Mohamed Aljarah

INX-08189, a Phosphoramidate Prodrug of 6-O-Methyl-2′-C-Methyl Guanosine, is a Potent Inhibitor of Hepatitis C Virus Replication with Excellent Pharmacokinetic and Pharmacodynamic Properties

ABSTRACT INX-08189 is an aryl-phosphoramidate of 6-O-methyl-2′-C-methyl guanosine. INX-08189 was highly potent in replicon assays, with a 50% effective concentration of 10 ± 6 nM against hepatitis C genotype 1b at 72 h. The inhibitory effect on viral replication was rapid, with a 50% effective concentration (EC50) of 35 ± 8 nM at 24 h. An intracellular 2′-C-methyl guanosine triphosphate (2′-C-MeGTP) concentration of 2.43 ± 0.42 pmol/106 cells was sufficient to achieve 90% inhibition of viral replication. In vitro resistance studies confirmed that the S282T mutation in the NS5b gene conferred an approximately 10-fold reduction in sensitivity to INX-08189. However, the complete inhibition of S282T mutant replicons still could be achieved with an EC90 of 344 ± 170 nM. Drug combination studies of INX-08189 and ribavirin indicated significant synergy in antiviral potency both in wild-type and S282T-expressing replicons. Genotype 1b replicons could be cleared after 14 days of culture when exposed to as little as 20 nM INX-08189. No evidence of mitochondrial toxicity was observed after 14 days of INX-08189 exposure in both HepG2 and CEM human cell lines. In vivo studies of rats and cynomolgus monkeys demonstrated that 2′-C-MeGTP concentrations in liver equivalent to the EC90 could be attained after a single oral dose of INX-08189. Rat liver 2′-C-MeGTP concentrations were proportional to dose, sustained for greater than 24 h, and correlated with plasma concentrations of the nucleoside metabolite 2′-C-methyl guanosine. The characteristics displayed by INX-08189 support its continued development as a clinical candidate for the treatment of chronic HCV infection.

In vitro and in vivo metabolism of INX-189, a potent HCV inhibitor, in rat and cynomolgus monkey hepatocytes [Abstract]

Hepatic fibrosis is characterized by the excessive production of collagen and extracellular matrix proteins. Among cytokines, Interferon gamma (IFNγ) is recognized as a potent anti-fibrotic cytokine in liver and lung fibrosis models. However, its poor pharmacokinetics resulted in lack of efficacy and failure of clinical trials. PEGylated IFNα (PEGINTRON, PEGASYS) is shown to be beneficial in liver diseases, but PEGylation of IFNγ is not explored yet. Apart from increasing solubility and stability, PEGylation prolongs circulation time leading to improved efficacy at lower doses or less frequent administrations. In this study, we modified IFNγ with different sized PEG molecules (5, 10 and 20KDa) and studied its pharmacokinetics and antifibrotic effects in the acute CCl4-induced liver injury model. Following synthesis, the biological activity of all PEG-IFNγ constructs versus IFNγ was assessed in RAW cells using NO release assay and activation of the signaling pathway was analyzed using a pGAS-Luc reporter plasmid assay. Effects of the constructs on fibrotic parameters were examined in 3T3 fibroblasts and in a liver injury mouse model using immuno-histochemistry and quantitative PCR. All PEGylated IFNγ were biologically active and activated IFNγ signaling pathway. In vitro, IFNγ and different PEGylated IFNγ induced a significant reduction of crucial anti-fibrotic parameters (collagen and α- SMA). In vivo, pharmacokinetics of radiolabeled [125I]-IFNγ- PEG conjugates demonstrated increased plasma circulation time and decreased renal clearance with increasing degree of PEGylation. In acute liver injury model, the assessed liver levels of IFNγ-PEG5 and IFNγ-PEG10 were higher than IFNγ while IFNγ-PEG20 showed significantly reduced levels. All PEGylated IFNγ induced potent anti-fibrotic effects compared to native IFNγ. Fibrotic parameters (collagen, α-SMA and Desmin) were drastically decreased with increased PEGylation. However, infiltration of inflammatory cells (neutrophils and macrophages) and MHCII expression was significantly augmented in livers with increasing PEGylation. Furthermore, MHCII expression was also increased in other organs (Kidneys, lungs and heart) with PEGylated IFNγ compared to native IFNγ. In addition, PEGylated IFNγ caused considerable loss in body weight, increased inflammation (increased plasma TNFα and IL-6) and severe leukopenia. In conclusion, PEGylation significantly improved the anti-fibrotic effects of IFNγ but also increased the adverse effects. Since the beneficial effects of PEG-IFNγ are significantly better than unmodified IFNγ, alternative strategies such as a cell-specific targeting are warranted for an effective treatment.