and 24 h postdose) in the 5 mg/kg oral pharmacokinetic study, 8 to 10 mL of blood was collected into Vacutainer cell preparation tubes (Becton Dickinson) for isolation of peripheral blood mononuclear cells (PBMC) and processed following manufacturer instructions. Small aliquots (10 μL) of isolated PBMC pellets diluted in 0.9% NaCl were maintained at room temperature and used to determine cell count. 500 μL of 70% methanol was added to the remaining PBMC pellets and, together with plasma samples, stored at −80 °C until shipment for further processing and analysis. PBMC Sample Preparation. Cellular debris was removed from PBMC extracts by centrifugation at 15000g. The resulting supernatants were dried at 37 °C in a centrifuging evaporator (Mivac Duo, Genevac, Gardiner, NY) and reconstituted in 50% methanol. Samples were then applied to a Speedisk C18 cartridge (J.T. Baker, Center Valley, PA) for solid phase extraction with the resulting eluent evaporated and reconstituted in 20 mM tetrabutylammonium acetate (TBAA) containing D4AP-DP as an internal standard. A final spin (15000g, 5 min) was used to remove any remaining particulate matter from extracts before analysis for TFV-DP by LC−MS/ MS.

7340 can further improve upon the already favorable safety and tolerability profile of TDF. Oral prodrugs optimized for rapid intracellular cleavage must survive a daunting series of obstacles to reach the systemic circulation. They must first be absorbed from the gastrointestinal tract, passing through the intracellular compartment of enterocytes, and then escape extraction by the liver, which accounts for the majority of the metabolic capacity in the body. In addition to its expression in lymphoid cells, CatA has been reported to be expressed in the intestine and liver.17 GS-7340 has also been shown to be a substrate for a variety of serine and cysteine hydrolases including others that are likely to be expressed in these tissues.18 The localization of hydrolases capable of cleaving GS-7340 in the intestine and liver suggests the potential for substantial first-pass clearance that could limit oral bioavailability and subsequent loading of lymphoid cells and tissues. In order to understand the extent to which the disposition of GS-7340 is affected by first-pass metabolism, we studied GS7340 permeability through caco-2 cell monolayers, stability in intestinal and hepatic extracts, and pharmacokinetics in intact and portal vein cannulated dogs. Results reported here illustrate that GS-7340 is able to saturate efflux transport and metabolism in the intestine during absorption facilitated by its high solubility. While a majority of absorbed GS-7340 is extracted by the liver, sufficient material is able to survive first-pass extraction to allow for efficient loading of lymphoid cells and tissues.

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EXPERIMENTAL SECTION Materials. GS-7340, TFV, TFV diphosphate (DP), and analytical internal standards D4AP (9-[((2R,5R)-2,5-dihydro-5phosphonomethoxy)-2-furanyl]adenine), D4AP-DP, and bisalanine-n-butyl prodrug of D4AP were synthesized at Gilead Sciences, Inc. (Foster City, CA). All other reagents were the highest grade available from Sigma-Aldrich. Plasma Stability. GS-7340 was incubated at 2 μM in either dog or human plasma (obtained from Bioreclamation, Liverpool, NY) for 4 h at 37 °C. At specified time points, an aliquot from the incubation was quenched by addition of 9 volumes of 100% acetonitrile and internal standard. Following the last collection, samples were centrifuged at 3000g for 30 min and 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). Intestinal and Hepatic S9 Stability. GS-7340 was incubated at 10 μM with either dog or human intestinal and hepatic S9 fractions (obtained from In Vitro Technologies, Baltimore, MD) for 120 min at 37 °C in a 96-well plate format. At specified time points following compound addition, samples were quenched with 9 volumes of an aqueous solution containing 25% acetonitrile and 50% methanol. Plates were centrifuged at 3000g for 30 min, and 10 μL of the resulting solution was analyzed by LC−MS/MS. Data (analyte to internal standard peak area ratio) were plotted on a semi log scale and fitted using an exponential fit. Assuming first order kinetics, the half-life and rate of metabolism were determined. Predicted hepatic extraction was calculated from the half-life by reported methods using the well-stirred model for hepatic clearance.19 Caco-2 Permeability. Bidirectional permeability studies were done using confluent monolayers of the human colon 460

