J . vet. Phannacol. Therap. 12, 147-156, 1989.

Appetite-modulating drugs in dwarf goats, with special emphasis on benzodiazepine- induced hyperphagia and its antagonism by flumazenil and R"15-3505 A. S. J . P. A. M. VAN MIERT, M. KOOT & C. T. M. VAN DUIN Department of Veterinary Basic Sciences, Division of Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

van Miert, A.S.J.P.A.M., Koot, M. & van Duin, C.T.M. Appetite-modulating drugs in dwarf goats, with special emphasis on benzodiazepine-induced hyperphagia and its antagonism by flumazenil and R"15-3505. J. vet. Pharmacol. Therap. 12, 147-156. In dwarf goats fasted for 2 h, i.v. administration of the benzodiazepine (BZ) agonists diazepam (60 pg/kg), brotizolam (2 and 4 pg/kg) and climazolam (100 pg/kg) induced hyperphagic effects, whereas i.v. injections of the BZ-antagonist flumazenil (R"15-1788; 0.5 mg/kg), the anthelmintic ivermectin (0.1 mg/kg), the 5-HT2 antagonist ritanserine (0.1 mglkg), ACTH (10 pg/kg) and prednisolone (1 mglkg) were inactive in a 30-min feeding test. Both the BZ-antagonist R"15- 3505 (3 0.1 mg/kg) and the opiate receptor antagonist naloxone (0.1 mg/kg) had anorectic effects in dwarf goats given 30 min access to a palatable pelleted concentrate. The hyperphagic effects of climazolam and brotizolam were not antagonized by flumazenil, whereas similar doses of this drug completely reversed muscle incoordination and ataxia induced by much higher doses of these BZ-agonists. In the combination experiments with naloxone and BZ- agonists, naloxone antagonized the hyperphagic effects of both diazepam and brotizolam. Similarly, in the diazepam-li"l5-3505 study, there was a significant effect of diazepam and a significant inhibition of this effect by R"15-3505 (50 pg/kg). In the diazepam-ivermectin combination experiment no evidence for drug potentiation was found. These results and the mode of action of the above mentioned drugs are discussed in relation to feeding behaviour. A. S . J . P . A . M . van Miert, Department of Veterinaly Basic Sciences, Division PPT, P.O. Box 80176, 3508 TD Utrecht, The Netherlands.

INTRODUCTION

Weaning, social upheaval, transport, crowd- ing and mixing with resultant requirements to re-establish a social order may decrease feed intake and render animals more susceptible to infection. Furthermore, anorexia is a common sign of febrile diseases (Baile et al., 1981; Phillips, 1984; van Miert et al., 1986a). In

order to reduce the loss of production due to these factors, a better understanding of the benefits and detriments of both feed intake stimulants and anorexia is required.

The regulation of body weight under vari- ous states of energy need depends on an animal's ability to control feed intake to meet these needs. Based on the available evidence, it appears that in the central nervous system

147

148 A. S . J. P. A. M. van Miert, M. Koot 6 C. T. M. van Duin

the ventromedial and lateral hypothalamus are the areas involved in satiety and hunger, respectively. Other important areas are the paraventricular nucleus and rostra1 brain areas (Morley, 1987; Baile & McLaughlin, 1987). Parenteral administration of opioid antagonists, inverse benzodiazepine agonists and 5-hydroxytryptophan has inhibited feed- ing in rats (Morley, 1987; Cooper & Estall, 1985; Cooper, 1986; Fernstrom, 1985; Car- ruba et al., 1986), whereas intracerebro- ventricular injection of y-aminobutyric acid agonists and parenteral administration of serotonin antagonists and benzodiazepine agonists stimulated feed intake (Morley, 1987; Massi & Marini, 1987; Cooper & Estall, 1985). In veterinary medicine, corticosteroids have been advocated as appetite stimulants Uenkins, 1982).

