Stable Derivatives of Thromboxane A2 with Differential Effects on Platelets

Home , Prostaglandin H2, Thromboxane, Thromboxane A2

Proc. Natl. Acad. Sci. USA Vol. 86, pp. 5600-5604, July 1989 Medical Sciences Difluorothromboxane A2 and stereoisomers: Stable derivatives of thromboxane A2 with differential effects on platelets and blood vessels (receptors/contraction/aggregation/prostaglandins/10,10-difluorothromboxane A2 and analogues) THOMAS A. MORINELLI*, ANSELM K. OKWU*, DALE E. MAIS*, PERRY V. HALUSHKA*t, VARGHESE JOHNt, CHIEN-KUANG CHENt, AND JOSEF FRIEDt Departments of *Cell and Molecular Pharmacology and Experimental Therapeutics and of tMedicine, Medical University of South Carolina, Charleston, SC 29425; and tDepartment of Chemistry, The University of Chicago, Chicago, IL 60637 Contributed by JosefFried, April 13, 1989

ABSTRACT The present study reports on the selective this analogue is an antagonist (13). Other studies, also using effects on human platelets and canine saphenous veins of four different species, have shown differences in receptors in the stable difluorinated analogues and thromboxane A2 (TXA2), in two cell types (14, 15). Because different species were used which the characteristic 2,6-dioxa[3.1.1]bicycloheptane struc- as the source of platelets and blood vessels, these differences ture of TXA2 has been retained. The four compounds differ in may be species-selective rather than represent actual differ- their stereochemistry of the 5,6 double bond and/or the ences in the receptors. 15-hydroxyl group. Only 10,10-difluoro-TXA2 (compound I) Recent studies, however, have provided stronger evidence with the natural stereochemistry of TXA2 was an agonist in for different TXA2/PGH2 receptors in platelets and in blood both platelets and canine saphenous veins (EC50 = 36 ± 3.6 nM vessels (12, 16, 17). The platelet and vascular receptors also and 3.7 ± 0.8 nM, respectively). (15R)-10,10-Difluoro-TXA2 differ in their steric requirements for the orientation of the (compound II), (5E)-10,10-difluoro-TXA2 (compound IM), and 15-hydroxyl group (17). In saphenous veins orientation ofthe (5E,15R)-10,10-difluoro-TXA2 (compound IV) were antago- 15-hydroxyl group influenced the potency of the compounds nists of platelet aggregation stimulated by compound I (Kd = as antagonists in the vessels, whereas in platelets the orien- 98 ± 46 nM, 140 ± 42 nM, and 1450 ± 350 nM, respectively). tation had no influence. The platelet receptor has been However, compounds 11,1, and IV stimulated contraction of designated the (TXA2/PGH2)a receptor, a for aggregation, canine saphenous veins (EC50 = 36 ± 4.4 nM, 31 ± 6.8 nM, and and the vascular receptor (TXA2/PGH2), r for tone (12, 16, 321 ± 50 nM, respectively). All four compounds could displace 17). the TXA2/prostaglandin H2 antagonist 9,11-dimethylmeth- Most compounds used in these studies possessed the ano-11,12-methano-16-(3-'251-4-hydroxyphenyl)-13,14-dihy- [2.2.1]bicycloheptane skeleton of PGH2; less attention had dro-13-aza-15a13-w-tetranor-TXA2 from its platelet receptor been paid to analogues of TXA2, which possess the (Kd values = 100 ± 30 nM, compound I; 280 ± 60 nM, [3.1.1]bicycloheptane system. In both structural types in- compound II; 230 ± 70 nM, compound HI; and 1410 ± 1020 creased stability was imparted to the molecules by substitu- nM, compound IV). These results support the existence of two tion of one or both of the oxygen atoms by carbon, sulfur, or subtypes of TXA2/prostaglandin H2 receptors and emphasize nitrogen. However, these systems still represent significant the importance of the stereochemical requirements of these structural changes compared with TXA2 or PGH2. Indeed, TXA2 analogues for interaction with these receptors. These some of these analogues do not mimic the actions ofTXA2 in stable fluorinated TXA2 analogues should prove useful tools for platelets (13) and blood vessels. The current studies report on the further characterization of these and other TXA2/pros- the selective effects on human platelets and canine saphenous taglandin H2 receptors. veins of a difluoro analogue of TXA2 and three of its stereoisomers in which the 2,6-dioxabicyclo structure of TXA2 has Thromboxane A2 (TXA2), and prostaglandin H2 (PGH2) been retained except for the substitution of two hydrogen cause aggregation of platelets and constriction of vascular atoms by fluorine (18). smooth muscle (1-4) by means of cell-surface receptors. To study the specific events accompanying the interaction of these autacoids with their receptors, it is necessary to use MATERIALS AND METHODS stable analogues because both TXA2 and PGH2 are unstable, The syntheses of the four difluorinated TXA2 derivatives, their half-lives under physiological conditions being 30 sec compounds I-IV (Fig. 1), used in this study are described and 5 min, respectively. For these reasons many compounds elsewhere (18). 9,11-Dimethy-lmethano-11,12-methano- have been synthesized that are related in structure to both 16-(3-iodo-4-hydroxyphenyl)-13 ,14-dihydro-13-aza-15a,B- PGH2 and TXA2 and that act as either agonists or antagonists w-tetranor-TXA2 (I-PTA-OH) and [1251]PTA-OH were syn- for TXA2/PGH2 receptors on platelets and vascular smooth thesized as described (11). U-46619 was a gift from Upjohn. muscle (5-12). Platelet Aggregation. Blood was drawn via venipuncture Studies ofthese analogues have provided evidence that the from normal human volunteers, who had not taken any TXA2/PGH2 receptors on vascular smooth muscle differ from those of platelets. For example, a carbocyclic analogue Abbreviations: PGH2, prostaglandin H2; TXA2, thromboxane A2; of TXA2 [2,3(Z),3a-(lE,3R)-3-(3-hydroxy(1-octenyl)-bicyclo- CTA2, 2,B(Z),3a-(lE,3R)-3-(3-hydroxy(1-octenyl)-bicyclo[3.1.1I- [3.1.1]hept-2-yl-5-heptenoic acid; CTA2] has been shown to hept-2-yl-5-heptenoic acid; I-PTA-OH, 9,11-dimethylmethano- constrict cat coronary arteries, whereas in human platelets 11,12-methano-16-(3-iodo-4-hydroxyphenyl)-13,14-dihydro-13-aza- 15a13-co-tetranor-TXA2; U-46619, (15S)-hydroxy-lla,9a-(epoxy- methano)prosta-(5Z,13E)-dienoic acid; SQ 29548, -{(lS)-[la,2.8- The publication costs of this article were defrayed in part by page charge (5Z),3p,4a]}-7-[3-({2-[(phenylamino)carbonyl]hydrazino}methyl)- payment. This article must therefore be hereby marked "advertisement" 7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid; STA2, 9,11-epithio- in accordance with 18 U.S.C. §1734 solely to indicate this fact. 11,12-methano-TXA2- 5600 Downloaded by guest on September 25, 2021 Medical Sciences: Morinelli et al. Proc. Natl. Acad. Sci. USA 86 (1989) 5601

