R 68 070: Thromboxane A2 Synthetase Inhibition and Thromboxane A2/Prostaglandin Endoperoxide Receptor Blockade Combined in One Molecule - I. Biochemical Profile In Vitro

F De Clerck; J Beetens; D de Chaffoy de Courcelles; E Freyne; P A J Janssen

doi:10.1055/s-0038-1646523

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1989; 61(01): 035-042
DOI: 10.1055/s-0038-1646523

Original Article

Schattauer GmbH Stuttgart

R 68 070: Thromboxane A₂ Synthetase Inhibition and Thromboxane A₂/Prostaglandin Endoperoxide Receptor Blockade Combined in One Molecule - I. Biochemical Profile In Vitro

F De Clerck

The Department of Haematology, Janssen Research Foundation, Beerse, Belgium

,

J Beetens

The Department of Haematology, Janssen Research Foundation, Beerse, Belgium

,

D de Chaffoy de Courcelles

¹The Department of Biochemistry, Janssen Research Foundation, Beerse, Belgium

,

E Freyne

²The Department of Chemistry, Janssen Research Foundation, Beerse, Belgium

,

P A J Janssen

The Department of Haematology, Janssen Research Foundation, Beerse, Belgium

› Author Affiliations

Abstract

PDF Download

Summary

R 68 070 or (E)-5-[[[(3-pyridinyl)[3-(trifluoromethyl)phenyl]- methylen]amino]oxy] pentanoic acid (Janssen Research Foundation, Belgium) combines specific thromboxane A₂ (TXA₂) synthetase inhibition with TXA₂/prostaglandin endoperoxide receptor blockade in one molecule.

In vitro, the compound specifically inhibits the production of TXB₂ from [¹⁴C] arachidonic acid by washed human platelets (IC₅₀ = 8.2 × 10^-9 M) and by platelet microsomes (IC₅₀ = 3.6 × 10^-9 M), of MDA (IC₅₀ = 1.91 × 10^-8 M) and of TXB₂ (IC₅₀ = 1.47 × 10^-8 M) by thrombin-coagulated human platelet-rich plasma (P.R.P.) and whole blood respectively and increases the levels of PGD₂, PGE₂, PGF_2α and 6-keto-PGF_1α. The activity of cyclo-oxygenase-, prostacyclin synthetase-, 5-, 12- and 15-lipoxygenase-enzymes are not affected. Additionally, R 68 070 inhibits human platelet aggregation in P.R.P. induced by U 46619 3 × 10^-7 M to 2 × 10^-6 M (IC₅₀ = 2.08 × 10^-6 M to 2.66 × 10^-5 M), collagen 0.5 to 2 μg/ml (IC₅₀ = 2.85 × 10^-6 M to 4.81 × 10^-5 M), arachidonic acid 7.5 × 10^-4 M to 2 × 10^- M (IC₅₀ = 2.1 × 10^-8 M to 3.3 × 10^-8 M) and the U 46619 (1 × 10^-7 M)-induced accumulation of [32P] phosphatidic acid (IC₅₀ = 5.24 × 10^-7 M) in washed human platelets. Collagen (0.75 μg/ml)-induced ATP release (IC₅₀ = 4.1 × 10^-6 M), ADP (1 to 2.5 × 10^-6 M)-induced second wave aggregation (IC₅₀ = 3.19 × 10^-6 M) in P.R.P. as well as the collagen (1 μg/ml)-induced adhesion/aggregation reaction in human whole blood (IC₅₀ = 1.02 × 10^-5 M) are reduced as well by the compoun.

Primary platelet reactions induced by serotonin, ADP, PAF, or A 23187, platelet adenylate cyclase- and cAMP phosphodiesterase-activity, and platelet inhibitory activities of PGD₂, PGI₂, PGE₂, PGE₁ are not modified by R 68 070.