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Quantification of Plasma Concentrations of GS-7340 and TFV. Dog plasma samples from pharmacokinetic studies were subject to protein precipitation by addition of acetonitrile and formic acid to final concentrations of 66% and 0.2%, respectively, containing the nucleotide analogue D4AP and its prodrug as internal standards. Plasma analytes were separated on a 4 μm 75 × 2 mm Synergi Hydro column (Phenomenex, Torrance, CA) using a mobile phase containing 0.2% formic acid and a linear gradient from 0.5% to 95% acetonitrile at a flow rate of 250 μL/min over 4 min. Seven point standard curves prepared in blank plasma covered concentrations of 3 orders of magnitude and showed linearity in excess of an r2 value of 0.99. The lower limits of GS-7340 and TFV quantification in plasma were 1.4 nM. Separately prepared quality control samples of 10 and 2,000 nM in plasma were analyzed at the beginning and end of each sample set to ensure accuracy and precision within 20%. Quantification of PBMC Concentrations of TFV-DP. TFV-DP quantification used a previously described LC−MS/ MS ion pairing nucleotide detection method.23 Briefly, analytes were separated using a 3.0 μm 1.0 × 100 mm Luna C18 column (Phenomenex, Torrance, CA) using an ion pairing buffer containing 2 mM ammonium phosphate and 0.2 mM TBAA and a multistage linear gradient from 5% to 50% acetonitrile at a flow rate of 40 μL/min over 24 min. Six point standard curves prepared in blank PBMC matrices covered concentrations of over 2 orders of magnitude and showed linearity in excess of an r2 value of 0.99. The lower limit of quantification was 82.5 nM for TFV-DP. Separately prepared quality control samples of 500 and 10,000 nM were analyzed at the beginning and end of each sample set to ensure accuracy and precision within 20%. Intracellular concentrations were calculated assuming a mean cell volume of 0.2 μL/million PBMC.24 LC−MS/MS Instrumentation. Pharmacokinetic samples were analyzed using an HTS PAL autosampler with cooled sample storage stacks set at 6 °C (Leap Technologies, Carrboro, NC) and an LC-20ADXR ternary pump system (Shimadzu Scientific Instruments, Columbia, MD). Dog plasma and PBMC LC−MS/MS analyses were done in positive ion and multiple reaction monitoring modes using a Sciex API4000 mass spectrometer (Applied Biosystems, Foster City, CA). In vitro stability assay samples were analyzed using an HTS PAL autosampler with cooled sample storage stacks set at 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 (Waters, Milford, MA), and GS-7340 was retained using a 2 μm 20 × 2.1 mm Mercury RP C18 (Phenomenex, Torrance, CA), maintained at 60 °C with a mobile phase flowing at 1 mL/min, consisting of 0.2% formic acid and a linear gradient from 0 to 85% acetonitrile over 1 min. Pharmacokinetic Data Analyses. Noncompartmental pharmacokinetic parameters were calculated using WinNonlin 5.01 (Pharsight Corporation, Mountain View, CA).

Table 1. GS-7340 Stability in Extracts from Dogs and Humansa hepatic S9 species

intestinal S9 t1/2 (min)

t1/2 (min)

EH (%)

plasma t1/2 (min)

dog human

31.0 ± 7.0 33.5 ± 1.7

31.0 ± 7.0 18.0 ± 5.4

60.9 ± 0.0 78.8 ± 4.8

92.0 ± 23.1 116.9 ± 34.5

EH = estimated hepatic extraction. Values are the mean ± standard deviation of n = 4 independent experiments. a