The present study was performed to evalu- ate the effects of ACTH, prednisolone, some benzodiazepine (BZ)-agonists (diazepam, cli- mazolam and brotizolam), BZ-antagonists (flumazenil and R"15-3505; Fig. l), ivermectin (containing not less than 80% of the hydro- genation product of avermectin-B1,) and ritanserin on the dwarf goat's eating be-

( a ) &q C00CH2CH3

N \ c' 0 CH3

FIG. 1. Chemical structure of 2 benzodiaze- pine antagonists (a) R"15-3505 (Ethyl 7-chloro- 5,6-dihydro-5-methyl-6-oxo-4H-imidazo [ 1,5-a] [ 1,4] benzodiazepine-3-carboxylate) and (b) R"15-1788 (Ethyl 8-fluoro-5,6-dihydro-5-methyl-6-0~0-4H-imi- dazo [ 1,5-a][ 1.41 benzodiazepine-3-carboxylate; flumazenil).

haviour. Avermectin-B,,, an anthelmintic macrocyclic lactone, has been found to possess y-aminobutyric (GABA)-ergic activity (Pong et al., 1981). Ritanserin is a new serotonin antagonist with high selectivity for 5-HT2 receptors; this drug easily crosses the blood- brain barrier (Leysen et al., 1981).

The advantage of our model is that it is sensitive to both increases in pelleted concen- trate intake induced by drugs (hyperphagia) and to decreases in feed intake (hypophagia) produced by anorexic agents and pyrogens (van Miert et al., 1986a). For example, 60 pg/kg diazepam increased the level of consumption of a palatable concentrate from about 240 g to 430 g, within a 30-min test period. On the other hand, it has been shown that 100 pg/kg naloxone induced substantial reduction in feed intake from about 240 g to approximately 80 g during the observation time.

MATERIALS AND METHODS

Animals

Thirty-three healthy dwarf goats, female and castrated males, were used; they weighed 21-38 kg. All animals were kept indoors and fed a.diet of hay and pelleted concentrate, which was given at 8.00 a.m. Water was provided ad libitum.

Experimental procedures

All goats were conditioned to the restraint box, feeding schedule and handling by the investigator for at least 3 days before any experimentation. T o ensure that the animals ate during the course of an experiment, they were fasted for 2 h (9.00-11.00 a.m.). Immediately after drug administration, a weighed amount of pelleted concentrate was presented to them for 30 min; in the ACTH and prednisolone experiments feed was pre- sented 3 h after the start of the drug infusion. Feed consumption was determined by sub- tracting the remaining feed and spillage from the initial amount. In some experiments, this procedure was repeated after 2.5 h. In each

Appetite-modulating drugs in dwarf goats 149

series, a cross-over design was used, in which some goats received one of the drugs, whereas the other animals in that group received saline; the goats were allowed a 3-day recov- ery period, and the treatment was then reversed. Feed intake responses were calcu- lated as g/kg metabolic weight (kg0.756). Results were expressed as mean k SEM. Statistical methods used were those previously described (van Miert et al., 1986a).

glucose 5% and distilled water to 100%). ACTH was given as an i.v. infusion - using a venous catheter placed in the jugular vein - at the rate of 0.66 pg/kg/min for 15 min (total dose 10 pg/kg); prednisolone was given i.v. at a rate of 66.66 pg/kg/min'for 15 min (total dose 1 mg/kg). All other drugs were given immediately prior to the feed consumption test.

RESULTS Drugs

The following compounds were used: diazepam, climazolam, flumazenil (R"15- 1788) and R"15-3505 (all from Hoffmann- La Roche, Bade, Switzerland), brotizolam (Boehringer, Ingelheim, FRG), naloxone- HCI (Endo Laboratories, New York, USA), ritanserine (Jarissen Pharmaceutica, Beerse, Belgium), ivermectin (Merck Sharp and Dohme-Agvet division, Haarlem, The Nether- lands), ACTH-1-24 (Cortrosyn, Organon, Oss, The Netherlands) and prednisolone sodium succinate (Upjohn Comp., Ede, The Netherlands).

