Abstract
Delivery of pharmacologically active nucleoside triphosphate analogs to sites of viral infection is challenging. In prior work we identified a 2'-C-methyl-1'-cyano-7-deaza-adenosine C-nucleotide analog with desirable selectivity and potency for the treatment of hepatitis C virus (HCV) infection. However, the prodrug selected for clinical development, GS-6620, required a high dose for meaningful efficacy and had unacceptable variability due to poor oral absorption as a result of suboptimal solubility, intestinal metabolism, and efflux transport. While obtaining clinical proof of concept for the nucleotide analog, a more effective prodrug strategy would be necessary for clinical utility. Here, we report an alternative prodrug of the same nucleoside analog identified to address liabilities of GS-6620. A phosphoramidate prodrug containing the nonproteinogenic amino acid methylalanine, an isopropyl ester and phenol in the (S) conformation at phosphorous, GS2, was found to have improved solubility, intestinal stability, and hepatic activation. GS2 is a more selective substrate for hepatically expressed carboxyl esterase 1 (CES1) and is resistant to hydrolysis by more widely expressed hydrolases, including cathepsin A (CatA) and CES2. Unlike GS-6620, GS2 was not cleaved by intestinally expressed CES2 and, as a result, was stable in intestinal extracts. Levels of liver triphosphate following oral administration of GS2 in animals were higher than those of GS-6620, even when administered under optimal conditions for GS-6620 absorption. Combined, these properties suggest that GS2 will have better oral absorption in the clinic when administered in a solid dosage form and the potential to extend the clinical proof of concept obtained with GS-6620.
Highlights
Nucleoside analogs are hydrophilic, require sequential intracellular phosphorylation to form pharmacologically active triphosphates, and often distribute suboptimally throughout the body, resulting in reduced activity and unwanted side effects
The poor performance of GS-6620 was likely due to extensive intestinal metabolism of the prodrug by carboxyl esterase 2 (CES2), an enzyme more highly expressed in the human gastrointestinal tract than in certain species evaluated during preclinical studies, including dogs [8]
We focused on identifying an alternative prodrug to GS-6620, with improved liver delivery following oral administration and minimal metabolism in the intestine
Summary
Nucleoside analogs are hydrophilic, require sequential intracellular phosphorylation to form pharmacologically active triphosphates, and often distribute suboptimally throughout the body, resulting in reduced activity and unwanted side effects. Nucleoside analogs became a focus in drug discovery for hepatitis C virus (HCV) due to their pan-genotype activity, high barrier to resistance selection, and a lack of preexisting virus with reduced susceptibility [2]. Its active 5=-triphosphate metabolite is a potent competitive inhibitor of HCV NS5B RNA-dependent RNA polymerase (RdRp) and has high selectivity for the viral polymerase over human DNA and RNA polymerases, including the mitochondrial RNA polymerase POLRMT [7]. Following ester cleavage and chemical release of the phenol moiety, another enzyme, histidine triad nucleotide binding protein 1 (HINT1), has been implicated in cleaving the P-N bond of some phosphoramidates [13]. Selective CES1 cleavage followed by efficient HINT1 cleavage are desirable characteristics for promoting liver delivery
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