F F * 'C."b F . | F F.0e ~~~~~~~COOH 00 0

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F ...' s COCH F ,.0 | 09

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. I COOH

0 0.

OH OH

U46619 FIG. 1. Structure of difluoro-TXA2 and three of its stereoisomers used in this study, along with the structures ofTXA2 and U-46619, a stable PGH2 analogue. Compound I, 10,10-difluoro-TXA2; compound II, (15R)-10,10-difluoro-TXA2; compound III, (5E)-10,10-difluoro-TXA2; and compound IV, (5E,15R)-10,10-difluoro-TXA2. medication for at least 10 days, into syringes containing 10 min. Incubation medium was 50 mM Tris/100 mM NaCl AuM indomethacin/5 mM EDTA (final concentrations). In- buffer containing -0.1 nM (-5 x 104 cpm) [125I]PTA-OH per formed consent was obtained from all subjects; this study was tube. The reaction was terminated by adding 4 ml of the approved by the Medical University of South Carolina In- ice-cold Tris/NaCl buffer at pH 7.4, followed by rapid stitutional Review Board for Human Research. Washed filtration through Whatman GF/C glass fiber filters. Filters platelets, prepared as described (12), were suspended in a 50 were washed three more times with 4 ml ofthe ice-cold buffer mM Tris/100 mM NaCl, pH 7.4, buffer containing 5 mM (11). The IC50 values obtained from log-logit transformation glucose. Before aggregation 250 /M CaCl was added to the of the competition binding data were defined as the concen- platelet suspension. Platelets were aggregated in a Chronolog trations of ligand required to produce a 50% displacement of model 300 aggregometer by using published methods (17). specifically bound [1251]PTA-OH from its binding site. The The washed platelets (450 ILI, 5.0 x 188 platelets per ml) were IC50 value was used to determine the Kd by using the added to individual glass cuvettes and preincubated for 1 min Cheng-Prusoff equation (20). Nonspecific binding was de- at 370C. Then difluoro-TXA2 (compound I) or the stable fined as that amount of radioactivity remaining in the pres- TXA2/PGH2 mimetic U-46619 (6) was added (final concen- ence of 750 nM I-PTA-OH (11). trations varied between 5 nM and 5 PM), and the aggregation Saphenous Veins. Medial saphenous veins were dissected response was recorded. Concentration-response curves from pentobarbital-anesthetized mongrel dogs (30 mg/kg) were constructed for U-46619 and difluoro-TXA2 (compound and placed into ice-cold Krebs-Henseleit bicarbonate buffer I), and the EC50 values were calculated directly from log-logit (118 mM NaCI/5.4 mM KCI/1.0 mM MgSO4/2.5 mM CaCl2/ transformations of the data. The EC50 value was defined as 1.1 mM NaH2PO4/25 mM NaHCO3/10 mM D-glucose/10 the concentration required to produce 50o of the maximum ,uM indomethacin). After removal of surrounding fascia the aggregation occurring 1 min after addition of the aggregating vein was cut into rings (5-6 mm long) and attached to an agent. isometric force-displacement transducer (Grass). The tissues In studies using the difluoro-TXA2 isomers, compounds II, were maintained at 2 g of resting tension and allowed to III, and IV, to inhibit difluoro-TXA2-stimulated platelet stabilize for =1 hr at 37°C in Krebs-Henseleit buffer (95% aggregation the following method was used. Isolated platelets 02/5% CO2, pH 7.4). U-46619 and difluoro-TXA2 and its were prepared as above and placed in aggregation cuvettes. isomers were diluted in buffer and added in 10- to 20-,ul Various concentrations of compounds II, III, and IV were volumes to the 10-ml tissue bath yielding the various con- added, incubated for 1 min at 370C, followed by addition of centrations. EC50 values for U-46619 and difluoro-TXA2 and various concentrations of difluoro-TXA2 (compound I). its isomers were calculated from the cumulative concentra- Schild analysis was performed on the data derived from these tion-response curves. The maximum concentrations induced concentration-response curves to determine the Kd values by both agonists were similar and were checked by crossover for the three antagonists (19). of agonists in the same vessel. Binding of ['25I]PTA-OH to Washed Human Platelets. In- Statistics. Values are presented as the mean ± SEM cubations (200 IlI) containing 5 x 107 platelets were per- Student's t test was performed to determine whether signif- formed in silanized (12 x 75 mm) glass tubes at 37°C for 30 icant differences between mean values existed. Downloaded by guest on September 25, 2021 5602 Medical Sciences: Morinelli et al. Proc. Natl. Acad. Sci. USA 86 (1989)

I 100 - z I + IV cn cn i I + III LU Un CL) z + z oc s 0- I II 0 a. ti Cl) 40- Lw I + cr OR I-PTA-OH 50 -

