Neurochemical Investigation on the Effects of a New Diphenylpiperazine Calcium Antagonist, KB-2796, on the Central Dopaminergic System of Rats
Total Page：16
File Type：pdf, Size：1020Kb
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.