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J. Biol. Chem., Vol. 282, Issue 41, 29812-29820, October 12, 2007

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Characterization of the Metabolic Activation of Hepatitis C Virus Nucleoside Inhibitor -D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) and Identification of a Novel Active 5'-Triphosphate Species^*

From the Roche Palo Alto LLC, Palo Alto, California 94304

Received for publication, June 27, 2007 , and in revised form, August 6, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
-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, -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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 (1). The current standard treatment for hepatitis C consisting of pegylated interferon- and ribavirin only results in about a 50% sustained virological response in patients infected with genotype 1 hepatitis C virus (HCV),² the most predominant genotype in the United States and Europe (2–4). 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 (5, 6). 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 (7). 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 (8–13). 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 (14, 15). The corresponding nucleotide triphosphate analogs are substrates for HCV polymerase NS5B and inhibit RNA synthesis activity of HCV NS5B in vitro. The incorporation of the nucleotide analogs into nascent HCV RNA strongly reduces the efficiency of further RNA elongation by NS5B, resulting in termination of the nascent RNA product. Therefore, these nucleoside analogs are non-obligatory chain terminators despite the presence of a 3'-hydroxyl group.

Recently, -D-2'-deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) has been identified as a potent and selective inhibitor of HCV replication in the subgenomic replicon system with little or no cytotoxicity in various human cell lines or bone marrow precursor cells (16). The corresponding triphosphate of PSI-6130 is an inhibitor of HCV NS5B competitive with natural CTP (17). Conversion to the active 5'-triphosphate form by cellular kinases is an important part of the mechanism of action for nucleoside analogs. In this study we determined the metabolism of -D-2'-deoxy-2'-fluoro-2'-C-methylcytidine in primary human hepatocytes isolated from several donors. We show that -D-2'-deoxy-2'-fluoro-2'-C-methylcytidine was converted to -D-2'-deoxy-2'-fluoro-2'-C-methylcytidine 5'-triphosphate and -D-2'-deoxy-2'-fluoro-2'-C-methyluridine 5'-triphosphate via deamination of the phosphorylated cytidylates. Furthermore, we determined the kinetics of the formation of the two active triphosphates and the potency of the two triphosphates against the native HCV replicase and NS5B as well as the molecular mechanism of action of the two triphosphates.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Compounds—-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) was provided by Pharmasset, Inc. (18). A stock solution of 10 mM PSI-6130 was prepared in Dulbecco's phosphate-buffered saline and stored at –20 °C. Tritium-labeled PSI-6130 was synthesized at Roche Palo Alto LLC. The tritiated compound was dissolved in 50% (v/v) ethanol at the concentration of 0.97 mCi/ml with a specific activity of 25.78 Ci/mmol. The stock solution was stored at –20 °C. The phosphorylated derivatives of PSI-6130, namely PSI-6130-MP, -DP, and -TP, were provided by Pharmasset, Inc. -D-2'-Deoxy-2'-fluoro-2'-C-methyluridine (RO2433) was synthesized at Roche Palo Alto LLC. RO2433-MP, -DP, and -TP were synthesized by TriLink BioTechnologies (San Diego, CA). Compound stock solutions were prepared in nuclease-free H₂O and stored at –20 °C. 3'-dCTP was purchased from TriLink BioTechnologies.

Cell Culture of Primary Human Hepatocytes—Plated fresh human hepatocytes or hepatocyte suspensions were obtained either from CellzDirect, Inc. or from In Vitro Technologies, Inc. Fresh human hepatocytes obtained from each company were plated or cultured on 6-well collagen coated plates (BD Biosciences #356400) at 1.5 million cells per well using complete serum containing medium obtained from the respective companies. Cells were allowed to recover for at least 18 h before the addition of the compound. All incubations were carried out at 37 °C in a humidified 5% CO₂ atmosphere.

To determine the time course of uptake and phosphorylation of PSI-6130, human primary hepatocytes were incubated with ³H-labeled PSI-6130 at a final concentration of 2 µM and 10 µCi/ml. The compound was added 72, 48, 24, 16, 6 and 1 h before cell harvesting. All time points and untreated cell controls were set up in duplicates.

