Characterization of the Metabolic Activation of Hepatitis C Virus Nucleoside Inhibitor β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) and Identification of a Novel Active 5′-Triphosphate Species

Abstract

β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) replication in the subgenomic HCV replicon system, and its corresponding 5′-triphosphate is a potent inhibitor of the HCV RNA polymerase in vitro. In this study the formation of PSI-6130-triphosphate was characterized in primary human hepatocytes. PSI-6130 and its 5′-phosphorylated derivatives were identified, and the intracellular concentrations were determined. In addition, the deaminated derivative of PSI-6130, β-d-2′-deoxy-2′-fluoro-2′-C-methyluridine (RO2433, PSI-6026) and its corresponding phosphorylated metabolites were identified in human hepatocytes after incubation with PSI-6130. The formation of the 5′-triphosphate (TP) of PSI-6130 (PSI-6130-TP) and RO2433 (RO2433-TP) increased with time and reached steady state levels at 48 h. The formation of both PSI-6130-TP and RO2433-TP demonstrated a linear relationship with the extracellular concentrations of PSI-6130 up to 100 μm, suggesting a high capacity of human hepatocytes to generate the two triphosphates. The mean half-lives of PSI-6130-TP and RO2433-TP were 4.7 and 38 h, respectively. RO2433-TP also inhibited RNA synthesis by the native HCV replicase isolated from HCV replicon cells and the recombinant HCV polymerase NS5B with potencies comparable with those of PSI-6130-TP. Incorporation of RO2433-5′-monophosphate (MP) into nascent RNA by NS5B led to chain termination similar to that of PSI-6130-MP. These results demonstrate that PSI-6130 is metabolized to two pharmacologically active species in primary human hepatocytes. β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) replication in the subgenomic HCV replicon system, and its corresponding 5′-triphosphate is a potent inhibitor of the HCV RNA polymerase in vitro. In this study the formation of PSI-6130-triphosphate was characterized in primary human hepatocytes. PSI-6130 and its 5′-phosphorylated derivatives were identified, and the intracellular concentrations were determined. In addition, the deaminated derivative of PSI-6130, β-d-2′-deoxy-2′-fluoro-2′-C-methyluridine (RO2433, PSI-6026) and its corresponding phosphorylated metabolites were identified in human hepatocytes after incubation with PSI-6130. The formation of the 5′-triphosphate (TP) of PSI-6130 (PSI-6130-TP) and RO2433 (RO2433-TP) increased with time and reached steady state levels at 48 h. The formation of both PSI-6130-TP and RO2433-TP demonstrated a linear relationship with the extracellular concentrations of PSI-6130 up to 100 μm, suggesting a high capacity of human hepatocytes to generate the two triphosphates. The mean half-lives of PSI-6130-TP and RO2433-TP were 4.7 and 38 h, respectively. RO2433-TP also inhibited RNA synthesis by the native HCV replicase isolated from HCV replicon cells and the recombinant HCV polymerase NS5B with potencies comparable with those of PSI-6130-TP. Incorporation of RO2433-5′-monophosphate (MP) into nascent RNA by NS5B led to chain termination similar to that of PSI-6130-MP. These results demonstrate that PSI-6130 is metabolized to two pharmacologically active species in primary human hepatocytes. Hepatitis C is a major health problem affecting ∼170 million people worldwide of which around 3 million chronically infected patients reside within the United States (1Alter M.J. Kruszon-Moran D. Nainan O.V. McQuillan G.M. Gao F. Moyer L.A. Kaslow R.A. Margolis H.S. N. Engl. J. Med. 1999; 341: 556-562Crossref PubMed Scopus (2435) Google Scholar). The current standard treatment for hepatitis C consisting of pegylated interferon-α and ribavirin only results in about a 50% sustained virological response in patients infected with genotype 1 hepatitis C virus (HCV), 2The abbreviations used are: HCV, hepatitis C virus; MP, 5′-monophosphate; DP, 5′-diphosphate; TP, 5′-triphosphate; HPLC, high performance liquid chromatography. 