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 microM), midazolam (0-200 microM), nifedipine (0-200 microM), and 7-benzoxy-4-trifluoromethylcoumarin (0-200 microM) 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 microM). 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.
Carr
Brian
B
Department of Drug Metabolism, Merck Research Laboratories, West Point, PA, USA. brian_carr@merck.com
Norcross
Ryan
R
Fang
Yulin
Y
Lu
Ping
P
Rodrigues
A David
AD
Shou
Magang
M
Rushmore
Tom
T
Booth-Genthe
Catherine
C
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D003374:Coumarins / Q000378:metabolism; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007654:Ketoconazole / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008253:Macaca mulatta / Q000235:genetics* / Q000378:metabolism; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D008874:Midazolam / Q000378:metabolism; D008969:Molecular Sequence Data; D009543:Nifedipine / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D018411:Spodoptera; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism
D003374:Coumarins; D065607:Cytochrome P-450 Enzyme Inhibitors; D018076:DNA, Complementary; D011994:Recombinant Proteins; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; D009543:Nifedipine; D008874:Midazolam; D007654:Ketoconazole