Comparative Metabolism and Disposition of the Novel Anti-Tuberculosis Agent PA-824 in Mycobacteria, Mammals and Phagocytes

Abstract

PA-824, a novel 4-nitroimidazole prodrug, is currently in phase II clinical trial for tuberculosis (TB) therapy. It is bioactivated by the deazaflavin dependent nitroreductase (Ddn) from Mycobacterium tuberculosis to form a major des-nitro metabolite that releases toxic reactive nitrogen species responsible for the antimycobacterial activity. However, there are no published reports regarding the metabolism of this prodrug by host (mammalian) enzymes or its cellular accumulation in phagocytes, where this mycobacterium can reside. Therefore, the aim of this thesis was to investigate the metabolism and disposition of PA-824 in mammals (mouse in vitro and in vivo plus human liver in vitro) as well as phagocytes compared with purified Ddn enzyme and mycobacteria (M. tuberculosis and Mycobacterium smegmatis). Purified Ddn enzyme and M. tuberculosis catalysed the formation of seven metabolites of PA-824, including the previously reported des-nitro metabolite. Six of these metabolites, but not the des-nitro metabolite, were observed with M. smegmatis. Mammalian liver S9 preparations (mouse and human) were able to form only four of these metabolites and did not catalyse the des-nitrification or nitroreduction of PA-824. Structure elucidation of the products led to the following proposed metabolic pathways of PA-824. Reductive metabolism by hepatic enzymes via imidazole ring reduction results in a C3-hydride metabolite that yields two additional unstable products. Oxidative cleavage of the benzyl linker chain of PA-824 form (6S)-2-nitro-6,7-dihydro- 5H-imidazo[2,1-b][1,3]oxazin-6-ol. Hydration of the imidazole ring at the C3 position appears to be a non-enzymatic reaction with ferrous ions. Following an oral dose of PA-824 to mouse, only four metabolites were observed in plasma and tissues: the C3-hydrate, (6S)-2-nitro-6,7-dihydro-5H-imidazo[2,1- b][1,3]oxazin-6-ol, and the two unidentified metabolites. Hence, PA-824 appears to undergo both oxidative and reductive metabolism in vivo. High intracellular concentrations of PA-824 were achieved within the phagocytes via a passive mechanism. The drug concentrations in this sanctuary site of mycobacterial infection were at least 4,200-fold higher than the reported minimum inhibitory concentrations required for the activity of this drug. Thus, species selective activation and high concentrations of PA-824 at the site of infection may indicate a potential for PA-824 to be the first new drug for TB therapy in almost 50 years.