| Haloperidol (HP), an antipsychotic agent, is used for the treatment of acute and chronic schizophrenia and associated with extrapyramidal side effects which may be due to altered pharmacokinetics or metabolism of HP. HP is extensively metabolized in humans with 60% by glucuronidation and 23% by a reduction pathway. This study proposes to investigate the in vitro glucuronidation and oxidation of HP in mixed species liver S9 and microsomes. Using a sensitive LC/MS method, the metabolites identified included HP O-glucuronide, reduced HP, chlorophenylpiperidinol, hydroxyl-HP, HTPT, and HPP+. Two new metabolites identified were 3-hydroxy HPP+ and HP N+-glucuronide. A NADPH dependent reactive electrophilic metabolite 3-hydroxy HPP+ was trapped with glutathione and N-acetylcysteine. HP O-glucuronide was formed in liver microsomes from all species except in the dog. The HP N+-glucuronide was formed only in human liver microsomes (HLM). HP O-glucuronide, HP N +-glucuronide and 3-hydroxy HPP+ were biosynthesized from HLM and their structures identified by 1D and 2D 1H-NMR. The incubation of HP with a panel of eleven human cDNA expressed UGTs found that UGT2B7, UGT1A9 and UGT1A4 catalyzed HP O-glucuronidation, whereas UGT1A4 catalyzed HP N+-glucuronidation. Although the O-glucuronidation of HP in HLM exhibited an atypical Eadie-Hofstee graph, the apparent kinetics were calculated using single enzyme Michaelis-Menten equation. The K m and Vmax in HLM were calculated to be 100 +/- 9.9 muM and 3.61 +/- 0.12 nmoles/mg/min, respectively. For UGT2B7, the K m and Vmax were determined to be 32.8 +/- 2.0 muM and 2.06 +/- 0.03 nmoles/mg/min. For UGTIA9, the Km and Vmax were calculated to be 139 +/- 6.5 muM and 4.12 +/- 0.08 nmoles/mg/min. UGTIA4, which exhibited atypical homotropic cooperativity in a Eadie-Hofstee graph, the Km, Vmax, and Hill coefficient (n) were determined to be 120 +/- 9.5 muM, 0.74 +/- 0.03 nmoles/mg/min and 1.7, respectively. The relative contributions by UGT2B7, UGT1A9, and UGT1A4 for HP O-glucuronidation in HLM were 65%, 24% and 13%, respectively, which were determined with the relative activity factor method. HP and the HP glucuronides were found not to be substrates for efflux proteins. |
