Comparison of the satiating effect of the CCKA receptor agonist A71378 with CCK-8S

Peptides, Vol. 17, No. 2, pp. 355-357, 1996 Coovrieht 0 1996 Elsevier Science Inc. sit2 in the USA. All rights reserved 01%.9781/96 $15.00 + .OO

SSDI 01%~9781(95)02131-O

ELSEVIER

BRIEF COMMUNICATION

Comparison of the Satiating Effect of the CCKA Receptor Agonist A7 1378 With CCK-8S M. VOITS, ’ J.-P. VOIGT,

M. BOOMGAARDEN,

P. I-IENKLEIN

AND

H. PINK

Institute of Pharmacology and Toxicology, Charitk, Medical Faculty of the Humboldt University, D-ZOO98 Berlin, Germany Received

18 September

1995

VOITS, M., J.-P. VOIGT, M. BOOMGAARDEN, P. HENKLEIN AND H. FINK. Comparison offhe sutii~ring eficr ofthe CC& receptor agonist A71378 with CCK-8s. PEPTIDES 17(2) 355357,1996.-The satiating effect of the selective cholecystokinin, (CCK,) receptor agonist A71378 and the mixed A and B receptor agonist CCK3S were compared in 24-h food-deprived rats. After systemic application of 1.6.8.0, and 40 pglkg A71378 or CCK-IS, respectively, food intake was measured for 24 h. During the first hour A71378 and CCK-IS decreased food intake similarly. Two and 4 h after treatment, the satiating effect of A71378 continued. In contrast, 2 h after administration of CCK-IS a slight effect was observed at the highest dose (40 pg/kg), which totally disappeared after 4 h. In summary, the effect of A71378 on food intake is longer lasting compared to CCK-8s. Cholecystokinin (CCK)

A71378

CCK-IS

A-agonist

THE peptide cholecystokinin (CCK) is an important intestinal hormone and one of the most widely distributed neuropeptides in the brain, where it may act as a neurotransmitter or neuromodulator (3). Several molecular forms, ranging from 4 to 58 amino acids, are found in the brain and peripheral organs. The sulfated octapeptide CCK-8S is the most abundant fragment in the brain. CCK is assumed to be involved in a variety of physiological and behavioral actions [ (4) for review]. Several studies have been performed to differentiate the effects mediated at the known receptor subtypes (CCKA and CCKa receptor). For example, it is suggested that the CCKa receptor mediates anxiety-related behavior, whereas the CCKA receptor is thought to be involved in the regulation of food intake [ (4) for review]. Most of this knowledge is derived from studies using the mixed agonist CCK-8S and selective antagonists. Many investigations have been conducted with agonists selective for the CCKa receptor, whereas only a few have been done with selective CCKA agonists. A71378, a CCK-7 analogue, acts as a potent and selective ligand at the CCKA receptor with agonistic activities (8). It was shown that A71378 has a high potency in eliciting pancreatic amylase secretion and ileal muscle contraction from guinea pig, in contrast to a relatively weak effect in mobilizing intracellular calcium from NCI-H345 cells, which express CCKa/gastrin receptors (8). It takes 700 times more A71378 to displace 50% of labeled CCK from the B receptor as it does from the A receptor

Food intake

Rat

(8). Only in a few studies has A7 1378 been used to discriminate between CCKA- and CCKs-mediated effects like stimulation of phosphoinositol hydrolysis and pepsinogen release in guinea pig gastric glands (7)) to determine the properties of CCK receptors on rabbit vagus nerve (9), and to demonstrate the pharmacology of CCKA receptors on chief cells ( 10). To OUTknowledge, only one study was performed to test A71378 in vivo for appetitesuppressant activity in rats compared to other new CCK-7 analogues with CCKA agonistitc activity (6). In the present study, we compared the satiating effect of the selective CCK,, agonist A71378 with the mixed A and B receptor agonist CCK-8S, which is known to reduce food intake potently. METHOD

Male Wistar rats (n = 69, Winkelmann, Borchen) weighing 220-250 g were used for the experiments. The animals were housed in groups of five under a 12-h lightdark schedule with lights on at 0600 h. The rats had free access to standard rodent food ( Altromin, Lage) and tap water. A71378 [ desamino-Tyr ( SOIH) -Nle-Gly-Trp-Nle(Nmethyl) Asp-Phe-NH, ; mol.wt. 9901 and CCK-IS [AspTyr( S03H)-Met-Gly-Trp-Met-Asp-Phe-NH2 ; mol.wt. 11041 were synthesized by solid-phase synthesis and purified by HPLC in our institute. Immediately before use the peptides were dissolved in 0.9% NaCl and diluted to the final concentration.

