Metabolism and pharmacokinetics of the anti-hepatitis C virus nucleotide prodrug GS-6620.

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.

Sensitivity of mitochondrial transcription and resistance of RNA polymerase II dependent nuclear transcription to antiviral ribonucleosides.

Ribonucleoside analogues have potential utility as anti-viral, -parasitic, -bacterial and -cancer agents. However, their clinical applications have been limited by off target effects. Development of antiviral ribonucleosides for treatment of hepatitis C virus (HCV) infection has been hampered by appearance of toxicity during clinical trials that evaded detection during preclinical studies. It is well established that the human mitochondrial DNA polymerase is an off target for deoxyribonucleoside reverse transcriptase inhibitors. Here we test the hypothesis that triphosphorylated metabolites of therapeutic ribonucleoside analogues are substrates for cellular RNA polymerases. We have used ribonucleoside analogues with activity against HCV as model compounds for therapeutic ribonucleosides. We have included ribonucleoside analogues containing 2'-C-methyl, 4'-methyl and 4'-azido substituents that are non-obligate chain terminators of the HCV RNA polymerase. We show that all of the anti-HCV ribonucleoside analogues are substrates for human mitochondrial RNA polymerase (POLRMT) and eukaryotic core RNA polymerase II (Pol II) in vitro. Unexpectedly, analogues containing 2'-C-methyl, 4'-methyl and 4'-azido substituents were inhibitors of POLRMT and Pol II. Importantly, the proofreading activity of TFIIS was capable of excising these analogues from Pol II transcripts. Evaluation of transcription in cells confirmed sensitivity of POLRMT to antiviral ribonucleosides, while Pol II remained predominantly refractory. We introduce a parameter termed the mitovir (mitochondrial dysfunction caused by antiviral ribonucleoside) score that can be readily obtained during preclinical studies that quantifies the mitochondrial toxicity potential of compounds. We suggest the possibility that patients exhibiting adverse effects during clinical trials may be more susceptible to damage by nucleoside analogs because of defects in mitochondrial or nuclear transcription. The paradigm reported here should facilitate development of ribonucleosides with a lower potential for toxicity.

Synthesis and antiviral activity of a series of 1'-substituted 4-aza-7,9-dideazaadenosine C-nucleosides.

A series of 1'-substituted analogs of 4-aza-7,9-dideazaadenosine C-nucleoside were prepared and evaluated for the potential as antiviral agents. These compounds showed a broad range of inhibitory activity against various RNA viruses. In particular, the whole cell potency against HCV when R=CN was attributed to inhibition of HCV NS5B polymerase and intracellular concentration of the corresponding nucleoside triphosphate.

Discovery of GS-9131: Design, synthesis and optimization of amidate prodrugs of the novel nucleoside phosphonate HIV reverse transcriptase (RT) inhibitor GS-9148.

GS-9148 [(5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl)phosphonic acid] 4 is a novel nucleoside phosphonate HIV-1 reverse transcriptase (RT) inhibitor with a unique resistance profile toward N(t)RTI resistance mutations. To effectively deliver 4 and its active phosphorylated metabolite 15 into target cells, a series of amidate prodrugs were designed as substrates of cathepsin A, an intracellular lysosomal carboxypeptidase highly expressed in peripheral blood mononuclear cells (PBMCs). The ethylalaninyl phosphonamidate prodrug 5 (GS-9131) demonstrated favorable cathepsin A substrate properties, in addition to favorable in vitro intestinal and hepatic stabilities. Following oral dosing (3mg/kg) in Beagle dogs, high levels (>9.0microM) of active metabolite 15 were observed in PBMCs, validating the prodrug design process and leading to the nomination of 5 as a clinical candidate.

Novel nucleotide human immunodeficiency virus reverse transcriptase inhibitor GS-9148 with a low nephrotoxic potential: characterization of renal transport and accumulation.

