Effect of synthetic enantiomeric cannabinoids on platelet aggregation I. NATHAN^
AND
G. AGAM
Department of Clinical Biochemistry, Ben Gurion University of the Negev, P. 0.Box 653, Beer-Sheva, Israel 84105
R. MECHOULAM Faculty of Medicine, Hebrew* University of Jerusalem, Jerusalem, Israel
A. DVILANSKY Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by CONCORDIA UNIV on 12/09/14 For personal use only.
Department of Herata?okogy, Ben Gurion University of the Negev, P.O. Box 65.3, Beer-Shew, Israel 84105 AND
A. A. E I V N E ~ Department of Ltfe Sciences, Ben Gurion University of the Negev, P. 0.Box 653, Beer-Sheva, Israel 8410.5 Received February 29, 1991 NATHAN,I., AGAM,G., ~ E C H O W L AW., M , DQILANSKY, A., and LIVNB,A. A. 1992. Effect of synthetic enantiomeric cannabinoids on platelet aggregation. Can. J. Physiol. Pharmacsl. "1: 1305- 1308. The effect of a synthetic pair of enantiomeric cannabinoids on platelet function was evaluated. The nonpsychotropic enantiomer, the 1,1-dimethylheptyl homolog of (+)-(3S,4S)-7-hydroxy-A-6-tetrahydrocannabinol (HU-21 l), was found to be more active in inhibiting ADP-induced platelet aggregation than the highly psychotropic (-)-enantiomer (HU-210). The related (+)-(3R,4R) cannabinoid, HU-213, which lacks the 7-hydroxy moiety, exerted its inhibitory effect within a wider range of concentrations. The results indicate a differentiation between psychotropic activity and inhibition of platelet aggregation in the cannabinoid group of compsunds. Key words: enantiomeric cannabinoids, platelet aggregation. NATHAN,I., AGAM,G.,MBCHBULAM, R., BVILANSKY, A., et LHVME, A. A. 1992. Effect of synthetic enantiomeric cannabinoids on platelet aggregation. Can. J. Physisl. Pharmacol. 70 : 1305- 1308. On a CvaluC l'effet de deux cambino'ides knantiomkres synthktiques sur la fonction plaquettaire. L'knantiomkre non psychotrope, l'homologue, 1,l-dimtthylheptyl du (+)-(3S,4S)-7-hydroxy-A-6tCtrahydrocannabinol (HU-211): a inhibt plus efficacement l'agrkgation plaquettaire induite par 1'ABP que l'inantiomkre-(-) kautement psychotrope (HU-2 10). Le cannabinside connexe (+)-(3R,4R), HU-213, qui est amputt du fragment 7-hydroxy, a exerct son effet inkibiteur 2 l'intkrieur d'une plus vaste plage de concentrations. Les rCsultats indiquent une diffkrenciation entre l'aetivitk psyehotrope et l'inhibition de I'agrCgation plaquettaire chez les cannabino'ides. MOPScl&s : cannabino'ides Cnantiomkres, agrkgation plaquettaire. [Traduit par la rkdaction]
Introduction We have previously studied various interactions of cannabinoids with platelets. Cannabinoids inhibit platelet aggregation in a specific manner (Formukesng et a&.1989; Nathan et ul. 1986). They display an inhibitory effect on the various components sf 5-hydroxytryptamine (5-HT) uptake. The active uptake, the passive transport, and the saturation level sf 5-HT are differentially inhibited by different cannabinoid derivatives (Nathan et al. 1982). Recently, the pure enantiomers of the synthetic cannabinoid, 7-hydrsxy-A-6-tetrahydrocanmabin01 I ,1-dimethylkeptyl homolog, were prepared (Mechsulam el al. 1990). The psychotropic activity was shown to reside solely in the ( -)-(3W ,4R)enantiorner (code named WU-210); the (+)-(3S.4S) enantiomer (code named HU-2 11) was inactive (Mechoulam et al. 1988; Little et al. 1989)). MU-210 was found to bind to the cannabinoid receptor (Ki = 234 pM in homologous displacement studies in rat brain preparations). HU-211 was approximately 1506 times less potent (Hswlett et al. 1998). In the present study, we have examined the activity of these new synthetic drugs on platelet aggregation. Materials and methods Reagents Albumin (bovine, essentially fatty acid free, from fraction V albumin) was obtained from Sigma (St. Louis, Mo.); fibrinogen (human, 'Author for correspondence. 2Author deceased. Pr~ntedIII Canada / 1rnpr:mi nu Canada
97 % clottable) was from Cutter (Berkeley, Calio. Cannabinoids were synthesized as described (Mechoulam et a!. 1990).