To determine the dose response of the phosphorylation of PSI-6130, human primary hepatocytes were incubated with ³H-labeled PSI-6130 at 0, 2, 10, 25, 50, 100, and 250 µM for 24 h. Final concentrations of PSI-6130 were achieved by supplementing ³H-labeled PSI-6130 with non-radiolabeled PSI-6130. Duplicate cell samples were harvested after 24 h of incubation.

To determine the half-life of the triphosphates of PSI-6130 and RO2433, human primary hepatocytes were incubated for 24 h with ³H-labeled PSI-6130 at 2 µM and 10 µCi/ml. The cell monolayer was washed once with the cell culture medium without PSI-6130 and then incubated with fresh medium without PSI-6130 at 0-, 0.5-, 1-, 2-, 4-, 6-, 8-, 24-, 48-, and 72-h time points after the removal of PSI-6130. Duplicate cell samples were set up for each time point. The viable cell numbers of the untreated cell controls for each experiment were determined at the end of the experiment using the trypan blue exclusion method.

Preparation of Cell Extract for High Performance Liquid Chromatography (HPLC) Analysis—At the time of cell harvest the cell culture medium was aspirated, and the cells were washed once with cold phosphate-buffered saline. The cells were scraped into 1 ml of pre-chilled 60% (v/v) methanol and extracted in methanol for 24 h at –20 °C. The extracted samples were then centrifuged at 10,000 x g for 15 min to remove cell debris. The supernatant was transferred to new tubes and evaporated in a speed vacuum at room temperature. The pellets were stored at –80 °C until analysis.

The dried pellets of cell extracts were dissolved in H₂O and filtered though a nanosep MF centrifugal device (Pall Life Sciences #ODM02C34). Before HPLC analysis, cell extract samples were spiked with unlabeled reference standards PSI-6130, RO2433, and their phosphorylated derivatives.

HPLC—The phosphorylated derivatives of PSI-6130 were separated by ion exchange HPLC with a Whatman Partisil 10 SAX (4.6 x 250 mm) column coupled to a radiometric detector (-RAM, IN/US Systems, Inc.). The mobile phase gradient changed linearly from 0% buffer A (H₂O) to 100% buffer B (0.5 M KH₂PO₄ + 0.8 M KCl) between 4 and 8 min. 100% buffer B ran from 8 to 18 min and changed back to 100% A in 1 min. Buffer A ran until 25 min. The flow rate was 1 ml/min. A ratio of 5:1 Flo Scint IV or Ultima-Flo^TM AP (PerkinElmer Life Sciences) to column eluent was used for the detection of radiolabeled species in the -RAM detector (IN/US Systems, Inc.).

The separation of PSI-6130 and RO2433 was performed by reverse phase chromatography with a Zorbax SB-C8 column (4.6 x 250 mm, 5 µm) coupled to a radiometric detector (-RAM). The gradient changed linearly from 100% buffer A (0.01 M heptane sulfonic acid, sodium salt, 0.1% (v/v) acetic acid in water) to 10% buffer B (0.01 M heptane sulfonic acid sodium salt, 0.1% (v/v) acetic acid in 1:1 methanol water) between 0 and 3 min and then changed linearly from 10% buffer B to 95% buffer B between 3 and 18 min. 95% buffer B ran from 18 to 22 min and changed back to 100% A in 0.1 min. Buffer A ran until 25 min. The flow rate was 1 ml/min. PSI-6130 and its intercellular metabolites were identified by comparison of the retention times of the intracellular species in the radiochromatogram with the retention times of nonradioactive reference standards spiked in the cell extract samples and detected by UV absorption at 270 nm.

Acid Phosphatase Treatment of Cell Extracts—Hepatocyte cell extracts were incubated with acid phosphatase (Sigma #P-0157) at a final concentration of 0.05 mg/ml (23.9 units/ml) at 37 °C for 2.5 h to dephosphorylate any phosphorylated metabolites of PSI-6130. After digestion the samples were analyzed by reversed phase HPLC.

HCV Replicon Assay—The 2209-23 cell line containing a bicistronic HCV subgenomic replicon (genotype 1b, Con1 strain), which expresses a Renilla luciferase reporter gene as an index of HCV RNA replication, has been described before (9). The analysis of inhibition of HCV replication by nucleoside analogs and IC₅₀ determinations were performed as described (12).

