Cytochrome P450-dependent transformations of 15R- and 15S-hydroperoxyeicosatetraenoic acids: stereoselective formation of epoxy alcohol products.

Although there are many reports of epoxy alcohol synthesis from lipoxygenase products (fatty acid hydroperoxides) in mammalian tissues, there are no well-defined examples of the stereoselective synthesis of individual epoxy alcohol diastereomers. An earlier report on the metabolism of 15S-hydroperoxyeicosatetraenoic acid (15S-HPETE) in rat liver microsomes suggested such a specific reaction [Weiss, R. H., et al. (1987) Arch. Biochem. Biophys. 252, 334-338]. To characterize this reaction further, we set out to determine the precise structures and mechanism of biosynthesis of the epoxy alcohol products. We compared the products formed from 15R- and 15S-HPETE by hematin (a nonenzymatic reaction), by liver microsomes isolated from control and phenobarbital-treated rats, and by purified cytochrome P450 2B1. Eight epoxy alcohol isomers were identified by mass spectrometry and 1H NMR. In the hematin reaction, the major products are four epoxy alcohols with the epoxide in the trans configuration, diastereomers are formed in similar amounts, and the 15-HPETE enantiomers give indistinguishable patterns of products. By contrast, the liver microsomes and P450 2B1 enzyme form predominantly single diastereomers, and the configuration of the epoxide is dependent on the stereochemistry of the substrate. The main product formed from 15S-HPETE is 11S-hydroxy-14S,15S-trans-epoxyeicosa-5Z,8Z,12E- trienoic acid, and the amounts increase upon phenobarbital induction. The main products from 15R-HPETE are 11-hydroxy-14S,15R-epoxyeicosa-5Z,8Z,12E-t rienoic acid from microsomes from control rats and 13-hydroxy-14S,15R-cis-epoxyeicosa-5,8,11-trienoic acid in microsomes from phenobarbital-induced rats. The P450 2B1 enzyme gave products similar to those from the phenobarbital-induced microsomes. Analysis of an incubation using the 18O-labeled 15S-HPETE substrate demonstrated 97.6% retention of both hydroperoxy oxygens in the major product with progressively lower 18O retentions in the minor products (74-32%), possibly reflecting degrees of enzymatic control of these reactions. These results establish a precedent for the stereoselective synthesis of epoxy alcohols by mammalian cytochrome P450s.

Preferential vasoconstriction to cysteinyl leukotrienes in the human saphenous vein compared with the internal mammary artery. Implications for graft performance.

BACKGROUND: Platelet aggregation with the release of their vasoactive mediators is an important factor contributing to the patency of coronary bypass grafts. However, the role of leukocyte-derived mediators on graft performance is unclear. Leukotrienes (LTs) are proinflammatory mediators released from a variety of leukocytes that possess both vasoactive and mitogenic properties. We have therefore compared the effects of the cysteinyl LTs (C4, D4, and E4) on the human saphenous vein (SV) and human internal mammary artery (IMA). METHODS AND RESULTS: Human SVs from 43 patients (mean age, 58 years) and IMAs from 33 patients (mean age, 57 years) were obtained from individuals undergoing coronary artery bypass surgery for coronary artery disease. The samples were set up in organ baths to record changes in vessel wall tension. In undistended SVs the cysteinyl LTs elicited concentration-dependent contractions. The Emax for LTE4 (4.23 +/- 1.0 mN; n = 6) was significantly less than that observed with either LTC4 (25.7 +/- 4.01 mN; n = 7; P < .001) or LTD4 (26.19 +/- 3.16 mN; n = 7; P < .001). In addition, the LTD4 receptor antagonist ICI 198615 (30 nmol/L) significantly inhibited the LTD4 concentration-response curve but not the LTC4 responses. Furthermore, treatment of the SV with acivicin (0.05 mmol/L), a gamma-glutamyl transpeptidase inhibitor, caused a significant rightward displacement of the LTC4 concentration-response curve. In contrast, LTC4 and LTD4 produced a response in IMAs from only 3 of 29 patients. LTC4 and LTD4 produced small contractions, of which the maximum responses were 3.28 +/- 1.92 mN (n = 5) and 3.12 +/- 1.38 mN (n = 5). LTE4 produced no responses in the IMA. Experiments in which the SV was pretreated with L-NG-monomethyl-L-arginine (L-NMMA; 10(-4) mol/L) or indomethacin (10(-5) mol/L) or was denuded of endothelium had no significant effect on the Emax values for LTE4. Also, the IMA remained unresponsive to cysteinyl leukotrienes after treatment with L-NMMA or indomethacin or endothelium removal. In vitro autoradiography localized specific [3H]-LTC4 and [3H]-LTD4 binding sites (putative receptors) to the smooth muscle cells of both SV and IMA, with greater binding to the SV. CONCLUSIONS: Our data show that there is a preferential contraction to LTs in SV compared with IMA. This difference in smooth muscle cell reactivity to the cysteinyl LTs suggests that endogenous LT production from circulating or infiltrating leukocytes may be an important factor contributing to graft function.

