Metabolism and clearance characterization of novel HBV RNase H inhibitors

Abstract ID 91472 Poster Board 277 Hepatitis B Virus (HBV) chronically infects ∼300 million people worldwide, resulting in > 880,000 deaths annually from HBV-associated liver complications. Current treatment employs a combination of nucleos(t)ide analogs and interferon-α. The NAs dominate therapy, however, in most patients, treatment cessation results in viremia recurrence, and thus lifelong treatment is required. HBV replicates by reverse transcription, which is catalyzed by the reverse transcriptase (RT) and ribonuclease H (RH) domains of the polymerase (P) protein. We have determined that the RH active site is an attractive drug target and have ∼300 RH inhibitors within a growing library of >2500 experimental compounds. Inhibitors primarily belong to 3 chemotypes: The α-Hydroxytropolones (αHT), the N-hydroxypyridinediones (HPD), and the N-hydroxynapthyridinones (HNO), and while we have determined compounds within these chemotypes have sub-micromolar effective concentrations 50% (EC₅₀) with minimal toxicity in liver cell lines, we have never characterized them pharmacologically. Here we assess the metabolism and clearance parameters of the compounds with the best selectivity indexes (cytotoxicity/efficacy) in each chemotype to guide an ongoing medicinal chemistry campaign. To assess factors that affect a compound's rate of clearance, we measured the effect of plasma protein concentration on compound potency for 10 compounds in each chemotype by our established HBV replication inhibition assay with and without supplemental bovine serum albumin. We determined that the potency of 4 αHTs, 2 HPDs, and 1 HNO was significantly decreased in increased albumin levels. We next determined the half-life of 10 compounds from each chemotype by a microsome stability assay. All compounds tested with detectable analyte peaks by liquid chromatography tandem mass spectrometry had half-lives >30 min. To assess drug-drug interaction potential, 10 compounds from each chemotype were first screened at 10 μM against the 5 major CYP isoforms: 3A4, 2C19, 2C9, 1A2, and 2D6 in biochemical competitive inhibition assays and no compounds substantially inhibited any isoform (<50%). Finally, the potential to induce CYP3A4 expression was assessed for 2 compounds in each chemotype by reverse transcriptase qPCR, and we determined that CYP3A4 expression increased after incubation with both αHTs, 1 HPD, and 1 HNO at 10 μM for 72 hr. These data indicate that the most promising anti-HBV RNase H compounds have pharmacological characteristics that reflect approved drugs and inform future structure activity relationships during drug design.