HCV Replicase Assay—The membrane-associated native HCV replication complexes were isolated from 2209-23 replicon cell lines as described (6). The inhibition of the RNA synthesis activity of the HCV replicases by PSI-6130-TP was determined as described (6) except that 5 µl of cytoplasmic replicase complex (2.5 x 10⁶ replicon cell equivalent) was added to a 20-µl reaction for 60 min. The inhibition of the RNA synthesis activity of the HCV replicases by RO2433-TP was determined in reactions containing 6.25 µl of cytoplasmic replicase complex (3.1 x 10⁶ replicon cell equivalent), 50 mM HEPES, pH 7.5, 10 mM KCl, 10 mM dithiothreitol, 5 mM MgCl₂, 20 µg/ml actinomycin D, 1 mM ATP, GTP, and CTP, 24 µCi of (0.4 µM) [-³³P]UTP (PerkinElmer #NEG607H), 1 units/µl SUPERase. In (Ambion), 10 mM creatine phosphate, 200 µg/ml creatine phosphokinase with or without the nucleotide triphosphate inhibitor in a final volume of 20 µl for 90 min.

HCV Polymerase Assay—The inhibition potency of PSI-6130-TP on the RNA-dependent RNA polymerase activity of recombinant NS5B570-Con1 (genotype 1b, GenBank^TM accession number AJ242654 [GenBank] ) was measured as the incorporation of radiolabeled nucleotide monophosphate into acid-insoluble RNA products as described (6) with the following modifications; IC₅₀ determinations were carried out using 200 nM in vitro transcribed complementary internal ribosome entry site RNA template, 1 µCi of tritiated UTP (42 Ci/mmol), 500 µM ATP, 500 µM GTP, 1 µM CTP, 1x TMDN buffer (40 mM Tris-HCl, pH 8.0, 4 mM MgCl₂, 4 mM dithiothreitol, 40 mM NaCl) and 200 nM NS5B570-Con1. The inhibition potency of RO2433-TP was determined as described above with the following modification of NTP concentrations: 1 µCi of tritiated CTP (39 Ci/mmol), 500 µM ATP, 500 µM GTP, 1 µM UTP. The compound concentration at which the enzyme-catalyzed rate is reduced by 50% (IC₅₀) was calculated using equation,

(Eq. 1)

where Y corresponds to the relative enzyme activity, % Min is the residual relative activity at saturating compound concentration, % Max is the relative maximum enzymatic activity, and X corresponds to the compound concentration.

The apparent Michaelis constants (K_m(app)) for UTP or CTP were measured using assay conditions above with the following modifications; K_m(app) for CTP was measured using 2 µCi of tritiated UTP (0.93 µM), 4.07 µM unlabeled UTP, 50 µM ATP, 50 µM GTP, and 5 nM to 50 µM CTP; K_m(app) for UTP was measured using 2 µCi of tritiated CTP (1.67 µM), 3.33 µM unlabeled CTP, 50 µM ATP, 50 µM GTP, and 5 nM to 50 µM UTP. Apparent K_m(app) values were calculated by nonlinear fitting using Equation 2,

(Eq. 2

where Y corresponds to the rate of RNA synthesis by NS5B, V_max(app) is the maximum rate or RNA synthesis at saturating substrate concentration, and X corresponds to CTP or UTP concentration.

K_i values were derived from the Cheng-Prusoff Equation (Equation 3) for competitive inhibition,

(Eq. 3)

where [NTP] is the concentration of CTP or UTP, and K_m(app) is the apparent Michaelis constant for CTP or UTP. Mean K_i values were averaged from independent measurements at 0.2, 1, 5, and 25 µM CTP or UTP concentrations in triplicate experiments.

