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Potency of L-364,718 as an antagonist of the behavioral effects of peripherally administered cholecystokinin
Life Sciences, Vol. 42, pp. 153-159 Printed in the U.S.A.
Pergamon Journals
POTENCY OF L-364,718 AS AN ANTAGONISTOF THE BEHAVIORAL EFFECTS OF PERIPHERALLY ADMINISTERED CHOLECYSTOKININ Sareena Khosla and Jacqueline N. Crawley Unit on Behavioral Neuropharmacology Clinical Neuroscience Branch National Institute of Mental Health, Bethesda, MD 20892 USA (Received in final form November i0, 1987)
Summary A new antagonist of the peripheral cholecystokinin receptor, L-364,718, was found to block the reductions in food intake and exploratory activity induced by intraperitoneal administration of cholecystokinin octapeptide sulfate. L-364,718 significantly reversed the cholecystokinin-induced reduction in feeding at doses of 10 ug/kg - 10 mg/kg i.p. L-364,718 significantly reversed the cholecystokinin-induced reduction in exploratory activity at doses of 500 ng/kg - 10 mg/kg i.p. The time course of antagonist activity of L-364,718 was immediate to 90 minutes after intraperitoneal administration. L-364,718 had no significant effect on food intake or exploratory activity when administered alone, over the dose range of 100 ng/kg-lO mg/kg i.p. This compound appears to be at least one hundred times more potent than proglumide or benzotript as an antagonist of the behavioral effects of peripherally administered cholecystokinin. A new class of cholecystokinin octapeptide sulfate (CCK) receptor antagonists has been developed, which shows a high degree of potency and selectivity for the peripheral CCK binding site in pancreas and gastric glands (1,2,3,4,5). One of these compounds, L-364,718, 3S-(-)-N-(2,3-dihydro-1methyl-2-oxo-5-phenyl-lH- 1,4-benzodiazepine-3-yl)-lH-indole-2-carboxamide, acts as a competitive antagonist, as determined by Scatchard analysis, with an a f f i n i t y of 81 pM for binding of 1251-CCK-8 to guinea pig pancreatic membranes (2). I n vivo, administered orally, L-364,718 antagonized CCK-induced inhibition of gastric emptying in rats (ED50 = 140 Hg/kg), and antagonized CCK-induced reductions in food consumption in rats (ED50 = 321 ug/kg), for a time period of 2-5 hours (5). Specificity of L-364,718 for the physiological actions of CCK was demonstrated bY its lack of activity on phenylalanine-, tryptophan-, or atropine-induced inhibition of gastric emptying, or on motilin-induced gallbladder contractions or secretin-induced pancreatic secretion. In addition, L-364,718 was found to have no effects alone at doses of 0.2 - 125 mg/kg p.o. on spontaneous locomotor activities, analgesia, salivation, or righting reflex (5). These data suggest that L-364,718 is considerably more potent than the standard CCK antagonist, proglumide (4), and that L-364,718 is a selective antagonist of the CCK binding site in the gut. We have previously employed simple feeding and exploratory paradigms 0024~3205/88 $3.00 + .00 Copyright (c) 1988 Pergamon Journals Ltd.