This biochemical profile is compatible with a dual mechanism of action of R 68 070, namely TXA₂ synthetase inhibition at low concentrations, plus additionally TXA₂/prostaglandin endoperoxide receptor blockade at higher concentrations

Key words

R 68 070 - TXA₂-synthetase - Receptor blockade - Man - Experimental models

PDF (447 kb)

References

References
1 Needleman P, Moncada S, Bunting S, Vane JR, Hamberg M, Samuelsson B. Identification of an enzyme in platelet microsomes which generates thromboxane A₂from prostaglandin endoperoxides. Nature. 1976; 261: 558-560
2 Samuelsson B, Goldyne H, Granström E, Hamberg M, Hammarström S, Malmsten C. Prostaglandins and thromboxanes. Annu Rev Biochem 1978; 47: 997-1029
3 FitzGerald GA, Reilly IA G, Pedersen AK. The biochemical pharmacology of thromboxane synthetase inhibition in man. Circulation 1985; 72: 1194-1201
4 Vermylen J, Carreras LO, Van Schaeren J, De Freyn G, Machin SJ, Verstraete M. Thromboxane synthetase inhibition as anti-thrombotic strategy. Lancet 1981; 1: 1073-1075
5 De Freyn G, Deckmyn H, Vermylen J. A thromboxane synthetase inhibitor reorients endoperoxide metabolism in whole blood towards prostacyclin and prostaglandin E₂ . Thromb Res 1982; 26: 389-400
6 Gresele P, Deckmyn H, Huybrechts E, Vermylen J. Serum albumin enhances the impairment of platelet aggregation with thromboxane synthetase inhibition by increasing the formation of prostaglandin D₂ . Biochem Pharmacol 1984; 33: 2083-2088
7 Bertelé V, Cerletti C, Schieppati A, Diminnio G, de Gaetano G. Inhibition of thromboxane synthetase does not necessarily prevent platelet aggregation. Lancet 1981; 1: 1057-1058
8 Hornby EJ, Skidmore IF. Evidence that prostaglandin endoperoxides can induce platelet aggregation in the absence of thromboxane A₂production. Biochem Pharmacol 1982; 31: 1153-1160
9 Bertelé V, Tomasiak M, Falanga A, Cerletti C, de Gaetano G. Aspirin inhibits platelet aggregation but not because it prevents thromboxane synthesis. Lancet 1982; 2: 775
10 Rybicki JP, Le Breton GC. Prostaglandin H₂directly lowers human platelet cAMP levels. Thromb Res 1983; 30: 407-417
11 Gresele P, Amout J, Deckmyn H, Vermylen J. Endogenous antiaggregatory prostaglandins can contribute to inhibition of hemostasis: a pharmacological study in vivo in humans. Adv Prostaglandin Thromboxane Leukotriene Res 1987; 17: 248-253
12 Gresele P, Van Houtte E, Arnout J, Deckmyn H, Vermylen J. Thromboxane synthetase inhibition combined with thromboxane receptor blockade: a step forward in antithrombotic strategy. Thromb Haemostas 1984; 52: 364
13 Bertelé V, de Gaetano G. Potentiation by dazoxiben, a thromboxane synthetase inhibitor, of platelet aggregation inhibitory activity of a thromboxane receptor antagonist and of prostacyclin. Eur J Pharmacol 1982; 85: 331-333
14 Rajtar G, Cerletti C, Castagnoli MN, Bertelé V, de Gaetano G. Prostaglandins and human platelet aggregation. Implications for the anti-aggregating activity of thromboxane-synthetase inhibitors Biochem Pharmacol 1985; 34: 307-310
15 FitzGerald DJ, Fragetta J, Fenelon LC, FitzGerald GA. Thromboxane synthetase inhibition and thromboxane/endoperoxide receptor antagonism in a chronic canine model of coronary thrombosis. Adv Prostaglandin Thromboxane Leukotriene Res 1987; 17: 496-500
16 Beetens JR, Van Hoydonck AE, Herman AG. Stimulation of prostacyclin production by vitamin C in ram seminal vesicle microsomes: possible mode of action. Arch Int Pharmacodyn Ther 1985; 277: 56-65
17 Beetens JR, Loots W, Somers Y, Coene MC, De Clerck F. Ketoconazole inhibits the biosynthesis of leukotrienes in vitro and in vivo. Biochem Pharmacol 1986; 35: 883-891