Waethohis Preparation of platelet suspension and assay of aggregation were performed as described (Funder ef a / . 1988). Hn brief, venous blood was drawn from healthy volunteers, who had been without medication for the previous 10 days. The blood was anticoagulated with acid -citrate -dextrose solution at a volume ratio of 6: 1. Platelet-rich plasma (PRP) was obtained by centrifugation at 120 X g for 10 min and had a pH of 6.5. The platelets were gel filtered through a Sepharose 2B column (6 x 0.76 cm). The solution used to equilibrate the column and elute the platelets contained (mM) 140 NaC1, 5 KCl, 0.42 NaH,PO,, 10 glucose. and 20 Hepes and 3.6 mg bovine albumin/mL, pH 7.35. Platelet aggregation of a stirred platelet suspension (5 x 107/mL) was measured at 37OC in a final volume of 0.45 mL, supplemented with 0.1 mg fibrinogen. For the light transmission measurement, the course of aggregation was followed by light transmission in a Chronolog aggregometer. Platelets were incubated for 1 rnin with the tested cannabinoids or with the vehicle at 37"C, prior to the addition of ADP (10 yM). The extent of aggregation was measured as the change in light transmission (A%LT) at 5 rnin after the addition of the inducer. Stock solutions of the cannabinoids were prepared in 70 5% ethanol, and an aliquot of 3 yL was added to the assay mixture. For the evaluation of aggregation by platelet counts, 58-pH, aliquots from the aggregation mixture were drawn at 8, 2.5, and 5 min after the addition of the inducer of aggregation into 50 pL of 2% formaldehyde in the gel-filtration solution; platelet counts were carried out microscopically and only nonaggregated single platelets were counted.
CAN. J. PHYSIOL. PHARMACOL. VOL. 70, 1992
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(B)
FIG. 1. Effect of HU-211 on platelet aggregation. (A) Inhibitory effect of 10 pM ADP-induced aggregation by gation induced by lo-' M of the drug.
ha HU-211. (B) Aggre-
FIG. 2. Dose response of the inhibition of ADP-induced platelet aggregation by synthetic cannakinoids. The resuats depict a representative experiment.
SCHEME 1. Structural forn~uiacof HW-210, HU-2 11, and HU-213.
Platelet serotonin release was measured as previously described (Biran et nk. 1973). The structural formulae of the synthetic cannabinoids presently studied are presented in Scheme 1.
Results The effect of the new cannabinoid derivatives HU-210 and HU-21 H on ADP-induced platelet aggregation was examined. Both drugs affected ADP-induced platelet aggregation in a bimodal manner: inhibition at a low concentration M) of platelet aggregation or induction of aggregation at a higher concentration M). Figure H depicts aggregation tracings of a representative experiment showing both the inhibitory effect of 10-% HU-2 11 upon ABP-induced platelet aggregation and the drug-induced aggregation at a concentration of 1 0 - W of the enantismer. To verify a direct effect of the cannabinoids on platelet aggregation rather than on their refractive index, platelet counts were carried out microscopically during the course of aggregation. Under the conditions shown in Fig. 1A, single platelet
counts decreased by 73% after 5 min in the control in which aggregation was induced by BO pM ADP. When 18-% HU-211 was added B min prior to ABP, single platelet counts decreased only by 45 % , reflecting 38 7% inhibition s f aggregation. Under the conditions shown in Fig. 1B, M HU-211 decreased single platelet counts by 67% after 5 min of aggregation. Aggregation induced by thrombin (0.1 U/mL) was not inhibited by HU-211 at and 10-%. In addition, thrombin-induced serotonin release was not affected by the cannabinoids, under the same conditions. The comparison among the two enantiorners, HU-210 and HU-2 1 1, and ( +)-A-6-tetrahydrocannabinol, l ,1-dimethylhegtyl homolog (code named HU-2 13) revealed that HU-2 1 1 was more potent in inhibiting platelet aggregation than HU-2 18, at all concentrations tested (Fig, 2). Furthermore, the maximal inhibitory effect s f HU-2 10 under the experimental conditions was 60%.At concentrations > 3 pM,both HU-2 10 and HU-2 1 1 induced platelet aggregation, irrespective of the addition s f ABP, but HU-213 did not induce platelet aggregation. Therefore, it was possible to examine HU-2 13 at a concentration of 36) pM. The results indicate a high potency (90% inhibition) at this concentration. In view of the drug-induced aggregatory activity, it was
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NATHAN ET AL.
