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Japan. J. Pharmacol. 33, 749-755 (1983) 749

SELECTIVE BINDING OF YM-09151-2, A NEW POTENT NEUROLEPTIC, TO D2- RECEPTORS

Michio TERAI,Shinji USUDA, Izumi KUROIWA,Osamu NOSHIRO and Hiroo MAENO Departmentsof Biochemistryand Pharmacology, Central Research Laboratories, YamanouchiPharmaceutical Co., Ltd., Azusawa, Itabashi-ku, Tokyo 174, Japan AcceptedFebruary 28, 1983

Abstract-Effects of YM-09151-2 and five other neuroleptics (, spiperone, , and ) on the binding of [3H]-ligands to nine different receptors (a,-, a2-adrenergic, Q-adrenergic, muscarinic, D2-dopaminergic, H1-histaminergic, 5HT,-, 5HT2-serotonergic and opiate receptors) and on -sensitive adenylate cyclase were determined using brain membranes in the rat, guinea-pig and dog. The affinity of YM-091 51-2 for D2-receptors with a K, value of 0.1 nM was more than 1000-times higher than that for the other receptors and dopamine sensitive adenylate cyclase, and it was the greatest among the nueroleptics tested.

A close relationship between antidopami Therefore, a highly D2-selective dopamine nergic and antischizophrenic effects of neu blocker is desirable for clinical use as well as roleptics has been demonstrated by phar for neurochemical studies. Sulpiride, a macological, electrophysiological, histo derivative seems to be a relatively chemical and biochemical studies (1). Recent specific blocker of the D2-receptor, but shown studies have also shown multiplicity of the to hardly cross the blood brain barrier; dopaminergic receptors, referred to as D, whereas YM-09151-2, N-[(2RS, 3RS)-1 (adenylate cyclase-linked), D2 (non benzyl-2-methyl-3-pyrrolidinyl] -5-chloro-2 adenylate cyclase-linked), D3 (non-adenylate methoxy-4-methylaminobenzamide, which is cyclase-linked) and D4 (inversely adenylate also a benzamide substitute, can readily pass cyclase-linked) according to the terminology through the barrier and exhibits potent proposed by Seeman (1, 2), in the brain; and neuroleptic effects in animals (3, 4). The the antischizophrenic effect seems to be at present paper describes the extremely high least partly attributable to a blockade of a specificity of YM-09151-2 for the D2 particular type (D2) of the dopaminergic receptor. receptors. However, usefulness of the drugs in the treatment of schizophrenic patients is Materials and Methods often limited, partly due to insufficient Materials: Dopamine hydrochloride, (-) specificity in the affinity for the D2 bitartarate and (+) dopaminergic receptors, which occasionally hydrochloride were obtained from Sigma, St results in undesirable side effects such as Louis, MO, U.S.A. [3H] Mepyramine (24.1 orthostatic hypotension and thirst. Lack of Ci/mmole) and [3H] (20.2 Ci/ specificity of neuroleptics for the D2-receptor mmole) were from Amersham, England. also makes it difficult to attribute their [3H] (23.8 Ci/mmole), [3H] (-) dopaminergic functions to the D2-receptor. (31.5 Ci/mmole), [3H] 750 M. TERAI ET AL.