Accumulation of antiviral nucleotides in renal proximal tubules is controlled by their basolateral uptake via the human renal organic anion transporters type 1 (hOAT1) and 3 (hOAT3) and apical efflux via the multidrug resistance protein 4 (MRP4). GS-9148 is a novel ribose-modified nucleotide human immunodeficiency virus (HIV) reverse transcriptase inhibitor, and its oral prodrug GS-9131 is currently being evaluated in the clinic as an anti-HIV agent. To assess the potential of GS-9148 for nephrotoxicity, its mechanism of renal transport, cytotoxicity, and renal accumulation were explored in vitro and in vivo. In comparison with the acyclic nucleotides cidofovir, adefovir, and tenofovir, GS-9148 showed 60- to 100-fold lower efficiency of transport (V(max)/K(m)) by hOAT1 and was 20- to 300-fold less cytotoxic in cells overexpressing hOAT1, indicating its lower hOAT1-mediated intracellular accumulation and reduced intrinsic cytotoxicity. GS-9148 was also relatively inefficiently transported by hOAT3. Similar to acyclic nucleotides, GS-9148 was a substrate for MRP4 as evidenced by its reduced intracellular retention in cells overexpressing the efflux pump. Consistent with these molecular observations, GS-9148 was inefficiently taken up by fresh human renal cortex tissue in vitro and showed a limited accumulation in kidneys in vivo following oral administration of [(14)C]GS-9131 to dogs. Compared to acyclic nucleotide analogs, GS-9148 was also found to have lower net active tubular secretion in dogs. Collectively, these results suggest that GS-9148 exhibits a low potential for renal accumulation and nephrotoxicity.

Design, synthesis, and anti-HIV activity of 4'-modified carbocyclic nucleoside phosphonate reverse transcriptase inhibitors.

A diphosphate of a novel cyclopentyl based nucleoside phosphonate with potent inhibition of HIV reverse transcriptase (RT) (20, IC(50)=0.13 microM) has been discovered. In cell culture the parent phosphonate diacid 9 demonstrated antiviral activity EC(50)=16 microM, within two-fold of GS-9148, a prodrug of which is currently under clinical investigation, and within 5-fold of tenofovir (PMPA). In vitro cellular metabolism studies using 9 confirmed that the active diphosphate metabolite is produced albeit at a lower efficiency relative to GS-9148.

Effect of nucleoside and nucleotide reverse transcriptase inhibitors of HIV on endogenous nucleotide pools.

BACKGROUND: Alterations in endogenous nucleotide pools as a result of HIV therapy with nucleoside and nucleotide reverse transcriptase inhibitors (N[t]RTIs) is a proposed mechanism for therapy-related adverse events and drug interactions resulting in treatment failure. In vitro studies were performed in order to understand the effect of N(t)RTIs on endogenous nucleotide pools. METHODS: The T-cell line CEM-CCRF was treated with control antimetabolites or the N(t)RTIs abacavir, didanosine, lamivudine, tenofovir (TFV) and zidovudine (AZT), either alone or in combination. The levels of natural 2'-deoxynucleoside triphosphates (dNTP) and ribonucleoside triphophosphates were determined by liquid chromatography coupled with triple quadrupole mass spectrometry. RESULTS: Antimetabolites altered nucleotide pools in a manner consistent with their known mechanisms of action. AZT was the only N(t)RTI that significantly altered dNTP pools. incubation of 10 microM AZT, either alone or in combination with other N(t)RTIs, increased 2'-deoxyadenosine triphosphate, 2'-deoxyguanosine triphosphate and thymidine triphosphate levels by up to 1.44-fold the concentrations observed in untreated cells. At higher than pharmacological concentrations of AZT, evidence for inhibition of 2'-deoxycytidylate deaminase and enzymes involved in the salvage of thymidine was also observed. Phosphorylated metabolites of TFV are known to inhibit purine nucleoside phosphorylase (PNP). However, in contrast to a potent PNP inhibitor, TFV was unable to alter intracellular dNTP pools upon addition of exogenous 2'-deoxyguanosine. CONCLUSIONS: N(t)RTIs have the potential to alter nucleotide pools; however, at the pharmacologically relevant concentrations, tested N(t)RTI or their combinations did not have an effect on nucleotide pools with the notable exception of AZT.