first-pass hepatic extraction based on stability in hepatic S9 from dogs and humans. Human hepatic S9 suggests somewhat higher hepatic extraction relative to dog (estimated to be 60.9% and 78.8%, respectively). The similarity in stability of GS-7340 in extracts from dog and human suggests that the dog is an appropriate preclinical species to study GS-7340 pharmacokinetics. Permeability through Caco-2 Cell Monolayers. The concentration dependent permeability of GS-7340 through monolayers of caco-2 cells was studied to understand absorption potential. With increasing concentration from 10 to 1,000 μM, GS-7340 showed higher apical (A) to basolateral (B) permeability corresponding to saturation of efflux transport (Figure 1A). The efflux ratio of GS-7340 (B to A divided by A to B) decreased from 20 at 10 μM to 5.4 at 1,000 μM. Addition of the transport inhibitor CsA lowered the efflux ratio to 1, while causing a marked increase in A to B permeability (mean Papp values at 10 μM GS-7340 of 0.34 × 10−6 cm/s and 1.51 × 10−6 cm/s, respectively). Plasma Pharmacokinetics. GS-7340 was administered to male beagle dogs in a number of different studies to assess parameters of absorption, distribution, metabolism, and elimination. Consistent with results obtained in extracts in vitro, GS-7340 was rapidly cleared following intravenous administration at a rate (2.2 L/h/kg) greater than hepatic blood flow in the dog (reported to be 1.85 L/h/kg in dog).25 Clearance in excess of hepatic blood flow is consistent with extrahepatic elimination of the labile prodrug. As observed in caco-2 studies, the oral bioavailability of GS-7340 was found to be dose-dependent, ranging from 1.7% at 2 mg/kg to 24.7% at 20 mg/kg (Table 2). The oral bioavailability observed at high dose GS-7340 approached the theoretical maximum value of approximately 30% based on the estimate of approximately 70% hepatic extraction from hepatic S9 incubations (Figure 1B). When GS-7340 was administered orally at the lowest dose to dogs that had been pretreated with CsA, a marked increase in exposure was observed, resulting in calculation of an oral bioavailability of 16.6%. The increase in exposure observed upon CsA coadministration correlated primarily with an increase in maximal concentration with no change in half-life suggesting a change in the extent of intestinal absorption. CsA administration caused emesis in all animals, likely contributing to the high level of variability observed between animals and reducing the magnitude of the increase in exposure. Determination of Intestinal and Hepatic Extraction in Portal Vein Cannulated Dogs. The half-life of GS-7340 during incubations with dog intestinal S9 suggests the potential for metabolism during intestinal absorption. In order to better understand the nature of metabolism occurring during intestinal absorption, metabolite formation was qualitatively monitored following oral administration of GS-7340 in samples collected from the jugular and portal veins (Figure 2). High

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RESULTS Metabolic Stability in Human and Dog Matrices. The stability of GS-7340 was assessed in plasma and S9 fractions from the intestine and liver isolated from dogs and humans. As summarized in Table 1, GS-7340 was relatively stable in plasma and had a similar half-life in intestinal S9 from the two species. Approximately 70% of the dose is estimated to be subject to 461

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Figure 1. (A, left) Effect of drug concentration and efflux transport inhibition on apparent permeability of GS-7340 through monolayers of caco-2 cells. GS-7340 was incubated alone at 10, 100, or 1,000 μM or at 10 μM in the presence of 10 μM cyclosporin A (CsA). With increasing concentration or in the presence of a transport inhibitor the apical to basolateral (A to B) permeability (Papp) of GS-7340 increased and the B to A Papp decreased, corresponding to a concomitant decrease in efflux ratio (B to A divided by A to B; indicated above bars for each treatment condition). Data represents the mean ± standard deviation of 4 independent experiments each done in duplicate. (B, right) Effect of dose or coadministration with the efflux transport inhibitor CsA on the oral bioavailability of GS-7340 in dogs following a single oral administration at doses of 2, 5, 10, or 20 mg/kg or at 2 mg/kg to animals pretreated with 75 mg of CsA. The oral bioavailability of GS-7340 increased with dose or upon coadministration with a transport inhibitor. Data fit arbitrarily to show proposed sigmoidal relationship between dose and oral bioavailability. Values represent the mean ± standard deviation of 3 dogs at all doses except at 10 mg/kg where 4 dogs were studied.

