HCV NS5B polymerase; Mericitabine targets the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp). The active triphosphate metabolite acts as a non-obligate RNA chain terminator, competitively binding to the active site of the NS5B enzyme and preventing the elongation of the viral RNA transcript. This inhibition blocks viral RNA replication and halts the production of new virus particles.

PSI-6130 is an analogue of cytidine. Mericitabine (RG 7128; R-7128) is an oral prodrug of PSI-6130. According to preclinical data, PSI-6130 exhibits an EC90 value in the HCV replicon test of 4.6±2 μM. Mericitabine (RG 7128; R-7128) has little cytotoxicity, doesn't damage mitochondrial DNA, and is very selective for HCV. Cellular kinases phosphorylate PSI-6130, resulting in the production of an active 5'-triphosphate metabolite that inhibits HCV's NS5B RNA polymerase. Mecitabine (RG 7128; R-7128) has a notable anti-HCV-1 effectiveness and a comparatively acceptable safety profile [2]. Mericitabine is a novel type of nucleoside polymerase inhibitor (NPI) that needs to be absorbed inside the cells and phosphorylated twice to produce two active triphosphates. Oral cytidine nucleoside analog prodrug meretibine (RG 7128; R-7128) has strong antiviral activity against HCV polymerase in all HCV genotypes [3].

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As the active intracellular metabolite of mericitabine, the 5'-triphosphate of PSI-6130 inhibits the HCV NS5B polymerase. In replicon assays, mericitabine itself and its parent compound PSI-6130 exhibit potent inhibition of HCV genotypes 1a and 1b, with mean EC₅₀ values of 0.31 μM and 0.51 μM, respectively. Preclinical data have shown that PSI-6130 has an EC₉₀ value of 4.6 ± 2 μM in the HCV replicon assay. The compound demonstrates high specificity for HCV, minimal cytotoxicity, and does not affect mitochondrial DNA.

Mericitabine (RG7128) is a nucleoside polymerase inhibitor (NPI), which requires intracellular uptake and phosphorylation to two active triphosphates. Mathematical modeling has provided important insights for characterizing hepatitis C virus (HCV) RNA decline and estimating in vivo effectiveness of antiviral agents; however, it has not been used to characterize viral kinetics with NPIs. HCV RNA was frequently measured in 32 treatment-experienced patients infected with HCV genotype 1 during and after mericitabine monotherapy for 14 days with 750 mg or 1500 mg administered once (qd) or twice daily (bid). The initial decline of HCV RNA was typically slower than with interferon-α or protease inhibitors, and 12 patients presented a novel pattern of HCV RNA kinetics characterized by a monophasic viral decline. Viral kinetics could be well fitted by assuming that the effectiveness in blocking viral production gradually increased over time to reach its final value, ε(2), consistent with previous accumulation time estimates of intracellular triphosphates. ε(2) was high with bid dosing (mean 750 mg and 1500 mg: 98.0% and 99.8%, respectively; P = 0.018) and significantly higher than in patients treated qd (mean qd versus bid: 90% versus 99%, P < 10(-7)). Virus rebounded rapidly upon drug discontinuation, which was attributed to the elimination of active drug and the subsequent decline of drug effectiveness, with mean t(1/2) = 13.9 hours in the bid regimens[3].

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In HCV-infected patients, mericitabine monotherapy at 14 days with twice-daily dosing (1500 mg BID) achieved an estimated 99.8% effectiveness in blocking viral production. Mathematical modeling of viral kinetics showed that the effectiveness of blocking viral production gradually increased over time as intracellular triphosphate levels accumulated, reaching final effectiveness (ε(2)) of 98.0% and 99.8% for 750 mg and 1500 mg BID regimens, respectively. In Phase II trials, patients receiving triple therapy with mericitabine showed a 20% increase in virologic response over pegylated interferon α plus ribavirin alone. The S282T resistance mutation was not selected during short-term mericitabine monotherapy.