Each drug was injected into the jugular vein; ivermectin was given as a 0.05% aqueous solution (in ethyl alcohol 20%, Tween 80 2%,

Following the pre-feeding procedure, dwarf goats injected with sterile pyrogen-free saline alone displayed a stable level of pelleted concentrate consumption in the 30-min test period (Table I). Ritanserine (0.1 mg/kg i.v.) induced vasodilatation and tachycardia (not shown), but no statistically significant modifi- cation of feed intake. As can be seen in Table I, naloxone induced a significant decrease in feed intake, whereas ivermectin had no effect. Three hours after the start of infusion, both ACTH and prednisolone did not produce significant changes in concentrate intake in a 30-min test period (Table I). Diazepam (60 kg/kg i.v.), brotizolam (2 and 4 pglkg i.v.) and climazolam (0.1 mg/kg i.v.) induced signi- ficant increases in feed intake (Table 11, Figs

TABLE I. Feed intake response (i f SEM in g/kg0.756) following intravenous injection of test agents in goats fasted for 2 h

Testagent Dose/kg b.w. (i.v.) Feed intake? Experiments (n) Animals (n) Body weight (kg)

Saline Iverrnectin Saline Naloxone

Saline Ritanserine

Saline ACTH

Saline Prednisolone

0.1 ml 0.1 mg 0.1 ml 0.1 mg

0.1 ml 0.1 mg

0.1 ml 0.01 mg$ 0.1 ml 1 mg$

24.7 f. 1.9 12 12 32.71 f 1.77 25.2 k 3.3

30.6 f 4.3 12 12 31.58 f 1.52 18.8 k 4.1*

30.1 & 4.2 6 6 30.44 f 1.71 32.7 k 4.7

24.2 f 3.0 6 6 23.94 f 1.03 24.5 k 4.0

27.1 k 2.8 11 11 29.43 k 2.19 26.1 k 2.9

*P < 0.05. thmediately after drug administration, a weighed amount of pelleted concentrate was presented to the

$Given in 15 rnin; the concentrate was presented 3 h after the start of the infusion. goats for 30 min.

150 A. S . J. P . A . M . van Miert, M . Koot 6' C. T. M . van Duin

TABLE 11. Feed intake response (2 f SEM in g/kg0.756) following intravenous injection of test agents in goats fasted for 2 h

Testagent Dose/kg b.w. (i.v.) Feed intake? Experiments (n) Animals (n) Body weight (kg)

Saline Didzepam

Saline Diazepam

Saline Climazolam

Saline Brotizolam

Saline Flumazenil

Saline R"15-3505

Saline R"15-3505

Saline R"15-3505

Saline Diazepam

0.1 ml 0.03 mg 0.1 ml 0.06 mg

0.1 ml 0.1 mg

0.1 ml 0.002 mg

0.1 ml 0.5 mg 0.1 ml 0.15 mg

0.1 ml 0.10 mg

0.1 ml 0.05 mg

0.1 ml 0.06 mg

20.8 f 1.7 23.5 f 1.8

21.6 f 2.8 32.6 f 3.5*

28.0 f 3.7 39.1 f 3.9*

28.7 f 3.8 38.8 L 3.7*

18.3 f 3.2 21.9 f 2.8

15.4 f 1.3 8.5 f 1.7*

12.2 f 1.3 5.6 _+ 1.6*

10.3 f 2.8 8.6 f 1.9

13.7 f 1.9 24.2 f 3.3'

16

17

18

24

12

8

8

8

8

8

1 1

13

12

12

8

8

8

8

25.90 f 1.20

27.15 f 1.02

27.47 f 1.10

25.30 f 1.21

26.10 f 1.28

27.44 f 1.45

27.43 f 1.40

27.38 f 1.42

27.56 f 1.48

*Significantly different from control value (P < 0.05). t Immediately after drug administration, a weighed amount of pelleted concentrate was presented to the

goats for 30 min.