Drug concn. QpM) FIG.3. The biphasic effect of synthetic cannabinoids on ADP-induced platelet aggregation. Results are mean I S E of up to 9 experiments. a , occurrence of experiments in which drug-induced aggregation was observed. b , not determined. necessary to carry out additional experiments, as presented in Fig. 3. Both enantiomers, HU-210 and HU-211, display a biphasic effect: they induce aggregation at relatively high concentrations, r 3 pM, and inhibit ADP-induced platelet aggregation at lower concentrations. At a concentration of 3 pM, HU-2 18 and HU-2 1 1 induced aggregation by themselves in 3 of 10 and 3 s f 7 experiments, respectively. In contrast, HU-213 did not induce aggregation but was inhibitory under the experimental conditions at concentrations up to 30 pM. At low concentrations, all three drugs inhibited platelet aggregation. The most prominent difference is observed at a concentration of 0.3 pM, where the (3S74S) enantiomer, HU-211, was much more active than the (3R,4R) enantismer, HU-210 (22 f 6.8 and 2 _+ 1.5 % inhibition? respectively).
Discussion This study describes the effect of a pair of new synthetic enantiomers of tetrahydrocannabinol (THC)-like compounds on ADP-induced platelet aggregation. Results obtained by aggregation tracings were further confirmed qualitatively by microscopic platelet counts. The results reveal that the (+)(3S,4S) enantismer, HU-211, was more active than the (-)(3R,4R) HU-2 10. (+)-Dimethylheptyl-THC, HU-2 13, was shown to be inhibitory over a wide concentration range, whereas the new pair induced platelet aggregation at high concentrations, 2 3 pM. Thus, (+)-dimethylheptyl-THC may possibly have some stabilization effect on the platelet membrane which prevents Bysis and induction of aggregation at a similar concentration range. HU-2 11 possesses a hydroxyl group at @-7,which is known to increase THC activity. 7-Hydroxy derivatives of both A-6 and A- 1 THC are major active metabolites of these compounds (Ben-Zvi et a / . 1970; Nilsson et ak. 1978; Wall et a%.1970). Another structural change of A-6 THC introduced in HU-211 is the replacement of the PT-pentyl side chain with the 1 , l dimethylheptyl group, which is also known to increase biological activity (Mechoulam and Feigenbaum 1987). This change markedly potentiates the inhibitory effect of cannabinoids on platelet aggregation (Nathan et al. 1986). The results support our previous findings, which indicate that (+) isomers
1307
are more potent than the (-1 owes, and extend the results to show that the new compound (HU-211), which possesses an additional O H group at C-7, is highly active. It should be noted that this compound, being crystalline, has been obtained in essentially absolute enantiomeric purity. This drug has recently been shown to possess antiemetic activlty (Feigenbaum et al. 1989~4)and to act as a functional N-methyl-saspartate receptor blocker (Feigenbaum st a!. H989b). It was recently documented that various cannabinoids antagonize platelet aggregation induced by adrenalin, ADP, and the platelet-activating factor (PAP;), measured in PRP (Formukong et al. 1989). These authors claimed that primary aggregation was only partially inhibited by cannabinoids. In our study, the percentage of inhibition measured by the cell-counting technique, a more sensitive method (Frojmovic et al. 1989), is lower than that measured by the aggregometer. It is also possible that part of the effect measured by the aggregometer may be attributed to changes in light transmittance and not to aggregation, thus supporting Formukong's claim s f partial inhibition. The mode of action of these drugs at the molecular level is not clear. Recently, the receptor for THC was identified (Devane et a / . 