2The abbreviations used are: HCV, hepatitis C virus; MP, 5′-monophosphate; DP, 5′-diphosphate; TP, 5′-triphosphate; HPLC, high performance liquid chromatography. the most predominant genotype in the United States and Europe (2Manns M.P. McHutchison J.G. Gordon S.C. Rustgi V.K. Shiffman M. Reindollar R. Goodman Z.D. Koury K. Ling M. Albrecht J.K. Lancet. 2001; 358: 958-965Abstract Full Text Full Text PDF PubMed Scopus (5902) Google Scholar, 3Fried M.W. Shiffman M.L. Reddy K.R. Smith C. Marinos G. Goncales Jr., F.L. Haussinger D. Diago M. Carosi G. Dhumeaux D. Craxi A. Lin A. Hoffman J. Yu J. N. Engl. J. Med. 2002; 347: 975-982Crossref PubMed Scopus (5884) Google Scholar, 4Hadziyannis S.J. Sette Jr., H. Morgan T.R. Balan V. Diago M. Marcellin P. Ramadori G. Bodenheimer Jr., H. Bernstein D. Rizzetto M. Zeuzem S. Pockros P.J. Lin A. Ackrill A.M. Ann. Intern. Med. 2004; 140: 346-355Crossref PubMed Scopus (2754) Google Scholar). New treatment options with improved clinical efficacy and greater tolerability are urgently needed. Novel antiviral agents targeting essential processes of HCV replication as part of optimized combination regimens could achieve increased clinical efficacy and potentially improved adverse event profiles as well as shortened treatment duration as compared with the current standard of care. HCV RNA replication is mediated by a membrane-associated multiprotein replication complex (5Hardy R.W. Marcotrigiano J. Blight K.J. Majors J.E. Rice C.M. J. Virol. 2003; 77: 2029-2037Crossref PubMed Scopus (72) Google Scholar, 6Ma H. Leveque V. De Witte A. Li W. Hendricks T. Clausen S.M. Cammack N. Klumpp K. Virology. 2005; 332: 8-15Crossref PubMed Scopus (59) Google Scholar). The HCV NS5B protein, the RNA-dependent RNA polymerase, is the catalytic subunit of the HCV replication complex and is responsible for the synthesis of the RNA progeny and, hence, is a prime target of anti-viral inhibition. Nucleoside analogs have been established as successful antiviral agents targeting the active site of DNA polymerases for the treatment of other viral diseases, including human immunodeficiency virus, hepatitis B virus, and herpes simplex virus (7Vivet-Boudou V. Didierjean J. Isel C. Marquet R. Cell. Mol. Life Sci. 2006; 63: 163-186Crossref PubMed Scopus (60) Google Scholar). The majority of marketed antiviral nucleoside analogs need to be converted to the active 5′-triphosphate forms in the target cells. These nucleotide triphosphate analogs then serve as alternative substrates for the viral DNA polymerases and compete with the incorporation of the corresponding natural nucleotide triphosphates. Upon incorporation by the viral DNA polymerases, the lack of the 3′-hydroxyl group in the deoxyribose moiety leads to the termination of the nascent viral DNA (chain termination). In the past few years a number of ribonucleoside analogs with 2′-C-methyl, 2′-O-methyl, or 4′-azido substituents on the ribose moiety have been reported to be inhibitors of HCV replication in the subgenomic replicon system (8Carroll S.S. Tomassini J.E. Bosserman M. Getty K. Stahlhut M.W. Eldrup A.B. Bhat B. Hall D. Simcoe A.L. LaFemina R. Rutkowski C.A. Wolanski B. Yang Z. Migliaccio G. De Francesco R. Kuo L.C. MacCoss M. Olsen D.B. J. Biol. Chem. 2003; 278: 11979-11984Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 9Klumpp K. Leveque V. Le Pogam S. Ma H. Jiang W.R. Kang H. Granycome C. Singer M. Laxton C. Hang J.Q. Sarma K. Smith