A novel cytochrome P450 enzyme responsible for the metabolism of ebastine in monkey small intestine.
Small intestinal microsomes of cynomolgus monkeys were found to catalyze hydroxylation and dealkylation of an H(1)-antihistamine prodrug, ebastine. To identify the main enzyme responsible for ebastine hydroxylation, which has been hitherto unknown, we purified two cytochrome P450 isoforms, named P450 MI-2 and P450 MI-3, from the intestinal microsomes on the basis of the hydroxylation activity. P450 MI-2 and P450 MI-3 showed the respective apparent molecular weights of 56,000 and 53,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The internal amino acid sequence of P450 MI-2 had high similarity with those of human CYP4F2, CYP4F3, and CYP4F8. The first 27 amino acid residues of P450 MI-3 were highly homologous with those of monkey CYP3A8 and human CYP3A4/5/7. Furthermore, P450 MI-2 and P450 MI-3 were recognized by anti-CYP4F and anti-CYP3A antibodies, respectively, in immunoblot analysis and catalyzed leukotriene B(4) omega-hydroxylation and testosterone 6beta-hydroxylation, which are known to be mediated by CYP4F and CYP3A, respectively. Although both enzymes had ebastine hydroxylation activity, the V(max) value of P450 MI-2 was much higher than that of P450 MI-3 (37.0 versus 0.406 nmol/min/nmol of P450), and the former K(M) (5.1 microM) was smaller than the latter K(M) (10 microM). Anti-CYP4F antibody inhibited the hydroxylation in small intestinal microsomes strongly (70%), but anti-CYP3A antibody did not. These results indicate that P450 MI-2 belongs to the CYP4F subfamily and is mainly responsible for hydroxylation of ebastine in monkey small intestinal microsomes. This suggests that the small intestinal CYP4F enzyme, P450 MI-2, can play an important role in the metabolism of drugs given orally.
Hashizume
T
T
Developmental Research Laboratories, Dainippon Pharmaceutical Co., Ltd., Osaka, Japan. takanori-hashidume@dainippon-pharm.co.jp
Mise
M
M
Matsumoto
S
S
Terauchi
Y
Y
Fujii
T
T
Imaoka
S
S
Funae
Y
Y
Kamataki
T
T
Miyazaki
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002090:Butyrophenones / Q000493:pharmacokinetics*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006634:Histamine H1 Antagonists / Q000493:pharmacokinetics*; D006900:Hydroxylation; D007421:Intestine, Small / Q000201:enzymology / Q000378:metabolism*; D008252:Macaca fascicularis; D008297:Male; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D010880:Piperidines / Q000493:pharmacokinetics*; D017386:Sequence Homology, Amino Acid
D002090:Butyrophenones; D006634:Histamine H1 Antagonists; D010880:Piperidines; D003577:Cytochrome P-450 Enzyme System; C058249:ebastine