Investigating the role of reactive metabolites and parent compound in drug induced liver injury

Adverse drug reactions (ADRs) are a major problem for drug companies and healthcare providers alike. Although ADRs can present anywhere in the body, the liver frequently effected, due to the relatively large concentrations of drugs it encounters. Drug induced liver injury (DILI) can occur through several different mechanisms. Bioactivation of drugs to reactive metabolites is believed to be a crucial step in the development of many cases of DILI. Nefazodone, an anti-depressant which was withdrawn due to hepatotoxicity, has been shown to be bioactivated to a reactive quinone-imine. The role of the mitochondria and their involvement in DILI is being increasingly recognised. The biguanides are known mitochondrial toxins, and phenformin and buformin were removed from the market due to unacceptably high incidents of lactic acidosis. The aims of this thesis were two-fold; to assess the bioactivation and irreversible binding of nefazodone and it safer analogue buspirone and to use the biguanides to assess mitochondrial toxicity in primary hepatocytes. In liver microsomes, both nefazodone and buspirone demonstrated NADPH-dependent irreversible binding, however, nefazodone irreversible binding was 9-fold that of buspirone. The metabolism of both nefazodone and buspirone was extensive and consisted mainly of hydroxylation and N-dealkylation reactions. In rat and human liver microsomes supplemented with GSH, nefazodone formed GSH conjugates with m/z 791 and m/z 807. This implied that the conjugates were formed from bioactivation of para-hydroxy nefazodone and dihydroxy-nefazodone. In rat liver microsomes, buspirone did not form any GSH conjugates. Further investigations of nefazodone and buspirone were carried out in freshly isolated rat hepatocytes. Metabolism of nefazodone and buspirone was investigated and revealed extensive metabolism of both compounds; however, GSH conjugates of neither compound were discovered. At 6 hours, both nefazodone and buspirone demonstrated significant irreversible binding (117.54±15.32 pmol equiv./mg protein and 84.43±30.93 pmol equiv./mg protein respectively) but only nefazodone demonstrated a significant decrease in cell viability (19.25±18.26 % control viability). Inhibition studies, using ABT, significantly reduced the irreversible binding of nefazodone (49.34±4.64 pmol equiv./mg protein) but did not decrease cytotoxicity. This indicated that in rat hepatocytes the parent compound may be responsible for toxicity. Mitochondrial toxicity was investigated using the model mitochondrial toxins the biguanides. Initially, studies in cultured primary rat hepatocytes demonstrated that phenformin was the most potent mitochondrial toxin and dissipated the mitochondrial membrane potential, as measured by TMRM, within 24 hours (1.028±0.39% control fluorescence). This was taken forward to investigate mitochondrial toxicity in primary rat hepatocytes. Investigations into phenformin in rat hepatocytes demonstrated a high turnover to glucuronide metabolites and the novel metabolites [O, OMe] phenformin glucuronide and [2O] phenformin glucuronide were identified. Inhibition studies of CYP450 2D were undertaken using quinine (100µM) and this demonstrated significant inhibition of phenformin up to 200µM (AUC 47.30±47.30 without quinine vs AUC 648.80±121.28 with quinine). Despite increased phenformin concentrations, inhibition of phenformin metabolism did not produce overt cytotoxicity, however, lactate concentrations correlated with increased phenformin concentration. The work presented here highlights the need for a greater understanding of the role of bioactivation and irreversible binding in hepatotoxicity. It also demonstrates that whilst irreversible binding can help inform decisions as to whether a compound progresses into clinical trials, it should be made in the context of other safety assessments. Investigations into phenformin mitochondrial toxicity, illustrates the need to assess drugs and systems fully, to establish model compounds to investigate mechanisms of ADRs. A greater understanding of in vitro systems and the tools utilised to assess them, will benefit drug discovery and development. Ultimately, understanding these in vitro tests and the model compounds used to assess them, will help bridge the gap to man.