PRELIMINARY COMMUNICATION BETAHISTINE INHIBITS FOOD INTAKE IN RATS

Copyright 2001 by Institute of Pharmacology Polish Academy of Sciences Polish Journal of Pharmacology Pol. J. Pharmacol., 2001, 53, 701 707 ISSN 1230-6002 PRELIMINARY COMMUNICATION BETAHISTINE INHIBITS FOOD INTAKE IN RATS Adam Szel¹g, Ma³gorzata Trocha, Anna Merwid-L¹d Department of Pharmacology, Medical University of Wroclaw, Mikulicza-Radeckiego 2, PL50-345 Wroc³aw, Poland Betahistine inhibits food intake in rats. A. SZEL G, M. TROCHA, A. MERWID- -L D. Pol. J. Pharmacol., 2001, 53, 701 707. Betahistine, administered intraperitoneally, decreased, in a dose-dependent manner and in a statistically significant degree, total food intake in different experimental models in rats. Key words: betahistine, food intake, H 3 receptors, intraperitoneal injection INTRODUCTION Four groups of receptors are of great importance in food intake regulation: serotoninergic, histaminergic, adrenergic and dopaminergic receptors. 5-HT 1a and 2 receptor activation increases while 5-HT 1b/d, 5-HT 2c, 1, 2,H 1 or H 3 and D 1 or D 2 activation decreases food intake [7]. The role of 3 and 5-HT 3 receptors in food intake regulation is still unclear [6, 10, 14, 27 30]. Because of many adverse effects of drugs acting at the central nervous system level, there is a need to synthesize new peripherally acting groups of drugs reducing body weight [8]. Brain histamine has been shown to influence various hypothalamic functions, such as feeding, chewing processes, drinking, neuroendocrine secretion, the sleep-wakefulness cycle and thermoregulation [9]. It has been suggested that H 1 receptor activation and/or H 3 receptor inhibition decreases food intake [22, 23]. Clinical practice indicates that H 1 antihistaminic drugs (first generation), taken continuously, cause body weight increase. In rats, the stimulation of H 1 receptors inhibits food intake [22]. A lot of H 1 and H 3 receptors are located in the hypothalamus, especially in the regions connected with food intake regulation. H 3 receptors mediate synthesis of histamine and its release from histaminergic nerve endings [3 5]. Released histamine can subsequently influence other receptors, e.g. H 1. Thioperamide (selective H 3 receptor antagonist) increases histamine synthesis and release and in this way increases histaminergic neuron activity [1]. It correspondence

A. Szel¹g, M. Trocha, A. Merwid-L¹d has been demonstrated that thioperamide, at a dose of 100 nmol, inhibits food intake after intracerebroventricular (icv) microinjections. Chlorpheniramine (H 1 receptor antagonist), after intraperitoneal (ip) administration at a dose of 26 mol/kg, abolishes this effect of thioperamide, but administered alone at the same dose has no effect on food intake [20]. The data suggest that H 1 receptors, located in the ventromedial and paraventricular hypothalamic nuclei, receive stimuli inhibiting food intake from the tuberomammilar nucleus through histaminergic neurons. It has been demonstrated that these histaminergic neurons regulate feeding circadian rhythm throughout H 1 but not H 2 receptors. Chlorpheniramine attenuates thioperamide activity, which inhibits food intake during the early dark period, when histaminergic neuron activity is low and rats normally feed. -Fluoromethyl-histidine (histidine decarboxylase inhibitor), given icv, increased food intake in the early light period when histaminergic neuron activity is high [9, 13, 16, 17, 20, 21, 24, 25]. In the hypothalamus, histamine is a mediator of satiety additionally increased by food chewing. The tuberomammilar nucleus is linked with the nucleus of the trigeminal nerve. The motor nucleus of the trigeminal nerve receives stimuli from the musseter muscle and the peridental ligament. It is suggested that histamine turnover in the nucleus of trigeminal nerve starts parallelly with the beginning of chewing while histamine turnover in the ventromedial nucleus is inhibited when food consumption is finished [9, 12, 22]. Histamine concentration is increased in the brains of rats deprived of food for 24 h. This may explain the lack of appetite after the period of fasting [22]. In the hypothalamus, the activation of histaminergic mechanisms in response to deficiency of energy, may play an important role in the homeostatic control in the brain [22]. It is known that high temperature decreases food intake and increases water consumption. -Fluoromethyl-histidine administered directly into the hypothalamus abolishes abilities to adapt to the