D.B. Heindl D. Hobbs C.J. Merrett J.H. Symons J. Cammack N. Martin J.A. Devos R. Najera I. J. Biol. Chem. 2006; 281: 3793-3799Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 10Migliaccio G. Tomassini J.E. Carroll S.S. Tomei L. Altamura S. Bhat B. Bartholomew L. Bosserman M.R. Ceccacci A. Colwell L.F. Cortese R. De Francesco R. Eldrup A.B. Getty K.L. Hou X.S. LaFemina R.L. Ludmerer S.W. MacCoss M. McMasters D.R. Stahlhut M.W. Olsen D.B. Hazuda D.J. Flores O.A. J. Biol. Chem. 2003; 278: 49164-49170Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 11Eldrup A.B. Prhavc M. Brooks J. Bhat B. Prakash T.P. Song Q. Bera S. Bhat N. Dande P. Cook P.D. Bennett C.F. Carroll S.S. Ball R.G. Bosserman M. Burlein C. Colwell L.F. Fay J.F. Flores O.A. Getty K. LaFemina R.L. Leone J. MacCoss M. McMasters D.R. Tomassini J.E. Von Langen D. Wolanski B. Olsen D.B. J. Med. Chem. 2004; 47: 5284-5297Crossref PubMed Scopus (190) Google Scholar, 12Le Pogam S. Jiang W.R. Leveque V. Rajyaguru S. Ma H. Kang H. Jiang S. Singer M. Ali S. Klumpp K. Smith D. Symons J. Cammack N. Najera I. Virology. 2006; 351: 349-359Crossref PubMed Scopus (118) Google Scholar, 13Smith D.B. Martin J.A. Klumpp K. Baker S.J. Blomgren P.A. Devos R. Granycome C. Hang J. Hobbs C.J. Jiang W.R. Laxton C. Pogam S.L. Leveque V. Ma H. Maile G. Merrett J.H. Pichota A. Sarma K. Smith M. Swallow S. Symons J. Vesey D. Najera I. Cammack N. Bioorg. Med. Chem. Lett. 2007; 17: 2570-2576Crossref PubMed Scopus (88) Google Scholar). Prodrugs of two nucleoside analogs, 2′-C-methylcytidine (NM107) and 4′-azidocytidine (R1479), have successfully progressed into clinical development and shown efficacy in HCV-infected patients (14Afdhal, N., Rodriguez-Torres, M., Lawitz, E., Godofsky, E., Chao, G., Fielman, B., Knox, S., and N., B. (2005) in 40th Annual Meeting of the European Association for the Study of the Liver, April 13–17, 2005, European Association for the Study of the Liver (EASL), Paris, abstract 93Google Scholar, 15Roberts s. Cooksley G. Dore G. Robson R. Shaw D. H. M. S. G. D. Klumpp K. M. C. 2006; Scholar). The corresponding nucleotide triphosphate analogs are substrates for HCV polymerase NS5B and RNA synthesis of HCV NS5B in vitro. The incorporation of the nucleotide analogs into nascent HCV RNA the of RNA by in termination of the nascent RNA nucleoside analogs are chain the of a 3′-hydroxyl (PSI-6130) been identified as a potent and inhibitor of HCV replication in the subgenomic replicon system with or in human or cells T.R. J. L. S. T. J. Bennett P.A. M.J. Chem. 2006; 17: PubMed Scopus Google Scholar). The corresponding triphosphate of PSI-6130 is inhibitor of HCV NS5B with natural H. M. T.R. T. A. M.J. P.A. 2007; PubMed Scopus Google Scholar). to the active 5′-triphosphate by is part of the of for nucleoside In this study the of in primary human hepatocytes isolated from that was converted to 5′-triphosphate and β-d-2′-deoxy-2′-fluoro-2′-C-methyluridine 5′-triphosphate of the phosphorylated the of the formation of the two active and the of the two the native HCV replicase and NS5B as well as the of of the two triphosphates. (PSI-6130) was by L. S. T.R. M.J. P.A. J. Med. Chem. 2005; PubMed Scopus Google Scholar). of PSI-6130 was in and at PSI-6130 was at The was in 50% at the of with a of The was at The phosphorylated derivatives of PSI-6130, and were by was at and were by were in and at was from of human hepatocytes or were from or from In human hepatocytes from were or on at million cells well from the were to for at the of the were at in a the time of and of PSI-6130, human primary hepatocytes were with PSI-6130 at a of and The was and 1 time and were up in the response of the of PSI-6130, human primary hepatocytes were with PSI-6130 at and for