Table 2. Pharmacokinetic Parameters for GS-7340 in Dogs Following Either Intravenous or Oral Administration at Various Dosesa

a

dose (mg/kg)

route

N

Cmax (μM)

0.5 2 5 10 20 2

iv po po po po po + CsA

3 3 3 4 3 3

1.00 0.11 1.58 1.81 5.74 0.81

± ± ± ± ± ±

0.11 0.06 0.47 1.33 0.62 0.53

t1/2 (h) 0.12 0.21 0.17 0.32 0.34 0.15

± ± ± ± ± ±

0.02 0.13 0.06 0.12 0.15 0.05

AUC0−t (μM·h)

F (%)

± ± ± ± ± ±

NA 1.7 ± 0.4 8.6 ± 0.8 14.3 ± 12.5 24.7 ± 6.5 16.6 ± 13.0

0.48 0.03 0.41 1.38 4.65 0.31

0.05 0.01 0.04 1.20 1.23 0.24

NA = not applicable. CsA = cyclosporin A. F = oral bioavailability.

metabolites are subject to extraction by liver, although to a lesser extent than GS-7340. Intravenous infusion studies in portal vein cannulated dogs were completed to gain a more quantitative understanding of the extent of liver extraction occurring in vivo in dogs. The clearance of GS-7340 was determined following infusion into the jugular or portal veins while sampling plasma from the systemic circulation (Figure 3). Substantially higher GS-7340 clearance was calculated following portal infusion relative to jugular infusion (6.45 L/h/kg and 2.23 L/h/kg, respectively). These values yield an estimated hepatic extraction of 65%. The hepatic extraction determined by relative exposure determined following portal and jugular infusion is similar to that predicted based on incubation with dog hepatic S9 in vitro (60.9%) and is not unlike that observed following oral administration to portal vein cannulated dogs discussed above. Plasma and PBMC Pharmacokinetic Profile. Having characterized the contribution of first-pass intestinal and hepatic extraction in dogs, we next studied the relationship between the systemic pharmacokinetic profile of GS-7340 and TFV, and PBMC levels of TFV-DP following oral administration of 5 mg/kg GS-7340 to dogs (Figure 4). GS-7340 was rapidly absorbed, achieving a maximal plasma concentration of 1.58 μM 0.14 h postdose. The disappearance of GS-7340 was rapid (undetectable following 2 h postdose) and associated with a concomitant increase in TFV in plasma. The transient plasma exposure of GS-7340 resulted in the rapid accumulation

relative amounts of GS-7340 were observed in the portal vein and were greatly reduced in the systemic circulation (based on jugular sampling). Consistent with estimates based on hepatic S9 incubations, quantification of GS-7340 over time from portal and jugular samples resulted in the estimation of GS-7340 hepatic extraction of ∼70% (data not shown). The formation of metabolites during intestinal absorption and their subsequent presence in the portal vein was monitored in samples taken during the absorption phase of GS-7340 using predicted LC− MS/MS parameters. Evidence for formation of metabolites formed by cleavage of the phenol (metabolite Y) or isopropyl (metabolite X and TFV) moieties by intestinal enzymes was detected, but no premature cleavage of the amino acid ester promoiety was detected based on the lack of detection of a mass corresponding to the putative monophenol TFV metabolite. While the presence of metabolites was not confirmed by synthesis of authentic standards for this study, masses were not detected in blank plasma and the retention times of the metabolites were consistent with their predicted retention on the reverse phase column based on charge and lipophilicity. The appearance of metabolites in the portal vein during the absorption phase of GS-7340 is consistent with the instability observed in intestinal S9 fractions in vitro and suggests that intestinal metabolism is taking place in dogs and serving to reduce the oral bioavailability of intact GS-7340. Relative LC−MS/MS response in samples simultaneously collected from the portal and jugular veins suggests that these 462

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Figure 2. Formation of metabolites during the process of intestinal absorption caused by premature cleavage of the phenyl or ester promoieties of GS-7340. LC−MS/MS response for samples taken from the portal (A) and jugular (B) veins following oral administration of 5 mg/kg GS-7340 to portal vein cannulated dogs. GS-7340 (m/z 477 to 270), metabolite Y (Met Y, des-phenyl GS-7340; m/z 401 to 176), metabolite X (Met X, desisopropyl and des-phenyl; m/z 359 to 176), and tenofovir (TFV; m/z 288 to 176) were monitored by their respective parent to daughter mass transitions. Metabolites were detected in plasma 15 min following oral administration. Results are from pooled plasma samples from n = 3 animals.