Molecular assessment of the potential for renal drug interactions between tenofovir and HIV protease inhibitors.

BACKGROUND: Active renal secretion of tenofovir (TFV) across proximal tubules occurs via uptake by human organic anion transporters 1 and 3 (hOAT1 and hOAT3) coupled with efflux by multidrug resistance protein 4 (MRP4). Co-administration of some HIV protease inhibitors (PIs) with tenofovir disoproxil fumarate (TDF), an oral prodrug of TFV, has been shown to increase systemic levels of TFV, leading to a hypothesis that PIs may affect tubular secretion of TFV and potentially alter the renal safety of TDF. METHODS: The effect of PIs on the transport of TFV by hOAT1, hOAT3 and MRP4 was assessed using in vitro cell-based transport models. RESULTS: At concentrations equal to their therapeutic peak plasma levels (Cmax) all PIs showed <20% inhibition of TFV transport by hOAT1. hOAT3 was more sensitive to Pls with ritonavir (RTV) and lopinavir being the most potent inhibitors of TFV transport (62% and 37% inhibition, respectively, at their Cmax). In the absence of human serum, RTV at concentrations exceeding its therapeutic Cmax also exhibited a minor effect on the cellular efflux of TFV by MRP4 (<30% inhibition at 20 microM). However, no effects of PIs on hOAT1, hOAT3 or MRP4 were detected in the presence of human serum with the exception of RTV that inhibited hOAT3 by approximately 35% at its Cmax. In addition, PIs did not affect the cytotoxicity of TFV or TDF in MRP4- or MRP2-overexpressing cells. CONCLUSION: These data indicate a low potential of PIs to interfere with the active tubular secretion of TFV and to alter the clinical renal safety profile of TDF.

Simultaneous quantitation of the nucleotide analog adefovir, its phosphorylated anabolites and 2'-deoxyadenosine triphosphate by ion-pairing LC/MS/MS.

The nucleotide analog adefovir is an important therapy for hepatitis B viral infection. The study of nucleoside/tide pharmacology has been hampered by difficulties encountered when trying to develop LC/MS/MS methods for these polar analytes. In an attempt to identify a more convenient, selective and sensitive alternative to the analysis of the metabolism of radiolabeled parent nucleotide traditionally used for in vitro cell culture studies, an LC/MS/MS method was developed for the quantitative detection of adefovir and its phosphorylated metabolites in cellular samples. Ion-pairing reversed phase LC using tetrabutylammonium (TBA) and ammonium phosphate had the best compromise between chromatographic separation and positive mode MS/MS detection. Using microbore reverse phase columns and a low flow acetonitrile gradient it was possible to quantitate adefovir, its metabolites and 2'-deoxyadenosine triphosphate. A cross-validation showed comparable levels of adefovir and its metabolites were determined using either LC/MS/MS or radioactivity detection. However, initial methods were conducted at high pH and utilized an acetonitrile step gradient causing unacceptable column life and unpredictable equilibration. Further method optimization lowered the concentration of TBA and phosphate, decreased pH and applied a linear gradient of acetonitrile. This work resulted in a method that was found to have sensitivity, accuracy and precision sufficient to be a useful tool in the study of the intracellular pharmacology of adefovir in vitro and may be more broadly applicable.

In vitro evaluation of the anti-HIV activity and metabolic interactions of tenofovir and emtricitabine.