2-4). Especially during the first observation period (0-30 min after drug administration), diazepam stimulated feed {dnsumption when compared with control 'values (Fig. 4). The hyperphagic effect of this BZ-agonist was dose-related (Table 11, Fig. 4). In the diazepam-ivermectin combination experi- ment, no evidence for drug potentiation was found (Fig. 4).

The animals did not display signs of overt sedation and muscle incoordination at the dose levels of diazepam and climazolam used. The hyperphagic effect of brotizolam (2 pg/ kg i.v.) was unaccompanied by signs of seda- tion or ataxia, although following treatment with 4 pglkg of brotizolam, dwarf goats showed muscle-relaxation and sleep. In con- trast to the BZ-agonists, the BZ-antagonist flumazenil (R"15-1788; tested at 0.5 mg/kg i.v.) had no effect on the level of feed consumption (Table 11). Interestingly, the hyperphagic effects of climazolam and broti-

zolam were not antagonized by flumazenil (Fig. 2), whereas similar i.v. doses of this drug completely reversed - within 10 s - muscle incoordination and ataxia induced by much higher doses of these BZ-agonists (brotizolam 20 pg/kg Lv., climazolam 1.2 mg/kg i.v., diazepam 300 pg/kg i.v.).

In the interaction experiments using the opiate antagonist naloxone and the BZ- agonists (Fig. 3), naloxone (0.1 mg/kg i.v.) antagonized the hyperphagic effect of both diazepam (60 pg/kg i.v.) and brotizolam (4 pg/kg i.v.). Similarly, in the diazepam R"15-3505 study (Fig. 3), there was a signifi- cant effect of diazepam and a significant inhibition of this effect by R"15-3505 (g' iven at a dose of 50 pg/kg i.v.). Strikingly, the BZ- antagonist R"15-3505 by itself had a highly significant inhibitory effect on the intake of pelleted concentrate at higher dose levels (Table 11). However, R"15-3505 did not induce convulsions and anxiety symptoms

Appetite-modulating drugs in dwarf goats 15 1

s ~~ x .- c 0 c

p 20 L

10

* ** 1 1 *

1 *T*

0-30 0-30

Time (rnin)

FIG. 2. Effect of flumazenil (R"15-1788; 0.5 mg/kg i.v.) on the hyperphagic effects induced by clima- zolam (C; 0.1 rnglkg i.v.) and brotizolam (B; 4 pg/ kg i.v.) in dwarf goats fasted for 2 h. Flumazenil (F) was administered 15 rnin before injection of benzo- ' diazepine (BZ) agonists. The consumption of pel- leted concentrate was measured immediately after ' BZ-agonist or saline (S; 0.1 ml/kg i.v.) administra- tion during a 30-min test period. The experiments with climazolam were performed with a group of six goats, whereas with brotizolam 10 goats were used. Results are expressed as means 5 SEM; *P < 0.05 for BZ-agonists versus saline. **P < 0.05 for BZ-agonists plus flumazenil versus saline, but not significantly different from BZ-agonists alone.

which are characteristic for 'inverse BZ- agonists'.

DISCUSSION

The influence of the reticulo-rumen on feed intake includes primarily physical factors such as smooth muscle tension, and rate and strength of extrinsic forestomach contractions (Forbes, 1986). Previous work in our labora- :ory has demonstrated that opioid antagonists and benzodiazepines have moderate effects on the extrinsic rumen contractions. When feed was withheld both diazepam (van Miert et al., 1985; van Miert et al., 1986a) and elfaze- pam (Della-Fera et al., 1977) decreased con- traction rates by 30-35%, whereas naloxone and naltrexone increased the frequency of

0-30 0-30

* 1

0-30 Time (rnin)