changes in ambient temperature. That is why rectal temperature increases or decreases in relation to the changes in ambient temperature. Therefore, in the hypothalamus, the enhancement of histamine level decrease disturbs homeostasis [11]. Previous studies concerning the role of histamine H 3 receptors in the food intake regulation were mostly carried out after injections of H 3 histamine receptor modulators into the cerebral ventricle or into different regions of the hypothalamus. The results of this research confirm that the regulation of histamine release may be of practical importance. There is no strong evidence of efficacy of these modulators when they are administered not directly to the brain but systemically. This study has been carried out on various groups of rats which were given betahistine (H 3 receptor antagonist and H 1 receptor agonist) systemically (ip), and its influence on food intake has been examined. MATERIALS and METHODS Animals The study was carried out on adult male Wistar rats (280 310 g) obtained from the Experimental Animal Farm at the Pharmaceutical Company Jelfa, Jelenia Góra, Poland. The rats were housed individually in chambers with a 12:12 h light-dark circle and temperature maintained at 21 23 C. Before the experiment animals had free access to standard food and water. All experiments were performed after at least two weeks of adaptation to this environment. Chemicals and drugs Betahistine dihydrochloride (Betaserc, tablets 8 mg, Solvay Duphar B.V., the Netherlands) and Tween 80, liquid (Loba, Feinchemie, Austria) were used in the study. Betahistine was suspended in 1% Tween 80 solution, in the volume of 4 ml/kg of body weight. Experiment Animals were divided into three experimental groups. The first one was on a normal diet, the second was deprived of food and the third group was accustomed to tasty food. During experiments animals were kept individually in chambers with free access to water. After the period of adaptation to the experimental conditions rats had free access (through short tunnels with grid floor) to chow contained in bins placed atop two electronic balances (exact to 0.1 g). The balances were connected to 702 Pol. J. Pharmacol., 2001, 53, 701 707

BETAHISTINE AND FOOD INTAKE a microcomputer that measured cumulative food intake throughout the experiment. The first experiment investigated the influence of betahistine on food intake in the group of rats kept on a normal diet. After 24 h of adaptation to new conditions, the rest of chow was removed and new portions of food were weighed out exact to 0.1 g. The rats were divided into four groups and they were given food just after ip betahistine administration at a dose of 1 mg/kg (first group, n = 10) and a dose of 24 mg/kg (second group, n = 10), and 30 min after intragastrical betahistine administration at a dose of 24 mg/kg (third group, n = 10). Quantity of food consumed after ip drug administration was assessed 1, 2, 4 and 24 h after food had been given. After intragastrical drug administration, quantity of the consumed food was assessed 4 and 24 h after food had been given. The rats in the control group (fourth group, n = 10) were given 1 % Tween 80 solution ip or po, respectively. Other conditions of the experiment were unchanged. The second experiment investigated the influence of betahistine on food intake in the group of rats accustomed to tasty diet. Tasty food was prepared from standard food mixed with sweet, condensed milk and distilled water in proportion 200 g : 50 ml : 200 ml, respectively. This kind of food, in 40 g portions, was given to the animals for 30 min every day between 9:00 and 9:30. During this time the standard food, in pelets, was removed. The rats had free access to water. The experiment was carried out after 10 days of adaptation, when the quantity of the consumed food was on a steady level. The rats were divided into three groups: two examined and one control group. Betahistine was administered ip at doses of 1 mg/kg (first group, n = 8) and 8 mg/kg (second group, n = 8). Thirty minutes after ip drug administration, the standard food was removed and the tasty food was given. Food intake was assessed after the next 30 min. The control group (n = 10) was given 1% Tween 80 solution in the same volume as betahistine (4 ml/kg). Other conditions of the experiment were unchanged. The third experiment tested the influence of betahistine on food intake in the group of rats deprived of food, trained for ten days. The rats were fasted for 20 