BACKGROUND: Tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) are nucleoside reverse transcriptase inhibitors (NRTIs) with once-daily dosing approved for use in HIV-1 infection. We evaluated this combination for anti-HIV activity and intracellular metabolic interactions in vitro. METHODS: Intracellular anabolism of combinations of FTC and tenofovir (TFV) were studied in the human T leukaemic cell line CEM and human peripheral blood mononuclear cells (PBMCs). The anti-HIV activity of the combination was studied in the human T leukaemic MT-2 cell line against wild-type or mutant virus (K65R and M184V) and in PBMCs against wild-type virus. Antiviral synergy was quantitated by isobologram and MacSynergy methods. RESULTS: Both TFV and FTC were efficiently converted to their active metabolites in PBMCs and CEM cells. In CEM cells, there was a statistically significant increase in the levels of TFV diphosphate (P=0.047) and FTC triphosphate (P=0.0069) following a 24 h incubation with the combination compared with the levels seen with the individual drugs. In PBMCs, similar levels of active metabolites were observed for each drug when they were incubated iindividually or in combination. The combination of TFV and FTC displayed additive to synergistic activity against HIV replication in PBMCs and resulted in strongly synergistic anti-HIV activity in MT-2 cells against both wild-type and mutant virus. CONCLUSIONS: The combination of TFV and FTC showed additive to synergistic anti-HIV activity in vitro, which correlated with the levels of intracellular phosphorylation observed. These results support the use of these drugs as a dual NRTI backbone in combination therapy for the treatment of HIV-1.

Lack of a metabolic and antiviral drug interaction between tenofovir, abacavir and lamivudine.

OBJECTIVE: An anti-HIV regimen composed of the nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) tenofovir (TFV) disoproxil fumarate (TDF), abacavir (ABC) and lamivudine (3TC) has performed poorly in patients. This study evaluated the combination of TFV, ABC and 3TC for metabolic or antiviral antagonism in vitro. DESIGN: Procedures were developed to evaluate the in vitro metabolism and antiviral activity of drug combinations of TFV, ABC and 3TC in cell types relevant for HIV infection. METHODS: Anabolism of combinations of TFV and ABC were studied over a 24 h period in the human T leukaemic CEM lymphoblast cell line and human primary peripheral blood mononuclear cells (PBMCs) stimulated with human interleukin-2 and phytohaemagglutinin. The anti-HIV activity of combinations of TFV and ABC in the presence or absence of 3TC was studied in stimulated PBMCs infected with the HXB2 strain of HIV-1. RESULTS: Levels of the active metabolites produced from TFV and ABC after incubation with CEM or PBMCs showed no significant change upon introduction of the other NRTI. Moreover, the pool sizes for the natural substrates of 2'-deoxyadenosine triphosphate and 2'-deoxyguanosine triphosphate were also unchanged. In anti-HIV assays in PBMCs, the combination of TFV and ABC was found to be additive with respect to inhibition of HIV replication. Addition of 3TC to the combination did not result in synergistic or antagonistic effects. CONCLUSIONS: The poor efficacy of the triple NRTI regimen of TDF, ABC and 3TC is probably not due to a metabolic drug interaction resulting in antagonism of antiviral activity.

Effective metabolism and long intracellular half life of the anti-hepatitis B agent adefovir in hepatic cells.

Adefovir dipivoxil (ADV) is esterolytically cleaved to the 2'-deoxyadenosine monophosphate (dAMP) analog adefovir, subsequent phosphorylation leads to the formation of the anti-Hepatitis B virus (HBV) agent adefovir-DP. To better understand the mechanism of action of ADV, metabolism studies were done in Hep G2, Huh-7 and primary human hepatocytes. Separation of radiolabeled adefovir metabolites after incubation in Hep G2 cells suggested that adefovir in its mono- and di-phosphorylated forms are the only metabolites formed from adefovir. Incubation of 10 microM adefovir with hepatic cell lines and fresh monolayers of primary human hepatocytes from two donors and analysis of intracellular metabolites by liquid chromatography coupled to tandem mass spectrometry resulted in adefovir-DP levels of approximately 10 pmol/million cells. Adefovir was more efficiently phosphorylated in primary hepatocytes than cell lines with adefovir-DP accounting for 44% versus 26% of total intracellular adefovir after 24 h. Egress studies showed adefovir-DP to have a half-life of 33 +/- 3 h, 10 +/- 1 h, 48 +/- 3 h and 33 +/- 2 h in Hep G2, Huh-7, and primary hepatocytes from two separate donors, respectively. The markedly shorter half-life in Huh-7 cells was inferred to be transport dependent based on its sensitivity to the transport inhibitor MK-571. Effective phosphorylation coupled with a long intracellular half-life and small competing dATP pool sizes in primary hepatocytes forms the cellular metabolic basis for the efficacy of adefovir dipivoxil in the treatment of chronic hepatitis B.

