Platelets by Burimamide G

Br. J. Pharmac. (1980), 71, 157-164 INHIBITION OF THROMBOXANE A2 BIOSYNTHESIS IN HUMAN PLATELETS BY BURIMAMIDE G. ALLAN, K.E. EAKINS*, P.S. KULKARNI* & R. LEVI Department of Pharmacology, Cornell University Medical College, New York, N.Y. and *Departments of Opthalmology and Pharmacology, Columbia University College of Physicians & Surgeons, New York, N.Y., U.S.A. 1 Burimamide selectively inhibited the formation of thromboxane A2 from prostaglandin endoperoxides by human platelet microsomes in a dose-dependent manner (IC50 = 2.5 x 10-5 M). Burima- mide was found to be equipotent to imidazole as a thromboxane synthetase inhibitor. 2 Metiamide, cimetidine and a series of compounds either bearing a structural or pharmacological relationship to histamine caused little or no inhibition of thromboxane A2 biosynthesis by human platelet microsomes. 3 Burimamide (5 x 10-4 to 2.3 x 10- M) did not inhibit either the cyclo-oxygenase or the prostacyclin synthetase of sheep seminal vesicles or the prostacyclin synthetase of dog aortic microsomes. 4 Burimamide (2.5 x 10'- to 1.2 x 10-4 M) inhibited sodium arachidonate-induced human platelet aggregation; the degree of inhibition was dependent upon the concentration of arachidonic acid used to aggregate the platelets. Introduction The prostaglandin endoperoxides (prostaglandin G2, (Gryglewski, Zmuda, Korbut, Krecioch & Bieron, H2), the first cyclo-oxygenated products of arachido- 1977). nic acid metabolism can be converted enzymatically Various imidazole derivatives were studied by into either primary prostaglandins such as prosta- Moncada et al. (1977) and of these, only one-methyl glandin E2, F2, or D2, the non-prostanoate throm- imidazole was found to be a potent inhibitor of boxane A2 or prostacyclin (Moncada, Gryglewski, thromboxane A2 biosynthesis. Further studies by Tai Bunting & Vane, 1976). Non-steroidal anti- & Yuan (1978) demonstrated one-alkyl substituted inflammatory agents such as aspirin and indometha- imidazoles to be the most potent thromboxane syn- cin inhibit the initial cyclo-oxygenase reaction which thetase inhibitors, inhibitory activity being abolished converts arachidonic acid to the prostaglandin endo- when the substituents appeared in other positions of peroxides (Vane, 1971). Since there are great differ- the imidazole ring. The histamine H2-receptor antag- ences in the biological activities of the products gener- onists, metiamide, cimetidine and burimamide, are ated from the prostaglandin endoperoxides, selective also structural derivatives of imidazole, side chain inhibitors of the formation of individual end products substitution occurring at the four (five) position of the of endoperoxide metabolism would be desirable as imidazole ring (Brimblecombe, Duncan, Durant, pharmacological tools. Emmett, Ganellin & Parsons, 1975). Several compounds have been found which inhibit In a preliminary series of experiments burimamide selectively thromboxane generation from prosta- was found to inhibit thromboxane A2 biosynthesis in glandin endoperoxides, these include: benzydamine human platelet microsomes (Allan & Eakins, 1978). In (Moncada, Needleman, Bunting & Vane, 1976) imi- the present study we have compared this activity of dazole (Needleman, Raz, Ferrendelli & Minkes, 1977; burimamide with imidazole and the histamine Moncada, Bunting, Mullane, Thorogood, Vane, Raz H2-receptor antagonists, metiamide and cimetidine, & Needleman, 1977), N-0164, a phenyl phosphonate and some other chemically related compounds. We derivative of phloretin phosphate (Eakins & Kul- have also investigated the selectivity of this inhibitory karni, 1977), 9, 1 1-epoxy methanoprostanoic acid, action of burimamide: thus we have studied the (Sun, 1977) and 2-isopropyl-3-nicotinyl-indole (L8027) effects of burimamide on cyclo-oxygenase and prosta- 0007-1188/80/130157-08 $01.00 C) Macmillan Publishers Ltd 1980 158 G. ALLAN, K.E. EAKINS, P.S. KULKARNI & R. LEVI cyclin synthetase activities. In addition, because of the ["4C]-arachidonic acid (1 jig) was incubated with postulated involvement of thromboxane A2 as the sheep seminal vesicle microsomes (6.1 mg protein) for endogenous trigger of platelet aggregation, we have 20 min at room temperature in the presence of 2 mm studied the effects of burimamide on arachidonic adrenaline in a total volume of 2 ml Tris buffer (50 acid-induced human platelet aggregation. mM pH 7.4). Acid lipid extraction was followed by paper chromatographic separation of products on Sil- ica Gel G paper using the organic phase of ethyl acet- Methods ate; isoctane: acetic acid: water (11:5:2:10). Radio- scan of the products of paper chromatography was Thromboxane synthetase activity carried out with a Vanguard Autoscanher Model 930. Areas of radioactivity coinciding with the