18 Takeguchi C, Kohno E, Sih CJ. Mechanism of prostaglandin biosynthesis I. Characterization and assay of bovine prostaglandin synthetase. Biochemistry 1971; 10: 2372-2376
19 De Clerck F, Van Nueten JM. Platelet-mediated vascular contractions. Inhibition of flunarizine, a calcium-entry blocker Biochem Pharmacol 1983; 32: 765-771
20 Beetens JR, Van Den Bossche R, Herman AG. Production of 6-oxo-PGF_1αby aorta rings in the presence of ascorbic acid. Arch Int Pharmacodyn Ther 1982; 256: 151-152
21 McMillan RM, MacIntyre DE, Gordon JC. Simple sensitive fluorimetric assay for malondialdehyde production by blood platelets. Thromb Res 1977; 11: 425-428
22 Patrono C. et al Low dose aspirin and inhibition of thromboxane B₂production in healthy subjects. Thromb Res 1980; 17: 317-327
23 Salomon Y. Adenylate cyclase assay. Adv Cyclic Nucleotide Res 1979; 10: 35-55
24 Bult H, Beetens J, Herman A. Blood levels of 6-oxo-prostaglandin F_lαduring endotoxin-induced hypotension in rabbits. Eur J Pharmacol 1980; 63: 47-56
25 De Chaffoy de CourcellesD, Leysen JE, De Clerck F, Van Belle H, Janssen PA J. Evidence that phospholipid turnover is the signal transducing system coupled to serotonin-S₂receptor sites. J Biol Chem 1985; 260: 7603-7608
26 Mills DC B, Smith JB. The influence on platelet aggregation of drugs that affect the accumulation of adenosine 3’:5’-cyclic monophosphate in platelets. Biochem J 1971; 121: 185-196
27 De Clerck F, Xhonneux B, Tollenaere JP, Janssen PA J. Dependence of the antagonism at human platelet 5-HT₂receptors by ketanserin on the reaction pH. Thromb Res 1985; 40: 581-596
28 De Clerck F, Van Gorp L, Beetens J, Reneman RS. Platelet- mediated vascular permeability in the rat: a predominant role for 5-hydroxytryptamine. Thromb Res 1985; 38: 321-339
29 Feinman RD, Lubowsky J, Charo I, Zabinski M. The lumiaggregometer: a new instrument for simultaneous measurement of secretion and aggregation by platelets. J Lab Clin Med 1977; 90: 125-129
30 De Clerck F, Somers Y, Van Gorp L. Platelet-vessel wall interactions in haemostasis: implication of 5-hydroxytryptamine. Agents Actions 1984; 15: 627-635
31 Gresele P, Arnout J, Janssens W, Deckmyn H, Lemmens J, Vermylen J. BM 13177, a selective blocker of platelet and vessel wall thromboxane receptors, is active in man. Lancet 1984; : 991-994
32 Brittain RT, Boutal L, Carter MC, Coleman RA, Collington EW, Geisow HP, Hallett P, Hornby EJ, Humphrey PP A, Jack D, Kennedy I, Lumley P, McCabe PJ, Skidmore IF, Thomas M, Wallis CJ. AH 23848 a thromboxane receptor-blocking drug that can clarify the pathophysiologic role of thromboxane A₂ . Circulation 1985; 72: 1208-1218
33 Smith JB, Jubil W. OKY-1581 a selective inhibitor of thromboxane synthesis in vivo and in vitro. Prostaglandins 1981; 22: 353-363
34 Ruppert D, Weithman KU. HL 725, an extremely potent inhibitor of platelet phosphodiesterase and induced platelet aggregation in vitro. Life Sci 1982; 31: 2037-2043
35 Coleman RA, Humphrey PP A, Kennedy I, Levy GP, Lumley P. U 46619, a selective thromboxane A₂-like agonist. Br J Pharmacol 1980; 68: 1278
36 Coleman RA, Humphrey PP A, Kennedy I, Levy GP, Lumley P. Comparison of the actions of U 46619, a prostaglandin H₂-analogue, with those of prostaglandin H₂and thromboxane A₂on isolated smooth muscle preparations. Br J Pharmacol 1981; 73: 773-778
37 Draper NR, Smith M. Applied Regression Analysis. Wiley & Sons; New York: 1981
38 Pedersen EK, Watson ML, FitzGerald GA. Inhibition of thromboxane biosynthesis in serum; limitations of the measurements of immunoreactive 6-keto-PGF_1α . Thromb Res 1983; 33: 99-103