h per day (water ad libitum). After 10 days of training, when food intake was stabilized, the rats were divided into three groups. The first group of rats (n = 8) was administered betahistine at a dose of 8 mg/kg ip and the second one (n = 8) 24 mg/kg ip. Standard food was given 30 min thereafter. Food intake was assessed 1, 2, 4 h after food had been given. The control group (n = 10) was given 1% Tween 80 solution in a volume of 4 ml/kg ip. Statistical analysis All data were statistically analyzed by Student s t-test and a p value of 0.05 was considered statistically significant. The data are presented as a mean and standard deviation. RESULTS Table 1 shows that in the group of rats kept on the normal diet betahistine decreased total food intake in a dose-dependent manner. The dose of 1 mg/kg ip reduced food intake by: 74.7% (p 0.01), 72.9% (p 0.005), 64.4% (p 0.05), 6.6% (NS) after 1, 2, 4 and 24 h, respectively. Except for the last result (after 24 h) all other results were statistically significant. Betahistine at the dose of 24 mg/kg revealed stronger activity, reducing food intake by: 93.1% (p 0.001), 88.1% (p 0.001), 86.7% (p 0.001) and 81.0% (p 0.001) after 1, 2, 4 and 24 h, respectively. All results were statistically significant. After drug administration at 24 mg/kg food intake was decreased by 72.9% (NS), 56% (NS), 62.2 (p 0.05), 79.7% (p 0.001), after 1, 2, 4 and 24 h, respectively, comparing to corresponding values obtained with the dose of 1 mg/kg. The differences were statistically significant only after 4 and 24 h. In the third group of rats (Tab. 2) betahistine, given intragastrically at the dose of 24 mg/kg, reduced food intake only by 21.7% (NS). After 24 h, the tested substance was more potent and decreased food intake by 45.2% (p 0.001). Table 3 shows that in all groups accustomed to tasty food betahistine significantly reduced total food intake in a dose-dependent manner. The drug dose of 1 mg/kg reduced quantity of the consumed food by 40.3% (p 0.02). At the higher dose (8 mg/kg), the tested substance decreased food intake considerably (by 75.8%, p 0.001). Both results were statistically significant. Table 4 shows that betahistine also decreased food intake in a dose-dependent manner in groups of rats deprived of food and trained for 10 days. ISSN 1230-6002 703

A. Szel¹g, M. Trocha, A. Merwid-L¹d Betahistine, administered at the dose of 8 mg/kg ip, reduced the quantity of the consumed food by 67.5%, 70.4%, 56.9% and at 24 mg/kg by 86.9%, 87.0% and 75.0% after 1, 2 and 4 h, respectively. All results were highly statistically significant (p 0.001). After drug administration at 24 mg/kg, food intake was decreased by 59.7% (p 0.02), 56.1% (p 0.005), 42.0% (p 0.01), after 1, 2 and 4 h, respectively, comparing to corresponding values obtained with the dose of 8 mg/kg. All differences were statistically significant. Table 1. The influence of betahistine, given ip, on food intake in the groupof rats kept on a normal diet Food intake (g) after: Treatment 1h 2 h 4 h 24 h n = number Control group: 1 % Tween 80 4 ml/kg ip 2.77 ± 0.47 3.69 ± 0.56 5.33 ± 0.74 23.21 ± 1.96 10 100 100 100 100 Betahistine 1 mg/kg ip : 2 Betahistine 24 mg/kg ip 2 : 0.7 ± 0.31* 1.0 ± 0.35** 1.9 ± 0.53** 21.68 ± 0.96 10 25.3 27.1 35.6 93.4 0.19 ± 0.02*** 0.44 ± 0.03*** 0.71 ± 0.05*** 4.4 ± 1.03*** 10 6.9 11.9 13.3 19,, 2 mean values; * p 0.01, ** p 0.005, *** p 0.001 Table 2. The influence of betahistine, given intragastrically, on food intake in the groupof rats kept on a normal diet Food intake (g) after: Treatment 1h 2h 4h 24h n = number Control group: 1 % Tween 80 4 ml/kg po 2.21 ± 0.21 3.02 ± 0.28* 5.03 ± 0.39** 22.6 ± 1.41** 10 100 100 100 100 in relation to 1 h 137% 239% 1159% Betahistine 24 mg/kg po : 3.94 ± 1.07 12.38 ± 1.62** 10 78.3 54.8, mean values; * p 0.05, ** p 0.001 704 Pol. J. Pharmacol., 2001, 53, 701 707