migration Thromboxane A2-like activity was generated using of authentic standards were located and radioactivity the coupled enzyme preparation developed by Mon- counted in a Packard Model 2405 Liquid Scintillation cada et al. (1976a,b). Sheep seminal vesicle micro- Counter. Burimamide was incubated for 5 min at somes (200 to 300 jg protein) were incubated without 22°C with the sheep seminal vesicle microsomes co-factors with sodium arachidonate (1 jg) in a total before the addition of radiolabelled arachidonic acid volume of 100 gl Tris buffer (50 mm pH 7.5) at room and the inhibitory effect of this compound on product temperature (22'C) for 1 min; 50 Ill samples were then formation was determined. tested for prostaglandin endoperoxide-like activity by The effect of burimamide on the prostacyclin syn- bioassay. The remainder of this suspension was incu- thetase activity of dog aorta microsomes was also bated with indomethacin-treated human platelet studied by bioassay. Prostacyclin-like activity was microsomes (150 to 250 jig protein) for 2 min at 0°C; produced by incubating prostaglandin H2 (50 ng) the thromboxane A2-like activity thus generated was with dog aorta microsomes (20 to 50 gig protein) for 1 then tested by bioassay. Biological characterization of min at 22°C (Kulkarni, Eakins, Saber & Eakins, endoperoxides and thromboxane was made by bioas- 1977). Prostacyclin was extracted three times in ether, say on spiral strips of rabbit thoracic aorta continu- the ether was evaporated under nitrogen and the resi- ously superfused at 10 ml/min with oxygenated Krebs due reconstituted in 50 mM Tris buffer and tested for solution at 37°C. The assay tissues were treated with biological activity. Prostacyclin was assayed by its a combination of antagonists, infused to give final ability to inhibit arachidonic acid-induced platelet concentrations of diphenhydramine, atropine and aggregation. Washed rabbit platelets (WRP) were pre- phenoxybenzamine of 0.1 jg/ml, propranolol 2 jig/ml pared by the method of Hamberg, Svensson, Waka- and methysergide 0.2 gg/ml. These antagonists ren- bayashi & Samuelsson (1974). Aggregation of WRP dered the assay tissues insensitive to histamine, acetyl- was studied with 0.4 ml. samples of WRP in a chrono- choline, catecholamines and 5-hydroxytryptamine. log platelet aggregometer. Aggregation was induced Indomethacin 1 jig/ml was also added to the super- by the addition of sodium arachidonate; a concen- fusion medium to prevent prostaglandin biosynthesis tration of sodium arachidonate yielding threshold by the assay tissues. A series of compounds bearing a aggregation was chosen for each batch of WRP. structural resemblance to imidazole were tested for Extracts of prostacycin-like activity were incubated their ability to inhibit the formation of thromboxane with WRP for 2 min before the addition of the aggre- A2-like activity from prostaglandin endoperoxides by gating agent. Burimamide was allowed to incubate human platelet microsomes. The test drugs were pre- with dog aorta microsomes for 2 min at 0°C before incubated with the human platelet microsomes for 2 the addition of prostaglandin H2 and inhibition of min at 0°C before the addition of the crude endoper- prostacyclin formation determined. oxide substrate (Allan & Eakins, 1978). The effect of burimamide on the cyclo-oxygenase activity of sheep seminal microsomes was also studied Cyclo-oxygenase and prostacyclin synthetase activity by bioassay. Prostaglandin endoperoxides were generated and characterized as described earlier. Burima- Radiochemical determination of the formation of the mide was incubated with the sheep seminal vesicle cyclo-oxygenase product, prostaglandin E2, and the microsomes for 2 min at 0°C before the addition of stable degradation product of prostacyclin, 6-keto sodium arachidonate. Using the same experimental prostaglandin F1l, was carried out by the method of conditions, inhibition of the enzyme by indomethacin Cottee, Flower, Moncada, Salmon & Vane (1977). (Flower, 1974) was also studied. These workers had demonstrated that at low substrate/enzyme ratios, in the presence of adrenaline, Platelet aggregation studies the major prostaglandins biosynthesized by the sheep seminal vesicle cyclo-oxygenase from arachidonic acid Human blood was collected by venipuncture from were 6-keto prostaglandin Flc, and prostaglandin E2. volunteers (who had not taken aspirin for at least 14 INHIBITION OF TXA2 BIOSYNTHESIS BY BURIMAMIDE 159 a b c d e tt t + + t t SSV IPM SSV IPM SSV IPM SSV IPM SA SA Bur SA Cim SA Met (1002g/mm)m100g/m) (2mm00Cg/ml) 2min OOC 2min OOC 2min OOC Figure I Effect of histamine H2-receptor antagonists on the generation of thromboxane A2-like activity. Bioassay of rabbit aorta contracting

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