Neurochemical Investigation on the Effects of a New Diphenylpiperazine Calcium Antagonist, KB-2796, on the Central Dopaminergic System of Rats

Akira Ikegami, Akio Ozaki, Hideaki Hara * , Takayuki Sukamoto, Akira Yamashita and Keizo Ito

New Drug Research Laboratories, Kanebo, Ltd., 1-5-90, Tomobuchi-cho, Miyakojima-ku, Osaka 534, Japan

Received October 26, 1991 Accepted December 26, 1991

ABSTRACT-The effects of KB-2796, a new diphenylpiperazine calcium antagonist, on the striatal dopaminergic system of rats were investigated in comparison with var ious calcium antagonists and the dopamine antagonist chlorpromazine. The inhibiting effect of KB-2796 on [3H]spiperone binding to striatal membranes in vitro was weaker than those of chlorpromazine and the other diphenylpiperazine analogues, flunarizine and cinnarizine, and more potent than those of verapamil and nicardipine. Diltiazem and nifedipine were inactive. KB-2796 (30, 100 mg/kg, p.o.) had no effect on Kd and Bmaxvalues of in vitro [3H]spiperone specific binding to striatal membranes obtained from the rat at 36 hr and 7 days after repeated administration for 18 days, whereas flu narizine (30 mg/kg, p.o.) and chlorpromazine (3 mg/kg, p.o.) increased Bmax values by 47% and 31%, respectively, at 36 hr, but not at 7 days after the final administra tion. At 1 hr after the single administration, KB-2796 (30, 100 mg/kg, p.o.) had no effect on the content of dopamine and its metabolites in the striatum, whereas flunar izine (30 mg/kg, p.o.) and chlorpromazine (3 mg/kg, p.o.) increased the level of homovanillic acid. These results indicate that flunarizine may affect dopaminergic neurotransmission by partially blocking dopamine D2 receptors, while KB-2796 has negligible in vivo effect on the dopaminergic system.