Inhibition by cyclosporin A of adenosine triphosphate-dependent transport from the hepatocyte into bile.

BACKGROUND: Immunosuppressive treatment with cyclosporin A may be associated with impaired hepatobiliary elimination of bile salts and with cholestasis. Inhibition by cyclosporin A of the primary-active adenosine triphosphate (ATP)-dependent transport systems responsible for excretion of bile salts and cysteinyl leukotrienes across the hepatocyte canalicular membrane into bile may explain the cholestatic side effect. METHODS: ATP-dependent transport of bile salt and of cysteinyl leukotrienes was studied in human liver plasma membrane vesicles and additionally in rat liver plasma membrane vesicles enriched in canalicular membranes. RESULTS: Inhibition of ATP-dependent taurocholate transport in human liver by 50% was measured at 3 mumol/L cyclosporin A and at 4 mumol/L fujimycin. Kinetic analyses in rat liver indicated non-competitive inhibition by cyclosporin A with respect to ATP and competitive inhibition with respect to taurocholate with inhibition constant (Ki) values of 1.0 and 0.3 mumol/L, respectively. CONCLUSIONS: The ATP-dependent export carriers for bile salts and cysteinyl leukotrienes in the hepatocyte canalicular membrane are novel targets for inhibitory side effects of cyclosporin A. Inhibition of ATP-dependent bile salt transport may induce cholestasis.

Studies of in vitro skin permeation and retention of a leukotriene antagonist from topical vehicles with a hairless guinea pig model.

A leukotriene antagonist [Ro 23-3544; 6-acetyl-7-[5-(4-acetyl-3-hydroxy-2-propylphenoxy)pentyloxy] -3,4-dihydro-2H-1-benzopyran-2-carboxylic acid; 1] was studied in vitro for its permeation through and retention in hairless guinea pig skin from various topical vehicles. Both the free acid and the sodium salt forms of the drug were used. The vehicles evaluated were polyethylene glycol 400, propylene glycol, dimethyl sulfoxide (DMSO), C12-C15 alcohol lactates, dimethyl isosorbide, butyrolactone, methylpyrrolidone, hexyl laurate, isopropyl myristate, and caprylic/capric triglyceride (Neobee M5). For the salt form of the drug, the highest permeability coefficient and retention were obtained from DMSO and methylpyrrolidone, respectively. For the acid form, however, the highest permeability coefficient and retention were obtained from hexyl laurate and DMSO, respectively. The highest permeation and retention values were not obtained from the same vehicle for either the salt or the acid form of the drug. This observation questions the validity of using permeation (flux) measurements to screen topical drugs and formulations. Although the precise reasons for this lack of correlation between permeation and retention are not known at this time, this study has shown that the solubility parameters of the drug and the vehicles used may play an important role. It seems logical to conduct skin retention studies rather than flux measurements in evaluating drug delivery from dermatological products.