’ Requests for reprints should be addressed to Dr. Mechthild Voits, Institute of Pharmacology and Toxicology, Char&e, Medical Faculty of the Humboldt University, D-10098 Berlin, Germany.

355

356

VOITS

Rats were singly housed 5 days before and during the experiment. After 24 h of food deprivation, rats received IP A71378 or CCK-8S at doses of 1.6, 8.0, and 40 pug/kg, respectively, or 0.9% NaCl for control using an injection volume of 5 ml/kg. Food intake was measured 1,2,4, and 24 h after drug treatment. Because normality failed in some experimental groups, statistical analysis was performed by Kruskal-Wallis analyses of variance (one-way ANOVA on ranks) followed by Dunn‘s test for comparisons of treatment groups vs. control group. Significance level was set at p < 0.05. Data out of t1.6 SD were excluded from analysis.

10

DISCUSSION The

selective CCKA agonist A71378 was shown to have potent activity in suppression of food intake in rats to less than 30% of controls. To date, little is known about the properties of selective CCK, agonists. To our knowledge, only one study was performed to investigate the appetite-suppressive effect of A71378 (6). In that study the pharmacological properties of several CCK-7 analogues were compared, whereby A7 1378 was the most effective (6). In our study we compared the efficacy of A71378 with the well-characterized, naturally occurring CCK fragment, CCK-8S, which acts at both receptor subtypes. During the first hour, A71378 and CCK-8S reduced food intake similarly. However, the decrease of food intake by A71378 was longer lasting compared to CCK-8s. Four hours after application of A71378, the drug was still effective at a dose of 40 pg/kg. At the same time, no differences were observed between rats treated with 40 pg/kg CCK-8S and saline. In a former study, A71378 was reported to suppress food intake 6-7 h after application, but the methods were not described in detail (6). A71623, the prototype of CCK4 analogues with CCKA agonistic activity, was potent to suppress food intake in rats, mice, beagle dogs, and cynomolgous monkeys ( 1,2). With exception of the dog, the hypophagia seen after CCK-8S was considerably more shortlived than that of A71623 as reflected in the relative changes in EDs0 over time ( 1,2). The longer duration of action of A71378 on food intake might be due to it having greater stability than CCK-8s. However, another CCK-7 analogue, with no long-lasting effect on food intake, was more stable than A71378 in both kidney and liver homogenates (6). Thus, increased stability does not necessarily correlate with an increased duration of time over which food intake is suppressed. Differences between CCK-8S, hepta- and tetrapeptide analogues can be attributed to many fac-

u NaCl

lh

611A71378

0

CCK-8s

I 2h

RESULTS

The effects of A71378 and CCK-8S on food intake at 1, 2, and 4 h after treatment are shown in the Fig. 1. During the first hour both A71378 and CCK-8S decreased food intake similarly to less than 30% of control. Two and 4 h after treatment, the satiating effect of A71378 continued in a dose-dependent manner. Compared to control, 2 h after treatment food intake was decreased by A71378 to 32% (8 hg/kg) and 17% (40 pglkg) and was still reduced to 54% (8 pg/kg) and 44% (40 pg/kg) after 4 h. In contrast, 2 h after application of CCK8S, a slight reduction of food intake was found at the highest dose of 40 pglkg (69% of control), which totally disappeared after 4 h. Twenty-four hours after treatment with A71378 or CCK-8S, no alteration of food intake was observed when compared to controls (not shown). Overall, these data show that the effect of A7 1378 on food intake is longer lasting compared to CCK-8s.