h. concentrations of PSI-6130 were by PSI-6130 with PSI-6130. were after of the of the of PSI-6130 and human primary hepatocytes were for with PSI-6130 at and The was with the PSI-6130 and then with PSI-6130 at and time after the of PSI-6130. were up for time The of the for were at the of the the of for the time of the was and the cells were with The cells were into 1 of and in for at The were then at for to The was to and in a at The were at The of were in and a Life were with PSI-6130, and phosphorylated phosphorylated derivatives of PSI-6130 were by with a to a The from to B and B from to and to in 1 The was 1 of or Life to was used for the of species in the The of PSI-6130 and RO2433 was by with a to a The from in to B in and 3 and then from B to B 3 and B from to and to in The was 1 PSI-6130 and its metabolites were identified by of the of the intracellular species in the with the of in the and by at of were with at a of at for to phosphorylated metabolites of PSI-6130. the were by HCV a HCV subgenomic replicon which a as of HCV RNA been K. Leveque V. Le Pogam S. Ma H. Jiang W.R. Kang H. Granycome C. Singer M. Laxton C. Hang J.Q. Sarma K. Smith D.B. Heindl D. Hobbs C.J. Merrett J.H. Symons J. Cammack N. Martin J.A. Devos R. Najera I. J. Biol. Chem. 2006; 281: 3793-3799Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). The of of HCV replication by nucleoside analogs and were as Pogam S. Jiang W.R. Leveque V. Rajyaguru S. Ma H. Kang H. Jiang S. Singer M. Ali S. Klumpp K. Smith D. Symons J. Cammack N. Najera I. Virology. 2006; 351: 349-359Crossref PubMed Scopus (118) Google Scholar). HCV membrane-associated native HCV replication were isolated from replicon as H. Leveque V. De Witte A. Li W. Hendricks T. Clausen S.M. Cammack N. Klumpp K. Virology. 2005; 332: 8-15Crossref PubMed Scopus (59) Google Scholar). The of the RNA synthesis of the HCV by PSI-6130-TP was as H. Leveque V. De Witte A. Li W. Hendricks T. Clausen S.M. Cammack N. Klumpp K. Virology. 2005; 332: 8-15Crossref PubMed Scopus (59) Google that of replicase complex replicon was to a for The of the RNA synthesis of the HCV by RO2433-TP was in of replicase complex replicon 1 and of 1 In with or the nucleotide triphosphate inhibitor in a of for HCV of PSI-6130-TP on the RNA-dependent RNA polymerase of recombinant number was as the incorporation of nucleotide into RNA as H. Leveque V. De Witte A. Li W. Hendricks T. Clausen S.M. Cammack N. Klumpp K. Virology. 2005; 332: 8-15Crossref PubMed Scopus (59) Google with the were site RNA 1 of 1 and The of RO2433-TP was as with the of 1 of 1 The at which the is by 50% was to the is the at is the and to the The for or were with the for was of and to for was of and to were by to the of RNA synthesis by is the or RNA synthesis at and to or were from the for is the of or and is the for or were from at and or concentrations in Incorporation RNA nucleotide incorporation and of nucleotide and nucleotide triphosphate analogs by HCV polymerase was with a RNA as K. Leveque V. Le Pogam S. Ma H. Jiang W.R. Kang H. Granycome C. Singer M. Laxton C. Hang J.Q. Sarma K. Smith D.B. Heindl D. Hobbs C.J. Merrett J.H. Symons J. Cammack N. Martin J.A. Devos R. Najera I. J. Biol. Chem. 2006; 281: 3793-3799Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). The incorporation and of and analogs were with and nucleotide at the The incorporation and of and analogs were with the RNA and nucleotide at the The RNA were on a and of from primary human hepatocytes with (PSI-6130) were by PSI-6130 and metabolites from PSI-6130 were identified by the of species with the of shown in PSI-6130 and the 5′-phosphorylated derivatives and PSI-6130-TP were identified in human hepatocytes with PSI-6130. In addition, metabolites with corresponding