naloxone (40.0 Ci/mmole), [3H]quinuclidinyl pH 7.5. The washed pellets were suspended benzilate (33.1 Ci/mmole), [3H] in the binding reaction buffer and frozen at (26.4 Ci/mmole) and [3H]spiperone (27.6 -80°C . A hypotonic lysate (M,) of the crude Ci/mmole) were purchased from New mitochondrial fraction of canine caudate England Nuclear, Boston, MA, U.S.A. The nucleus was prepared as described before following materials were also commercially (5). obtained: atropine (Nakarai, Kyoto, Japan), Receptor binding assays: All of the binding chlorpromazine (Rhone-Poulenc, Paris, assays described here were conducted ac France) (Kongo Chem., cording to known methods: [3H] Prazosin Toyama, Japan), haloperidol (Dainippon, (0.4 nM) binding to a,-adrenergic receptors Osaka, Japan), morphine (Sankyo, Tokyo, in the rat brain was determined by the Japan), (Takeda, Osaka, method of Greengrass and Bremner (6); Japan), serotonin (Tokyo Kasei, Tokyo, 1.0 nM [3H]clonidine binding to a2 Japan), spiperone (Eisai, Tokyo, Japan), adrenergic receptors in the rat cerebral sulpiride (Fujisawa, Osaka, Japan) and cortex by the method of U'Prichard et al. (7); Whatman GF/B filters (Whatman, Kent, 1.0 nM [3H]dihydroalprenolol binding to 8 U.K.). We acknowledge generous gifts of adrenergic receptors in the rat cerebral the following compounds: cis- (H. cortex by the method of Bylund and Snyder Lundbeck & Co., Copenhagen, Denmark), (8); 0.06 nM [3H]quinuclidinyl benzilate methysergid (Sandoz, Basel, Switzerland), binding to muscarinic cholinergic receptors (Organon, Oss, Netherlands) and in the rat brain by the method of Yamamura prazosin (Pfizer, New York, NY, U.S.A.). and Snyder (9); 0.4 nM [3H]spiperone YM-09151-2, clonidine and clozapine were binding to D2-dopaminergic receptors in the prepared by Dr. S. lwanami in our laboratories. rat striatum in the presence of 0.3 W YM-09151-2 was dissolved in 99.5% ethanol mianserin, a 5-HT2 blocker (10), by the at the concentration of 2 mM and then diluted method of Leysen et al. (11); 0.5 nM [3H] with 10% ethanol; the final ethanol concen mepyramine binding to H, -histaminergic tration in the reaction mixture was kept receptors in the guinea-pig cerebellum by below 0.5%. Other drugs hardly soluble in the method of Tran et al. (12); 0.6 nM water were also dissolved similarly. [3H] naloxone binding to opiate receptors in Membrane preparation: Male Wistar rats the rat frontal cortex by the method of Chang (7 to 9 weeks old) or Hartley guinea-pigs and Cuatrecases (13). Both 1.5 nM [3H] (300 to 400 g) were killed by decapitation. serotonin binding to 5HT,-serotonergic Brain without cerebellum, cerebral cortex, receptors in the rat cerebral cortex and 0.4 nM frontal cortex, striatum and cerebellum were [3H]spiperone binding to 5HT2-serotonergic quickly removed and chilled in ice-cold receptors in the rat frontal cortex were deter 0.85% NaCl. The tissues were homogenized mined by the method of Peroutka and Snyder in a motor-driven Teflon homogenizer in 9 (14). Specific binding was defined as the volumes of 0.32 M sucrose. The homogenates excess over blank in the presence of 6 /,M were centrifuged at 900xg for 10 min and prazosin for a, -receptors, 3 W clonidine for the precipates were washed once. The a2-receptors, 10 fM (-)alprenolol for 3 combined supernatant fluids were further receptors, 1 ,uM atropine for muscarinic centrifuged at 12,000xg for 20 min. The receptors, 10 eM cis-flupentixol for D2 pellets were washed once with 0.32 M receptors, 10 ,uM diphenhydramine for H, sucrose and then twice with 5 mM Tris-HCI, receptors, 10 flM levallorphan for opiate D2-SPECIFICITY OF YM-09151-2 751 receptors, 10 uM serotonin for 5HT, (Yamasa, Choshi, Japan) or by the protein receptors and 10 ,uM methysergid for 5HT2 binding assay of Brown et al. (16) using receptors. The IC50 values, the concen Millipore filters (17). trations required to inhibit specific binding Protein amount: Protein was determined by 50%, were computed by the logit-log by the method of Lowry et al. (18), using analysis (15); and the inhibition constant bovine serum albumin as a standard. (K;) was obtained according to the following equation: Results When plotted in a logit-log scale as a K;=1C50 1 + K /~ [3H]-lig_and„ of [3H]-ligandconcentration function of drug concentration (-log concen in which K„ is the dissociation constant. tration), the inhibitions of [3H]spiperone Dopamine-sensitive adenylate cyclase: binding to rat striatal membranes (D2 Dopamine-sensitive adenylate cyclase of the dopaminergic receptor) by YM-091 51-2 and membranes from canine caudate nucleus was five other neuroleptics were linearly increased determined as described previously (5). The within the range of concentrations tested, as standard reaction mixture contained (in a shown in Fig. 1. Of the neuroleptics tested, final volume of 0.5 ml) 80 mM Tris-maleate YM-09151-2 was the most potent in the buffer, pH 7.4; 8 mM MgSO4; 2 mM ATP; inhibition of [3H]spiperone binding with an 0.02% ascorbic acid; 0.6 mM EGTA; 0.5 mM IC50 of 0.28 nM, followed in order of potency 3-isobutyl-1-methylxanthine; 50 /tM GTP by spiperone, haloperidol, chlorpromazine, and an appropriate amount of membranes sulpiride and clozapine. Similar inhibitions by with and without 20 tcM dopamine. The the neuroleptics of [3H]spiperone binding to reaction was carried out at 30°C for 6 min, the canine caudate nucleus membranes were and the amount of cyclic AMP was deter also obtained (Table 1). For an easy com mined either by a radioimmunoassay kit parison of the neuroleptics in their affinity

