leukotriene-a4 and zileuton

Leukotriene (LT) A(4) hydrolase is a dual function enzyme that is essential for the conversion of LTA(4) to LTB(4) and also possesses an aminopeptidase activity. SC-57461A (3-[methyl[3-[4-phenylmethyl)phenoxy]propyl]amino]propanoic acid HCl) is a potent inhibitor of human recombinant LTA(4) hydrolase (epoxide hydrolase and aminopeptidase activities, K(i) values = 23 and 27 nM, respectively) as well as calcium ionophore-induced LTB(4) production in human whole blood (IC(50) = 49 nM). In the present study, we investigated its action in several animal models. Oral activity was evident from the ability of the compound to inhibit mouse ex vivo calcium ionophore-stimulated blood LTB(4) production with ED(50) values at 1.0 and 3.0 h of 0.2 and 0.8 mg/kg, respectively. A single oral dose of 10 mg/kg SC-57461A blocked mouse ex vivo LTB(4) production 67% at 18 h and 44% at 24 h, suggesting a long pharmacodynamic half-life. In a rat model of ionophore-induced peritoneal eicosanoid production, SC-57461 inhibited LTB(4) production in a dose-dependent manner (ED(50) = 0.3-1 mg/kg) without affecting LTC(4) or 6-keto-prostaglandin F(1alpha) production. Oral pretreatment with SC-57461 in a rat reversed passive dermal Arthus model blocked LTB(4) production with an ED(90) value of 3 to 10 mg/kg, demonstrating good penetration of drug into skin. Plasma level of intact SC-57461 (3 h after oral gavage dosing with 3 mg/kg) was 0.4 microg/ml, which corresponds to >80% inhibition of dermal LTB(4) production. Oral or topical pretreatment with SC-57461A 1 h before challenge with arachidonic acid blocked ear edema in the mouse. SC-57461A is a competitive, selective, and orally active inhibitor of LTA(4) hydrolase in vivo, making it useful to explore the contribution of LTB(4) to a number of inflammatory diseases.

Topics: Administration, Oral; Administration, Topical; Animals; Arthus Reaction; beta-Alanine; Dermatitis; Edema; Eicosanoids; Enzyme Inhibitors; Epoxide Hydrolases; Hydroxyurea; In Vitro Techniques; Leukotriene A4; Leukotriene Antagonists; Lipoxygenase Inhibitors; Mice; Peritoneum; Rats; Skin

5-Lipoxygenase is the first committed enzyme in the leukotriene biosynthetic pathway and is known to catalyze not only the first oxygenation of arachidonate to form 5(S)-hydroperoxyeicosatetraenoic acid (5(S)-HPETE), but also dehydration of this intermediate into leukotriene A4 (LTA4) by an activity termed leukotriene A4 synthase. Inhibition of cytosolic 5-lipoxygenase prepared from human blood granulocytes with zileuton (100 microM) was virtually complete, but LTA4 synthase activity was only inhibited by 47%. Structural characterization of eicosanoids synthesized in these preparations revealed an abundance of 15-lipoxygenase metabolites including 15-HETE when arachidonate was used as substrate and 5(S),15(S)-dihydroxy-6,8,11,13(E,E,Z,Z)-eicosatetraenoic acid when 5(S)-HPETE was used as substrate. When neutrophils were prepared that contained less than 1% eosinophil contamination, zileuton was found to almost completely inhibit all 5-lipoxygenase, as well as LTA4 synthase products. Immunochemical analysis of the supernatants from purified neutrophils and eosinophils confirmed the previous observation that neutrophils do not express 15-lipoxygenase. Incubation of 5(S)-HPETE with recombinant mammalian 15-lipoxygenase resulted in the formation of 6-trans-LTB4 and 6-trans-12-epi-LTB4 as LTA4 products, as well as the 12-lipoxygenase product 5(S),12(S)-diHPETE. The mechanism of action of 15-lipoxygenase acting as an LTA4 synthase is proposed to involve removing the pro-R hydrogen atom at carbon-10 of 5(S)-HPETE, which is antarafacial to the hydroperoxy group to yield LTA4.

Topics: Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Cell-Free System; Cytosol; Dose-Response Relationship, Drug; Eosinophils; Granulocytes; Humans; Hydroxyeicosatetraenoic Acids; Hydroxyurea; Leukotriene A4; Leukotrienes; Lipoxygenase Inhibitors; Models, Chemical; Neutrophils; Recombinant Proteins

We report that leukotriene A4, the electrophilic product of 5-lipoxygenase catalysis, irreversibly inactivates the enzyme. Leukotriene A4 inhibits 5-hydroxyeicosatetraenoic acid formation by human neutrophils and differentiated granulocytic HL-60 cells in a concentration-dependent manner with IC50 values = 22.4 +/- 2.5 and 29.0 +/- 8.0 microM, respectively. Recovery of cellular enzymatic activity is negligible (< 6%) following inactivation. Leukotriene A4 inactivates cellular 5-lipoxygenase without inhibiting its translocation from the cytosol to the membrane, suggesting that it impairs catalysis without impairing formation of the complex between 5-lipoxygenase and its membrane-associated activating protein. Consistent with this, leukotriene A4 inactivates purified 5-lipoxygenase from human neutrophils, via saturable, pseudo first-order kinetics with a rate constant, ki = 0.14 min-1 and a dissociation constant, Ki = 2.1 +/- 0.7 microM. Purified 5-lipoxygenase incubated with [3H]arachidonic acid incorporated a radiolabeled species that was not removed by electrophoresis under reduced denaturing conditions. Preincubation with leukotriene A4 diminished the incorporation of radiolabeled material, consistent with irreversible modification of 5-lipoxygenase by its metastable product, leukotriene A4. This unusual product inactivation mechanism may contribute to the decline in 5-lipoxygenase activity observed during catalysis.

Topics: Arachidonate 5-Lipoxygenase; Cell Differentiation; Cell Line; Humans; Hydroxyeicosatetraenoic Acids; Hydroxyurea; Kinetics; Leukemia, Promyelocytic, Acute; Leukocytes; Leukotriene A4; Lipoxygenase Inhibitors; Neutrophils; Tumor Cells, Cultured