Gel-based Nucleotide Incorporation Assay—The RNA template-directed nucleotide incorporation and extension of nucleotide triphosphates and nucleotide triphosphate analogs by HCV polymerase was performed with a 19-nucleotide RNA oligo (5'-AUGUAUAAUUAUUGUAGCC-3') under assay conditions as described (9). The incorporation and extension of CTP and CTP analogs were determined with 5'-end-radiolabeled GG primer and nucleotide triphosphates at the indicated concentrations. The incorporation and extension of UTP and UTP analogs were performed similarly with the same RNA oligo template, 5'-end-radiolabeled GGC primer and nucleotide triphosphates at the indicated concentrations. The radio-labeled RNA products were separated on a TBE-urea acrylamide gel and analyzed using phosphorimaging (GE Healthcare).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Metabolic Profile of PSI-6130—Cellular extracts from primary human hepatocytes incubated with tritium-labeled -D-2'-deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) were resolved by ion exchange HPLC. PSI-6130 and metabolites derived from PSI-6130 were identified by comparing the retention times of radiolabeled species with the retention times of unlabeled reference compounds (Fig. 1A). As shown in Fig. 1B, PSI-6130 (3.0 min) and the 5'-phosphorylated derivatives PSI-6130-DP (13.2 min) and PSI-6130-TP (16.8 min) were identified in human hepatocytes incubated with PSI-6130. In addition, metabolites with retention times corresponding to those of the deaminated product of PSI-6130, -D-2'-deoxy-2'-fluoro-2'-C-methyluridine (RO2433, 3.8 min) and its corresponding 5'-phosphates RO2433-DP (12.5 min) and RO2433-TP (14.8 min), were detected (Fig. 1B). The monophosphates of the cytidine and uridine analogs PSI-6130-MP and RO2433-MP were not separated sufficiently under the chromatography conditions and, therefore, co-eluted as a single radioactive peak at 10.6 min. It has been reported that 2'-O-methylcytidine was extensively metabolized to CTP and UTP due to deamination coupled with demethylation of the 2'-substituent or base swapping after glycosidic bond cleavage (19). None of the intracellular metabolites of PSI-6130 was eluted with retention time corresponding to those of 2'-C-methyl-CTP, CTP, and UTP (data not shown). Therefore, there was no evidence for metabolism of PSI-6130 at the 2'-position or evidence for hydrolysis at the glycosidic bond. These data suggest that the primary routes of PSI-6130 metabolism in human hepatocytes were phosphorylation at the 5'-position and deamination at the base.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Ion exchange HPLC profile of an extract of primary human hepatocytes incubated with PSI-6130. A, HPLC separation and retention times of reference compounds PSI-6130 (a, 2.5 min), RO2433 (b, 3.4 min), RO2433-MP (c, 10.2 min), PSI-6130-MP (d, 10.4 min), RO2433-DP (e, 12.2 min), PSI-6130-DP (f, 12.8 min), RO2433-TP (g, 14.4 min), and PSI-6130-TP (h, 16.3 min). B, HPLC profile of an extract from hepatocytes incubated with 3H-labeled PSI-6130 at 2 µM for 24 h. The retention times in min of the intracellular species are indicated above the radioactive peaks. Intracellular PSI-6130 and its metabolites were identified by comparing the retention times of the radioactive peaks with those of the UV absorption peaks of reference compounds. There is a calibrated 0.3–0.4-min delay in the retentions times of radioactive trace compared with those of UV trace due to sample traveling from UV detector to radiometric detector.

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Identification of PSI-6130 and its metabolites by reversed phase HPLC and acid phosphatase treatment. A, reversed phase HPLC separation of standard compounds: PSI-6130 (a, 20 min), RO2433 (b, 11.7 min), PSI-6130-MP (c, 7.5 min), (d, PSI-6130-DP, PSI-6130-TP, RO2433-MP, RO2433-DP, and RO2433-TP). B, reversed phase HPLC profile of an extract from hepatocytes incubated with 25 µM extracellular PSI-6130 for 24 h. C, reversed phase HPLC profile of the same extract after acid phosphatase treatment.

Kinetics of Phosphorylation of PSI-6130 in Primary Human Hepatocytes—To determine the steady state level of the two triphosphates in hepatocytes after exposure to PSI-6130, primary human hepatocytes from 4 different donors were incubated with 2 µM PSI-6130 for up to 72 h. The uptake of PSI-6130 by human hepatocytes was fast, and total intracellular activity reached steady state levels at 1 h of PSI-6130 incubation, the earliest time point in this study (Table 2). PSI-6130-TP was detectable in hepatocytes from all 4 donors at 6 h after PSI-6130 incubation and increased with time to reach steady state levels at 24–48 h. The formation of the triphosphate of the uridine metabolite, RO2433-TP, demonstrated a delayed time course relative to that of PSI-6130-TP. RO2433-TP was detectable in hepatocytes from only 2 of 4 donors at 6 h and in hepatocytes of all 4 donors at 16 h. RO2433-TP formation reached steady state levels at 48–72 h. RO2433-TP concentrations were lower than those of PSI-6130-TP at time points earlier than 16 h but surpassed those of PSI-6130-TP at 24 h and beyond. The mean steady state level concentrations of PSI-6130-TP and RO2433-TP after incubation with 2 µM PSI-6130 were 1.3 ± 0.6 and 2.0 ± 1.1 pmol/10⁶ cells at 48 h, respectively. Unchanged PSI-6130 was the major intracellular species at all time points tested after incubation of human hepatocytes with radiolabeled PSI-6130 (Fig. 4).