vI 1 2 3 TIME (MIN) FIG. 2. Inhibition of difluoro-TXA2 (compound I)-stimulated aggregation by compound II, compound III, compound IV, and -9.5 -8.5 -7.5 -6.5 I-PTA-OH. Platelets were stimulated using 1 AM difluoro-TXA2 (compound I); the extent of aggregation (increase in light transmis- LOG CONCENTRATION sion units) was followed for 2 min. Platelets were incubated for 1 min with 1 gM compound II, compound III, compound IV, or 0.2 tM FIG. 3. Contraction of isolated canine saphenous veins by U- I-PTA-OH before stimulation by difluoro-TXA2. 46619 and the stereoisomers of difluoro-TXA2. Saphenous veins were prepared as described and stimulated by various concentrations RESULTS of agonists. Cumulative concentration-response curves from a rep- Platelet Aggregation Studies. Difluoro-TXA2 (compound I) resentative experiment are shown. *, U-46619; r, compound I; *, compound II; o, compound III; and X, compound IV. and its three stereoisomers were evaluated for their ability to initiate platelet aggregation. Only difluoro-TXA2 (compound the isomers, followed by compounds II, III, and IV (Table 1). I) induced aggregation of isolated human platelets and was a To determine whether the compounds were interacting at the full agonist when compared with U-46619 (EC50 = 36 + 3.6 TXA2/PGH2 receptor, the effects of SQ 29548, a specific nM, n = 11) (Fig. 2). Compound II (1 ,uM) sporadically TXA2/PGH2 receptor antagonist (22), on the contractile produced a small reversible aggregation of isolated platelets. responses were evaluated. Canine saphenous veins were However, compound II consistently inhibited the aggrega- contracted by compound I, and once the maximum contraction responses induced by compound I (Fig. 2, Table 1). tion was reached, SQ 29548 was added in cumulative con- Compound II was also tested for its specificity in inhibiting centrations that relaxed the contracted vessels (Fig. 4). platelet aggregation; compound II did not alter the EC50 value I-PTA-OH also inhibited the contraction stimulated by com- for thrombin stimulation of platelet aggregation (data not pounds I and III (data not shown). shown). Thus, compound II did not nonspecifically inhibit Radioligand Binding Assays. The four difluorinated com- compound I-induced platelet aggregation. Compounds III pounds, along with U-46619, were tested for their ability to and IV did not induce aggregation to concentrations of 4 /LM compete with [1251]PTA-OH in binding to washed human but antagonized difluoro-TXA2 (compound I)-stimulated ag- platelets. Table 1 summarizes the IC50 values determined gregation (Fig. 2). Compound II was the most potent inhibitor from log-logit transformations of competition curves. Diflu- of difluoro-TXA2-induced aggregation, with a Kd value = 98 oro-TXA2 (compound I) was the most potent of the four ± 46 nM (n = 3), followed by compounds III and IV (Kd = compounds followed by compounds II and III, with com- 140 ± 42 nM, n = 3, and 1450 ± 350 nM, n = 3, respectively). pound IV being the least potent (Table 1). Compound I had I-PTA-OH (0.2 ,uM), a TXA2/PGH2 receptor antagonist (11), a pseudo Hill coefficient of -0.84 ± 0.07 (n = 8 not inhibited the aggregation response caused by difluoro-TXA2 significantly different from -1). Compounds II, III, and IV (Fig. 2), indicating that it stimulated aggregation by interact- had pseudo Hill coefficients of -0.77 ± 0.06 (n = 6), -0.62 ing with the TXA2/PGH2 receptor. ± 0.07 (n = 7), and -0.41 ± 0.07 (n = 3), respectively, all of Contraction of Saphenous Veins. Fig. 3 shows a repre- which differed significantly from -1 (P < 0.05). sentative set of concentration-response curves for the four compounds in isolated canine saphenous veins. U-46619, a stable agonist (6), is included for comparison. All four DISCUSSION compounds stimulated contractions in a concentration- This study describes the pharmacology of 10,10-difluoro- dependent manner. Difluoro-TXA2 was the most potent of TXA2 (compound I) and three closely related compounds Table 1. Platelet aggregation, contraction of isolated canine saphenous veins, and competition binding studies with [1251]PTA-OH EC50, nM Kd, nM Saphenous vein Inhibition of platelet Platelet radioligand Agent Platelet aggregation contraction aggregation binding U-46619 64 ± 6* 1.7 ± 0.3 (11) 120 ± 10 (13) 10,10-Difluoro-TXA2 (compound I) 36 ± 3.6 (11) 3.7 ± 0.8t (9) 100 ± 30t (8) Compound II 36 ± 4.4 (5) 98 ± 46 (3) 280 ± 60 (6) Compound III 31 ± 6.8 (8) 140 ± 42 (3) 230 ± 70 (7) Compound IV 321 ± 50 (4) 1450 ± 350t (3) 1410 ± 1020 (3) All values are expressed as mean ± SEM. Number of experiments is indicated in parentheses. *Data obtained from ref. 21. tP < 0.05 compared with compounds II, III, and IV. fP < 0.05 compared with compound II. Downloaded by guest on September 25, 2021 Medical Sciences: Morinelli et al. Proc. Natl. Acad. Sci. USA 86 (1989) 5603