Calcium antagonists are drugs widely pre mine D2 receptors in the striatum (3, 4). scribed for cardiovascular and cerebrovascular Therefore, interaction of these calcium antago disorders. Their therapeutic effectiveness re nists with the dopaminergic transmission sys lies on their abiliy to block calcium entry tem is thought to be a possible mechanism of through voltage sensitive calcium channels. occurrence of the side effects. Recently, side effects such as extrapyramidal In recent years, several investigators have symptoms and depression have been observed demonstrated that some calcium antagonists in patients, especially in elderly patients, treat inhibit dopamine release (5, 6) and directly in ed with calcium antagonists such as flunarizine teract with dopamine recognition sites, there and cinnarizine (1, 2). Neuroleptics such as by possibly affecting dopaminergic transmis chlorpromazine are known to elicit extrapyram sion (7). Flunarizine, a diphenylpiperazine cal idal side effects by blocking postsynaptic dopa cium antagonist, inhibits dopamine release from rat striatal slices in vitro (8) and [3H]spiperone binding to rat striatal homoge nate (9). Govoni et al. (10) have reported that Drug Research Laboratories of Kanebo. The flunarizine and nimodipine, a dihydropyrizine following drugs were purchased from Sigma: calcium antagonist, increase the numbers of flunarizine dihydrochloride, cinnarizine, nife [3H]spiroperidol binding sites with a reduction dipine, nicardipine hydrochloride, chlorproma of the binding affinity after repeated adminis zine hydrochloride, pargyline hydrochloride tration in rats. Furthermore, the binding affin and caffeic acid. (+)-[Phenyl-4-3H]spiperone ity of flunarizine for dopamine D2 receptors (specific activity, 1.07 TBq/mmol) was obtained has been shown to be higher than that for the from Amersham; (+)-butaclamol, from Re Ca2+ channel sites (11). search Biochemicals; and sodium octyl sulfate, KB-2796, 1-[bis(4-fluorophenyl)methyl] -4 from Nacalai Tesque. (2,3,4-trimethoxybenzyl)piperazine dihydro chloride, is a new diphenylpiperazine calcium Preparation of membrane and binding assay antagonist which exhibits selective vasodilator Preparation of membrane and [3H]spiperone activity on cerebral vessels (12). KB-2796 dis binding assays were performed according to places [3H]nitrendipine binding to homoge the method reported by Govoni et al. (10) nates of guinea pig cortex (13) and canine ves with minor modifications. Rats were decapi sels (14). We have previously reported that tated and their brains were quickly removed. KB-2796 does not inhibit apomorphine Striatal tissues were dissected and frozen in liq induced stereotyped behaviors such as climb uid nitrogen and kept at -80°C until assayed. ing and circling in mice, but it inhibits Tissues were homogenized in 30 volumes of methamphetamine-induced locomotion and ice-cold 50 mM Tris-HC1 buffer (pH 7.4), circling at a high dose (15). However, the washed by centrifugation at 30,000 X g, and neurochemical mechanism of KB-2796 on the finally resuspended in 40 volumes of 50 mM central dopaminergic system is not well Tris-HC1 buffer, pH 7.4 (buffer B) containing known. There are only a few reports on the 120 mM NaCl, 5 mM HCl, 2 mM CaC12, 1 MM effect of the in vivo chronic treatment with MgC12, 0.1% ascorbic acid and 10,uM pargy calcium antagonists on dopaminergic receptors line. (10). In this study, therefore, we examined the For determining the in vitro effects of drugs binding profile of KB-2796 to dopamine D2 on dopamine D2 receptors, binding assays receptors and its effect on dopamine metabo were performed by incubating 1.9 ml of buffer lism in the striatum of rats to determine its B containing [3H]spiperone (final concentra possible influence on dopaminergic transmis tion: 0.4 nM) and each drug at various con sion. centrations with 0.1 ml of crude synaptic mem branes (final concentration: 1.25 mg-tissue/ml) MATERIALS AND METHODS at 37°C for 20 min. (+)-Butaclamol (10 pM) was used to define non-specific binding. The Animals binding assay was terminated by rapid vacuum Male Wistar rats (Japan SLC, Inc.) filtration through Whatman GF/C glass fiber weighing 200 220 g were used. Animals were filters. housed in a temperature and humidity-con For determining the in vivo chronic effects trolled room (12 hr light and 12 hr dark cycle) of drugs on dopamine D2 receptors, drugs sus with free access to food and water. pended in 5% arabic gum were orally adminis tered to a group of 5 rats once a day for 18 Drugs days. The animals were killed 36 hr and 7 days KB-2796 was synthesized and verapamil and after the final administration of drugs, accord diltiazem were extracted from Wasoran tablets ing to the method reported by Govoni et al. (Eisai Co. Ltd.) and Helvessor tablets (10). Striatal tissues and membrane fractions (Tanabe Co. Ltd.), respectively, by the New were obtained, and binding assays were per formed by the same method described above, RESULTS except that 6 concentrations of [3H]spiperone ranging from 0.0125 to 0.7 nM were used. The In vitro effects of calcium antagonists on dissociation constant (Kd) and the density of [3HJspiperone binding binding sites (Bmax) were determined by As shown in Fig. 1, among the calcium Scatchard analysis (16). Protein content was antagonists tested, KB-2796, flunarizine, cin determined according to Lowry et al. (17). narizine, verapamil and nicardipine inhibited [3H]spiperone binding to striatal membranes Determination of the content of dopamine and in a concentration-dependent manner. Diltia its metabolites zem also slightly inhibited binding; however, A group of 5 fasted rats were orally admin the K; value was greater than 15,000nM. Nife istered drugs suspended in 5% arabic gum. dipine had almost no effect on displacement of One hour after administration, the animals binding, with inhibition of 7% at 10-4 M. were killed by decapitation. Their brains were The K; values and Hill coefficients for each removed, and the striatal tissues were dis drug are shown in Table 1. Flunarizine, the sected on ice, frozen in liquid nitrogen, and most potent drug among the calcium antago stored at -80°C until assayed. nists tested, was approximately 5 times less The striatal content of dopamine and its potent in inhibiting [3H]spiperone binding, metabolites was measured by the method of and KB-2796 was approximately 35 times less Warnhoff (18). The tissues were homogenized potent than chlorpromazine. The Hill coeffi in 10 volumes of 0.05 M perchloric acid con cient for KB-2796, flunarizine, cinnarizine and taining 0.0032% EDTA-2Na and 1/10 volumes verapamil were close to 1, whereas the coeffi of caffeic acid (3,uM) as an internal standard, cient for nicardipine was less than 1. and the homogenate was centrifuged at 20,000 X g at 4°C for 15 min. High pressure liquid Effects of repeated administration of KB-2796, chromatography (HPLC) was performed on 20 flunarizine and chlorpromazine on f3HJspiperone ,ul of supernatant, using an electro-chemical binding detector (ECD). Figures 2 and 3 show representative data of

