Metabolism of amiodarone (Part III): identification of rabbit cytochrome P450 isoforms involved in the hydroxylation of mono-N-desethylamiodarone

Abstract

1. Amiodarone (AMI) is a potent anti-arrhythmic drug and mono-N-desethylamiodarone (MDEA) is its only known metabolite. It was found recently that in rabbit liver microsomes MDEA was biotransformed to n-3-hydroxybutyl-MDEA (3OH-MDEA). 2. In liver microsomes isolated from the untreated rabbit, the formation of 3OH-MDEA obeyed Michaelis-Menten enzyme kinetics with Km = 6.39 +/- 1.07 microM and Vmax = 0.56 +/- 0.21 nmolmin(-1) mg(-1) protein. 3. Furthermore, (1) among chemicals usually used as inhibitors of cytochrome P450, only midazolam (MDZ), cyclosporin A and ketoconazole inhibited the MDEA hydroxylase activity significantly (>60% inhibition), (2) MDZ, a substrate of CYP3A, inhibited the 30OH-MDEA formation competitively (Ki = 10 +/- 5 microM), (3) the formation rates of 3OH-MDEA correlated positively with those of 1'OH-MDZ (r = 0.81; n = 6), and (4) MDEA hydroxylase activity of microsomes isolated from rabbit rifampicin-induced cultured hepatocytes was 4-fold more active than the control. 4. Since CYP3A6 is mainly induced by rifampicin in rabbit-cultured hepatocytes, the data suggest that this isoform is involved in the biotransformation of MDEA to 3OH-MDEA. 5. Since alpha-naphthoflavone, cimetidine and quinidine also partially inhibited the MDEA hydroxylase activity, it is possible that other CYPs, such as 1A, 2C and 2D, may also be active in the metabolism of amiodarone.

Metabolite identification studies on amiodarone in in vitro (rat liver microsomes, rat and human liver S9 fractions) and in vivo (rat feces, urine, plasma) matrices by using liquid chromatography with high‐resolution mass spectrometry and multiple‐stage mass spectrometry: Characterization of the diquinone metabolite supposedly responsible for the drug's hepatotoxicity

“…The previously known metabolites of amiodarone were O ‐dealkyl amiodarone, monohydroxy metabolites, carboxy amiodarone, benzfuron dihydoxylated amiodarone, deaminated amiodarone, N ‐oxide amiodarone, N ‐desethyl amiodarone, N ‐desethyl carboxy amiodarone, N ‐desethyl monohydroxy amiodarone, N ‐desethyl dihydroxy amiodarone, di‐ N ‐desethyl amiodarone, deiodo amiodarone, monohydroxy deiodo amiodarone, di‐deiodinated N ‐desethyl amiodarone, and glucuronide conjugates of hydroxy, carboxyl and N ‐desethyl monohydroxy amiodarone . Hence, the first task was to sort out their presence among 26 metabolites detected in this study.…”

“…The previously known metabolites of amiodarone were O-dealkyl amiodarone, monohydroxy metabolites, carboxy amiodarone, benzfuron dihydoxylated amiodarone, deaminated 33 Known [3] M2-c Known [3] M2-e Known [3] M6-e Known [3] M8-a Known [3] M9-a amiodarone, N-oxide amiodarone, N-desethyl amiodarone, N-desethyl carboxy amiodarone, N-desethyl monohydroxy amiodarone, N-desethyl dihydroxy amiodarone, di-N-desethyl amiodarone, deiodo amiodarone, monohydroxy deiodo amiodarone, di-deiodinated N-desethyl amiodarone, and glucuronide conjugates of hydroxy, carboxyl and N-desethyl monohydroxy amiodarone. [3][4][5][6][7][8][9][10][11][12] Hence, the first task was to sort out their presence among 26 metabolites detected in this study. The presence of known metabolites was substantiated through matching of observed accurate masses with the reported/calculated masses, similarity of calculated chemical formula, and appearance of justifying mass fragments in each case ( Table 3 and Supplementary Figs.…”

“…The values range between 17 to 60% in rats and 65 ± 22% in humans . The drug is eliminated from the body through several metabolic pathways, among which N ‐desethylation is crucial, while desethyl monohydroxylation, desethyl dihydroxylation, and monohydroxylation are minor routes of metabolism …”

The metabolism of amiodarone by various CYP isoenzymes of human and rat, and the inhibitory influence of ketoconazole

“…A paradoxical increase in DEA formation occurred for CYP3A1 at lower KTZ concentrations ranging from 20 to 1000 nM. Preferential inhibition of metabolism of the formed DEA by the same isoenzyme could have caused an increase in DEA formation in the presence of KTZ (44). A second explanation could be KTZ-associated inhibition of formation of other metabolite(s) of AM besides DEA, thus leading to increased amount of AM available to undergo DEA biotransformation.…”

Effect of Quinidine on the 10-Hydroxylation ofR-Warfarin: Species Differences and Clearance Projection

“…Although inhibitory antibodies against rabbit CYP3A were not available to us at the time these experiments were performed, the 10-hydroxylation of R-warfarin in rabbit liver microsomal incubations was nearly completely inhibited by ketoconazole, and to a lesser extent, by troleandomycin (Table 1). Both ketoconazole and troleandomycin are selective inhibitors of human CYP3A and also were shown to inhibit rabbit CYP3A6 when carbamazepine and N-desethylamiodarone were evaluated as substrates (Mesdjian et al, 1999;Kozlik et al, 2001). These data collectively suggest that CYP3A enzymes are responsible for the 10-hydroxylation of R-warfarin in rats, rabbits, and monkeys.…”