BETAHISTINE AND FOOD INTAKE DISCUSSION Table 3. The influence of betahistine, given ip, on food intake in the groupof rats accustomed to a tasty diet Control group: 1 % Tween 80 4 ml/kg ip 100 Betahistine 1 mg/kg ip : 2 Betahistine 6 mg/kg ip 2 : Food intake (g) after 1 h n = number 11.56 ± 1.59 10 6.9 ± 0.76* 8 59.7 2.8 ± 0.51** 8 24.2,, 2 mean values; * p 0.02, ** p 0.001 In all examined groups, betahistine administered ip decreased food intake. It has been suggested that the inhibition of H 3 receptor activity increases histamine synthesis and release. Histamine subsequently increases histaminergic neuron activity via H 1 receptors and in this way it inhibits food intake [22]. It should be taken into consideration that in the groups of rats on a normal diet and accustomed to tasty food, food chewing (free access to food) increases histamine concentration in the hypothalamus, inhibiting food intake [9, 12, 22]. Similar changes in histamine concentration were observed in the groups of rats deprived of food, when fasting also decreased food intake [19, 26]. Analysis of the influence of H 3 receptor antagonists on food intake points to two mechanisms increasing histamine level thereby decreasing food consumption: 1) free access to food or deprivation of food; 2) increasing histamine synthesis and release as a result of H 3 receptor inhibition after betahistine administration. Taking the above conditions into consideration, the results of the experiments concerning the role of histamine in food intake after administration of H 1 receptor antagonists directly into the brain (the hypothalamus, cerebral ventricles) differ from the results obtained after systemic administration of these substances. There are differences between a rat circadian rhythm and that of human. That is why data from the studies on the action of H 1 receptor antagonists in rats are different from those obtained Table 4. The influence of betahistine, given ip, on food intake in the groupof rats deprived of food, trained ten days Food intake (g) after: 1 h 2 h 4 h n = number Control group: 1 % Tween 80 ip 9.23 ± 0.26 16.63 ± 0.95 24.06 ± 1.55 10 100 100 100 Betahistine 8 mg/kg ip : 2 Betahistine 24 mg/kg ip 2 : 3.0 ± 0.54* 4.92 ± 0.7* 10.36 ± 1.19* 8 32.5 29.6 43.1 1.21 ± 0.43* 2.16 ± 0.47* 6.01 ± 0.91* 8 13.1 13.0 25.0,, 2 mean values; * p 0.001 ISSN 1230-6002 705

A. Szel¹g, M. Trocha, A. Merwid-L¹d in men. A more potent anorectic action of H 1 receptor antagonists is revealed during the activity period, i.e. the day in men and the night in rats. As mentioned above, histamine regulates circadian rhythm of food intake through H 1 receptors, the stimulation of which decreases food intake in the daytime and increases at night. Therefore, H 1 receptor antagonist administration increases food consumption in men, because people are accustomed to having meals during the day (the lack of inhibitory action of endogenous histamine in the daytime) and decreases food intake in rats, because they feed mostly at night (inhibitory action of endogenous histamine at night). The possibility that the influence of histamine on food intake may be independent of day-night cycle but may depend on feeding-fasting time should also be taken into consideration. Thus, histamine might act on humans in the daytime similarly as it acts on rats at night. The lack of influence of antazoline and promethazine (H 1 receptor antagonists), at doses acting centrally, on betahistine-evoked inhibition of food intake undermines the important role of H 1 receptors in the mechanism of betahistine action [15]. Betahistine inhibits food intake in the group of fasted rats and in the group of rats accustomed to tasty food, when administered ip at very small doses. Other in vitro works showed betahistine binding to H 1 receptors after very high doses: 150 300 mg/kg ip [2]. That is why it has been suggested that betahistine acts via H 3 receptor inhibition, but histamine does not have to be a mediator. H 3 histamine receptor modulation can influence the release of other mediators such as serotonin or noradrenaline which are also connected with food intake regulation. Serotonin might inhibit food intake through 5-HT 1C receptor. However, the role of released serotonin (after H 3 receptor inhibition by betahistine) was excluded since ritanserine (5-HT 1C receptor antagonist) at 0.5 0.6 mg/kg did not influence food intake [18]. The results of different research suggest the important role of histamine and histamine H 3 receptors in food intake regulation. However, almost all of them were carried out after intraventricular microinjections. Our experiment indicates that after systemic (ip) administration of betahistine in rats, the quantity of the consumed food was decreased. It cannot be excluded that after intragastrical administration of this substance, food intake will also be reduced. Lack of the influence of betahistine on food intake 4 h after intragastrical administration of the tested drug could be explained by the fact that betahistine increased hydrochloric acid release. It can abolish the central anorectic activity of betahistine. 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