0022-3565/02/3012-618–624$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 301, No. 2 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 4765/979886 JPET 301:618–624, 2002 Printed in U.S.A. A New Family of Thromboxane Receptor Antagonists with Secondary Thromboxane Synthase Inhibition CECIL R. PACE-ASCIAK,1 DENIS REYNAUD, PETER DEMIN, RUKSHANA ASLAM, and ANDREA SUN Programme in Integrative Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada Received November 16, 2001; accepted January 18, 2002 This article is available online at http://jpet.aspetjournals.org ABSTRACT ϭ ϫ We report herein a novel class of thromboxane receptor (TP formation and aggregation evoked by collagen with an IC50 8 receptor) antagonists modeled on unstable natural lipids that we 10Ϫ7 M. Other PBT (hepoxilin cyclopropane) analogs so far tested identified several years ago, the hepoxilins. These antagonists were 5- to 10-fold less active, and the native hepoxilins were have been rendered chemically and biologically more stable than about 500-fold less active. Neither PBT-3 nor the other analogs the natural compounds through structural modification by chem- inhibited 12-lipoxygenase, phospholipase A2, or cyclooxygenase ical synthesis. We demonstrate that the analogs inhibit the aggre- 1 or 2, and weakly stimulated adenyl cyclase (threshold stimula- gation of human platelets in vitro evoked by the thromboxane tion at 10Ϫ7 M and little selectivity for each of the PBT com- receptor agonists, I-BOP ([1S-[1␣,2␣(Z),3␤(1E,3S*),4␣]]-7-[3-[3- pounds). TP antagonism by PBT-3 was further demonstrated in 125 hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabi-cyclo[2.2.1]hept- receptor binding studies through use of I-BOP, where the IC50 2-yl]5-heptenoic acid) and U46619 (9,11-dideoxy-9␣,11␣- for PBT-3 was 8 ϫ 10Ϫ9 M, approximately 16-fold less than for methanoepoxy-prosta-5Z,13E-dien-1-oic acid). The most potent I-BOP itself. These findings identify a new mode of action of of the analogs described, PBT-3 [10(S)-hydroxy-11,12-cyclopro- PBT-3 and other related analogs as primarily TP antagonists. pyl-eicosa-5Z,8Z,14Z-trienoic acid methyl ester], has an IC50 ver- These studies identify a new family of compounds useful in further sus aggregation by I-BOP ϭ 0.6 ϫ 10Ϫ7 M and versus U46619 ϭ development as novel therapeutics for thromboxane-mediated 7 ϫ 10Ϫ7 M, representing one of the most potent anti-aggregating diseases. substances so far described. PBT-3 also inhibits thromboxane Platelet aggregation is an important component of the diate, prostaglandin endoperoxide, common to all the hemostatic mechanism that prevents undesired bleeding, in prostaglandins and thromboxane. On the other hand extreme which a platelet plug forms at the site of injury to the blood cases of aggregation can lead to serious outcome as in septic vessel, leading to cessation of bleeding. Several aggregation shock and thrombosis (Parellada and Planas, 1977; Randall pathways have been described (Packham, 1993), one of which and Wilding, 1982; Fiedler et al., 1989; Silver et al., 1995; is the thromboxane pathway (Hamberg et al., 1974, 1975; Wolkow et al., 1997; Zaitsu et al., 1999). By comparison, Diczfalusy and Hammarstrom, 1979; Hammarstrom and Dic- NSAIDs such as aspirin reduce platelet aggregation through zfalusy, 1980). The active mediator in this pathway is throm- inhibition at the early stage of cyclooxygenase within the boxane A2, a powerful unstable pro-aggregating substance platelet but, also, in other cells/tissues, such as the blood and a vasoconstrictor of blood vessels. It is formed in plate- vessel wall, prevent the formation of all prostaglandins, some lets from arachidonic acid, a fatty acid present in mem- of which are anti-aggregatory and therefore beneficial, e.g., branes, by the enzyme, cyclooxygenase, through an interme- prostacyclin and prostaglandin E2 (Vane, 1978; Willis, 1978; Bunting et al., 1983; Harada et al., 1998). Therefore, proper This study was supported in part by a grant (MT-4181) to C.R.P.-A. from the Canadian Institutes of Health Research and from The Hospital for Sick Chil- management of thromboxane-mediated disease is desirable dren. at the level of thromboxane (synthesis and/or receptor ac- 1Associated with the Department of Pharmacology, University of Toronto, Toronto, ON M5S 1A8, Canada. tion), as this leaves the beneficial prostaglandins in place ABBREVIATIONS: NSAIDs, nonsteroidal anti-inflammatory drugs; TP receptor, thromboxane A2 receptor; ADAM, 9-anthryldiazomethane; TSI, throm- boxane synthase inhibitor; TRA, thromboxane receptor antagonist; TxB2, thromboxane B2; AA, arachidonic acid; HPLC, high-performance liquid chromatography; 12-HETE, 12(S)-hydroxyeicosa-5Z,8Z,10E,14Z-trienoic acid; HHT, 12-hydroxy-heptadeca-5Z,8E,10E-trienoic acid; PBT, hepoxilin cyclopropane analog; PBT-1, 