Cynomolgus Monkey CYP2D44 Newly Identified in Liver, Metabolizes Bufuralol, and Dextromethorphan

Abstract

The cynomolgus monkey is used in drug metabolism studies, because of its evolutionary closeness to human, including cytochrome P450. Cynomolgus monkey CYP2D17, highly homologous to human CYP2D6, has been identified and characterized. Here, we report characterization of another CYP2D, CYP2D44, identified in cynomolgus monkey liver. The CYP2D44 cDNA contained an open reading frame of 497 amino acids sharing high sequence identity (87-93%) with other primate CYP2Ds. CYP2D44 mRNA was predominantly expressed in liver, similar to CYP2D17 mRNA. CYP2D17 and CYP2D44 form a gene cluster in the genome, similar to human CYP2Ds. Metabolic assays of the CYP2D17 and CYP2D44 proteins heterologously expressed in Escherichia coli indicated that CYP2D44 metabolized human CYP2D6 substrates, bufuralol and dextromethorphan (bufuralol 1'-hydroxylation and dextromethorphan O-demethylation) but to a lesser extent than CYP2D17. Kinetic analysis of dextromethorphan metabolism indicated that the apparent K(m) and V(max) of CYP2D17 and CYP2D44 catalyzed O-demethylation were similar, and, the V(max) values of CYP2D17 and CYP2D44 catalyzed N-demethylation (which human CYP2D6 catalyzes much less effectively) were similar, but the apparent K(m) of the CYP2D44 reaction was higher. Western blot analysis showed that CYP2D proteins were expressed in cynomolgus and rhesus monkey liver as well as in human and marmoset liver. Similar to CYP2D6, CYP2D44 copy number varied among the eight cynomolgus monkeys and four rhesus monkeys used in this study. These results indicated that CYP2D44, together with CYP2D17, had functional characteristics similar to those of human CYP2D6 but measurably differed in dextromethorphan N-demethylation, suggesting its importance for CYP2D-dependent drug metabolism in macaque.