piperidines and 17-octadecynoic-acid

The mechanism-based inactivation of human CYP2J2 by three terminal acetylenic compounds:

Topics: Alkynes; Amides; Butyrophenones; Chromatography, Liquid; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Danazol; Enzyme Activation; Fatty Acids, Unsaturated; Heme; Humans; Kinetics; Piperidines; Tandem Mass Spectrometry

CYP4F enzymes, including CYP4F2 and CYP4F3B, were recently shown to be the major enzymes catalyzing the initial oxidative O-demethylation of the antiparasitic prodrug pafuramidine (DB289) by human liver microsomes. As suggested by a low oral bioavailability, DB289 could undergo first-pass biotransformation in the intestine, as well as in the liver. Using human intestinal microsomes (HIM), we characterized the enteric enzymes that catalyze the initial O-demethylation of DB289 to the intermediate metabolite, M1. M1 formation in HIM was catalyzed by cytochrome P450 (P450) enzymes, as evidenced by potent inhibition by 1-aminobenzotriazole and the requirement for NADPH. Apparent K(m) and V(max) values ranged from 0.6 to 2.4 microM and from 0.02 to 0.89 nmol/min/mg protein, respectively (n = 9). Of the P450 chemical inhibitors evaluated, ketoconazole was the most potent, inhibiting M1 formation by 66%. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, inhibited M1 formation in a concentration-dependent manner (up to 95%). Immunoinhibition with an antibody raised against CYP4F2 showed concentration-dependent inhibition of M1 formation (up to 92%), whereas antibodies against CYP3A4/5 and CYP2J2 had negligible to modest effects. M1 formation rates correlated strongly with arachidonic acid omega-hydroxylation rates (r(2) = 0.94, P < 0.0001, n = 12) in a panel of HIM that lacked detectable CYP4A11 protein expression. Quantitative Western blot analysis revealed appreciable CYP4F expression in these HIM, with a mean (range) of 7 (3-18) pmol/mg protein. We conclude that enteric CYP4F enzymes could play a role in the first-pass biotransformation of DB289 and other xenobiotics.

Topics: Amidines; Antibodies; Antiparasitic Agents; Arachidonic Acid; Benzamidines; Benzoflavones; Butyrophenones; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP2J2; Cytochrome P-450 CYP3A; Cytochrome P-450 CYP4A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 4; Enzyme Inhibitors; Fatty Acids, Unsaturated; Humans; Hydroxyeicosatetraenoic Acids; Intestinal Mucosa; Intestines; Kinetics; Methylation; Microsomes; Oxygenases; Piperidines; Prodrugs; Recombinant Proteins; Stereoisomerism

1. The effects of the endocannabinoid, anandamide, and its metabolically stable analogue, methanandamide, on induced tone were examined in sheep coronary artery rings in vitro. 2. In endothelium-intact rings precontracted to the thromboxane A(2) mimetic, U46619, anandamide (0.01 - 30 microM) induced slowly developing concentration-dependent relaxations (pEC(50) [negative log of EC(50)]=6.1+/-0.1; R(max) [maximum response]=81+/-4%). Endothelium denudation caused a 10 fold rightward shift of the anandamide concentration-relaxation curve without modifying R(max). Methanandamide was without effect on U46619-induced tone. 3. The anandamide-induced relaxation was unaffected by the cannabinoid receptor antagonist, SR 141716A (3 microM), the vanilloid receptor antagonist, capsazepine (3 and 10 microM) or the nitric oxide synthase inhibitor, L-NAME (100 microM). 4. The cyclo-oxygenase inhibitor, indomethacin (3 and 10 microM) and the anandamide amidohydrolase inhibitor, PMSF (70 and 200 microM), markedly attenuated the anandamide response. The anandamide transport inhibitor, AM 404 (10 and 30 microM), shifted the anandamide concentration-response curve to the right. 5. Precontraction of endothelium-intact rings with 25 mM KCl attenuated the anandamide-induced relaxations (R(max)=7+/-7%), as did K(+) channel blockade with tetraethylammonium (TEA; 3 microM) or iberiotoxin (100 nM). Blockade of small conductance, Ca(2+)-activated K(+) channels, delayed rectifier K(+) channels, K(ATP) channels or inward rectifier K(+) channels was without effect. 6. These data suggest that the relaxant effects of anandamide in sheep coronary arteries are mediated in part via the endothelium and result from the cellular uptake and conversion of anandamide to a vasodilatory prostanoid. This, in turn, causes vasorelaxation, in part, by opening potassium channels.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 4-Aminopyridine; Animals; Apamin; Arachidonic Acid; Arachidonic Acids; Barium; Calcium Channel Blockers; Cannabinoid Receptor Modulators; Cannabinoids; Capsaicin; Coronary Vessels; Cytochrome P-450 Enzyme Inhibitors; Dose-Response Relationship, Drug; Endocannabinoids; Endothelium, Vascular; Enzyme Inhibitors; Fatty Acids, Unsaturated; Glyburide; In Vitro Techniques; Indomethacin; Miconazole; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Peptides; Phenylmethylsulfonyl Fluoride; Piperidines; Polyunsaturated Alkamides; Potassium; Potassium Channel Blockers; Potassium Channels; Pyrazoles; Receptors, Drug; Rimonabant; Sheep; Tetraethylammonium; Vasoconstrictor Agents; Vasodilation