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to investigate the postprandial-like syndrome f i r s t described by Smith and co-workers (6). CCK inhibited the consumption of a palatable meal in a t h i r t y minute test session in mice (7,8,9). In addition, CCK reduced the number of approaches to a novel object, the number of approaches to an unfamiliar female mouse, and the total number of movements, while increasing the time spent in pauses of behavioral i n a c t i v i t y and percentage of time spent in the corner and perimeter of an open f i e l d (7,10,11). We have reported on the potency of several CCK antagonists in blocking these actions of CCK. The minimal effective dose for proglumide was 100 mg/kg i . p . , for benzotript was 10 mg/kg i . p . , and for CR 1409 was 1 mg/kg i.p. in mice (7,12). The following studies were designed to test the efficacy and potency of L-364,718 on these standard behavioral assays for intraperitoneally administered CCK. Methods Male Swiss-Webster mice, NIH substrain, 20-25 grams, were housed in groups of 10 in a temperature and humidity controlled animal vivarium, on a 7 AM - 7 PM lighting schedule. Twenty hours before behavioral testing, food was removed from the cages. Water remained available constantly. L-364,718, g i f t kindly supplied by Merck Sharp and Dohme Research Laboratories, West Point, PA, was suspended in 1% Emulphor EL-620 (GAF Corporation, New York, NY) and sonicated for intraperitoneal injection in an volume of 5 ml/kg. Sulfated cholecystokinin 26-33 (Bachem Co., Torrance, CA) was dissolved in saline for intraperitoneal injection of 5 ~g/kg in a volume of 5 ml/kg. L-364,718 100 ng/kg, 500 ng/kg, 2 ~g/kg, 10 ug/kg, 50 ug/kg, 200 ~g/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, or vehicle was injected into the l e f t side of the abdominal region, five minutes before CCK or saline was injected into the right side of the abdominal region. Immediately after the second injection, each mouse was individually placed in an 18 x 28 x 12 cm polypropylene cage, containing a square of fringed cardboard taped to one side. As previously described (7), exploratory behaviors were scored by a human observer uninformed of the treatment condition, over a five minute session. The parameter which produced the most consistent results was the cumulative duration of pauses, in which the total time spent in the pause mode was t a l l i e d . A pause was defined as total immobility for a minimum of three seconds duration. Immediately after the five minute exploration test, each mouse was placed in another clean cage containing a plastic weighing dish with a wet mash of one vanilla wafer cookie (Nilla Wafers, Nabisco Company, East Hanover, NJ) and three m i l l i l i t e r s of d i s t i l l e d water. Access to the palatable food source was available for a t h i r t y minute period, The dish containing the wet mash was weighed before and after the t h i r t y minute food consumption test by an observer uninformed of the treatment condition. Each mouse was used only once. Eight mice were used for each dose of each treatment. Time course analysis was performed by injecting L-364,718 or vehicle intraperitoneally O, 10, 20, 30, 60, 90, 120, or 180 minutes before intraperitoneal injection of CCK, 5 ~g/kg, and the beginning of the behavioral testing. Each mouse was used only once. Four mice were used for each dose of each treatment. Food intake and cumulative pause duration values were analyzed for significance of treatment and time effects by Analysis of Variance, followed by Newman-Keuls test for significance of individual means. Results L-364,718 significantly blocked the a b i l i t y of CCK to reduce food consumption (Figure 1). At doses from 10 ~g/kg - 10 mg/kg, L-364,718 significantly increased the amount of food consumed after CCK administration,
Vol. 42, No. 2, 1988
L-364,718, Cholecystokinin Antagonist
155