View larger version (70K): [in this window] [in a new window]   FIGURE 3. PSI-6130-TP and RO2433-TP are substrates of HCV polymerase and chain-terminators. A, sequence of the RNA template and primers. B, the incorporation of CTP and CTP analogs was initiated with GG primer according as described under "Experimental Procedures." The nucleotide triphosphates included in the reactions were as follows: 10 µM CTP, ATP, and UTP (lane 1); 2, 10, 100, and 1000 µM UTP (lanes 2–5); 10 µM CTP without UTP (lane 6) or with 2, 10, 100, and 1000 µM UTP (lanes 7–10); 10 µM PSI-6130-TP without UTP (lane 11) or with 2, 10, 100, and 1000 µM UTP (lanes 12–15); 10 µM 3'-dCTP without UTP (lane 16) or with 2, 10, 100, and 1000 µM UTP (lane 17–20). FL, full-length. C, incorporation of UTP and UTP analogs initiated with GGC primer. The nucleotide triphosphates included in the reactions were as follows: 100 µM CTP, ATP, and UTP (lane 1); 20, 50, and 200 µM ATP (lanes 2–5); 100 µM UTP without ATP (lane 6) or with 2, 20, 50, and 200 µM ATP (lanes 7–10); 100 µM RO2433-TP without ATP (lane 11) or with 20, 50, and 200 µM ATP (lanes 12–15); 100 µM 3'-dUTP without ATP (lane 16) or with 20, 50, and 200 µM ATP (lanes 17–20).

View larger version (16K): [in this window] [in a new window]   FIGURE 4. Time course of uptake and metabolism of PSI-6130. A, time course of uptake of PSI-6130 and formation of PSI-6130-TP and RO2433-TP. B, time course of formation of PSI-6130-MP + RO2433-TP, PSI-6130-DP, and RO2433-TP. The data show mean values from four experiments performed with primary hepatocytes from four donors.

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Dose response of the formation of PSI-6130-TP and RO2433-TP. Primary human hepatocytes were incubated with PSI-6130 for 24 h at 2, 10, 25, 50, 100, 250 µM concentrations. The data plotted represent the mean values and S.D. of experiments with hepatocytes from three donors.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The analysis of radiolabeled PSI-6130 incubated with primary human hepatocytes demonstrated the conversion of PSI-6130 into its 5'-phosphorylated derivatives (mono-, di-, and triphosphate) and to the uridine analog R2433 and its corresponding 5'-phosphorylated derivatives. Unmodified PSI-6130 remained the major intracellular species at all time points studied. These results suggest that PSI-6130 is only subjected to intracellular 5'-phosphorylation and base deamination, with no evidence for metabolism at other positions. In contrast, another cytidine analog, 2'-O-methylcytidine, demonstrated intracellular conversion with significant efficiency to CTP and UTP instead of 2'-O-methyl-CTP due to deamination combined with extensive demethylation at the 2' position or base swapping after deglycosylation (8, 19). The intracellular instability of 2'-O-methylcytidine most likely accounts for the poor potency of this cytidine analog in the HCV replicon system (8, 19). Importantly, both triphosphates of PSI-6130 and RO2433 were potent inhibitors of the RNA synthesis activity of the native HCV replicase and recombinant NS5B (Table 1), suggesting that the antiviral potency of PSI-6130 is not compromised by intracellular deamination.

View larger version (14K): [in this window] [in a new window]   FIGURE 6. Intracellular stability of PSI-6130-TP and RO2433-TP. Primary human hepatocytes were incubated with PSI-6130 at 2 µM for 24 h, at which point extracellular PSI-6130 was removed, and PSI-6130-TP and RO2433-TP levels were determined at 1, 2, 4, 6, 8, 24, 48, and 72 h after extracellular PSI-6130 removal. Representative kinetic profiles of PSI-6130-TP (A) and RO2433-TP (B) elimination in hepatocytes from one donor are shown.