Nucleotide analogue prodrug tenofovir disoproxil enhances lymphoid cell loading following oral administration in monkeys.

The antiviral drug tenofovir (TFV) is orally administered as the fumarate salt of its disoproxil prodrug (TFV disoproxil fumarate (TDF)). TFV is a dianion at physiological pH and, as a result, has poor lipid membrane permeability. Administration of the lipophilic and cell-permeable prodrug, TFV disoproxil, enhances the oral absorption of TFV. In order to determine whether oral administration of TDF also increases distribution to sites of viral infection, the plasma and circulating lymphoid cell pharmacokinetics of TFV and its phosphorylated metabolites were assessed following a single oral TDF or subcutaneous TFV administration at doses yielding equivalent plasma exposures to TFV in macaques. Despite TFV disoproxil's lack of plasma stability and undetectable levels in the first plasma samples taken, oral administration of TDF resulted in 7.9-fold higher peripheral blood mononuclear cell exposures to the active metabolite, TFV-diphosphate. The apparent plasma terminal half-life (t(1/2)) of TFV was also longer following oral TDF relative to subcutaneous TFV administration (median t(1/2) of 15.3 and 3.9 h, respectively), suggesting broader distribution to cells and tissues outside of the central plasma compartment. In conclusion, the disoproxil pro-moiety enhances not only the oral absorption of TFV but also tissue and lymphoid cell loading. These results illustrate that administration of even a fleeting prodrug can increase target tissue loading and give valuable insight for future prodrug development.

Intracellular metabolism of the nucleotide prodrug GS-9131, a potent anti-human immunodeficiency virus agent.

GS-9131 is a phosphonoamidate prodrug of the novel ribose-modified phosphonate nucleotide analog GS-9148 that demonstrates potent anti-human immunodeficiency virus type 1 (HIV-1) activity and an excellent resistance profile in vitro. Prodrug moieties were optimized for the efficient delivery of GS-9148 and its active diphosphate (DP) metabolite to lymphoid cells following oral administration. To understand the intracellular pharmacology of GS-9131, incubations were performed with various types of lymphoid cells in vitro. The intracellular accumulation and antiviral activity levels of GS-9148 were limited by its lack of cellular permeation, and GS-9131 increased the delivery of GS-9148-DP by 76- to 290-fold relative to that of GS-9148. GS-9131 activation was saturable at high extracellular concentrations, potentially due to a high-affinity promoiety cleavage step. Once inside the cells, GS-9148 was efficiently phosphorylated, forming similar amounts of anabolites in primary lymphoid cells. The levels of GS-9148-DP formed in peripheral blood mononuclear cells infected with HIV-1 were similar to that in uninfected PBMCs, and approximately equivalent intracellular concentrations of GS-9148-DP and tenofovir (TVF)-DP were required to inhibit viral replication by 90%. Once it was formed, GS-9148-DP was efficiently retained in activated CD4(+) cells, with a half-life of 19 h. In addition, GS-9131 showed a low potential for drug interactions with other adenine nucleoside/nucleotide reverse transcriptase inhibitors, based on the lack of competition for anabolism between suprapharmacologic concentrations of GS-9148 and TVF and the lack of activity of GS-9131 metabolites against purine nucleoside phosphorylase, an enzyme involved in the clearance of 2',3'-dideoxyinosine. Together, these observations elucidate the cellular pharmacology of GS-9131 and illustrate its efficient loading of lymphoid cells, resulting in a prolonged intracellular exposure to the active metabolite GS-9148-DP.

