2-Aminoisobutyric Acid Ethyl Ester (AIBEE) Phosphoramidate Prodrugs Deliver High Concentrations of Nucleoside 5’-Triphosphate in Human Hepatocytes and Dog Liver Biopsy Studies: Application in the Discovery of a Novel 2’-Dihalogenated Nucleoside HCV Polymerase Inhibitor ABBV-168

John Randolph

Phosphoramidate prodrugs have played an important role in research to identify nucleoside inhibitors of HCV polymerase due to their ability to deliver the parent drug to the liver for efficient conversion to the active triphosphate metabolite. A research program to investigate novel HCV-active nucleosides identified 2’-dihalogenated uridine analogs with good potency in genotype 1 replicon assays. However, an early lead containing the L-alanine isopropyl ester phosphoramidate prodrug moiety used with success in the clinic to deliver multiple HCV nucleoside inhibitors, including sofosbuvir (SOF), were found to provide low levels of the active nucleoside 5’-triphosphate (NTP) in liver when dosed orally in dogs. Alternative phosphoramidate prodrugs were screened using an assay developed to measure NTP concentrations in human hepatocytes to assess both bioactivation efficiency and persistence of the active species. This method identified 2-aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrugs which provide high NTP concentrations in comparison to nucleosides bearing the standard prodrug moiety. Activity of AIBEE prodrug analogs in replicon assays were low in comparison to L-alanine-containing phosphoramidate prodrug analogs due to the low expression levels of CES-1 enzymes in replicon cells, which are required for AIBEE prodrug bioactivation. PK studies in dog, collecting liver biopsy samples at 4 and 24 hours after oral dosing, found that AIBEE prodrugs provide high NTP concentrations at both time points in comparison to other phosphoramidate prodrugs. This research identified ABBV-168 that provided NTP concentrations in dog liver that were several fold higher than sofosbuvir at an equivalent dose.

 

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