Fig. 1. Displacement curves for [3H]spiperone (0.4 nM) specific binding to rat striatal membranes in the presence of chlorpromazine (•), flunarizine (LI), cinnarizine (•), KB-2796 (0), verapamil (A) and nicar dipine (o). Specific binding was defined using 10-s M (+)-butaclamol. Each value represents the mean of 3 experiments performed with duplicate samples. Table 1. Inhibition by various drugs of [3H]spiperone Scatchard analysis on [3Hjspiperone binding to (0.4 nM) specific binding to rat striatal membranes rat striatal membranes obtained 36 hr and 7 days, respectively, after repeated administra tion of KB-2796, flunarizine and chlorproma zine for 18 days. Tables 2 and 3 represent the average Kd and Bmax values obtained from 4 experiments. KB-2796 at 30 and 100 mg/kg did not affect Kd and Bmaxvalues. Flunarizine at 30 mg/kg and chlorpromazine at 3 mg/kg significantly increased Bmax values by 47% (P < 0.01) and 31% (P < 0.01), respectively, af ter 36 hr of washing out the drugs, whereas Kd values were not changed by administration. After 7 days of washing out the drugs, all changes in the binding parameters had dis appeared.

Fig. 2. Effect of KB-2796 (-•A• 30mg/kg, --0 100 mg/kg), flunarizine ( • 30 mg/kg) and chlor promazine (--El- 3 mg/kg on [3H]spiperone specific binding at 36 hr after repeated administration for 18 days. Each point is the mean of duplicate determina Fig. 3. Effect of KB-2796, flunarizine and chlorpro tions in a representative experiment. The lines deter mazine on [3H]spiperone binding at 7 days after re mined by linear regression analysis of Scatchard plots peated administration for 18 days. For further explana are indicated. -0 control. tions, see Fig. 2. Table 2. Effect of KB-2796, flunarizine and chlorpro cantly increased HVA content by 26% (P < mazine on [3H]spiperone binding at 36 hr after re 0.05) and 24% (P < 0.05), respectively. peated administration for 18 days DISCUSSION

