Characterization of the rhesus monkey CYP3A64 enzyme: Species comparisons of CYP3A substrate specificity and kinetics using baculovirus-expressed recombinant enzymes

The rhesus monkey ( Macaca mulatta) is a primate species used extensively as a preclinical safety species in drug development. In this report, we describe the cloning, expression, and characterization of CYP3A64 ( AY334551), a CYP3A4 homolog expressed in rhesus liver. The deduced amino acid sequence was found to be 93% similar to human CYP3A4, 83% similar to human CYP3A5, and identical to the previously reported cynomolgus monkey CYP3A8 ( Komori et al., 1992). The substrate specificity of CYP3A64 for testosterone ( 0-250 mu M), midazolam ( 0-200 mu M), nifedipine ( 0-200 mu M), and 7-benzoxy-4-trifluoromethylcoumarin ( 0-200 mu M) were compared with recombinant enzymes from rat ( CYP3A1, CYP3A2), dog ( CYP3A12, CYP3A26), rabbit ( CYP3A6), and human ( CYP3A4, CYP3A5). Immunoinhibition and chemical inhibition of CYP3A64 was demonstrated using the inhibitory monoclonal antibody ( MAb) 10-1-1 ( anti-3A4) and ketoconazole ( 0-10 mu M). The utility of CYP3A64 to be used as a standard in monkey induction assays was shown and the concentration of CYP3A64 protein in rhesus liver microsomes was estimated to be 72 pmol/mg protein. In summary, these results support the utilization of rhesus monkey CYP3A64 for in vitro drug metabolism studies and provide a more complete understanding of CYP3A substrate specificities and species differences in metabolic capabilities.