Drug Concentration (-log M)

Fig. 1. Logit-log inhibition plot (Hill plot) of [3H]spiperone binding to the D2-receptors in rat striatum membranes in the presence of 0.3 f-iM mianserin. B„=total binding, Bi=total binding in the presence of the drugs, B,,=nonspecific binding in the presence of 10 IiM cis-flupentixol. The abscissa indicates -log concentration of the drugs . Inhibition of the binding by YM-09151-2 (00), spiperone (O - 0), haloperidol (AA), chlorpromazine (A---- -A), sulpiride (0-0) and clozapine (-L7] n) were determined under the standard conditions. Slopes of the line give the Hill coefficient. Each point is the mean±standard error of six experiments. 752 M. TERAI ET AL.

Table 1. Inhibition by neuroleptics of [3H]spiperone binding to and dopamine-sensitive adenylate cyclase in the membranes from canine caudate nucleus and rat striatum

*: Not determined . **: Data from Sano et al. (5). The value indicates Ki in a micromolar concentration. Ki values were obtained from IC50 as described in the text using at least four different concentrations of the drugs. for the D2-receptors in different animal studied by the same kinetic analysis as that species, the K; values are presented in for D2-receptor binding. The pK, values Table 1. (-log K,) and the Hill coefficients (the slope YM-091 51-2 was studied for its inhibitory of line in the logit-log plot) were calculated effects on dopamine-sensitive adenylate by a linear regression of the data and cyclase (D1-dopaminergic receptors) in the presented in Table 2. The receptors tested membranes from the rat striatum and canine were a1-adrenergic, a2-adrenergic, (3 caudate nucleus. As shown in Table 1, the adrenergic, muscarinic-cholinergic, H1 K, value for the rat membrane was computed histaminergic, 5HT1-serotonergic, 5HT2 to be 0.7 saM, in agreement with the result of serotonergic and opiate receptors. Chlor Jenner and Marsden (19). For the five , clozapine, spiperone and different membrane preparations from haloperidol were potent inhibitors of a1 mongrel dogs, the K; value of YM-091 51-2 binding with the K; varied from 0.07 ,uM to 0.33 jiM with a mean values of 10 nM or below, whereas the value of 0.2 /M. Thus, there was no sub affinity of YM-09151-2 for the a1 stantial species difference in the inhibition of receptor (K,=0.2 ,aM) was only one the cyclase by YM-09151-2 between the twentieth that of haloperidol. The affinity of dog and the rat. Under the same experimental YM-09151-2 and haloperidol for the conditions, sulpiride was ineffective on the 5HT2-receptor (K,=0.1 jiM for both) was dopamine-stimulated activity of adenylate much smaller than that of chlorpromazine cyclase at 10-5 M. YM-09151-2 and (K,=7.6 nM) and spiperone (K,=1.3 nM). spiperone were equipotent, but 10-times Chlorpromazine and clozapine exhibited a less active than haroperidol in the inhibition high affinity with the K, values of lower than of dopamine-sensitive adenylate cyclase in 40 nM for muscarinic and H1-receptors, canine caudate nucleus. These results in whereas micromolar concentrations of YM dicated that YM-09151-2 in contrast to 09151-2, haloperidol and spiperone were other neuroleptics acted rather selectively on required for 50% inhibition in both receptor the D2 type of the two dopaminergic binding assays. Affinities of the neuroleptics receptors. with an exception of clozapine for a1-, (3-, The inhibition of binding of other [3H] 5HT1 and opiate receptors were relatively ligands to their specific receptors was also low or negligibly small at such concentrations D2-SPECIFICITY OF YM-09151-2 753

Table 2. Comparison in the ability of YM-09151-2 and other neuroleptics to bind to nine different types of receptors