Figure 3. Systemic plasma profile of GS-7340 following 30 min intravenous infusion at 0.5 mg/kg via the jugular vein (closed diamonds) or 0.4 mg/kg via the portal vein (open diamonds). Based on dose normalized exposure a hepatic extraction (EH) of 65% was estimated. Values represent the mean ± standard deviation of 3 dogs dosed in either the jugular or portal infusion studies.

Figure 4. Oral administration of GS-7340 results in transient exposure to GS-7340, low levels of TFV in the systemic circulation, and efficient loading of PBMC with TFV-DP. Pharmacokinetic profiles of plasma GS-7340 (open circles) and TFV (×) and PBMC TFV-DP (open diamonds) following a single oral administration of GS-7340 at 5 mg/ kg to dogs. Values represent the mean ± standard deviation of 3 dogs.

of persistent levels of TFV-DP in PBMC, reaching 18 μM in PBMC at 1 h postdose that persisted over the 24 h sampling period.

metabolism is taking place during intestinal absorption. While degraded in hepatic S9 and extracted by the liver following portal vein infusion in dogs, GS-7340 was sufficiently stable to allow for greater than 20% of the absorbed dose to reach the systemic circulation intact. The observation that GS-7340 is not more rapidly degraded in intestinal and hepatic cells suggests some resistance to hydrolases that are highly expressed in these tissues. Carboxylesterase 1 and 2 (CES1 and CES2) are known to be highly expressed in the liver and intestine, respectively,26 and efficient cleavage by these enzymes would preclude

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DISCUSSION GS-7340 was found to be unstable in intestinal and hepatic cytoplasmic extracts from dogs and humans while maintaining stability in plasma from these species, results consistent with its optimization for selective intracellular hydrolysis. The appearance of metabolites in the portal vein observed after oral administration to dogs further suggests that GS-7340 463

dx.doi.org/10.1021/mp3002045 | Mol. Pharmaceutics 2013, 10, 459−466

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Prior studies have elucidated the mechanism for efficient lymphoid cell loading by GS-7340. The central role of CatA in the intracellular cleavage of GS-7340 in lymphoid cells was established by an approximate 8-fold reduction in TFV accumulation in fibroblasts isolated from patients with a genetic deficiency in CatA activity.14 Efficient loading of lymphoid cells in vitro has previously been shown to translate into higher drug levels in lymphoid cells and tissues in animals. For example, radiolabeled tissue distribution studies in dogs have shown approximately 10-fold higher dose-normalized levels in lymph nodes following oral GS-7340 administration relative to TDF.13 Oral administration of GS-7340 to macaques has also been reported to result in approximately 50-fold higher TFV-DP levels in PBMC relative to TDF.31 In order to relate absorption and plasma exposure to lymphoid cell loading in the context of this study, PBMC were analyzed from dogs administered a 5 mg/kg dose of GS-7340. Consistent with prior results, we observed high and persistent levels of TFV-DP in PBMC. TFV-DP levels in PBMC following a single administration of 5 mg/kg GS-7340 to dogs are approximately 40-fold higher than those reported at steady state in the clinic following administration of 300 mg of TDF.32−36 Combined, these results illustrate that sufficient GS-7340 is able to survive first-pass clearance to efficiently load PBMC with TFV-DP. While these studies illustrate that GS-7340 has sufficient oral bioavailability to result in substantial loading of lymphoid cells following oral administration, a majority of the dose is extracted by the liver. Results from hepatic S9 in vitro and following oral or intravenous administration to dogs in vivo consistently suggest that approximately 70% of the absorbed dose is extracted by the liver. In addition to its indication for HIV, TFV when administered as TDF is also approved for the treatment of the hepatitis B virus (HBV) where it has potent antiviral activity. The efficient delivery of GS-7340 to the liver may have implications for the hepatic formation of the pharmacologically active metabolite TFV-DP. The potential utility of GS-7340 in patients chronically infected with HBV is currently the subject of a dose-ranging clinical study. In conclusion, results from the current detailed study of the bioavailability of GS-7340 help to further the understanding of the pharmacology of this effective prodrug of TFV. Owing to its high solubility, GS-7340 is able to generate sufficient mass flux across the intestine to saturate efflux transport and limit intestinal metabolism. The relatively selective hydrolase cleavage of GS-7340, including efficient metabolism by CatA and minimized interactions with major hydrolases expressed in the intestine and liver, allows for a sufficient proportion of the dose to avoid first-pass extraction and efficiently load lymphoid cells.