to those of the deaminated of PSI-6130, β-d-2′-deoxy-2′-fluoro-2′-C-methyluridine (RO2433, and its corresponding and RO2433-TP were The of the and analogs and were the and, as a at been reported that was metabolized to and to with of the or after J.E. Getty K. Stahlhut M.W. S. Bhat B. Eldrup A.B. Prakash T.P. Carroll S.S. Flores MacCoss M. McMasters D.R. Migliaccio G. Olsen D.B. 2005; PubMed Scopus Google Scholar). of the intracellular metabolites of PSI-6130 was with time corresponding to those of and was for of PSI-6130 at the or for at the These that the primary of PSI-6130 in human hepatocytes were at the and at the The were also by to metabolites of PSI-6130. species with corresponding to PSI-6130 and its RO2433 were with PSI-6130 the predominant intracellular species was of formation of or These well with the and the of at the and at the The phosphorylated with the of were by and as a and treatment converted the intracellular metabolites to PSI-6130 and intracellular metabolites phosphorylated derivatives of PSI-6130 and These results established that PSI-6130 could be phosphorylated to its pharmacologically active 5′-triphosphate and that PSI-6130 its could be deaminated to the corresponding analogs in primary human hepatocytes. In of RO2433 and incubation of human hepatocytes with PSI-6130 in the formation of concentrations of the triphosphate of its the RO2433 could HCV replication targeting NS5B cells a subgenomic genotype HCV replicon were with RO2433 or PSI-6130 for h, and of was determined. RO2433 the HCV replication in the HCV subgenomic replicon system at concentrations up to 100 μm, PSI-6130 inhibited HCV replication with a mean of the The lack of in the replicon could be to the triphosphate of RO2433 the HCV RNA polymerase, the RNA synthesis of the native membrane-associated HCV replication isolated from the replicon cells was in the of RO2433-TP inhibited the RNA synthesis of HCV replicase with a mean of μm, PSI-6130-TP inhibited HCV replicase with a mean of RO2433-TP also inhibited the RNA synthesis of the recombinant HCV NS5B on a RNA from the of the of the HCV with of and of μm, as compared with of and of for PSI-6130-TP the These results established that both RO2433-TP and PSI-6130-TP are potent inhibitors of RNA synthesis by HCV of PSI-6130 and RO2433 and NS5B NS5B in a the of HCV polymerase by PSI-6130-TP and incorporation and chain The incorporation of nucleotide and nucleotide analogs by HCV polymerase NS5B was in a a RNA Incorporation of and was from a could be in the of the nucleotide PSI-6130-TP and could also serve as substrates for HCV NS5B and were into the nascent RNA and incorporation of or in the of the nucleotide was was at concentrations up to 1 and Incorporation of and was from a the RNA could be in the of the nucleotide RNA was in the of and of to by NS5B HCV NS5B was also to and was to the and in the of the nucleotide and The with and as well as and to of the suggesting the incorporation of and was results demonstrate that PSI-6130-TP and RO2433-TP serve as alternative substrates for HCV NS5B and as chain into nascent this was was reported that the incorporation of into the nascent RNA by HCV polymerase led to chain in with H. M. T.R. T. A. M.J. P.A. 2007; PubMed Scopus Google Scholar). of of PSI-6130 in the steady state of the two in hepatocytes after to PSI-6130, primary human hepatocytes from were with PSI-6130 for up to h. The of PSI-6130 by human hepatocytes was and intracellular reached steady state levels at 1 of PSI-6130 the time in this study PSI-6130-TP was in hepatocytes from at after PSI-6130 incubation and increased with time to steady state levels at h. The formation of the triphosphate of the demonstrated a time to that of PSI-6130-TP. RO2433-TP