*: Less than 15% inhibition at 10-5 M . DHA: Dihydroalprenolol. QNB: Quinuclidinyl benzilate . The values represent -log Ki and its confidence limits at P=0.05. Figures in parenthesis show the Hill coefficient. At least four different concentrations of the drugs were used to determine the inhibition of [3H]-ligand binding. Under the standard binding conditions, -log Ki values of representive positive control drugs for their specific receptors were: 8.23±0.04 for phentolamine in [3H]prazosin binding, 8.12±0.06 for phentolamine in [3H]clonidine binding, 8.82±0.07 for (±)propranolol in [3H] dihydroalprenolol binding, 9.34±0.06 for atropine in [3H]quinuclidinyl benzilate binding, 8.04±0.07 for diphenhydramine in [3H]mepyramine binding, 9.03±0.12 for morphine in [3H]naloxone binding, 6.92±0.08 for methysergid in [3H]serotonin binding and 8.02±0.05 for mianserin in [3H]spiperone binding (5HT2).

as to block D2-receptors. Clozapine blocked induced hypertension, histamine-induced a,-, a2-, muscarinic-cholinergic, H,-histami contraction of the ileum, acetylcholine nergic and 5HT2-serotonergic more potently induced contraction of the ileum or serotonin than D2-dopaminergic receptors. induced head twitch (manuscript in pre paration). Sulpiride which is a benzamide Discussion derivative like YM-09151-2 also exhibits The present experiments demonstrate that higher affinity for the D2-receptor, with a K, YM-09151-2 has a highly selective affinity of 68 nM, than for any other receptors tested. for D2-dopaminergic receptors with a K; of However, it must be mentioned that the 0.1 nM, which is rather similar to that of affinities of YM-091 51-2 and sulpiride for spiperone. The affinity of YM-09151-2 for D3-receptors were 0.98 uM and 1.2 tcM, other receptors was by at least three orders respectively, in our previous report (3) (in of magnitude smaller than that for the D2 reference 3, the term "D2" was used instead receptor. This selective binding was con of D3). Thus, the selectivity of YM-09151-2 sistent with the pharmacological data such between the D2 and D3-receptors is far as antagonism against greater than that of sulpiride. The other 754 M. TERAI ET AL. neuroleptics are not so selective in the the animal was sacrificed. binding to D,-, D2 and D3-recepotrs as In summary, YM-09151-2 is the most YM-09151-2 and also considerably block selective and potent blocker of the D2 a, -adrenergic, muscarinic, H, -histaminergic dopaminergic receptor. or 5HT2-serotonergic receptors at such con Acknowledgments: We are grateful to Dr. centrations as to block the D2-receptor. In M. Harada for his fruitful discussion and to general, YM-09151-2 resembles spiperone Miss A. Sato for her technical assistance. in the receptor binding, except that the former has much less affinity for the a, -adrenergic References receptor. YM-09151-2 also resembles sul 1) Seeman, P.: Brain dopamine receptors. Phar macol. Rev. 32, 229-519 (1980) piride, except for the selectivity between D2 2) Seeman, P.: Nomenclature of central and and D3-receptors. Thus, it is of much interest peripheral dopaminergic sites and receptors. to know what differences are obtained in the Biochem. Pharmacol. 31, 2563-2568 (1982) clinical efficacy between YM-091 51-2 and 3) Usuda, S., Nishikori, K., Noshiro, 0. and Maeno, H.: Neuroleptic properties of cis-N-(1-benzyl the other neuroleptics. Since the antischi 2-methylpyrrolidin-3-yl)-5-chloro-2-methoxy zophrenic effect of neuroleptics is generally 4-methylaminobenzamide (YM-09151-2) with correlated to a blockade of D2-dopaminergic selective antidopaminergic activity. Psycho receptors, a potent D2-selective blocker, YM pharmacology (Berlin) 73, 103-109 (1981) 4) Kurihara, H. and Tadokoro, S.: Effects of cis 09151-2, may be clinically effective with (1-benzyl-2-methylpyrrolidin-3-yl)-5-chloro less side effects resulting from the blockade 2-methoxy-4-aminobenzamide (YM-09151-2) of other receptors and may be useful for a on operant avoidance responses in rats. Folia better understanding of the mechanism for Pharmacol. Japon. 77, 521-530 (1981) (Abs. dopamine actions. in English) 5) Sano, K., Noshiro, 0., Katsuda, K., Nishikori, K. There is a discrepancy between the present and Meano, H.: Dopamine receptors and and previous data (3) with respect to the K; dopamine-sensitive adenylate cyclase in canine values of YM-09151-2 for dopamine caudate nucleus, characterization and solu bilization. Biochem. Pharmacol. 28, 3617 sensitive adenylate cyclase in the canine 3627 (1979) caudate nucleus membranes. The affinity of 6) Greengrass, P. and Bremner, R.: Binding the cyclase for YM-09151-2 in the present characteristics of 3H-prazosin to rat brain (r experiment is evidently lower than the adrenergic receptors. Eur. J. Pharmacol. 55, 323-326 (1979) previous value. There are some possible 7) U'Prichard, D.C., Greenberg, D.A. and Snyder, reasons for this difference. The cyclic AMP S.H.: Binding characteristics of a radiolabeled assay method using the crude binding protein agonist and antagonist at central nervous and charcoal may be partly responsible in the system alpha noradrenergic receptors. Mol. Pharmacol. 13, 454-473 (1977) particular YM-09151-2 inhibition experi 8) Bylund, D.B. and Snyder, S.H.: Beta adrenergic ment, as generally discussed by Oka and receptor binding in membrane preparations Kaneko (20). By this method, the reproducible from mammalian brain. Mol. Pharmacol. 12, inhibition by YM-091 51-2 has been found to 568-580 (1976) 9) Yamamura, H.I. and Snyder, S.H.: Muscarinic be occasionally difficult. Because of this cholinergic binding in rat brain. Proc. Natl. difficulty, we now use the membrane Acad. Sci. U.S.A. 71, 1725-1729 (1974) filtration technique for separation of bound 10) Withy, R.M., Mayer, R.J. and Strange, P.G.: from free cyclic AMP instead of charcoal, as Use of [3H]spiperone for labelling dopaminergic and serotonergic receptors in bovine caudate demonstrated in the present experiment. nucleus. J. Neurochem. 37, 1144-1 154 (1981) Other responsible factors may be ages of 11) Leysen, J.G., Gommeren, W. and Laduron, the mongrel dogs and the season in which P.M.: Spiperone: a ligand of choice for neu D2-SPECIFICITY OF YM-09151-2 755