1988) and shortly thereafter was cloned and its primary structure determined (Matsuda et al. 1998). Evidence for involvjng the Gi protein-coupled receptor, which inhibits adenylate cyclase activity, was put forward (Devane et al. 1988; Howlett et al. 1990). The described receptor was stereoselective for (-) enantiomeric forms (Howlett et al. 1990). Since the (t)enantiomers are more potent in inhibition of platelet aggregation than the (-1 ones, the antiaggregatory effect is not mediated by the cannabinoid receptor. The inhibitory effect on platelet aggregation could be attributed to effects of cannabinoids on prostaglandin levels caused by inhibition of phospholipase A activity, or cycIo-oxygenase and lipoxygenase pathways (Burstein and Raz 1972; Barrett et al. 1985; Evans et al. 1987). In view of the high potency of HU-211 and its lack of psychotropic action, this cannabinoid merits serious consideration as an antiaggregatory agent. Further studies are still needed prior to possible medical application. Barrett, M. E., Gordon, I., and Evans, J. 1985. Isolation from Cannabis saltiva E. of Cannflavin, a novel inhibitor of prostagladin production. Biochem. Pharrnacoi. 34: 2019 - 2024. Ben-Zvi, Z . , Mechsulam, R., and Burstein, S. 1970. Identification through a synthesis of an active delta-6-tetrahydrocannabinol metabolite. J. Am. Chem. 92: 3468 - 3469. Biran, M., Bvilansky, A., Nathan, I., and Eivne, A. 1973. Impairment of human aggregation and serotonin release caused in vitro by Echis colomta venom. Throm. Diath. Maemorrh. 3: 191 198.
Burstein, S., and Raz, A. 1972. Inhibition of PGE2 biosynthesis. Prostaglandins, 2: 369 - 374. Bevane, W. A., Dysarz, F. A., Johnson, M. R., Melvin, E. S., and Howlett, A. C. 1988. Determination and characterizationof a cannabinoid receptor in rat brain. Mol. Pharmacsl. 34: 605 -613. Evans, A. T., Formukong, E. A.. and Evans, J. 1987. Actions of cannabis constituents on enzymes of arachidonate metabolism. Antiinflammatory potential. Biochem. Pharmacol . 36: 2037 - 2040. Feigenbaum, J., Richmond, S. A., Weissman, Y., and Mechoulam, R. 1989a. Inhibition of cisplatin-induced emesis in the pigeon by a non-psychotropic synthetic cannabinoid. Eur. 9. Pharmacol. 169: 159- 165. Feigenbaum, J. J., Bergrnann, F., Richmond, S. A., Mechoulam, R. Nadler, V., MIoog, Y., and Sokolovsky, M. 1989b. A wonpsychotropic cannabinoid acts as a functional N-methylaspar art ate receptor blocker. Proc. Natl. Acad. Sci. U.S .A. $6: 9584 -9587.
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Hormukong, E. A., Evans, A. T., and Evans, F. J. 1989. The inhibitory effects of cannabinoids, the active constituents of Cannabis saliva L. on human and rabbit platelet aggregation. 3. Pharm. Pharmacol . 41 : 705 - 709. Frojmovic, M. M., Milton, J. G., and Gear, A. L. 1989. Platelet aggregation measured in vitro by microscopic and electronic particle counting. Methods Enzymol. 169: 134 - 149. Funder, J . , Herscho, L., Rothstein, A., and Livne, A. 1988. Na'/H+ exchange and aggregation of human platelets by ADP. Biochim. Biophys. Acta, 938: 425 -433. Howlett, A. C., Chan~pion,T. M., Wilken, G . H., and Mechoulam, W. 1990. Stereochemical effects of I l -OH-delta-8-tetrahy drocannabinoldirnethyBhepty1 to inhibit adenylate cyclase and bind to the cannabinoid receptor. Neuropharrnacology , 29: 161 - 165. Levy, W., Schurr, A . , Nathan, I., Dvilansky, A * , and Livne, A. 1976. Impairment sf ADP-induced platelet aggregation by hashish component. Thrornb. Haemostasis, 36: 634-639. Little, P. J., Csrnpton, D. R., Mechoulam, R., and Martin, B. R. 1989. Stereochemical effects of 11-OH-delta-8-THC-dimethylheptyl in mice and dogs. Pharmacol. Biochem. Behav. 32: 661 666. Matsuda, L., Lelai, S., Brownstein, M., Young, A., and Bonner, T. 1990. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature (London), 346: 561 -564. Meckoulam, R., and Feigenbaum, J. J. 1987. Towards cannabinoid drugs. Preg. Med. Chem. 24: 159-207.