Effect of exogenous arachidonic acid metabolites applied on round window membrane on hearing and their levels in the perilymph.

Our previous studies revealed that treatment with sodium salicylate or indomethacin caused hearing loss, a decrease in prostaglandin (PG) levels, and an increase in leukotriene (LT) levels of the arachidonic acid (AA) cascade in the perilymph. We suspected that decreased PG-levels and/or elevated LT-levels in the inner ear may be responsible for the salicylate ototoxicity. In order to test this hypothesis, effects of exogenous treatments with PGs, PG-analog, LTs, and other lipoxygenase products on hearing and levels of AA metabolites in the perilymph were studied in chinchillas. Cyclooxygenase products, PGI2, 6-keto-PGF1 alpha, Iloprost (PGI2 analog), PGE2, and LTB4, LTC4, and 15-hydroxyeicosatetraenoic acid (15-HETE) in the lipoxygenase products in the dose of 150 ng were applied on the round window membrane (RWM); cochlear function tested by auditory brainstem response (ABR) and samples of perilymph were collected at 0.5, 1, 2, and 4 hours after the application. Samples of perilymph were assayed for all spectra of AA metabolites by high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). PG-treated animals developed minimal or no hearing loss. LT-treated animals exhibited hearing loss of 20 to 40 dB, peaking at one hour after the treatment. Elevated levels of arachidonic acid metabolites were measured in the perilymph of the ears treated with respective AA metabolites, with peak levels at one hour from the application. The findings of this study indicate that hearing loss can be induced by altered levels of PGs or LTs in the perilymph. This is another strong evidence that salicylate induced ototoxicity can be mediated by abnormal arachidonic acid metabolism in the inner ear.

Pharmacokinetics, safety, and ability to diminish leukotriene synthesis by zileuton, an inhibitor of 5-lipoxygenase.

Zileuton is a potent and specific inhibitor of 5-lipoxygenase, the first dedicated enzyme in the metabolism of arachidonic acid to the leukotrienes. The leukotrienes have been implicated in numerous pathological conditions. Zileuton was given to normal human volunteers in single doses of 200 mg to 800 mg. Results of these studies indicated that zileuton was well absorbed orally with an elimination half-life of approximately 2.5 hours. Leukotriene B4 production by ex vivo calcium ionophone stimulated whole blood was inhibited by up to 80% of baseline and correlated with plasma concentrations. Zileuton did not significantly inhibit cyclooxygenase as demonstrated by thromboxane B2 levels. There were no safety concerns that would preclude development.

Effects of low concentrations of arachidonic acid derived mediators on the membrane potential and respiratory burst responses of human neutrophils as assessed by flow cytometry.

Hypaque-Ficoll purified (95%) neutrophils (PMN) from normal healthy subjects were assessed for FMLP-elicited membrane potential (delta psi) responses and dichlorofluorescein (DCF) fluorescence (a measure of intracellular hydrogen peroxide production) using flow cytometry and appropriate fluorescent probes. Superoxide (O2) production was measured spectrophotometrically as the superoxide dismutase-inhibitable reduction of cytochrome C. The modulatory effects of dilute solutions of the arachidonic acid-derived inflammatory mediators LTB4, LTC4, LTD4, PGE1, PGE2 and PGF2 alpha (10(-9)-10(-5) M) were assessed in these systems. While LTB4 enhanced the proportion of cells depolarizing to the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP) 2-3x with a maximum effect in the 10(-9)-10(-8) M range, LTC4 and LTD4 showed no such enhancement except at high concentrations (10(-6) M). Unlike LTB4, LTC4 and LTD4 were unable to enhance FMLP mediated PMN O2 or DCF responses at any concentration tested, implying a divergence between the effects of the leukotrienes on membrane potential and oxidant responses. Pre-incubation of PMN with prostaglandins E1 or E2 led to a dose dependent inhibition of the proportion of depolarizing PMN in response to FMLP; PGF2 alpha did not show such an effect. The present data indicate that LTB4, in addition to being a powerful direct neutrophil activator, may act in a priming capacity by increasing the proportion of subsequently FMLP responsive cells, while PGE's inhibit. These modulatory effects appear relatively specific for LTB4 and the E-series prostaglandins.

