leukotriene-b4 and ebastine

The purpose of the study was to elucidate human intestinal cytochrome P450 isoform(s) involved in the metabolism of an antihistamine, ebastine, having two major pathways of hydroxylation and N-dealkylation. The ebastine dealkylase in human intestinal microsomes was CYP3A4, based on the inhibition studies with antibodies against CYP1A, CYP2A, CYP2C, CYP2D, CYP2E, and CYP3A isoforms and their selective inhibitors. However, ebastine hydroxylase could not be identified. We then examined the inhibitory effects of anti-CYP4F antibody and 17-octadecynoic acid, an inhibitor of the CYP4 family, on ebastine hydroxylation in intestinal microsomes, since CYP4F was recently found to be the predominant ebastine hydroxylase in monkey intestine; and a novel CYP4F isoform (CYP4F12), also capable of hydroxylating ebastine, was found to exist in human intestine. However, the inhibitory effects were only partial (about 20%) and thus it was thought that, although human CYP4F was involved in ebastine hydroxylation, another predominant enzyme exists. Further screening showed that the hydroxylation was inhibited by arachidonic acid. CYP2J2 was selected as a candidate expressed in the intestine and closely related to arachidonic acid metabolism. The catalytic activity of recombinant CYP2J2 was much higher than that of CYP4F12. Anti-CYP2J antibody inhibited the hydroxylation to about 70% in human intestinal microsomes. These results demonstrate that CYP2J2 is the predominant ebastine hydroxylase in human intestinal microsomes. Thus, the present paper for the first time indicates that, in human intestinal microsomes, both CYP2J and CYP4F subfamilies not only metabolize endogenous substrates but also are involved in the drug metabolism.

Topics: Antibodies, Blocking; Arachidonic Acid; Aryl Hydrocarbon Hydroxylases; Butyrophenones; Cloning, Molecular; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dealkylation; Enzyme Inhibitors; Histamine H1 Antagonists; Humans; In Vitro Techniques; Intestinal Mucosa; Intestines; Kinetics; Leukotriene B4; Microsomes; Mixed Function Oxygenases; Oxygenases; Piperidines; Recombinant Proteins

A cDNA encoding a novel human CYP4F enzyme (designated CYP4F12) was cloned by PCR from a human small intestine cDNA library. RT-PCR analysis demonstrated that CYP4F12 is expressed in human small intestine and liver. This cDNA contains an entire coding region of a 524-amino-acid protein that is 81.7, 78.3, and 78.2% identical to CYP4F2, CYP4F3, and CYP4F8, respectively. When expressed in Saccharomyces cerevisiae, the P450 catalyzes leukotriene B(4) omega-hydroxylation and arachidonic acid omega-hydroxylation, typical reactions of CYP4F isoforms. Their activity levels are, however, much lower than those of CYP4F2. Interestingly, CYP4F12 catalyzes the hydroxylation of the antihistamine ebastine with significantly higher catalytic activity relative to CYP4F2 (385 vs 5 pmol/min/nmol P450). These results indicate that CYP4F12 has a different profile of substrate specificity from other CYP4F isoforms, enzymes responsible for metabolizing endogenous autacoids, therefore suggesting that it may play an important role in xenobiotic biotransformation in the human small intestine.

Topics: Amino Acid Sequence; Arachidonic Acid; Aryl Hydrocarbon Hydroxylases; Base Sequence; Butyrophenones; Catalysis; Cloning, Molecular; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 4; DNA, Complementary; Gene Library; Humans; Intestine, Small; Leukotriene B4; Liver; Mixed Function Oxygenases; Molecular Sequence Data; Piperidines; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; Saccharomyces cerevisiae; Sequence Analysis, DNA; Sequence Homology, Amino Acid