Human Recombinant P450 Isoforms Research Articles

In vitro metabolism of bencycloquidium bromide and its inhibitory effects on human P450 isoenzymes: implication of CYP2D6, CYP2C19 and CYP3A4/5

Bencycloquidium bromide (BCQB) is a novel selective muscarinic M1/M3 receptor antagonist with potent therapeutic effects on rhinitis and chronic obstructive pulmonary disease. The metabolism of BCQB has been investigated in human liver microsomes and human recombinant P450 to elucidate the P450 isozymes responsible for its metabolism in human. Also, the metabolism pathway and the potency of BCQB in inhibiting CYP's various isozymes in humans were investigated. The main biotransformation route of BCQB was NADPH-dependent oxidation. BCQB was metabolized oxidatively to four metabolites that were identified as monohydroxylated derivatives of BCQB at the phenyl and pentyl moieties of the molecule. The results from in vitro inhibition studies indicated that quinidine inhibited 86% of metabolism of BCQB, while ticlopidine and ketoconazole inhibited 39 and 29%, respectively. Inhibition studies with selective chemical inhibitors and incubations with human recombinant P450 isoforms demonstrated that the oxidative metabolism of BCQB is mediated by CYP2D6, CYP2C19 and CYP3A4/5, whereas BCQB had no inhibitory effect on any other P450 isoenzyme in humans.

In vitro liver metabolism of aclidinium bromide in preclinical animal species and humans: Identification of the human enzymes involved in its oxidative metabolism

The metabolism of aclidinium bromide, a novel long-acting antimuscarinic drug for the maintenance treatment of chronic obstructive pulmonary disorder, has been investigated in liver microsomes and hepatocytes of mice, rats, rabbits, dogs, and humans. Due to the rapid hydrolysis of this ester compound, two distinct radiolabeled forms of aclidinium were studied. The main biotransformation route of aclidinium was the hydrolytic cleavage of the ester moiety, resulting in the formation of the alcohol metabolite (M2, LAS34823) and carboxylic acid metabolite (m3, LAS34850), which mainly occurred non-enzymatically. By comparison, the oxidative metabolism was substantially lower and the metabolite profiles were similar across all five species examined. Aclidinium was metabolized oxidatively to four minor primary metabolites that were identified as monohydroxylated derivatives of aclidinium at the phenyl (M4) and glycolyl (m6 and m7) moieties of the molecule. The NADPH-dependent metabolite m4 involved the loss of one of the thiophene rings of aclidinium. Incubations with human recombinant P450 isoforms and inhibition studies with selective chemical inhibitors and antibodies of human P450 enzymes demonstrated that the oxidative metabolism of aclidinium is primarily mediated by CYP3A4 and CYP2D6. Additionally, up to eight secondary metabolites were also characterized, involving further hydrolysis, oxidation, or glucuronidation of the primary metabolites. Also, the liver oxidative metabolism of the alcohol metabolite (LAS34823) resulted in the production of one hydroxylated metabolite (M1) mediated by human CYP2D6, whereas the acid metabolite (LAS34850) was not metabolized enzymatically, although a minor non-enzymatic and NADPH-dependent reduction was observed.

Oxidation of N, N-dimethylformamide and N, N-diethylformamide by human liver microsomes and human recombinant P450s

N- N, dimethyl- (DMF) and N- N, diethyl-formamide (DEF) are two hepatotoxic solvents, whose metabolism has not been investigated in humans. To identify the P450 isoforms involved in the microsomal oxidation of these solvents we used (a) 12 human liver samples; (b) human recombinant P450 isoforms (1A1, 1A2, 2B6, 2C10, 2E1, 3A4); (c) chemical and immunological inhibitions. When correlation analyses were performed using enzymatic markers in human liver microsomes, the p-nitrophenol hydroxylation rate significantly correlated ( r=0.87) with the dealkylation rate of DMF but not with that of DEF. Among the tested recombinant P450s only 2E1 oxidised DMF, while DEF was oxidised by 2E1, 2C10 and 3A4. 4-Methylpyrazole and anti human 2E1 IgG strongly inhibited the DMF demethylation but only partially the DEF deethylation. These findings indicate that, in the DMF metabolism, the role of 2E1 is crucial and its expression may be an important factor in determining the susceptibility of human to this solvent.

Cytochrome P450 isoforms responsible for the N-deethylation and cyclohexane-hydroxylation of NS-21

1. Cytochrome P450 (P450) isoforms responsible for the N-deethylation and cyclohexane-hydroxylation of (±)-4-diethylamino-1,1-dimethylbut-2-yn-1-yl 2-cyclohexyl-2- hydroxy-2-phenylacetate monohydrochloride monohydrate (NS-21) have been identified in rat and man. 2. Anti-CYP2C11 antibody inhibited the N-deethylation of S- and R-NS-21 in rat hepatic microsomes by 84 and 66% respectively, indicating that CYP2C11 is mainly responsible for these activities in male rats. 3. Of several human recombinant P450 isoforms, CYP3A4 had the activities for the Ndeethylation of S- and R-NS-21. In addition, triacetyloleandomycin (TAO), an inhibitor of the CYP3A subfamily, significantly inhibited the N-deethylation of S- and R-NS-21 in human hepatic microsomes by 67 and 69%, respectively. CYP3A4 therefore contributes to it in man. 4. Quinine, an inhibitor of the rat CYP2D subfamily, significantly inhibited the cyclohexane-4-cis-hydroxylation of S-NS-21 by 48% in rat hepatic microsomes. In contrast, this inhibitor had little effect on the cyclohexane-4-trans-hydroxylation of S-NS21, and the cyclohexane-4-cis-and trans-hydroxylation of R-NS-21. 5. Human recombinant CYP3A4 catalysed the cyclohexane-4-trans-hydroxylation of S-NS-21, and CYP2D6 supported the cyclohexane-4-cis- and trans-hydroxylation of S-NS-21. Quinidine, an inhibitor of human CYP2D6, had little effect on these latter activities in human hepatic microsomes. TAO significantly inhibited the cyclohexane-4-trans-hydroxylation of S-NS-21 by 75%, indicating that CYP3A4 catalyses this reaction.