Among the phosphorylated species of PSI-6130 and RO2433, the rank of the phosphate levels from highest to lowest intracellular concentration was: triphosphates > diphosphates > monophosphates at all time points of compound incubation and all extracellular concentrations (Fig. 4, Table 3). This suggests that the first cellular activation step to monophosphate is the rate-limiting step among the three phosphorylation steps. Recently, it has been reported that the human cellular kinases responsible for the sequential three-step phosphorylation of PSI-6130 to triphosphate are 2'-deoxycytidine kinase, uridine/cytidine monophosphate kinase, and uridine/cytidine diphosphate kinase (17). In that study the efficiency of PSI-6130 as a substrate for 2'-deoxycytidine kinase was reported to be almost 2 orders of magnitude lower than that of PSI-6130-MP and PSI-6130-DP for their respective kinases. Therefore, the results of the reported substrate efficiencies of the three kinases involved in PSI-6130-TP formation agree well with relative formation of PSI-6130 phosphates in human hepatocytes. Importantly, the intracellular concentrations of PSI-6130-TP and RO2433-TP demonstrated an excellent linear relationship with the extracellular concentration of PSI-6130 up to 100 µM, demonstrating a high capacity of human hepatocytes to form the biologically active triphosphates from PSI-6130.

Despite the intrinsic potency of RO2433-TP against HCV polymerase, RO2433 was not active in the HCV replicon system at concentrations up to 100 µM. RO2433 was either not phosphorylated in the replicon cells or could not penetrate the cell membrane. However, RO2433, when formed intracellularly from radiolabeled PSI-6130, dissociated rapidly across the cell membrane with a half-life faster than 30 min. Therefore, RO2433 is most likely not efficiently phosphorylated to form RO2433-MP. Similarly, the uridine analog of the HCV replication inhibitor R1479 (4'-azidocytidine) was inactive in the replicon system. However, when delivered as a monophosphate prodrug, 4'-azidouridine could be converted into a potent inhibitor of HCV replication, demonstrating that a block of monophosphate formation resulted in lack of antiviral activity of 4'-azidouridine (21). Assuming a likely block of RO2433 phosphorylation to its monophosphate, RO2433-MP in human hepatocytes was most likely formed through the deamination of PSI-6130-MP by the cellular dCMP deaminase and subsequently further phosphorylated to RO2433-DP and -TP by uridine/cytidine monophosphate kinase and possibly nucleoside diphosphate kinase (22–24). The proposed metabolic pathway for PSI-6130 is illustrated in Fig. 7.

View larger version (10K): [in this window] [in a new window]   FIGURE 7. Proposed metabolic pathway of PSI-6130. Enzymes involved in the phosphorylation and deamination catalysis: deoxycytidine kinase (dCK), uridine/cytidine monophosphate kinase (YMPK), nucleoside diphosphate kinase (NDPK), deoxycytidylate deaminase (dCMP deaminase).

PSI-6130 is a potent and highly selective nucleoside inhibitor of HCV replication targeting NS5B polymerase in the HCV replicon system. Here we demonstrated that PSI-6130 was converted to two pharmacologically active triphosphate species, PSI-6130-TP and its uridine analog RO2433-TP, in primary human hepatocytes. It is worth noting that RO2433-TP demonstrated greater intracellular stability (t = 38 h) as compared with PSI-6130-TP (t = 4.7 h). PSI-6130 has recently entered clinical development for the treatment of HCV infected patients. The longer intracellular half-life of RO2433-TP may have pharmacologic relevance for maintaining more constant concentrations of the antiviral triphosphate over the dosing period in clinical studies.

	FOOTNOTES

 
^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Roche Palo Alto LLC, 3431 Hillview Ave., Palo Alto, CA 94304. Tel.: 650-852-3190; Fax: 650-354-7554; E-mail: han.ma{at}roche.com.

² The abbreviations used are: HCV, hepatitis C virus; MP, 5'-monophosphate; DP, 5'-diphosphate; TP, 5'-triphosphate; HPLC, high performance liquid chromatography.

	ACKNOWLEDGMENTS

 
We thank Dr. Jim Barnett and group members for providing recombinant NS5B protein and Nixy Zutshi and group members for providing HCV replicon cells. We thank Dr. Phillip A. Furman at Pharmasset, Inc. for providing PS1–6130 and the phosphorylated derivatives of PSI-6130.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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