FIG. 3. Effect of R"15-3505 (R"; 50 pg/kg i.v.) and naloxone (N; 0.1 mg/kg i.v.) on the hyperphagic effects induced by diazepam (D; 60 pg/kg i.v.) and brotizolam (B; 2 pg/kg i.v.). The experiments with diazepam-naloxone were performed with a group of 11 goats, whereas with diazepam-R"l5-3505 and brotizolam-naloxone eight goats (n = 15) and 12 goats were used, respectively. *P < 0.05 for BZ- agonists versus saline (S); **P < 0.05 for BZ- agonists plus BZ-antagonists versus BZ-agonists alone. See Fig. 1 for further details.

these contractions to approximately 135% of the baseline values (Maas, 1982; van Miert, 1987a; van Miert et al., 1985; van Miert et al., 1986a).

The effects of these and other drugs on rumen motility adds an interesting aspect to their activities in the central nervous system (CNS), since the CNS has been shown to regulate various aspects of forestomach mo- tility which, in turn, could alter feeding behaviour. After intracerebroventricular ad- ministration, corticotropin-releasing hormone is a potent anorexic agent in the rat (Morley, 1987). In the present study, i.v. infusion of ACTH and prednisolone did not change feed intake in dwarf goats fasted for 2 h. In a previous study, similar infusions with ACTH and prednisolone significantly diminished rumen contractions, being lowest at 3 4 h. Furthermore, both hormones caused hyper- glycaemia in these animals (van Miert, 1987b; van Miert et al., 1986b). Thus, the idea that reticulo-rumen motility is one of the major regulators of normal ruminant feeding needs

152 A. S. J . P . A . M . van Miert, M . Koot E3 C . T. M . van Duin

C 0.5 3-3.5 0-0.53-3.5 0-0.5 3-3.5 0-0.53-3.5

Time (min)

FIG. 4. Effect of diazepam (D6: 60 pg/kg, D3: 30 pg/kg i.v.) and ivermectin (I: 0.1 mg/kg Lv.) on feed intake measured during two observation periods (0-30 min and 180-210 min after drug or saline injection). Note that ivermectin did not potentiate the effect of a low dose of diazepam (30 pg/kg i.v.). The experiments with diazepam were performed with groups of eight goats (D6) and eight goats (D3: n = 16), respectively; with ivermectin 12 goats were used. In the experiment with diazepam (D3) versus diazepam (D3) plus ivermectin eight goats were used. *P < 0.05 for diazepam (D6) versus saline. See Fig. 1 for further details.

further evaluation. In ruminants, glucose does not seem to be a suitable stimulus for meal patterning since blood glucose levels are not related to spontaneous meals, and even severe unphysiological hypoglycaemia and hypergycaemia usually fail to affect feeding behaviour (De Jong, 1987). Nevertheless, corticosteroids have been advocated as appe- tite stimulants. Therefore, their effects may simply be to make the animal feel better rather than any specific action on the control of feed intake (Jenkins, 1982).

There is a lot of evidence which suggests that serotonin (5-HT) inhibits eating be- haviour, both by a central and a peripheral mechanism of action (Blundell, 1984; Fern- strom, 1985; Carruba et al., 1986; Garattini et al., 1986). Consistent with this hypothesis, a number of studies have shown that some 5HT-antagonists increase feed intake. In the rat, the selective 5-HT2 antagonist ritanserin blocked the anorectic effect of intraperitoneal 5-HT, but proved to be completely inactive against the effect of 5-HT injected into the hypothalamic paraventricular nucleus (PVN), the area which is highly sensitive to 5-HT (Massi & Marini, 1987). The present study investigated the effect of ritanserin on the

dwarf goat’s eating behaviour. The results obtained indicate that a single i.v. injection of ritanserin at a dose of 0.1 mg/kg does not modify feed intake in goats fasted for 2 h. Recently, the 5-HT-ergic regulation of feed- ing has been shown to be more complex; 5-HTIA agonists seem to be involved in 5-HT- ergic hyperphagia, whereas 5-HTlB agonists decrease feeding, suggesting that it is the 5-HTlB receptor that is responsible for the 5-HT effects seen in the PVN (Morley, 1987).

