n-hydroxy-n--(4-butyl-2-methylphenyl)formamidine and 17-octadecynoic-acid

Cytochrome P450 (CYP) omega-hydroxylases and their arachidonic acid metabolites play important roles in myocardial ischemia-reperfusion injury. In this study we investigated the effects of several selective CYP omega-hydroxylase inhibitors on myocardial ischemia/reperfusion-induced myocardial apoptosis. Rats were subjected 30 min of ischemia and 2 h of reperfusion. Groups received either 17-octadecynoic acid (17-ODYA, 0.3 or 3 mg/kg), N-methylsulfonyl-12, 12-dibromododec-11-enamide (DDMS, 0.4 or 0.8 mg/kg), N-hydroxy-N'-(4-butyl-2-methylphenyl) formamidine (HET0016, 0.1 or 1 mg/kg) or vehicle 10 min prior to ischemia. To further assess the role of mitogen-activated protein kinases (MAPKs) in the CYP omega-hydroxylase inhibitor-induced anti-apoptotic effect, rats also received PD98059 (1 mg/kg), SB203580 (1 mg/kg) or SP600125 (6 mg/kg) 15 min prior to ischemia, with subsets of rats also receiving HET0016 10 min prior to ischemia. Compared with vehicle group, 17-ODYA, DDMS and HET0016 significantly inhibited myocardial apoptosis as evidenced by decreased DNA ladder formation, terminal dUTP deoxynucleotidyltransferase nick end-labeling (TUNEL) positive nuclear staining. They also decreased caspase-3 activity and Bax protein expression but up-regulated the expression of Bcl-2. Conversely, exogenous 20-HETE administration exerted opposite effects. Moreover, HET0016 increased the activity of extracellular signal-related protein kinases 1 and 2 (ERK1/2) but had no significant effect on p38 MAPK or c-Jun N-terminal kinase (JNK) during ischemia/reperfusion. Pretreatment with PD98059, the inhibitor of ERK1/2, but not SB203580 or SP600125, almost completely blocked the effect exerted by HET0016. Taken together, these data suggest that CYP omega-hydroxylase inhibition exerts significant anti-apoptosis effects, at least in part, by activation of ERK1/2 in ischemia/reperfusion heart.

Topics: Amides; Amidines; Animals; Apoptosis; Caspase 3; Cytochrome P-450 CYP4A; Enzyme Activation; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocardial Reperfusion Injury; Myocytes, Cardiac; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Signal Transduction; Sulfones

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

This study examined the effects of blocking the formation of 20-hydroxyeicosatetraenoic acid (20-HETE) on the acute fall in cerebral blood flow after subarachnoid hemorrhage (SAH) in the rat. In vehicle-treated rats, regional cerebral blood flow (rCBF) measured with laser-Doppler flowmetry fell by 30% 10 min after the injection of 0.3 ml of arterial blood into the cisterna magna, and it remained at this level for 2 h. Pretreatment with inhibitors of the formation of 20-HETE, 17-octadecynoic acid (17-ODYA; 1.5 nmol intrathecally) and N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine (HET0016; 10 mg/kg iv), reduced the initial fall in rCBF by 40%, and rCBF fully recovered 1 h after induction of SAH. The concentration of 20-HETE in the cerebrospinal fluid rose from 12 +/- 2 to 199 +/- 17 ng/ml after SAH in vehicle-treated rats. 20-HETE levels averaged only 15 +/- 11 and 39 +/- 13 ng/ml in rats pretreated with 17-ODYA or HET0016, respectively. HET0016 selectively inhibited the formation of 20-HETE in rat renal microsomes with an IC(50) of <15 nM and human recombinant CYP4A11, CYP4F2, and CYP4F3 enzymes with an IC(50) of 42, 125, and 100 nM, respectively. These results indicate that 20-HETE contributes to the acute fall in rCBF after SAH in rats.

Topics: Amidines; Animals; Carbon Dioxide; Cerebrovascular Circulation; Enzyme Inhibitors; Erythrocytes; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Male; Microscopy, Video; Partial Pressure; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Subarachnoid Hemorrhage; Time Factors