Amobidaquine Clearance and Its Metabolism to N-Desenthylamodiaquine Is Mediated by CYP2C8: A New High Affinity and Turnover Enzyme-Specific Probe Substrate

摘要

Amodiaquine (AQ) metabolism to N-desethylamodiaquine (DEAQ) is the principal route of disposition in humans. Using human liver microsomes and two sets of recombinant human cytochrome P450 isoforms (from lymphoblastoids and yeast) we performed studies to identify the CYP isoform(s) involved in the metabolism of AQ. CYP2C8 was the main hepatic isoform that cleared AQ and catalyzed the formation of DEAQ. The extrahepiatic Pi450s, 1A1 and 1B1, also cleared AQ and catalyzed the formation of an unknown metabolite M2. The K_m and V_max values for AQ N-desenylation were 1.2 #Mu3M and 2.6 pmol/min/pmol of CYP2C8 for recombinant CYP2C8, and 2.4 #mu#M and 1462 pmol/min/mg of protein for human liver microsomes (HLMS), respetively. Relative contribution of CYP2C8 in the formation of DEAQ was estimated at 100% using the relative activity factor method. Correlation analyses between AQ metabolism and the activities of eight hepatic P450s were made on 10 different HLM samples. Both the formation of DEAQ and the clearance of AQ showed excellent correlations (r~2 = 0.98 and 0.95) with 6#alpha#-hydroxylation of paclitaxel, marker substrate for CYP2C8. The inhibition of DEAQ formation by quercetin was competitive with K_i values of 1.96 for CYP2C8 and 1.56 #mu#M for HLMs. Docking of AQ into the active site homology models of the CYPI2C isoforms showed favorable interactions with CYP2C8, which supported the likelihood of an N-desethylation reaction. These data show that CYP2C8 is the main hepatic isoform responsible for the metabolism of AQ. The specificity, high affinity, and high turnover make AQ desenthylation an excellent marker reation for CYP2C8 activity.