Benzodiazepines (BZ) have been shown to increase feed intake of many mammalian species under a variety of conditions (Baile & McLaughlin, 1979; Cooper, 1980; Ballarini, 1984; Macy & Gasper, 1985; Cooper & Estall, 1985). High affinity brain-specific BZ-re- ceptors represent a heterogenous sub-class of y-aminobutyric acid (GABA) receptors, Functional GABA-BZ ion-receptor complexes are aggregites consisting of different kinds of sub-units, which may be combined in different ways to yield different receptor complexes. These different GABA-BZ ion- receptor complexes probably subserve differ- ent physiological functions (Squires, 1983). Studies have shown that BZ-receptors can be affected not only by BZ-agonists (such as

Appetite-modulating drugs in dwarf goats 153

antagonize the hyperphagic properties of BZ- agonists cannot be ascribed to ineffective plasma concentrations of the drug, since a similar dose of flumazenil completely re- versed sedation, incoordination and ataxia induced by much higher doses of these BZ- agonists. In contrast to the lack of effect of flumazenil, R"15-3505 (50 pg/kg i.v.) re- versed diazepam-induced hyperphagia (Fig. 3). However, higher doses of this drug (3 0.1 mg/kg i.v.) produced highly significant anor- ectic effects (Table 11). It is interesting that R"15-3505 did not cause other clinical effects such as anxiety and convulsions. Therefore, R"15-3505 can perhaps be best described as a mixed inverse agonist-antagonist at BZ- receptors. This conclusion remains tentative till further work will clarify the basis of the anorectic activity of R"15-3505.

The binding of BZ to BZ-receptors is enhanced and reduced by agonists and anta- gonists of GABA, respectively. Avermectin- BI,, an anthelmintic macrocyclic lactone, has been found to cause a concentration- dependent increase in the in uitro binding of diazepam to BZ-receptors (Williams & Yarbrough, 1979; Paul et al., 1980; Pong et al., 1981), whereas in uiuo studies demonstrated that this drug also enhanced some of the pharmacological actions of diazepam (Wil- liams & Yarbrough, 1979). Moreover, it has been shown that sodium valproate, another drug which affects GABA-ergic activity en- hanced feed intake (Shephard & Estall, 1984). The present study demonstrated that iver- mectin, which contained at least 80% of the hydrogenation product of avermectin-B l a ,

did not itself affect feeding in dwarf goats fasted for 2 h (Table I, Fig. 4). Furthermore, no potentiation of the effect of a low dose of diazepam (30 pg/kg i.v.) was observed by concurrent treatment with ivermectin (Fig. 4). This lack of intrinsic activity may be due to hydrogenation of avermectin-Blp and, or, limited passage of the blood-brain barrier by this drug. On the other hand, some directly- acting GABA:agonists have been reported not to mimic effects of BZ or to potentiate the actions of BZ (Shephard & Estall, 1984). No consistent picture emerges, therefore, from these studies to show how GABA-mediated effects and those of BZ could interact at the level of feed intake regulation.

diazepam, climazolam and brotizolam) and BZ-antagonists (R"15- 1788, flumazenil) but also by a class of ligands which produce effects opposite to those of BZ-agonists. These so- called 'inverse agonists' induce convulsions, anxiety and anorexia (Cooper et al., 1985; Cooper, 1986). The present study demon- strated that low doses of diazepam, brotizolam and climazolam have hyperphagic effects in dwarf goats, and therefore confirm the gen- eral finding that compounds which are active as agonists at BZ-receptors stimulate addi- tional feed consumption.