was in hepatocytes from only of at and in hepatocytes of at h. RO2433-TP formation reached steady state levels at h. RO2433-TP concentrations were those of PSI-6130-TP at time those of PSI-6130-TP at and The mean steady state concentrations of PSI-6130-TP and RO2433-TP after incubation with PSI-6130 were and cells at 48 h, respectively. PSI-6130 was the major intracellular species at time after incubation of human hepatocytes with PSI-6130 of and of intracellular from as RO2433-TP was only in of at the time from as RO2433-TP was only in of at the time in a the of hepatocytes to PSI-6130 to increased of PSI-6130 and formation of PSI-6130-TP and primary human hepatocytes from 3 were with PSI-6130 at concentrations up to a of for h. intracellular by intracellular in the increased with the extracellular PSI-6130 up to The mean intracellular species at extracellular PSI-6130 reached with the PSI-6130 the predominant species The formation of both PSI-6130-TP and RO2433-TP increased with the extracellular PSI-6130 concentrations up to 100 The concentrations of PSI-6130-TP and RO2433-TP at 100 extracellular PSI-6130 were and respectively. extracellular PSI-6130, PSI-6130-TP and RO2433-TP levels were increased the levels at 100 extracellular PSI-6130, the relationship was PSI-6130-TP and RO2433-TP were the major intracellular phosphorylated species at extracellular PSI-6130 concentrations suggesting a high capacity of the hepatocytes to the to the triphosphate forms and of concentrations of extracellular PSI-6130, RO2433-TP levels were those of response of and of intracellular in a of PSI-6130-TP and human hepatocytes from were for with PSI-6130, at which extracellular PSI-6130 was and the concentrations of intracellular PSI-6130-TP and RO2433-TP were at time up to The PSI-6130-TP up to 1 after the of extracellular PSI-6130. PSI-6130-TP after a with a mean of 4.7 The PSI-6130-TP was at 48 after the of extracellular PSI-6130. The RO2433-TP increased to steady state at after extracellular PSI-6130 and at steady state for up to RO2433-TP with a mean of The levels of species PSI-6130 and RO2433 after of extracellular PSI-6130 and RO2433 were by within after extracellular PSI-6130 suggesting of nucleoside analogs the of PSI-6130-TP and in a The antiviral of a nucleoside inhibitor is the of of the nucleoside into the of the nucleoside to the active intracellular of the and the of the triphosphate to with the RNA synthesis of the viral In this study to of the in the of of The of PSI-6130 with primary human hepatocytes demonstrated the of PSI-6130 into its 5′-phosphorylated derivatives and and to the and its corresponding 5′-phosphorylated PSI-6130 the major intracellular species at time These results that PSI-6130 is only to intracellular and with for at other In demonstrated intracellular with to and of to with at the or after (8Carroll S.S. Tomassini J.E. Bosserman M. Getty K. Stahlhut M.W. Eldrup A.B. Bhat B. Hall D. Simcoe A.L. LaFemina R. Rutkowski C.A. Wolanski B. Yang Z. Migliaccio G. De Francesco R. Kuo L.C. MacCoss M. Olsen D.B. J. Biol. Chem. 2003; 278: 11979-11984Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, J.E. Getty K. Stahlhut M.W. S. Bhat B. Eldrup A.B. Prakash T.P. Carroll S.S. Flores MacCoss M. McMasters D.R. Migliaccio G. Olsen D.B. 2005; PubMed Scopus Google Scholar). The intracellular of most for the of this in the HCV replicon system (8Carroll S.S. Tomassini J.E. Bosserman M. Getty K. Stahlhut M.W. Eldrup A.B. Bhat B. Hall D. Simcoe A.L. LaFemina R. Rutkowski C.A. Wolanski B. Yang Z. Migliaccio G. De Francesco R. Kuo L.C. MacCoss M. Olsen D.B. J. Biol. Chem. 2003; 278: 11979-11984Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, J.E. Getty K. Stahlhut M.W. S. Bhat B. Eldrup