1.0 1.S 3.0 S. l~nM7.5 I difluoro-TXA2, compounds II, III, and IV, to promote contraction of canine saphenous veins, whereas the same compounds were antagonists in platelets. The difference between the platelet and vascular TXA2/PGH2 receptors seen in this study is not due to species differences because canine and human saphenous vein TXA2/PGH2 receptors have been previously shown to appear to be the same (12). The reversal ofactivity is similar to that seen in the studies using CTA2 (13) I 2 and derivatives of 13-azapinane TXA2 (17). In those studies, CTA2 and four 13-azapinane derivatives were antagonists in platelets and agonists in blood vessels. I 10 25 50 100nM SQ29S48 Other studies using stable analogues of TXA2/PGH2 to examine structure-activity relationships in platelets and blood vessels have yielded similar results. 13-Azaprostanoic acid analogues were tested in rat thoracic aorta and human platelets and showed a different rank order potency (15). However, this group ofworkers concluded that the receptors were not different. A more recent study reported the existence ofTXA2/PGH2 receptor subtypes based on the relative affinities and stimulatory potencies for U-46619, prostaglandin D2, prostaglandin E2, and prostaglandin F2a in human platelets and rat aortae.§ FIG. 4. (Upper trace) Concentration-dependent contractions of The present study further supports the existence ofTXA2/ canine saphenous veins by compound I. (Lower trace) SQ 29548 PGH2 receptor subtypes in platelets and vascular smooth concentration-dependent inhibition of difluoro-TXA2 (compound I) muscle and adds additional information about the steric (50 nM)-stimulated contraction of isolated canine saphenous veins. requirements of these receptors for interaction with their differing only in the stereochemistry ofthe 15-hydroxyl group ligands. Our study also provides a class of stable compounds and/or the 5,6-double bond. Of the four compounds, only that most closely resemble TXA2 and that should prove 10,10-difluoro-TXA2, which possesses stereochemistry iden- useful tools for future studies of TXA2/PGH2 receptors. tical with that ofTXA2, was a full agonist in both platelets and saphenous veins. The EC50 value for 10,10-difluoro-TXA2 to aggregate washed human platelets is 4-5 times lower than §Ogletree, M., Allen, G., O'Keefe, E., Liv, K. & Hedberg, A., Sixth International Conference on Prostaglandins, June 1986, Florence, that seen for synthetic TXA2 (163 nM) and comparable to that 350. of PGH2 (45 nM) (23). The Kd value for 10,10-difluoro-TXA2 Italy, p. (100 nM) in the competition assays with [125I]PTA-OH is very similar to that reported for TXA2 (125 nM) (23). Thus, This work was supported, in part, by National Institutes of Health 10,10-difluoro-TXA2 possesses an affinity at least equal to Grants HL36838, HL29566, HL07260, and KD 11499. P.V.H. is a that of TXA2, the natural agonist for the [TXA2/PGH2], Burroughs-Wellcome Scholar in Clinical Pharmacology. (platelet) receptor. This affinity is also comparable in potency to U-46619 in washed human platelets and saphenous veins. 1. Hamberg, M., Svensson, J., Wakabayashi, T. & Samuelsson, 10,10-Difluoro-TXA2 has a cis 5,6 double bond, and the B. (1974) Proc. Nad. Acad. Sci. USA 71, 345-349. 