Interestingly, diazepam did not induce sig- nificant changes in pelleted concentrate intake during the second observation period (3-3.5 h after drug administration). The lack of effect during this episode is probably not due to rapid elimination of the drug, because most of the BZ-agonists have a relatively long elimination half-life in ruminants when com- pared with monogastric species (Beglinger et al., 1982). For example, the terminal half-life of climazolam ranged from 2.5 h in pigs to about 11 h in cattle (Ludwig et al., 1985). In the rat, diazepam and brotizolam provoked a post-injection hyperphagia, followed by a compensatory hypophagia resulting in 24-h feed intakes not different from those of controls; conversely neither body weight nor weight of fat pads were increased (Seyrig et al., 1986). Higher doses of diazepam, brotizolam and climazolam induce sedation, incoordina- tion and ataxia. These effects can be reversed with imidazobenzodiazepinones such as R"15- 1788 (flumazenil) and R"15-3505 (Beglinger et al., 1982; Gutzwiller et al., 1984).

Pharmacological, electrophysiological and behavioural data identified flumanzenil as a specific BZ-receptor antagonist (see Cooper & Estall, 1985). As far as appetite is concerned, flumazenil-antagonized BZ-induced hyper- phagic effects in rats (Cooper & Gilbert, 1985; Cooper & Moores, 1985), rabbits (Mansbach et al., 1984) and rhesus monkeys (Foltin et al., 1985). In dwarf goats, we failed to demon- strate intrinsic activity of flumazenil when given alone (Table 11), which is consistent with earlier results published by other investigators using laboratory animal species. A striking observation was that flumazenil did not atten- uate BZ-induced hyperphagic effects in dwarf goats (Fig. 2). The inability of flumazenil to

154 A. S. J. P . A . M . van Miert, M . Koot L9 C . T . M . van Duin

One interesting development is that BZ- induced hyperphagia may be linked to endogenous opioid peptide mechanisms. En- dogenous opioid peptides have been impli- cated in the regulation of feed intake (Baile et al., 1981; Baile et al., 1983; Baile et al., 1986; Baile & McLaughlin, 1987; Morley, 1987), whereas parenteral administration of opiate receptor antagonists - such as naloxone and naltrexone - decreases appetite (van Miert et al., 1986; Morley, 1987; Baile et al., 1981). Moreover, several investigators have shown that diazepam-induced hyperphagia can be suppressed by concurrent treatment with opiate receptor antagonists (Birk & Noble, 1981; Boast et al., 1983; Cooper, 1983). The present study demonstrated that naloxone antagonizes the hyperphagic effects of dia- zepam and brotizolam in dwarf goats (Fig. 3) and therefore confirm this general finding.

Another interesting development is that BZ-induced hyperphagia may be linked to cholecystokinin (CCK)-mediated mechanisms. BZs have been reported to antagonize the anti-nociceptive, hypothermic and satiety ac- tion of CCK (Kubota et al., 1985; Sugaya et al., 1985; Kuboto et al., 1986). Moreover, both flumazenil and diazepam prevented the eleva- tion in the pain threshold induced by CCK, whereas flumazenil also inhibited the CCK- induced satiety (Sugaya et al., 1987). These results suggest that both BZ-agonists and BZ- antagonists may interefere with the short- term satiety factor CCK at the CCK-receptor. Further, it is likely that opioid peptides and CCK may interact, contributing to the overall control of feed intake (BuCno et al., 1983; Bade et a/., 1986). Thus, the mechanisms by which opioid peptides, CCK and benzodiaze- pines affect feeding behaviour remain to be fully elucidated.

ACKNOWLEDGMENTS

The donation of climazolam, flumazenil, R"15-3505, brotizolam, ritanserine and iver- mectin by Hoffmann-La Roche, Bade (Dr W. F. Rehm), Boehringer Ingelheim (Dr 0. Kern), Janssen Pharmaceutica, Beerse (Dr L. A. A. Ooms) and Merck Sharp and Dohme, Haarlem (Dr H. Heinrich), respectively, is hereby acknowledged. The authors wish to

thank Miss N. van der Laan and M r J. M. Eyndhoven €or technical assistance in prepar- ing the manuscript.

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