39 Carey F, Haworth D. Thromboxane synthetase inhibition: implications for prostaglandin endoperoxide metabolism. Prostaglandins 1986; 31: 47-59
40 Mehta J, Mehta P, Lawson DL, Ostrowski N, Brignon L. Influence of selective thromboxane synthetase blocker CGS-13080 on thromboxane and prostacyclin biosynthesis in whole blood: evidence for synthesis of prostacyclin by leukocytes from platelet-derived endoperoxides. J Lab Clin Med 1985; 106: 246-252
41 Uotila P, Matintalo M. Inhibition of thromboxane synthetase by OKY-1581 stimulates the formation of PGE₂, PGF_2α, PGD₂ and 6-keto-PGF_1αin human platelets. Prostaglandins Leukotrienes Med 1984; 14: 41-46
42 Ambler J, Butler KD, Ku EC, Maguire ED, Smith JR, Wallis RB. CGS 12970: a novel, long acting thromboxane synthetase inhibitor. Br J Pharmacol 1985; 86: 497-504
43 Terashita Z, Imura Y, Tanabe M, Kawazoe K, Nishikawa K, Kato K, Terao S. CV-4151 - a potent, selective thromboxane A₂synthetase inhibitor. Thromb Res 1986; 41: 223-237
44 Hiraku S, Taniguchi K, Wakitani K, Omawari N, Kira H, Miyamoto T, Okegawa T, Kawasaki A, Ujiie A. Pharmacological studies on the TXA₂synthetase inhibitor (E)-3-[p-(1H-imidazol-1-ylmethyl)phenyl]- 2-propenoic acid (OKY-046). Jpn J Pharmacol 1986; 41: 393-401
45 Beetens JR, Claeys M, Herman AG. Antioxidants increase the formation of 6-oxo-PGF_1α by ram seminal vesicle microsomes. Biochem Pharmacol 1981; 30: 2811-2851
46 Flower RJ, Cheung HS, Cushman DW. Quantitative determination of prostaglandins and malondialdehyde formed by the arachidonate oxygenase (prostaglandin synthetase) system of bovine seminal vesicle. Prostaglandins 1973; 4: 325-341
47 De Clerck F, Van Nueten JM. Platelet-mediated vascular contractions: inhibition of the serotonergic component by ketanserin. Thromb Res 1982; 27: 713-727
48 Patscheke H, Stegmeier K. BM 13.177 is a selective antagonist of prostaglandin H₂and thromboxane A₂in human platelets. IRCS Med Sci 1984; 12: 9-10
49 Ogletree ML, Harris DN, Greenberg R, Haslanger MF, Nakane M. Pharmacological actions of SQ 29548, a novel selective thromboxane antagonist. J Pharmacol Exp Ther 1985; 234: 435-441
50 Armstrong RA, Jones RL, Wilson NH. Ligand binding to thromboxane receptors on human platelets: correlation with biological activity. Br J Pharmacol 1983; 79: 953-964
51 De Clerck F, David JL. Pharmacological Control of platelet and red blood cell function in the microcirculation. J Cardiovasc Pharmacol 1981; 3: 1388-1412
52 Best LC, Holland TK, Jones PB B, Russell RG G. The interrelationship between thromboxane biosynthesis, aggregation and 5-hydroxytryptamine secretion in human platelets in vitro. Thromb Haemostas 1980; 43: 38-40
53 Carter AJ, Heptinstall S. Platelet aggregation in whole blood: the role of thromboxane A₂and adenosine diphosphate. Thromb Haemostas 1985; 54: 612-616
54 Heptinstall S, Bevan J, Cockbill SR, Hanley SP, Parry MJ. Effects of a selective inhibitor of thromboxane synthetase on human blood platelet behaviour. Thromb Res 1980; 20: 219-230
55 Heptinstall S, Bientz N, Cockbill SR, Hanley SP, Peacock I. Different effects of thromboxane synthetase inhibitors on platelets from different individuals. Lancet 1982; 2: 1156
56 Sills T, Heptinstall S. Effects of a thromboxane synthetase inhibitor and a cAMP phosphodiesterase inhibitor, singly and in combination, on platelet behaviour. Thromb Haemostas 1986; 55: 305-308
57 Parise LV, Venton DL, Lebreton GC. Arachidonic acid-induced platelet aggregation is mediated by a thromboxane A₂/prostaglandin H₂receptor interaction. J Pharmacol Exp Ther 1984; 228: 240-244