Heat shock induces alterations of the lipoxygenase pathway in human polymorphonuclear granulocytes.

We have investigated the effect of the heat shock response on the leukotriene generation, chemotaxis, and generation of oxygen radicals of human polymorphonuclear granulocytes (PMNs) by preincubating the PMNs at 42 degrees C. Subsequently, the different test systems were performed at 37 degrees C. As we confirmed by the release of lactate dehydrogenase and beta-glucuronidase the elevated temperatures did not result in cytotoxic or degranulating processes. After heat shock treatment the generation of leukotrienes induced by the Ca(++)-ionophore A23187, fMLP or opsonized zymosan was inhibited in a time and temperature dependent manner (preincubation phase) as was measured by HPLC-analysis. In contrast, the conversion of 14C-arachidonic acid revealed the generation of LTB4, 5-HPETE and 5-HETE solely as a result of the preincubation at 42 degrees C without any further stimulation. In addition, the chemiluminescence response induced by opsonized zymosan and the chemotaxis against C5a and LTB4 was clearly inhibited after heat shock treatment. With regard to enzyme activities of the heat treated PMNs the protein kinase C activities were enhanced whereas the LTD4-dipeptidase and the LTB4-omega-hydroxylase were not affected.

The in vivo production of peptide leukotrienes after pulmonary anaphylaxis in the rat.

Inbred hyper-reactive rats, actively sensitized to OVA, were anesthetized, cannulated, and ventilated with room air. Tracheal instillation of Ag (OVA) resulted in an elevation of airways pressure (14.4 +/- 0.6 cm H2O). Measurement of biliary peptide leukotriene levels before and after Ag challenge using reverse phase HPLC and RIA techniques showed significant elevations in leukotriene (LT) levels, the amounts released being LTC4 (3.65 +/- 0.78), LTD4 (2.8 +/- 1.11), and N-Ac LTE4 (3.87 +/- 1.15) expressed as ng/100 g of body weight, n = 13. Identification of these metabolites were confirmed by HPLC/RIA techniques and LTC4 was further characterized by UV spectroscopy and its enzymatic conversion by gamma-glutamyl transpeptidase to LTD4. [3H]LTC4 (16 ng) administration by tracheal instillation resulted in a 31.4 +/- 4.3% recovery of radioactivity through the bile over 4 h (n = 3) with the major identified metabolite being N-Ac LTE4. [3H]LTC4 (16 ng) plus synthetic LTC4 (5 micrograms) showed a 30.8 +/- 3.1% recovery through the bile after tracheal instillation (3-h collection, n = 4) with significant amounts of LTC4 as well as N-Ac LTE4 present. [3H]LTC4 administration by the portal vein resulted in a 37.4 +/- 8.8% biliary recovery over 60 min (n = 6), the metabolites present in the bile being LTC4, LTD4, LTE4, and N-Ac LTE4. Pretreatment with the 5-lipoxygenase inhibitor L-656,224 (15 mg/kg, 3.5 h pre-p.o.) before Ag challenge resulted in a significant inhibition (greater than 90%, p less than 0.05) of biliary leukotriene levels in this model. Our study demonstrates that peptide leukotrienes are produced in the anesthetized rat after pulmonary anaphylaxis and that biliary leukotriene measurement is suitable for showing the biochemical efficacy of leukotriene inhibitors in vivo. In vivo tracer experiments suggest that the biliary metabolic profile of the peptide leukotrienes is dependent on the site and levels of release as well as the efficiency of the vascular clearance of the various metabolites.