Mericitabine (RG7128) is an oral cytidine nucleoside analog prodrug that exhibited strong antiviral effectiveness against the HCV polymerase across all HCV genotypes (9–11), with no evidence of resistance reported in patients treated with mericitabine monotherapy for 14 days (12). Upon entering the hepatocyte, mericitabine is converted to a cytidine monophosphate, which is then further converted to both a cytidine and a uridine triphosphate. Both triphosphate forms are active, with the cytidine form predominating at least early following the initiation of treatment[3].

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While detailed protocols for mericitabine itself are not extensively detailed in the available literature, the activity of its triphosphate metabolite is assessed using purified recombinant HCV NS5B polymerase. The assay typically involves incubating the enzyme with a synthetic RNA template, ribonucleotide substrates (including radiolabeled tracers), and varying concentrations of the inhibitor. Incorporation of radiolabel into the newly synthesized RNA strand is then measured to determine the IC₅₀ and Ki values for the triphosphate metabolite against the NS5B polymerase.

The primary cell-based assay used to evaluate mericitabine's antiviral activity is the HCV subgenomic replicon assay. In this assay, human hepatoma Huh-7 cells stably replicating HCV subgenomic RNA are treated with serial dilutions of the compound for 3 days. After incubation, total RNA is extracted, and HCV RNA levels are quantified by real-time RT-PCR. The reduction in HCV RNA compared to untreated controls is used to calculate EC₅₀ and EC₉₀ values. Cytotoxicity is concurrently assessed in parallel cultures using standard assays (e.g., MTT) to determine the CC₅₀ and selectivity index.

Multiple oral doses of mericitabine were administered for 14 days to 32 HCV-infected patients, split into four cohorts (n=10 patients per cohort with 8 getting drug and 2 taking placebo) with regimens of 750-mg QD, 1500-mg QD, 750-mg BID and 1500-mg BID. Mean changes in HCV RNA per dosing group are displayed in Figure 1[3].

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In the INFORM-1 study, 73 patients with chronic hepatitis C virus infection received mericitabine plus danoprevir for up to 13 days. Seventy-two patients experienced a continuous decline in HCV RNA levels during treatment, and of these patients, 14 had viral loads that remained >1,000 IU/ml by day 13 and 1 met the definition for viral breakthrough. In-depth NS5B and NS3/4A population and clonal sequencing studies and mericitabine and danoprevir drug susceptibility testing were performed to assess the variability and quasispecies dynamics before and upon monotherapy or dual therapy. Sequence analysis of the viral quasispecies indicated that the mericitabine resistance mutation S282T was not present at baseline, nor was it selected (even at a low level) during treatment. Protease inhibitor resistance mutations, either as predominant or as minority species, were detected in 18 patients at baseline. No enrichment of minority protease inhibitor-resistant variants present at baseline was observed during treatment; viral population samples were fully susceptible to mericitabine and/or danoprevir, despite the presence within their quasispecies of minority variants confirmed to have reduced susceptibility to danoprevir or other protease inhibitors. It was also observed that certain NS3 amino acid substitutions affected protease inhibitor drug susceptibility in a compound-specific manner and varied with the genetic context. In summary, the slower kinetics of viral load decline observed in some patients was not due to the selection of danoprevir or mericitabine resistance during treatment. Over 2 weeks' therapy, mericitabine suppressed the selection of danoprevir resistance, results that could differ upon longer treatment periods.[1]

Mericitabine is rapidly absorbed and extensively converted to its parent nucleoside, PSI-6130, via ester cleavage. In humans, once absorbed, mericitabine itself has very little plasma exposure due to this rapid conversion. The active triphosphate metabolite accumulates intracellularly with a slow elimination half-life, consistent with the observed "monophasic" viral decline kinetics in patients. In a Phase I/II study, the pharmacokinetics, safety, and tolerability of mericitabine were evaluated in healthy volunteers and HCV patients, including assessments of food effect on absorption.