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FIG. 1 L-364,718 or vehicle (1% emulphor in 0.9 % physiological saline) was administered intraperitoneally, five minutes before cholecystokinin octapeptide sulfate (CCK), 5 ug/kg, or saline, to male Swiss-Webster mice, previously fasted overnight. Mice were individually exposed for t h i r t y minutes to a wet cookie mash, which was weighed before and after the test session. CCK reduced food consumption to approximately 30% of control values. L-364,718 antagonized the CCK-induced reduction in food intake, while having no effect alone on food intake. Data are presented as mean + standard error of the mean. N=8 for each dose of each treatment. *p < .05, **p < .01, as compared to vehicle + CCK, by Newman-Keuls analysis following significant ANOVA. as compared to vehicle + CCK (ANOVA F10.137 = 5.13, p < .01; Newman-Keuls p < .05 for 10 ug/kg, 200 ~g/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg; p < .01 for 50 ~g/kg). L-364,718 had no significant effect when given in combination with saline on food consumption at doses from 100 ng/kg - 10 mg/kg (ANOVA F7,63 = 1.31, NS). L-364,718 significantly blocked the a b i l i t y of CCK to induce pauses of behavioral i n a c t i v i t y (Figure 2). At doses from 500 ng/kg - 10 mg/kg, L-364,718 significantly decreased the cumulative duration of pauses after CCK administration, as compared to vehicle + CCK (ANOVA F10,125 = 10.52, p < .01; Nev~nan-Keuls p < .01 for 500 ng/kg, 2 ~g/kg, 10 ug/kg, 50 ~g/kg, 200 ug/kg, 1 mg/kg, 5 mg/kg and 10 mg/kg). L-364,718 had no significant effect when given in combination with saline on cumulative
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duration of pauses at doses from 100 ng/kg - 10 mg/kg (ANOVA F7,63 = 0.37, NS). The time course of action of L-364,718 was in the range of 0 - 90 minutes (Figure 3). L-364,718, 10 ug/kg i . p . , s i g n i f i c a n t l y blocked the a b i l i t y of CCK, 5 pg/kg i . p . , to reduce food intake when given 0 - 60 minutes before CCK (ANOVA F8,41 = 3.08, p < .01; Newman-Keuls p < .05 for t = O, 10, 30 and 60 minutes, as compared to vehicle + CCK at t = 0 minutes and to vehicle + CCK at t = 60 minutes). L-364,718, 10 ~g/kg i . p . , s i g n i f i c a n t l y blocked the a b i l i t y of CCK, 5 ~g/kg i . p . , to induce pauses of behavioral i n a c t i v i t y when given 0 - 90 minutes before CCK (ANOVA F9 46 = 4.68, p < .01; Newman-Keuls p < .01 for t = O, 10 and 20"minutes, p < .05 for t = 30, 60 and 90 minutes, as compared to vehicle + CCK at t = 0 minutes and to vehicle + CCK at t = 60 minutes).
PAUSES
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+ CCK
%
70
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uu
10
+SAL ~'N~i~... o
100rig 500no 2~g 10ug 50~g 2OSug I~Q L-364,718 (dose/kg)
..
5r~g 10~ng
FIG. 2 L-364,718 or vehicle was administered f i v e minutes before CCK, 5 ~g/kg, i . p . , to mice immediately before individual exposure to a novel environment. Cumulativenumber of seconds spent in pauses of complete behavioral i n a c t i v i t y was scored by a human observer uninformed of the treatment condition, over a f i v e minute test session. L-364,718 antagonized the CCK-induced pauses of behavioral i n a c t i v i t y , while having no effect alone in this paradigm. Data are presented as mean + standard error of the mean. N=8 for each dose of each treatment. **p < .01 as compared to vehicle + CCK, by Newman-Keuls analysis following significant ANOVA.
Vol. 42, No. 2, 1988
L-364,718, Cholecystokinin Antagonist
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CCK
• L.364,718 110/~¢1k01
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FIG. 3 L-364,718, 10 ~g/kg i . p . , or vehicle was administered O, 10, 20, 30, 60, 90, 120, or 180 minutes before CCK, 5 ~g/kg i . p . , to mice for behavioral testing as described in the legends to Figures 1 and 2. (Top panel) L-364,718 significantly blocked the effects of CCK on food intake when administered O, 10, 30, and 60 minutes before CCK (*p < .05 as compared to vehicle + CCK at t = 0 or to vehicle + CCK at t = 60 minutes; Newman-Keuls analysis following significant ANOVA). (Bottom panel) L-364,718 significantly blocked the effects of CCK on cumulative duration of pauses of behavioral inactivity when administered O, 10, 20, 30, 60 and 90 minutes before CCK (*p < .05, **p < .01, as compared to vehicle + CCK at t = 0 or to vehicle + CCK at t = 60 minutes; Newman-Keuls analysis following significant ANOVA). Data are presented as mean + standard error of the mean. N=4 for each time point of each treatment.