Mechoulam, W., Feigenbaurn. J. J., Lander. N., Segal, M., Jarbe, T. U . C., Hiltunen, A. J., and Consroe, B. 1988. Enantiomeric cannabinoids: stereo specifisity of psychotropic activity. Experientia, 44: 762-764. Mechoulam, R., Lander, N, Breuer, A., and Zahalka, J. 1990. Synthesis of the individual, pharn-iacologically distinct enantiorners of a tetrahydrocannabinol derivative. Tetrahedron: Asymmetry, H : 315-319. Nathan. I., Yarem, A., Dvilansky, A., Lander, N., and Livne, A. 1982. Cannabinoids and serotonin uptake by blood platelets: evidence for multiple sites of action. Biochem. Phamacsl. 31: 439-441. Nathan, I., Dvilansky, A., Lazarowitz, J., Lander, N,, and Livne, A. 1986. Specific impairment of ADP-induced platelet aggregation by cannabinoids. Int. J. Tiss. React. VIII(3): 193 - 198. Nilsson, I. M., Agurell, S., Nilsson, J. L., Bhlsson, A., Sandberg, F., and Wahlqvist, M. 1970. Delta-1-tetrahydrocannabinol: structure of a major metabolite. Science (Washington, D.C.), 168: 1228 - 1229. Wall, M. E., Brine, D. R., Brine, G. A., Pitt, C. G., Freudenthal. W. I., and Christensen, H. D. 1970. Isolation, structure and biological activity of several metabolites of delta-9-tetrahydrocannabinol. J. Am. Chem. Soc. 92: 3446-3468.
AND
G. AGAM
Department of Clinical Biochemistry, Ben Gurion University of the Negev, P. 0.Box 653, Beer-Sheva, Israel 84105
R. MECHOULAM Faculty of Medicine, Hebrew* University of Jerusalem, Jerusalem, Israel
A. DVILANSKY Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by CONCORDIA UNIV on 12/09/14 For personal use only.
Department of Herata?okogy, Ben Gurion University of the Negev, P.O. Box 65.3, Beer-Shew, Israel 84105 AND
A. A. E I V N E ~ Department of Ltfe Sciences, Ben Gurion University of the Negev, P. 0.Box 653, Beer-Sheva, Israel 8410.5 Received February 29, 1991 NATHAN,I., AGAM,G., ~ E C H O W L AW., M , DQILANSKY, A., and LIVNB,A. A. 1992. Effect of synthetic enantiomeric cannabinoids on platelet aggregation. Can. J. Physiol. Pharmacsl. "1: 1305- 1308. The effect of a synthetic pair of enantiomeric cannabinoids on platelet function was evaluated. The nonpsychotropic enantiomer, the 1,1-dimethylheptyl homolog of (+)-(3S,4S)-7-hydroxy-A-6-tetrahydrocannabinol (HU-21 l), was found to be more active in inhibiting ADP-induced platelet aggregation than the highly psychotropic (-)-enantiomer (HU-210). The related (+)-(3R,4R) cannabinoid, HU-213, which lacks the 7-hydroxy moiety, exerted its inhibitory effect within a wider range of concentrations. The results indicate a differentiation between psychotropic activity and inhibition of platelet aggregation in the cannabinoid group of compsunds. Key words: enantiomeric cannabinoids, platelet aggregation. NATHAN,I., AGAM,G.,MBCHBULAM, R., BVILANSKY, A., et LHVME, A. A. 1992. Effect of synthetic enantiomeric cannabinoids on platelet aggregation. Can. J. Physisl. Pharmacol. 70 : 1305- 1308. On a CvaluC l'effet de deux cambino'ides knantiomkres synthktiques sur la fonction plaquettaire. L'knantiomkre non psychotrope, l'homologue, 1,l-dimtthylheptyl du (+)-(3S,4S)-7-hydroxy-A-6tCtrahydrocannabinol (HU-211): a inhibt plus efficacement l'agrkgation plaquettaire induite par 1'ABP que l'inantiomkre-(-) kautement psychotrope (HU-2 10). Le cannabinside connexe (+)-(3R,4R), HU-213, qui est amputt du fragment 7-hydroxy, a exerct son effet inkibiteur 2 l'intkrieur d'une plus vaste plage de concentrations. Les rCsultats indiquent une diffkrenciation entre l'aetivitk psyehotrope et l'inhibition de I'agrCgation plaquettaire chez les cannabino'ides. MOPScl&s : cannabino'ides Cnantiomkres, agrkgation plaquettaire. [Traduit par la rkdaction]