roleptic receptors, 1. kinetics and characteristics 16) Brown, B.L., Albano, J.D.M., Ekins, R.P., of in vitro binding. Biochem. Pharmacol. 27, Sgherizi, A.M. and Tampion, W.: A simple and 307-316 (1978) sensitive saturation assay method for the 12) Tran, V.T., Chang, R.S.L. and Snyder, S.H.: measurement of adenosine 3:5'-cyclic mono Histamine H, receptors identified in mammalian phosphate. Biochem. J. 121, 561-562 (1971) brain membranes with [3H]mepyramine. Proc. 17) Kumon, A., Yamamura, H. and Nishizuka, Y.: Natl. Acad. Sci. U.S.A. 75, 6290-6294 (1978) Mode of action of adenosine 3',5'-cyclic 13) Chang, K.-J. and Cuatrecasas, P.: Multiple phosphate on protein kinase from rat liver. opiate receptors, enkephalins and morphine Biochem. Biophys. Res. Commun. 41, 1290 bind to receptors of different specificity. J. Biol. 1297 (1970) Chem. 254, 2610-2618 (1979) 18) Lowry, O.H., Rosebrough, N.J., Farr, A.L. and 14) Peroutka, S.J. and Snyder, S.H.: Multiple Randall, R.J.: Protein measurement with the serotonin receptors: differential binding of Folin phenol reagent. J. Biol. Chem. 193, 265 [3H]5-hydroxytryptamine, [3H]Iysergic acid di 275 (1951) ethylamide and [3H]spiroperidol. Mol. Phar 19) Jenner, P. and Marsden, C.D.: Substituted macol. 16, 687-699 (1979) benzamide drugs as selective neuroleptic 15) De Lean, A., Munson, P.J. and Rodbard, D.: agents. Neuropharmacology 20, 1285-1293 Simultaneous analysis of families of sigmoidal (1981) curves: application to bioassay, radioligand 20) Oka, H. and Kaneko, T.: Protein binding assay assay, and physiological dose-response curves. for cyclic AMP. Taisha (Metabolism and Am. J. Physiol. 235, E97-E102 (1978) Disease) 9, 715-720 (1972) (in Japanese)