ET AL.

10

4h

1.6 8 40

1.6 8 40

DOSE kg/kg1 FIG. 1. Comparison of the effects of A71378 and CCK-8S at doses of

I .6, 8, and 40 &kg IP, respectively, and controls (NaCl) on food intake 1, 2, and 4 h after treatment in 24-h food-deprived rats. Food intake (g) is expressed as median and interquartile range. Differences between treatment groups and control group (*p < 0.05) are based upon KruskalWallis ANOVA followed by Dunn’s test. Data out of ? 1.6 SD were excluded from analysis (n= 7-9).

tors, including pharmacokinetic/pharmacodynamic considerations like in vivo half-life and bioavailability, differences between pancreatic receptors and those responsible for suppression of food intake, and species differences (5,6). Our results confirm the hypothesis that a hypohagia observed after peripheral treatment with CCK fragments is mediated by type A receptors and there is no evidence for an involvement of CCKa receptors. Taken together, we have shown that the selective CCK, agonists A71378 reduced food intake in rats as potent as but longerlasting than the well-known mixed CCK, and CCK, agonist CCK-8s. In addition to the CCK4 analogue A71623, the heptapetide A71378 provides a suitable tool for discriminating effects at the receptor subtypes because of the receptor selectivity and high in vivo activity. Moreover, CCKA agonists with longlasting food-reducing activity like A71378 may represent novel hypophagic agents in the therapy of obesity. ACKNOWLEDGEMENT

This work was supported by grant 01 ZZ 9101 from tbe Bundesministerium ftir Forschung und technologie (BMFT)

SATIATING

EFFECT

OF A71378

357

AND CCK-8S

REFERENCES 1. Asin, K. E.; Bednarz, L.; Nikkel, A. L.; et al. Behavioral effects of A-71623, a highly selective CCK, agonist tetrapeptide. Am. J. Physiol. 263:Rl25-R135; 1992. 2. Asin, K. E.; Bednarz, L.; Nikkel, A. L.; Gore, P. A., Jr.; Nadzan. A. M. A-71623, a selective CCK, receptor agonist, suppresses food intake in the mouse, dog, and monkey. Pharmacol. Biochem. Behav. 42:699-704; 1992. 3. Bradwejn, J.; Koszycki, D.; Shiriqui, C. Enhanced sensitivity to cholecystokinin tetrapeptide in panic disorder. Clinical and behavioral findings. Arch. Gen. Psychiatry 48:603-610; 1991. 4. Crawley. J. N.; Corwin, R. L. Biological actions of cholecystokinin. Peptides 15731-755; 1994. 5. Elliott, R. L.; Kopecka, H.; Tufano, M. D.; et al. Novel Asp”-replacement tetrapeptide analogues as potent and selective CCKA agonists. J. Med. Chem. 37:1562-1568; 1994 6. Holladay, M. W.; Bennett, M. J.; Tufano, M. D.; et al. Synthesis and biological activity of CCK heptapeptide analogues. Effects of con-

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formational constraints and standard modifications on receptor subtype selectivity, functional activity in vitro, and appetite suppression in vivo. J. Med. Chem. 35:2920-2928; 1992. Lin, C. W.; Bianchi, B. R.; Miller, T. R.; Witte, D. G.; Wolfram, C. A. W. Both CCKA and CCK,/gastrin receptors mediate pepsinogen release in guinea pig gastric glands. Am. J. Physiol. 262:Gl113-G1120; 1992. Lin, C. W.; Holladay, M. W.; Wine, D. G.; et al. A71378: A CCK agonist with high potency and selectivity for CCK, receptors, Am. J. Physiol. 258:G648-G651; 1990. Lin, C. W.; Miller, T. R. Both CCK, and CCKalgastrin receptors are present on rabbit vagus nerve. Am. J. Physiol. 263:R591-R595; 1992. Pradhan, T. K.: Qian, J.-M.; Sutliff, V. E.; Mantey, S. A.; Jensen, R. T. Identification of CCKA receptors on chief cells with use of a novel, highly selective ligand. Am. J. Physiol. 268:G605-G612; 1995.