Introduction We have previously studied various interactions of cannabinoids with platelets. Cannabinoids inhibit platelet aggregation in a specific manner (Formukesng et a&.1989; Nathan et ul. 1986). They display an inhibitory effect on the various components sf 5-hydroxytryptamine (5-HT) uptake. The active uptake, the passive transport, and the saturation level sf 5-HT are differentially inhibited by different cannabinoid derivatives (Nathan et al. 1982). Recently, the pure enantiomers of the synthetic cannabinoid, 7-hydrsxy-A-6-tetrahydrocanmabin01 I ,1-dimethylkeptyl homolog, were prepared (Mechsulam el al. 1990). The psychotropic activity was shown to reside solely in the ( -)-(3W ,4R)enantiorner (code named WU-210); the (+)-(3S.4S) enantiomer (code named HU-2 11) was inactive (Mechoulam et al. 1988; Little et al. 1989)). MU-210 was found to bind to the cannabinoid receptor (Ki = 234 pM in homologous displacement studies in rat brain preparations). HU-211 was approximately 1506 times less potent (Hswlett et al. 1998). In the present study, we have examined the activity of these new synthetic drugs on platelet aggregation. Materials and methods Reagents Albumin (bovine, essentially fatty acid free, from fraction V albumin) was obtained from Sigma (St. Louis, Mo.); fibrinogen (human, 'Author for correspondence. 2Author deceased. Pr~ntedIII Canada / 1rnpr:mi nu Canada
97 % clottable) was from Cutter (Berkeley, Calio. Cannabinoids were synthesized as described (Mechoulam et a!. 1990).
Waethohis Preparation of platelet suspension and assay of aggregation were performed as described (Funder ef a / . 1988). Hn brief, venous blood was drawn from healthy volunteers, who had been without medication for the previous 10 days. The blood was anticoagulated with acid -citrate -dextrose solution at a volume ratio of 6: 1. Platelet-rich plasma (PRP) was obtained by centrifugation at 120 X g for 10 min and had a pH of 6.5. The platelets were gel filtered through a Sepharose 2B column (6 x 0.76 cm). The solution used to equilibrate the column and elute the platelets contained (mM) 140 NaC1, 5 KCl, 0.42 NaH,PO,, 10 glucose. and 20 Hepes and 3.6 mg bovine albumin/mL, pH 7.35. Platelet aggregation of a stirred platelet suspension (5 x 107/mL) was measured at 37OC in a final volume of 0.45 mL, supplemented with 0.1 mg fibrinogen. For the light transmission measurement, the course of aggregation was followed by light transmission in a Chronolog aggregometer. Platelets were incubated for 1 rnin with the tested cannabinoids or with the vehicle at 37"C, prior to the addition of ADP (10 yM). The extent of aggregation was measured as the change in light transmission (A%LT) at 5 rnin after the addition of the inducer. Stock solutions of the cannabinoids were prepared in 70 5% ethanol, and an aliquot of 3 yL was added to the assay mixture. For the evaluation of aggregation by platelet counts, 58-pH, aliquots from the aggregation mixture were drawn at 8, 2.5, and 5 min after the addition of the inducer of aggregation into 50 pL of 2% formaldehyde in the gel-filtration solution; platelet counts were carried out microscopically and only nonaggregated single platelets were counted.
CAN. J. PHYSIOL. PHARMACOL. VOL. 70, 1992
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(B)
FIG. 1. Effect of HU-211 on platelet aggregation. (A) Inhibitory effect of 10 pM ADP-induced aggregation by gation induced by lo-' M of the drug.
ha HU-211. (B) Aggre-
FIG. 2. Dose response of the inhibition of ADP-induced platelet aggregation by synthetic cannakinoids. The resuats depict a representative experiment.
SCHEME 1. Structural forn~uiacof HW-210, HU-2 11, and HU-213.