Design and profiling of GS-9148, a novel nucleotide analog active against nucleoside-resistant variants of human immunodeficiency virus type 1, and its orally bioavailable phosphonoamidate prodrug, GS-9131.

GS-9148 [(5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl)phosphonic acid] is a novel ribose-modified human immunodeficiency virus type 1 (HIV-1) nucleotide reverse transcriptase (RT) inhibitor (NRTI) selected from a series of nucleoside phosphonate analogs for its favorable in vitro biological properties including (i) a low potential for mitochondrial toxicity, (ii) a minimal cytotoxicity in renal proximal tubule cells and other cell types, (iii) synergy in combination with other antiretrovirals, and (iv) a unique resistance profile against multiple NRTI-resistant HIV-1 strains. Notably, antiviral resistance analysis indicated that neither the K65R, L74V, or M184V RT mutation nor their combinations had any effect on the antiretroviral activity of GS-9148. Viruses carrying four or more thymidine analog mutations showed a substantially smaller change in GS-9148 activity relative to that observed with most marketed NRTIs. GS-9131, an ethylalaninyl phosphonoamidate prodrug designed to maximize the intracellular delivery of GS-9148, is a potent inhibitor of multiple subtypes of HIV-1 clinical isolates, with a mean 50% effective concentration of 37 nM. Inside cells, GS-9131 is readily hydrolyzed to GS-9148, which is further phosphorylated to its active diphosphate metabolite (A. S. Ray, J. E. Vela, C. G. Boojamra, L. Zhang, H. Hui, C. Callebaut, K. Stray, K.-Y. Lin, Y. Gao, R. L. Mackman, and T. Cihlar, Antimicrob. Agents Chemother. 52:648-654, 2008). GS-9148 diphosphate acts as a competitive inhibitor of RT with respect to dATP (K(i) = 0.8 muM) and exhibits low inhibitory potency against host polymerases including DNA polymerase gamma. Oral administration of GS-9131 to beagle dogs at a dose of 3 mg/kg of body weight resulted in high and persistent levels of GS-9148 diphosphate in peripheral blood mononuclear cells (with a maximum intracellular concentration of >9 microM and a half-life of >24 h). This favorable preclinical profile makes GS-9131 an attractive clinical development candidate for the treatment of patients infected with NRTI-resistant HIV.

Mechanism of active renal tubular efflux of tenofovir.

Tenofovir (TFV) undergoes renal elimination by a combination of glomerular filtration and active tubular secretion. While transporter-mediated uptake of TFV from the blood into proximal-tubule cells has been well characterized, comparatively little is known about the efflux system responsible for transporting TFV into the lumen during active tubular secretion. Therefore, members of the ATP-binding cassette family of efflux pumps expressed at the apical side of proximal-tubule cells were studied for the ability to transport TFV. Studies in multiple independent in vitro systems show TFV not to be a substrate for P glycoprotein (Pgp) or multidrug resistance protein type 2 (MRP2). In contrast to Pgp and MRP2, TFV was observed to be a substrate for MRP4. TFV accumulated to fivefold lower levels in MRP4-overexpressing cells, and its accumulation could be increased by an MRP inhibitor. Furthermore, MRP4-overexpressing cells were found to be 2.0- to 2.5-fold less susceptible to cytotoxicity caused by TFV. ATP-dependent uptake of TFV was observed in membrane vesicles containing MRP4 but not in vesicles lacking the transporter. On the basis of these and previous results, the molecular transport pathway for the active tubular secretion of TFV through renal proximal-tubule cells involves uptake from the blood mediated by human organic anion transporters 1 and 3 and efflux into urine by MRP4. A detailed understanding of the molecular mechanism of TFV active tubular secretion will facilitate the assessment of potential renal drug-drug interactions with coadministered agents.