15-hydroxyl group is in the S orientation. This stereochem- 2. Hamberg, M., Svensson, J. & Samuelsson, B. (1975) Proc. istry is characteristic for both PGH2 and TXA2 as well as for Nati. Acad. Sci. USA 72, 2994-2998. the stable mimetics U-46619 (6) and the sulfur analogue of 3. Hamberg, M., Hedquist, P., Strandberg, K., Svensson, J. & TXA2, 9,11-epithio-11,12-methano-TXA2 (STA2) (10). Chang- Samuelsson, B. (1975) Life Sci. 16, 451-462. ing the stereochemistry of the 5,6-double bond from cis to 4. Needleman, P. M., Minkes, M. & Raz, A. (1976) Science 193, trans and that ofthe 15-hydroxyl groups from R to S imparted 163-165. antagonist properties to the resulting compounds for the 5. Corey, E. J., Nicolaou, K. C., Machida, Y., Malmsten, C. L. [TXA2/PGH2]a receptor. The rank order potency for com- & Samuelsson, B. (1975) Proc. Nad. Acad. Sci. USA 72, pounds II, III, and IV to inhibit platelet aggregation stimulated 3355-3358. by 10,10-difluoro-TXA2 and compete with [125I]PTA-OH for 6. Bundy, G. L. (1975) Tetrahedron Lett. 24, 1957-1960. the platelet receptor was 7. Nicolaou, K. C., Magolda, R. L., Smith, J. B., Aharony, D., similar, compound II (15R) 2 com- Smith, E. F. & Lefer, A. M. (1979) Proc. Nati. Acad. Sci. USA pound III (15S, 5,6-trans) > compound IV (15R, 5,6-trans), 76, 2566-2570. indicating that inhibition of aggregation and competition with 8. Nicolaou, K. C., Magolda, R. L. & Claremon, D. A. (1980) J. [125I]PTA-OH are both occurring at the putative [TXA2/ Am. Chem. Soc. 102, 1404-1409. PGH2]J,, receptor. The pseudo Hill coefficients for compounds 9. Katsura, M., Miyamoto, T., Hamanaka, N., Kondo, K., II, III, and IV were significantly less than -1, indicating that Terada, T., Ohgaki, Y., Kawasaki, A. & Tsuboshima, M. (1983) the compounds may also be interacting at more than one class Adv. Prostaglandin Thromboxane Leuk. Res. 11, 351-357. of receptors or nonspecifically. While single changes in these 10. Ohuchida, S., Hamanaka, N. & Hayashi, M. (1983) Tetrahe- positions decreased the affinity of the compounds for the dron 39, 4263-4268. [TXA2/PGH2], receptor, the compounds all still possessed 11. Mais, D., Knapp, D., Ballard, K., Hamanaka, N. & Halushka, agonist activity. Changing both positions to the unnatural P. (1984) Tetrahedron Lett. 25, 4207-4210. stereochemistry at the same time resulted in a compound with 12. Mais, D., Saussy, D., Chaikhouni, A., Kochel, P., Hamanaka, a N. & Halushka, P. (1985) J. Pharmacol. Exp. Ther. 223, 10-fold decrease in affinity for both receptors. Similarly, for 418-424. U-46619 and STA2, changing the configuration of the 15- 13. Lefer, A. M., Smith, E. F., Araki, H., Smith, J. B., Aharony, hydroxyl group results in loss ofagonist activity in platelets (6, D., Claremon, D. A., Magolda, R. L. & Nicolaou, K. C. (1980) 10). Proc. Nati. Acad. Sci. USA 77, 1706-1710. The evidence for distinct receptors in platelets and blood 14. Gorman, R. R., Shebuski, R. J., Aiken, J. W. & Bundy, G. L. vessels arises from the ability of the three isomers of 10,10- (1981) Fed. Proc. Fed. Am. Soc. Exp. Biol. 40, 1997-2000. Downloaded by guest on September 25, 2021 5604 Medical Sciences: Morinelli et al. Proc. Nati. Acad. Sci. USA 86 (1989)