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Discussion The data presented herein describe the efficacy and potency of a new cholecystokinin antagonist, L-364,718, in blocking the effects of peripherally administered CCK on food intake and exploration in mice. This compound had no effect alone on either behavior, over a wide range of doses. The lack of effect of L-364,718 alone raises questions about the role of endogenous CCK in feeding behaviors. However, these data support the published reports which showed that L-364,718 binds to the peripheral CCK binding site as a competitive antagonist, and blocks physiological actions of CCK, while i t s e l f being devoid of agonist properties (2,3,4). This compound was highly potent and efficacious in blocking the behavioral effects of peripherally administered CCK. The exploratory behavior effects of an intraperitoneal dose of CCK of 5 ~g/kg (approximately 5 nmol/kg) were completely blocked by an intraperitoneal dose of L-364,718 of 500 ng/kg (approximately 1.2 nmol/kg). The feeding behavior effects of CCK, 5 ~g/kg i . p . , were completely blocked by an intraperitoneal dose of L-364,718 of I0 ug/kg (approximately 25 nmol/kg). In contrast, in similar behavioral paradigms in the same strain of mice, other antagonists of the peripheral CCK binding s i t e required much higher doses for a similar level of antagonism: proglumide, I00 - 300 mg/kg [approximately 300 - go0 umol/kg ( 7 ) ] ; benzotript, 0.I mg/kg - i0 mg/kg [approximately 300 nmol/kg - 30 umol/kg ( 7 ) ] ; CR-1409, a proglumide analog, I mg/kg [approximately 3 umol/kg (12)]. Therefore, L-364,718 appears to be considerably more potent than other available antagonists of the peripheral CCK binding s i t e . The minimum effect dose of L-364,718 given intraperitoneally in the present feeding paradigm in mice was 10 ~g/kg. The ED 50 for L-364,718 given orally in rats was 321 ug/kg (5). This discrepancy may relate to a) species differences; b) route of administration; c) the higher dose of CCK employed (5 ~g/kg versus 12.4 ug/kg), or d) the time point employed (5 minutes before CCK and behavioral testing versus 1 hour and 10 minutes before CCK and 2 hours before behavioral testing). With respect to the last point, the time course of L-364,718 activity after intraperitoneal administration in an emulphor vehicle was 0 - 90 minutes in our paradigm, while the time course of L-364,718 activity after oral administration in a methocel vehicle was at least 4 hours in the previous report (5). The potency of L-364,718 is coupled with pharmacological s p e c i f i c i t y for CCK as opposed to other gut peptides, in that t h i s compound did not block motilin-induced gall bladder contractions or secretin-induced pancreatic secretion (5). These properties make L-364,718 an a t t r a c t i v e research tool for investigating the role of endogenous CCK in the regulation of feeding behaviors. In addition, considering the b i o a v a i l a b i l i t y of L-364-718 given o r a l l y , t h i s compound may also have potential for c l i n i c a l applications in gastrointestinal dysfunction. References 1. 2. 3. 4. 5.
R.S.L. CHANG, V.J. LOTTI, R.L. MONAGHAN,J. BIRNBAUM, E.O. STAPLEY, M.A. GOETZ, G. ALBERS-SCHONBERG,A.A. PATCHETT, J.M. LIESCH, O.D. HENSENS and J.P. SPRINGER, Science 230 177-179 (1985). R.S.L. CHANG and V. J. LOTTI, Proc. Natl. Acad. Sci. USA 83 4923-4926 (1986a). R.S.L. CHANG, V.J. LOTTI, T.B. CHEN and K.A. KUNKEL, Molecular Pharmacology 30 212-217 (1986b). V.-~J. LOTTI, D.L. CERINO, P.J. KLING and R.S.L. CHANG, Life Sciences 39 1631-1638 (1986). V.J. LOTTI, R.G. PENDLETON, R.J. GOULD, H.M. HANSON, R.S.L. CHANGand B.V. CLINESCHMIDT, J. Pharmacol. Exptl. Therap.~ 241:1, 103-109 (1987).
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J. ANTIN, J. GIBBS, J. HOLT, R.C. YOUNG, and G.P. SMITH, J. Comp. Phxsiol. Psychol. 89 784-790 (1975). 7. J.N. CRAWLEY, J.A. STIVERS, D.W. HOMMER,L.R. SKIRBOLL and S.M. PAUL, J. Phamacol Exptl. Therap. 236 320-330 (1986). 8. J.E. MORLEY, Life Sciences 30 479-493 (1982). 9. G.P. SMITH, J. GIBBS and P.J. KULKOSKY, The Neural Basis of Feeding and and Reward, ed. B.G. Hoebel and D. Novin, p. 149, Haer I n s t i t u t e , Brunswick, ME (1982). 10. J.N. CRAWLEY, S.E. HAYS, S.M. PAUL and F.K. GOODWIN, Physiol. Behav. 27 407-411 (1981). i i . J.N. CRAWLEY, S. ST. PIERRE and P. GAUDREAU, J. Pharmacol. Exptl. Therap. 230 438-444 (1984). 12. M.T. KALTWASSER, B. PETRACK and J.N. CRAWLEY, Neurochemistry International 10:4 547-553 (1987).