Mericitabine has demonstrated a relatively good safety and tolerability profile in clinical studies. It exhibits minimal cytotoxicity and no effect on mitochondrial DNA in preclinical assays, which is a known liability of some older nucleoside analogs. No severe adverse events were noted in Phase I/II trials, and the drug was generally well-tolerated. The S282T mutation in the NS5B gene is the primary resistance mechanism, significantly reducing the drug's activity, but this mutation has not been observed to emerge during short-term treatment and is associated with a high fitness cost for the virus.

[1]. Characterization of HCV quasispecies dynamics upon short term dual-therapy with the HCV NS5B nucleoside polymerase inhibitor mericitabine and the NS3/4 protease inhibitor danoprevir. Antimicrob Agents Chemother. 2012 Nov;56(11):5494-5.

[2]. Directly acting antivirals against hepatitis C virus. J Antimicrob Chemother. 2011 Aug;66(8):1673-86.

[3]. Hepatitis C viral kinetics with the nucleoside polymerase inhibitor mericitabine (RG7128). Hepatology. 2012 Apr;55(4):1030-7.

Mercitabine has been investigated for the treatment of chronic hepatitis C. Mercitabine is a polymerase inhibitor currently under development for the treatment of chronic hepatitis C. Mercitabine is a prodrug of PSI-6130, which has shown excellent efficacy in preclinical studies. PSI-6130 is a pyrimidine nucleoside analog that inhibits hepatitis C virus (HCV) RNA polymerase, an enzyme essential for HCV replication. Mercitabine is an oral prodrug of PSI-6130, a selective cytidine nucleoside analog and a non-cytotoxic HCV polymerase inhibitor. After intracellular uptake, mercitabine is phosphorylated into two active triphosphate metabolites, which bind to and inactivate HCV RNA-dependent RNA polymerase (non-structural protein 5B; NS5B). This leads to the inhibition of HCV viral RNA production, thereby suppressing viral replication. The drug has a high resistance barrier, but the substitution mutation (S282T) on HCV NS5B significantly reduces the activity of mericitabine.

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Mericitabine was designed as a prodrug to overcome the poor oral bioavailability of PSI-6130. It is a first-in-class nucleoside polymerase inhibitor (NPI) that, unlike protease inhibitors, has a high barrier to resistance and broad genotypic activity across HCV genotypes 1-6. The drug acts as a non-obligate chain terminator; upon incorporation into the growing RNA chain, it terminates further elongation. While development for hepatitis C has been superseded by more modern direct-acting antiviral combinations, mericitabine played a key role in establishing the clinical proof-of-concept for nucleoside analog NS5B inhibitors and contributed to the understanding of viral kinetics and resistance mechanisms.

C18H26FN3O6

399.4194

399.181

C, 54.13; H, 6.56; F, 4.76; N, 10.52; O, 24.03

940908-79-2

16122663

White to off-white solid powder

1.56

1

7

8

28

707

4

CC(C)C(=O)OC[C@@H]1[C@H]([C@@]([C@@H](O1)N2C=CC(=NC2=O)N)(C)F)OC(=O)C(C)C

MLESJYFEMSJZLZ-MAAOGQSESA-N

InChI=1S/C18H26FN3O6/c1-9(2)14(23)26-8-11-13(28-15(24)10(3)4)18(5,19)16(27-11)22-7-6-12(20)21-17(22)25/h6-7,9-11,13,16H,8H2,1-5H3,(H2,20,21,25)/t11-,13-,16-,18-/m1/s1

(2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4-fluoro-2-((isobutyryloxy)methyl)-4-methyltetrahydrofuran-3-yl isobutyrate

RG7128; RG-7128; RG 7128; R-7128; R7128; R 7128; PSI 6130 diisobutyrate; PSI 6130 diisobutyrate; RO-5024048; 3',5'-Diisobutyryl PSI 6130; 3',5'-Diisobutyryl PSI 6130; Mericitabine.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~250.37 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.26 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.26 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.26 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)