15. Akbar, H., Mukhopadhyay, A., Anderson, K., Navran, S., Chemother. 14, 48-58. Ramstedt, K., Miller, D. & Feller, D. (1985) Biochem. Phar- 20. Cheng, Y.-C. & Prusoff, W. H. (1973) Biochem. Pharmacol. macol. 34, 641-647. 22, 3099-3109. 16. Mais, D., Dunlap, C., Hamanaka, N. & Halushka, P. (1985) 21. Ogletree, M. L., Harris, D. N., Greenberg, R., Haslanger, Eur. J. Pharmacol. 111, 125-128. M. F. & Nakane, M. (1985) J. Pharmacol. Exp. Ther. 234, 17. Mais, D., DeHoll, D., Sightler, H. & Halushka, P. (1988) Eur. 435-441. J. Pharmacol. 148, 309-315. 22. Halushka, P., Kochel, P. & Mais, D. (1987) Br. J. Pharmacol. 18. Fried, J., John, V., Szwedo, M. J., Jr., Chen, Ch.-K., Mo- 91, 223-227. rinelli, T., Okwu, A., O'Yang, C. & Halushka, P. (1989) J. Am. 23. Mayeux, P. R., Morton, H. E., Gillard, J., Lord, A., Morinelli, Chem. Soc. 111, 4510-4511. T. A., Boehm, A., Mais, D. E. & Halushka, P. V. (1988) 19. Arunlakshana, 0. & Schild, H. 0. (1959) Br. J. Pharmacol. Biochem. Biophys. Res. Commun. 157, 733-739. Downloaded by guest on September 25, 2021