Platelet serotonin release was measured as previously described (Biran et nk. 1973). The structural formulae of the synthetic cannabinoids presently studied are presented in Scheme 1.
Results The effect of the new cannabinoid derivatives HU-210 and HU-21 H on ADP-induced platelet aggregation was examined. Both drugs affected ADP-induced platelet aggregation in a bimodal manner: inhibition at a low concentration M) of platelet aggregation or induction of aggregation at a higher concentration M). Figure H depicts aggregation tracings of a representative experiment showing both the inhibitory effect of 10-% HU-2 11 upon ABP-induced platelet aggregation and the drug-induced aggregation at a concentration of 1 0 - W of the enantismer. To verify a direct effect of the cannabinoids on platelet aggregation rather than on their refractive index, platelet counts were carried out microscopically during the course of aggregation. Under the conditions shown in Fig. 1A, single platelet
counts decreased by 73% after 5 min in the control in which aggregation was induced by BO pM ADP. When 18-% HU-211 was added B min prior to ABP, single platelet counts decreased only by 45 % , reflecting 38 7% inhibition s f aggregation. Under the conditions shown in Fig. 1B, M HU-211 decreased single platelet counts by 67% after 5 min of aggregation. Aggregation induced by thrombin (0.1 U/mL) was not inhibited by HU-211 at and 10-%. In addition, thrombin-induced serotonin release was not affected by the cannabinoids, under the same conditions. The comparison among the two enantiorners, HU-210 and HU-2 1 1, and ( +)-A-6-tetrahydrocannabinol, l ,1-dimethylhegtyl homolog (code named HU-2 13) revealed that HU-2 1 1 was more potent in inhibiting platelet aggregation than HU-2 18, at all concentrations tested (Fig, 2). Furthermore, the maximal inhibitory effect s f HU-2 10 under the experimental conditions was 60%.At concentrations > 3 pM,both HU-2 10 and HU-2 1 1 induced platelet aggregation, irrespective of the addition s f ABP, but HU-213 did not induce platelet aggregation. Therefore, it was possible to examine HU-2 13 at a concentration of 36) pM. The results indicate a high potency (90% inhibition) at this concentration. In view of the drug-induced aggregatory activity, it was
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NATHAN ET AL.
Drug concn. QpM) FIG.3. The biphasic effect of synthetic cannabinoids on ADP-induced platelet aggregation. Results are mean I S E of up to 9 experiments. a , occurrence of experiments in which drug-induced aggregation was observed. b , not determined. necessary to carry out additional experiments, as presented in Fig. 3. Both enantiomers, HU-210 and HU-211, display a biphasic effect: they induce aggregation at relatively high concentrations, r 3 pM, and inhibit ADP-induced platelet aggregation at lower concentrations. At a concentration of 3 pM, HU-2 18 and HU-2 1 1 induced aggregation by themselves in 3 of 10 and 3 s f 7 experiments, respectively. In contrast, HU-213 did not induce aggregation but was inhibitory under the experimental conditions at concentrations up to 30 pM. At low concentrations, all three drugs inhibited platelet aggregation. The most prominent difference is observed at a concentration of 0.3 pM, where the (3S74S) enantiomer, HU-211, was much more active than the (3R,4R) enantismer, HU-210 (22 f 6.8 and 2 _+ 1.5 % inhibition? respectively).
