(M0930-06-38) Quantitation of Bemnifosbuvir and Metabolites in Human Bronchoalveolar Lavage (BAL) by LC-MS/MS

Purpose: Bemnifosbuvir (BEM, AT-527) is a novel oral nucleotide antiviral drug candidate currently in Phase 3 clinical development for the treatment of COVID-19. The primary objective of this project was to determine the concentrations of AT-511, free base of AT-527, and its metabolites AT-551, AT-229, and AT-273 ELF after administration of multiple doses of BEM in healthy subjects. BAL collection consists of administering measured volumes of warmed saline solution into targeted pulmonary regions to collect ELF via aspiration of the lavage solution. The resulting BAL sample contained diluted (ca. 100-fold) ELF as an aqueous medium. Lipophilic compounds are prone to non-specific binding (NSB) within storage containers; other physicochemical disparities among the four analytes in terms of solubility, stability, and specificity were also key factors in the development of a rugged assay combining sample collection, extraction, chromatographic resolution, and mass spectrometric detection.

Methods: BAL samples were collected and mixed with an anti-adsorptive agent solution prior to storage. Following deproteinization with cold acetonitrile: methanol (1:3), samples were subsequently diluted for injection. Chromatographic separation involved gradient elution on an XBridge C₁₈ column (50 x 4.6mm, 3.5 µm) using ten mM ammonium bicarbonate and acetonitrile: methanol (1:1) as an organic modifier. The LC system was coupled to a SCIEX API 5000 mass spectrometer operated in positive electrospray ionization mode monitoring the MRM transitions 582.2 >376.2 (AT-511), 313.1 >148.2 (AT-229), 464.1 >375.2 (AT-551) and 300.1 >152.2 (AT-273). Analytical ranges were 0.200 - 200 ng/mL for AT-511 and AT-229 and 0.400 - 400 ng/mL for AT-551 and AT-273. Levels of AT-511 and metabolites in ELF were calculated by correcting for urea levels in BAL and plasma.

Results: AT-511 exhibited significant NSB, which was notably absent for AT-229, AT-551 and AT-273. As the bronchoalveolar lavage protocol resulted in significant dilution of the ELF, overall extract dilution was minimized to achieve targeted LOQs. Further, the strategy to counter NSB required compatibility between sample additive and chromatographic conditions. Due to polarity differences between analytes, extraction involved non-discriminatory protein precipitation. The addition of ethanol to BAL samples in a 1:1 volume ratio countered NSB whilst also maintaining acceptable sample composition for sample analysis. Antiviral drug levels of BEM were consistently achieved in the lungs with BEM 550 mg BID: mean ELF levels of AT-273, a surrogate of the active triphosphate metabolite of the drug, were 0.62 and 0.45 µM respectively at 4 and 12 h, exceeding/approaching the target 0.5 µM, EC90 of the drug inhibiting SARS-CoV-2 replication in human airway epithelial cells.

Conclusion: A multi-component assay for AT-511 and associated metabolites was successfully qualified, with sample analysis revealing that BEM 550 mg BID reached antiviral exposure in the lungs as measured by ELF levels of AT-273.

Calibration Curves of AT-229, AT-273, AT-511 and AT-551

Representative Chromatograms of an extracted LLOQ