8(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,9E,14Z-trienoic acid methyl ester; PBT-2, 8(R)-hydroxy-11,12-cyclopropyl- eicosa-5Z,9E,14Z-trienoic acid methyl ester; PBT-3, 10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid methyl ester; PBT-4, 10(R)- hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid methyl ester; 12-LOX, 12(S)-lipoxygenase; COX, cyclooxygenase; Plase A2, phospholipase ␣ ␣ ␤ ␣ A2; PG, prostaglandin; I-BOP, [1S-[1 ,2 (Z),3 (1E,S*),4 ]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo-[2.2.1]hept-2-yl]-5-heptenoic acid; U46619, 9,11-dideoxy-9␣,11␣-methanoepoxy-prosta-5Z,13E-dien-1-oic acid. 618 Novel Family of Thromboxane Receptor Antagonists 619 (Bunting et al., 1983). Recently, this approach has identified Binding of 125I-BOP to Platelets. Washed platelets were pre- a new class of compounds, oxazolecarboxamide-substituted pared as described above, except that the platelet suspension was alkenoic acids with dual TSI/TRA activities (Takeuchi et al., made up at a concentration of 10 ϫ 106 cells/0.5 ml (Dorn, 1991) in a 1998). clear medium (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, During the course of our studies on the metabolic conver- 10 mM sodium-free HEPES, and 10 mM glucose, pH 7.3). The bind- ing assay involved the addition of radioligand (30,000 cpm 125I-BOP) sion of arachidonic acid, we discovered a novel pathway with to all tubes in triplicates, containing either various concentrations of products, hepoxilins, that showed biological actions on a va- unlabeled I-BOP (10Ϫ9-10Ϫ7 M) or PBT-3 (10Ϫ9-10Ϫ7 M) or U46619 riety of systems (Pace-Asciak et al., 1983; Pace-Asciak and (10Ϫ9-10Ϫ7 M) in 1 ␮l ethanol. Additional tubes containing excess Martin, 1984; Pace-Asciak, 1994). Since these compounds unlabeled I-BOP were included to assess the extent of nonspecific were unstable biologically, we prepared by total chemical binding. Binding was allowed to take place during 30 min at 37°C; synthesis a family of analogs, PBTs, that were both chemi- free radioligand was removed by rapid vacuum filtration through cally and biologically suitable for in vivo studies (Demin and Whatman (Maidstone, UK) GF/B glass fiber filters prewashed with Pace-Asciak, 1993). Indeed, we found that different com- clear medium. The tubes and the filters were rapidly washed with pounds within this family acted on different in vivo systems, ice-cold clear medium (three times with 3 ml). The radioactivity on e.g., insulin secretion (Pace-Asciak et al., 1999), decrease in the filters containing the ligand-receptor complexes was counted in plasma glucose (unpublished observations), or inhibition of an LKB (Uppsala, Sweden) Compugamma CS counter. Measurement of Adenyl Cyclase Activity. Human washed lung fibrosis (Pace-Asciak et al., 2000). During more compre- platelets (350 ϫ 106 cells) in 1 ml of assay buffer were preincubated hensive screening for in vitro biological actions, we discov- during 10 min at 37°C. Dimethyl sulfoxide (1 ␮l) alone (control) or ered that one of these analogs, PBT-3, potently inhibited containing various concentrations of PBT-1, -2, -3, and -4 (2.8 ϫ collagen-evoked aggregation of human platelets through se- 10Ϫ7-10Ϫ5 M final concentration) was added, and the cells were lective blockade of the thromboxane synthetic pathway (Rey- incubated for a further 5 min. The reaction was stopped by the naud et al., 2001). The present results demonstrate a far addition of 12% trichloroacetic acid, and the samples were sonicated more important and powerful action of this compound as an (four times for 3 sec). The samples were left on ice for 60 min to antagonist of the TP receptor revealing a more likely mech- extract cAMP. After centrifugation at 2500g for 15 min, the super- anism for its inhibitory actions on platelet aggregation. natants were transferred and extracted five times with 5 ml of water-saturated diethyl ether to remove the trichloroacetic acid. The aqueous phase was transferred and lyophilized. cAMP was measured 125 Experimental Procedures using specific double antibody radioimmunoassay kits with I- labeled cAMP according to the instructions by the manufacturer Materials. The hepoxilin analogs, PBT-1, -2, -3, and -4, were (Amersham Biosciences, Piscataway, NJ). Results are expressed in prepared as previously described (Demin and Pace-Asciak, 1993). picomoles/350 million cells. Experiments were performed in tripli- Collagen (Chrono-Par) was purchased from Chronolog Corp. (Hav- cate for each point and repeated twice. ertown, PA). ADAM reagent (9-anthryldiazomethane) was from Re- Measurement of COX-1 and COX-2 Activity. COX-1 and search Organics Inc. (Cleveland, OH). U46619, I-BOP, and 125I-BOP COX-2 enzyme preparations were purchased from Cayman Chemical were from Cayman Chemical (Ann Arbor, MI). (Ann Arbor, MI). Preliminary studies established that 40 U of COX-1 Isolation of Human Platelets.