efficient oral delivery. GS-7340 has been shown to be a substrate for pig liver carboxylesterase, a close homologue of CES1.18 However, the reported specific activity for GS-7340 cleavage by this enzyme was low, orders of magnitude less than cleavage catalyzed by CatA. A recent presentation confirmed the role of CES1 in the metabolism of GS-7340 while showing little effect of CES2.27 The selectivity of GS-7340 cleavage by an enzyme that is highly expressed in lymphoid cells and limited interaction with enzymes mediating first-pass clearance likely plays an important role in the distribution of GS-7340 to target cells in vivo. We have previously proposed that the oral bioavailability of TDF is limited by the combined action of intestinal Pglycoprotein (Pgp) and hydrolases.28 Similar to the hypothesis that intestinal Pgp and cytochrome P450 3A4 act synergistically to limit the oral bioavailability of substrates they share in common,29,30 efflux mediated by Pgp may serve to prolong the residence time of prodrugs in intestinal tissue, allowing for further degradation by hydrolases expressed in enterocytes. Based on the in vitro observation of instability in intestinal S9 and efflux in caco-2 monolayers, the oral bioavailability of GS7340 is likely also limited by a similar interplay between intestinal efflux and metabolism. The importance of intestinal efflux transport as a barrier to GS-7340 absorption was evident in the effect of the transport inhibitor CsA observed both in vitro and in vivo. In caco-2 cell assays, efflux transport was completely inhibited by CsA and in dogs CsA markedly increased oral bioavailability. In the absence of CsA-induced emesis causing a reduction in the amount of GS-7340 available for absorption, it is reasonable to assume that the oral bioavailability would approach the maximum possible value dictated by hepatic extraction. While oral bioavailability is usually assumed to be independent of dose, dose-dependent pharmacokinetics were observed for GS-7340 in dogs. In Figure 1 we propose a sigmoidal relationship between GS-7340 oral bioavailability and dose: (i) At low doses GS-7340 is poorly absorbed. (ii) As the GS-7340 dose and luminal concentrations increase, barriers to intestinal absorption are saturated. (iii) Oral bioavailability increased rapidly with dose until reaching a plateau at an upper limit defined by hepatic extraction. While accuracy of the curve fitting and the confidence that any one model can be applied is limited by substantial error observed in the 10 mg/kg dose group, a sigmoidal fit seemed more pharmacologically relevant than linear or hyperbolic models that were also explored. The relatively high variability observed at 10 mg/kg may be related to being on the steep part of the dose−response curve. The results following oral administration to dogs are entirely consistent with the concentration-dependent permeability we observed in caco-2 cells. Over doses ranging from 2 to 20 mg/ kg, we observed over a 10-fold increase in oral bioavailability. Assuming constant hepatic extraction of approximately 65%, the fraction absorbed of intact GS-7340 from the gastrointestinal tract ranged from 4.9% at 2 mg/kg to 71% at 20 mg/ kg. While saturation of first-pass hepatic extraction cannot be ruled out as an explanation for these observations, saturation of hepatic enzymes seems less likely given the high capacity of hepatic hydrolases and the comparatively low concentrations of GS-7340 present in the portal vein relative to those attained in the lumen of the gastrointestinal tract. The observation of steep dose nonlinearity for GS-7340 may reflect the synergistic interaction of efflux transport and intestinal metabolism, processes that are both saturated at higher doses.

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AUTHOR INFORMATION

Corresponding Author

*Gilead Sciences, Inc., 333 Lakeside Dr., Foster City, CA 94404. Phone: (650) 522-5536. Fax: (650) 522-1892. E-mail: [email protected]. Notes

The authors declare the following competing financial interest(s): All authors are employed by Gilead Sciences, Inc. the marketer of TDF (Viread) and the sponsor of the active clinical development of GS-7340. 464

dx.doi.org/10.1021/mp3002045 | Mol. Pharmaceutics 2013, 10, 459−466

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ACKNOWLEDGMENTS We thank Eisuke Murakami, Eve-Irene Lepist, Marshall Fordyce and Michael Miller for thoughtful review of the manuscript.

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