Richard D. Towner

Purification and Characterization of Fatty Acid Cyclooxygenase from Human Platelets

The fatty acid cyclooxygenase (EC 1.14.99.1) that produces the prostaglandin, thromboxane, and prostacyclin precursor (PGH2), was solubilized from human platelet microsomes in 20 sucrose and 1.0% Triton X-100. The enzyme was purified 300-fold by electrofocusing, Sephadex G-200 gel filtration, and hydrophobic chromatography on ethyl agarose. The cyclooxygenase catalyzed the conversion of arachidonic acid to prostaglandin endoperioxide, PGH2, that was trapped at -25 degrees C and separated on TLC at -20 degrees C. PGH2 was hydrolyzed to HHT in acidic pH, or was chemically converted to PGE2 in slightly alkaline pH in the absence of cofactors. The enzyme showed a broad pH optimum in the range of 7-9. Hemin containing substances such as methemoglobin were absolutely required as cofactors, while tryptophan, epinephrine, phenol, and hydroquinone stimulated the PGH2 formation. Metal ions, such as ZN2+ and Cd2+ inhibited the enzyme reaction at 0.1 to 1 mM. The molecular weight of the purified enzyme was estimated at 79,432 by sodium dodecyl sulfate disc gel electrophoresis at pH 8.0. The properties of the human platelet enzyme was generally similar to the sheep vesicular enzyme in the method of solubilization, pH optimum, and molecular weight.

The anti-inflammatory effects of LY178002 and LY256548

LY178002 (5-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methylene-4-thiazolidinone) and its N-methyl analog, LY256548, inhibit the enzymatic activity of phospholipase A2, 5-lipoxygenase and fatty acid cycloxygenase. They also inhibit leukotriene B4 production from human polymorphonuclear leukocytes stimulated with the calcium ionophore A23187. Since products of the arachidonic acid cascade have been implicated as important mediators in a variety of inflammatory diseases including arthritis, LY178002 and LY256548 were studied in the Freunds Complete Adjuvant-Induced Arthritis (FCA) model in rats. The compounds were administered orally and inhibition of bone damage and paw swelling was assessed of both the injected and uninjected paws. At 50 mg/kg LY178002 inhibited soft tissue swelling in the uninjected paw by 81% while LY256548 exhibited 57% inhibition. Bone damage was also significantly inhibited by both compounds. A dose response was conducted. The minimum effective dose for LY178002 was 10 mg/kg p.o. In the established FCA model LY178002 at 50 mg/kg p.o. inhibited the uninjected paw swelling by 75% while LY256548 did not show this level of activity. These results suggest that LY178002 and LY256548 may be useful in the treatment of arthritis.

Fused bicyclic Gly-Asp β-turn mimics with potent affinity for GPIIb-IIIa. Exploration of the arginine isostere

6-[4-Amidinobenzoyl]amino]-tetralone-2-acetic acid is a potent antagonist of GPIIb-IIIa. Substitution in the meta position of the benzamidine, or replacement with a heteroaryl amidine was tolerated in this series. Use of an acyl-linked 4-alkyl piperidine as an arginine isostere also provided active compounds. Compounds from this series provided substantial systemic exposure in the rat following oral administration.

Cardiovascular effect and stimulus-dependent inhibition of superoxide generation from human neutrophils by tibenelast, 5,6-diethoxybenzo(b) thiophene-2-carboxylic acid, sodium salt (LY186655)☆

Tibenelast (LY186655), 5,6,-diethoxybenzo(b)thiophene-2-carboxylic acid, sodium salt, is an orally active anti-anaphylactic compound in guinea pigs, and has been shown to prevent bronchospasm in moderately severe asthmatic patients. Pharmacological studies with tibenelast demonstrated that it is a selective phosphodiesterase (PDE) inhibitor in that it is moderately active against the lung and stomach enzyme while being a very weak inhibitor of the heart enzyme. The compound was without cardiovascular effects at anti-anaphylactic doses. In contrast to theophylline, tibenelast did not have a direct inotropic effect in the cat papillary muscle system. The concentration that inhibited 50% of the enzymatic activity (IC50) for tibenelast was 20- to 30-fold lower for neutrophil PDE than for PDE of other tissues. It was 100 times more potent than aminophylline in inhibiting superoxide generation from platelet-activating factor (PAF)-primed polymorphonuclear leukocytes (PMNL) challenged with chemotactic factor, N-formyl-methionyl-leucyl-phenylalanine. However, tibenelast was less effective in the tumor necrosis factor-primed system, and did not inhibit superoxide generation during phagocytosis or when other soluble stimuli, such as phorbo-12-myristate-13-acetate or the calcium ionophore A23187, were used. Furthermore, tibenelast did not inhibit enzymes involved in arachidonic acid metabolism. These results suggest that tibenelast probably inhibits superoxide release from PMNL via a selective inhibition on PDE.

Anti-inflammatory effects of LY221068 and LY269415

LY221068, 5-[[3,5-bis(1,1-dimethylethyl)-4-hydroxy phenyl]methylene]-3-(dimethylamino)-4-thiazolidinone, and the monomethylamino analog, LY269415, are anti-oxidants and potent inhibitors of iron dependent lipid peroxidation and 5-lipoxygenase enzyme. Since oxygen radical species, lipid peroxides and products of the arachidonic acid cascade have been implicated as important mediators in a variety of inflammatory diseases including arthritis, LY221068 and LY269415 were studied in the Freunds Complete Adjuvant Induced Arthritis (FCA) model in rats. The compounds were administered orally and inhibition of bone damage and paw swelling of both the injected and uninjected paws was assessed. At 50 mg/kg p.o., LY221068 inhibited soft tissue swelling in the uninjected paw by 72% while LY269415 at 25 mg/kg p.o. exhibited 74% inhibition. Bone damage was also significantly inhibited by both compounds. In a dose response study, the minimum effective dose for LY221068 was 10 mg/kg p.o. for LY269415 was 5 mg/kg p.o. In the established FCA model in rats, LY221068 at 50 mg/kg p.o. inhibited the uninjected paw swelling by 71% while LY269415 at 25 mg/kg p.o. inhibited 70%. These results suggest that LY221068 and LY269415 may be useful in the treatment of arthritis.

Affinity Chromatography of 15-Hyoroxyprostaglandin Dehydrogenase from Dog Lung

Abstract A 15-hydroxyprostaglandin dehydrogenase has been purified from dog lung by NAD+-CH-Sepharose 4B affinity chromatography. The molecular weight of the enzyme determined by gel filtration is 46, 500. The biochemical properties of the dog lung enzyme are similar to that isolated from other sources.

Structurally novel antiarrhythmic / antioxidant quinazolines

Abstract We report a structurally novel series of quinazolines with in vivo antiarrhythmic and/or in vitro iron-dependent lipid peroxidation inhibitory activity. Two analogues, 7 and 12, were evaluated in a canine model of regional myocardial ischemia and reperfusion.