Identification and mechanistic study of novel drug metabolites by LC-MS.

Understanding the metabolic fate of drug candidates in vitro and in vivo is a key component of drug development. Structural characterisation of drug candidate metabolites is important early in drug discovery to identify unwanted metabolic liabilities such as reactive, active, toxic or human specific metabolites. Reactive metabolite (RM) liability is a major concern for pharmaceutical companies during drug design, with most of the industry running primary RM trapping screens.This thesis investigates Fenclozic acid, homopiperazine reactivity, homomorphyline reactive aldehyde and methanol adduct formation, where RMs were not detected through routine glutathione/cyanide trapping assays.Fenclozic acid was withdrawn from clinical development due to hepatotoxicity; the mechanism of hepatotoxicity was never determined. In vitro covalent binding studies indicated phase I bioactivation in human liver microsomes, however no RMs were identified from in vitro experiments. As a part of this PhD thesis the metabolism of Fenclozic acid was investigated in bile duct cannulated rats using modern analytical techniques. Several new metabolites including glutathione related adducts formed through an epoxide RM were identified.A series of homopiperazine compounds were found to react with endogenous formaldehyde during rat in vivo studies. This thesis describes a detailed investigation into the identification and mechanism of formation of the resulting product, a bridged homopiperazine formed through a reactive Schiff base intermediate.A cysteineglycine conjugate observed for a series of homomorpholine compounds trapped by glutathione in human liver microsomes has been investigated in this thesis. NMR, detailed MS and methoxyamine trapping confirmed formation of a thiazolidine glycine product via a reactive aldehyde and subsequent glutathione rearrangement.A compound series in an early drug discovery programme formed unusual methanol adducts post incubation in human liver microsomes. The work undertaken in this thesis revealed the generation of a reactive aldehyde metabolite that did not form an adduct with glutathione, but reacted with the methanol mobile phase to form a pair of hemiacetal diastereoisomers.These examples would not have been detected using routine glutathione RM screening assays, this thesis highlights limitations of a screening approach, and where detailed metabolite identification studies employing modern LC-MS techniques are critical in understanding RM formation.