Discussion This study describes the effect of a pair of new synthetic enantiomers of tetrahydrocannabinol (THC)-like compounds on ADP-induced platelet aggregation. Results obtained by aggregation tracings were further confirmed qualitatively by microscopic platelet counts. The results reveal that the (+)(3S,4S) enantismer, HU-211, was more active than the (-)(3R,4R) HU-2 10. (+)-Dimethylheptyl-THC, HU-2 13, was shown to be inhibitory over a wide concentration range, whereas the new pair induced platelet aggregation at high concentrations, 2 3 pM. Thus, (+)-dimethylheptyl-THC may possibly have some stabilization effect on the platelet membrane which prevents Bysis and induction of aggregation at a similar concentration range. HU-2 11 possesses a hydroxyl group at @-7,which is known to increase THC activity. 7-Hydroxy derivatives of both A-6 and A- 1 THC are major active metabolites of these compounds (Ben-Zvi et a / . 1970; Nilsson et ak. 1978; Wall et a%.1970). Another structural change of A-6 THC introduced in HU-211 is the replacement of the PT-pentyl side chain with the 1 , l dimethylheptyl group, which is also known to increase biological activity (Mechoulam and Feigenbaum 1987). This change markedly potentiates the inhibitory effect of cannabinoids on platelet aggregation (Nathan et al. 1986). The results support our previous findings, which indicate that (+) isomers
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are more potent than the (-1 owes, and extend the results to show that the new compound (HU-211), which possesses an additional O H group at C-7, is highly active. It should be noted that this compound, being crystalline, has been obtained in essentially absolute enantiomeric purity. This drug has recently been shown to possess antiemetic activlty (Feigenbaum et al. 1989~4)and to act as a functional N-methyl-saspartate receptor blocker (Feigenbaum st a!. H989b). It was recently documented that various cannabinoids antagonize platelet aggregation induced by adrenalin, ADP, and the platelet-activating factor (PAP;), measured in PRP (Formukong et al. 1989). These authors claimed that primary aggregation was only partially inhibited by cannabinoids. In our study, the percentage of inhibition measured by the cell-counting technique, a more sensitive method (Frojmovic et al. 1989), is lower than that measured by the aggregometer. It is also possible that part of the effect measured by the aggregometer may be attributed to changes in light transmittance and not to aggregation, thus supporting Formukong's claim s f partial inhibition. The mode of action of these drugs at the molecular level is not clear. Recently, the receptor for THC was identified (Devane et a / . 1988) and shortly thereafter was cloned and its primary structure determined (Matsuda et al. 1998). Evidence for involvjng the Gi protein-coupled receptor, which inhibits adenylate cyclase activity, was put forward (Devane et al. 1988; Howlett et al. 1990). The described receptor was stereoselective for (-) enantiomeric forms (Howlett et al. 1990). Since the (t)enantiomers are more potent in inhibition of platelet aggregation than the (-1 ones, the antiaggregatory effect is not mediated by the cannabinoid receptor. The inhibitory effect on platelet aggregation could be attributed to effects of cannabinoids on prostaglandin levels caused by inhibition of phospholipase A activity, or cycIo-oxygenase and lipoxygenase pathways (Burstein and Raz 1972; Barrett et al. 1985; Evans et al. 1987). In view of the high potency of HU-211 and its lack of psychotropic action, this cannabinoid merits serious consideration as an antiaggregatory agent. Further studies are still needed prior to possible medical application. Barrett, M. E., Gordon, I., and Evans, J. 1985. Isolation from Cannabis saltiva E. of Cannflavin, a novel inhibitor of prostagladin production. Biochem. Pharrnacoi. 34: 2019 - 2024. Ben-Zvi, Z . , Mechsulam, R., and Burstein, S. 1970. Identification through a synthesis of an active delta-6-tetrahydrocannabinol metabolite. J. Am. Chem. 92: 3468 - 3469. Biran, M., Bvilansky, A., Nathan, I., and Eivne, A. 1973. Impairment of human aggregation and serotonin release caused in vitro by Echis colomta venom. Throm. Diath. Maemorrh. 3: 191 198.
Burstein, S., and Raz, A. 1972. Inhibition of PGE2 biosynthesis. Prostaglandins, 2: 369 - 374. Bevane, W. A., Dysarz, F. A., Johnson, M. R., Melvin, E. S., and Howlett, A. C. 1988. Determination and characterizationof a cannabinoid receptor in rat brain. Mol. Pharmacsl. 34: 605 -613. Evans, A. T., Formukong, E. A.. and Evans, J. 1987. Actions of cannabis constituents on enzymes of arachidonate metabolism. Antiinflammatory potential. Biochem. Pharmacol . 36: 2037 - 2040. Feigenbaum, J., Richmond, S. A., Weissman, Y., and Mechoulam, R. 1989a. Inhibition of cisplatin-induced emesis in the pigeon by a non-psychotropic synthetic cannabinoid. Eur. 9. Pharmacol. 169: 159- 165. Feigenbaum, J. J., Bergrnann, F., Richmond, S. A., Mechoulam, R. Nadler, V., MIoog, Y., and Sokolovsky, M. 1989b. A wonpsychotropic cannabinoid acts as a functional N-methylaspar art ate receptor blocker. Proc. Natl. Acad. Sci. U.S .A. $6: 9584 -9587.
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