A.B. Prakash T.P. Carroll S.S. Flores MacCoss M. McMasters D.R. Migliaccio G. Olsen D.B. 2005; PubMed Scopus Google Scholar). both of PSI-6130 and RO2433 were potent inhibitors of the RNA synthesis of the native HCV replicase and recombinant NS5B suggesting that the antiviral of PSI-6130 is by intracellular The formation of the active PSI-6130-TP and RO2433-TP increased with time and reached steady levels at 48 after to PSI-6130. The mean steady state intracellular concentrations of PSI-6130-TP and RO2433-TP at extracellular PSI-6130 were and These are to about for PSI-6130-TP and for on a of human cells as by the Jr., A. J. Google Scholar). These steady state concentrations are the of PSI-6130-TP and RO2433-TP for HCV NS5B polymerase and are in with the of PSI-6130 in the HCV replicon system the phosphorylated species of PSI-6130 and the of the levels from to intracellular at time of incubation and extracellular concentrations that the to is the the been reported that the human responsible for the of PSI-6130 to triphosphate are and H. M. T.R. T. A. M.J. P.A. 2007; PubMed Scopus Google Scholar). In that study the of PSI-6130 as a for was reported to be of that of and for the results of the reported of the in PSI-6130-TP formation well with formation of PSI-6130 in human hepatocytes. the intracellular concentrations of PSI-6130-TP and RO2433-TP demonstrated linear relationship with the extracellular of PSI-6130 up to 100 μm, a high capacity of human hepatocytes to the active from PSI-6130. the of RO2433-TP HCV polymerase, RO2433 was active in the HCV replicon system at concentrations up to 100 RO2433 was phosphorylated in the replicon cells or could the from PSI-6130, the with a RO2433 is most phosphorylated to the of the HCV replication inhibitor was in the replicon as a could be converted into a potent inhibitor of HCV that a of formation in lack of antiviral of P. G. M.R. F. A. S. C. Rajyaguru S. Martin J.A. Leveque V. Le Pogam S. Najera I. Klumpp K. Smith D.B. C. J. Med. Chem. 2007; PubMed Scopus Google Scholar). a of RO2433 to its in human hepatocytes was most the of by the and phosphorylated to and by and nucleoside W.R. Mol. Google Scholar, V. W. PubMed Scopus Google Scholar, M. A. PubMed Scopus Google Scholar). The for PSI-6130 is in a nucleotide incorporation mediated by HCV demonstrated that the incorporation of both and in the of the nucleotide incorporation similar to that of the chain and B and the in chain on the and been that the chain termination of nucleotide analogs is to a of the group with the ribose of the nucleotide on of the NS5B complex from RNA-dependent RNA polymerase and NS5B G. Tomassini J.E. Carroll S.S. Tomei L. Altamura S. Bhat B. Bartholomew L. Bosserman M.R. Ceccacci A. Colwell L.F. Cortese R. De Francesco R. Eldrup A.B. Getty K.L. Hou X.S. LaFemina R.L. Ludmerer S.W. MacCoss M. McMasters D.R. Stahlhut M.W. Olsen D.B. Hazuda D.J. Flores O.A. J. Biol. Chem. 2003; 278: 49164-49170Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, S.S. Olsen D.B. 2006; PubMed Scopus Google Scholar). could with PSI-6130-TP and RO2433-TP after incorporation to the of the PSI-6130 is a potent and nucleoside inhibitor of HCV replication targeting NS5B polymerase in the HCV replicon demonstrated that PSI-6130 was converted to two pharmacologically active triphosphate PSI-6130-TP and its in primary human hepatocytes. is that RO2433-TP demonstrated greater intracellular 38 as compared with PSI-6130-TP 4.7 PSI-6130 clinical development for the treatment of HCV infected The intracellular of RO2433-TP have for concentrations of the antiviral triphosphate the in clinical and group for recombinant NS5B and and group for HCV replicon cells. A. at for and the phosphorylated derivatives of PSI-6130.