Preclinical evaluation of AT-527, a novel guanosine nucleotide prodrug with potent, pan-genotypic activity against hepatitis C virus

Abstract

Despite the availability of highly effective direct-acting antiviral (DAA) regimens for the treatment of hepatitis C virus (HCV) infections, sustained viral response (SVR) rates remain suboptimal for difficult-to-treat patient populations such as those with HCV genotype 3, cirrhosis or prior treatment experience, warranting development of more potent HCV replication antivirals. AT-527 is the hemi-sulfate salt of AT-511, a novel phosphoramidate prodrug of 2'-fluoro-2'-C-methylguanosine-5'-monophosphate that has potent in vitro activity against HCV. The EC50 of AT-511, determined using HCV laboratory strains and clinical isolates with genotypes 1-5, ranged from 5-28 nM. The active 5'-triphosphate metabolite, AT-9010, specifically inhibited the HCV RNA-dependent RNA polymerase. AT-511 did not inhibit the replication of other selected RNA or DNA viruses in vitro. AT-511 was approximately 10-fold more active than sofosbuvir (SOF) against a panel of laboratory strains and clinical isolates of HCV genotypes 1-5 and remained fully active against S282T resistance-associated variants, with up to 58-fold more potency than SOF. In vitro, AT-511 did not inhibit human DNA polymerases or elicit cytotoxicity or mitochondrial toxicity at concentrations up to 100 μM. Unlike the other potent guanosine analogs PSI-938 and PSI-661, no mutagenic O6-alkylguanine bases were formed when incubated with cytochrome P450 (CYP) 3A4, and AT-511 had IC50 values ≥25 μM against a panel of CYP enzymes. In hepatocytes from multiple species, the active triphosphate was the predominant metabolite produced from the prodrug, with a half-life of 10 h in human hepatocytes. When given orally to rats and monkeys, AT-527 preferentially delivered high levels of AT-9010 in the liver in vivo. These favorable preclinical attributes support the ongoing clinical development of AT-527 and suggest that, when used in combination with an HCV DAA from a different class, AT-527 may increase SVR rates, especially for difficult-to-treat patient populations, and could potentially shorten treatment duration for all patients.

Fig 1. Chemical structures of AT-511, sofosbuvir (SOF), PSI-938, PSI-661 and BMS-986094.

Fig 2. Putative metabolic pathway for AT-527.

When dissolved, AT-527 releases its free base AT-511. Sequential hydrolysis, catalyzed by human cathepsin A (CatA) and/or carboxylesterase 1 (CES1) followed by spontaneous cleavage of the then unstable phenolic moiety, produces the L-alanyl intermediate (M1). Removal of the amino acid moiety by histidine triad nucleotide-binding protein 1 (HINT1) results in metabolite M2 which can then be converted to M3 by adenosine deaminase like protein 1 (ADALP1). M3 is further anabolized sequentially by guanylate kinase 1 (GUK1) and nucleoside diphosphate kinase (NDPK) to the pharmacologically active triphosphate, AT-9010. Both M2 and M3 can be dephosphorylated by 5’-nucleotidase (5’-NTase) to their respective nucleosides M4 and AT-273.

Fig 3. Inhibition of viral replication in HCV GT1b replicons treated with AT-511 or SOF.

Huh-luc/neo-ET cells stably transfected with the HCV GT1b NS3-NS5B coding sequence were incubated with serial dilutions of AT-511 or sofosbuvir (SOF) in parallel and anti-HCV activity was measured as described in Methods. Data are expressed as mean ± SE, n = 8 assays in triplicate.

Fig 4. CYP3A4-mediated metabolism of PSI-661, BMS-986094, PSI-938 and AT-511.

Values indicate the extent of the 5 μM starting concentration of each antiviral that was converted to the indicated nucleobase after a 2-h incubation with rhCYP3A4.

Fig 5. Intracellular concentrations of AT-511 and its phosphorylated metabolites in liver cells.

(A) mixed gender human hepatocytes, (B) male rat hepatocytes, (C) male mouse hepatocytes, (D) male dog hepatocytes, (E) male cynomolgus monkey hepatocytes and (F) Huh-7 cells. Cells were incubated for 24 h with 10 μM AT-511 in triplicate, collected, lysed and the lysates measured for concentrations of AT-511, AT-9010, M2 and M3 by LC-MS/MS. Data are expressed as mean ± SD.

Fig 6. Mean profiles of AT-511 and its metabolites in rat plasma over 24 h.

Sprague-Dawley rats (3 male and 3 female) were administered a 300 mg/kg single oral dose of AT-527 and plasma concentrations of AT-511, AT-273, M1 and M4 were measured by LC-MS/MS. Data are mean ± SD of all 6 animals.

Fig 7. Mean profiles of AT-511 and its metabolites in monkey plasma over 72 h.

Cynomolgus monkeys (groups of 3 males per dose) were administered a single oral dose of AT-527 at (A) 30 mg/kg, (B) 100 mg/kg and (C) 300 mg/kg, and plasma concentrations of AT-511, AT-273, M1 and M4 were measured by LC-MS/MS. Data are mean ± SD for each group.