KB-2796 displaces [3H]spiperone binding to dopamine D2 receptors with an intermediate potency among the calcium antagonists tested. The potency of KB-2796 was weakest among the other diphenylpiperazine analogues such as flunarizine and cinnarizine. Furthermore, KB-2796 influenced neither the binding profile of [3H]spiperone after repeated administration for 18 days nor dopamine metabolism even at a high dose of 100 mg/kg, p.o. We have re ported that KB-2796 and flunarizine protect against KCN-induced death in mice, with ED50 values of 40 and 28 mg/kg, p.o. (19). Table 3. Effect of KB-2796, flunarizine and chlorpro Furthermore, we have reported that KB-2796 mazine on [3H]spiperone binding at 7 days after re at 100mg/kg, p.o. does not affect apomorphine peated administration for 18 days induced stereotyped behavior such as cage climbing and turning and the content of dopa mine and its metabolites and dopamine turn over rate in mice (15). Flunarizine at 30 mg/kg, p.o. inhibits these behavioral changes and increase the content of the dopaminergic system (15). The LD50 value for KB-2796 is 506 mg/kg, p.o. in male rats (T. Unno et al., unpublished data). These results may indicate that although KB-2796 indeed has weak bind ing affinity to dopamine D2 receptors in vitro, its interaction with dopamine D2 receptors is negligible after systemic administration in vivo. However, it remains to be determined why KB-2796 did not affect in vivo dopaminergic Effects of single administration of KB-2796, neurotransmission in spite of exhibiting a flunarizine and chlorpromazine on the content weak activity in displacing [3H]spiperone bind of dopamine and its metabolites ing to dopamine D2 receptors. Pharmacokinet Table 4 shows the content of dopamine and ic parameters of KB-2796 such as absorption, its metabolites in rat striatum 1 hr after a sin penetration into the brain, and metabolism gle administration of KB-2796, flunarizine or may be possible factors which influence the in chlorpromazine. KB-2796 did not significantly vivo action of the drug. Waki et al. (20) have affect the content of dopamine, dihydroxy reported that KB-2796 and flunarizine admin phenyl acetic acid (DOPAC), homovalinic istered orally penetrate into the brain of rats. acid (HVA) and 3-methoxytyramine (3-MT), In fact, KB-2796 as well as nicardipine, which even at a dose of 100 mg/kg. Flunarizine at 30 is the weakest displacer of [3H]spiperone bind mg/kg and chlorpromazine at 3 mg/kg signifi ing in the present experiment, inhibit metham Table 4. Effect of KB-2796, flunarizine and chlorpromazine on DA and DA metabolites con tents in rat striatum

phetamine-induced locomotion and circling in nerve function might be modified by in vivo mice (17). This kind of inhibitory effect has chronic blockade of calcium antagonist-sensi also been reported with other calcium antago tive, voltage-dependent calcium channels, nists such as flunarizine and nifedipine (21) since nimodipine which has no binding affinity and verapamil (22). In amphetamine-induced to dopamine D2 receptors increased Kd and circling, calcium is supposed to play an impor Bmax values for [3H]spiperone binding. In the tant role (22). These facts may indicate that present experiment, KB-2796 did not affect KB-2796 given orally can penetrate into the the binding characteristics of [3H]spiperone af brain. However, the concentration in the brain ter chronic administration. KB-2796 is about after oral administration of KB-2796 could not three times more potent than flunarizine in reach a sufficient level to exert its effects on displacing [3H]nitrendipine binding to guinea dopamine D2 receptors. pig cortex membrane (13). Although we did Of the calcium antagonists tested in the pres not measure the brain concentration of KB ent study, flunarizine was found to be one of 2796 in the present experiments, as aforemen the most potent displacers of [3H]spiperone tioned, KB-2796 can penetrate into rat brain binding to dopamine D2 receptors in vitro and and is expected to exert its calcium antago found to increase Bmax values after repeated nistic activity in vivo. Therefore, the change in administration without changing Kd values as dopaminergic transmission by in vivo chronic observed with chlorpromazine. On the other administration of calcium antagonists cannot hand, Govoni et al. (10) also observed an in be simply explained by the blockade or the crease in the Bmax and Kd values with re modification of calcium antagonist-sensitive, peated administration of flunarizine. This dis voltage-dependent calcium channels. On this crepancy in the Kd value cannot be explained point, we need further studies to determine at present. The Kd value may be possibly in the role of calcium channels in the modulation fluenced by the amount of drug remaining in of dopaminergic neurotransmission. the brain after some washout periods. These results indicated that KB-2796 may Moreover, a difference in the strain of rats have negligible influence on in vivo central used may be a possible cause of the discrepan dopaminergic neurotransmission and may be cy. expected to cause less extrapyramidal side Govoni et al. (10) supposed that normal effects than flunarizine. Acknowledgments Edited by Amery, W.K., Van Neuten, J.M. and The authors wish to thank Ms. Y. Sakita and Ms. Y. 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