Pergamon Journals
POTENCY OF L-364,718 AS AN ANTAGONISTOF THE BEHAVIORAL EFFECTS OF PERIPHERALLY ADMINISTERED CHOLECYSTOKININ Sareena Khosla and Jacqueline N. Crawley Unit on Behavioral Neuropharmacology Clinical Neuroscience Branch National Institute of Mental Health, Bethesda, MD 20892 USA (Received in final form November i0, 1987)
Summary A new antagonist of the peripheral cholecystokinin receptor, L-364,718, was found to block the reductions in food intake and exploratory activity induced by intraperitoneal administration of cholecystokinin octapeptide sulfate. L-364,718 significantly reversed the cholecystokinin-induced reduction in feeding at doses of 10 ug/kg - 10 mg/kg i.p. L-364,718 significantly reversed the cholecystokinin-induced reduction in exploratory activity at doses of 500 ng/kg - 10 mg/kg i.p. The time course of antagonist activity of L-364,718 was immediate to 90 minutes after intraperitoneal administration. L-364,718 had no significant effect on food intake or exploratory activity when administered alone, over the dose range of 100 ng/kg-lO mg/kg i.p. This compound appears to be at least one hundred times more potent than proglumide or benzotript as an antagonist of the behavioral effects of peripherally administered cholecystokinin. A new class of cholecystokinin octapeptide sulfate (CCK) receptor antagonists has been developed, which shows a high degree of potency and selectivity for the peripheral CCK binding site in pancreas and gastric glands (1,2,3,4,5). One of these compounds, L-364,718, 3S-(-)-N-(2,3-dihydro-1methyl-2-oxo-5-phenyl-lH- 1,4-benzodiazepine-3-yl)-lH-indole-2-carboxamide, acts as a competitive antagonist, as determined by Scatchard analysis, with an a f f i n i t y of 81 pM for binding of 1251-CCK-8 to guinea pig pancreatic membranes (2). I n vivo, administered orally, L-364,718 antagonized CCK-induced inhibition of gastric emptying in rats (ED50 = 140 Hg/kg), and antagonized CCK-induced reductions in food consumption in rats (ED50 = 321 ug/kg), for a time period of 2-5 hours (5). Specificity of L-364,718 for the physiological actions of CCK was demonstrated bY its lack of activity on phenylalanine-, tryptophan-, or atropine-induced inhibition of gastric emptying, or on motilin-induced gallbladder contractions or secretin-induced pancreatic secretion. In addition, L-364,718 was found to have no effects alone at doses of 0.2 - 125 mg/kg p.o. on spontaneous locomotor activities, analgesia, salivation, or righting reflex (5). These data suggest that L-364,718 is considerably more potent than the standard CCK antagonist, proglumide (4), and that L-364,718 is a selective antagonist of the CCK binding site in the gut. We have previously employed simple feeding and exploratory paradigms 0024~3205/88 $3.00 + .00 Copyright (c) 1988 Pergamon Journals Ltd.
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to investigate the postprandial-like syndrome f i r s t described by Smith and co-workers (6). CCK inhibited the consumption of a palatable meal in a t h i r t y minute test session in mice (7,8,9). In addition, CCK reduced the number of approaches to a novel object, the number of approaches to an unfamiliar female mouse, and the total number of movements, while increasing the time spent in pauses of behavioral i n a c t i v i t y and percentage of time spent in the corner and perimeter of an open f i e l d (7,10,11). We have reported on the potency of several CCK antagonists in blocking these actions of CCK. The minimal effective dose for proglumide was 100 mg/kg i . p . , for benzotript was 10 mg/kg i . p . , and for CR 1409 was 1 mg/kg i.p. in mice (7,12). The following studies were designed to test the efficacy and potency of L-364,718 on these standard behavioral assays for intraperitoneally administered CCK. Methods Male Swiss-Webster mice, NIH substrain, 20-25 grams, were housed in groups of 10 in a temperature and humidity controlled animal vivarium, on a 7 AM - 7 PM lighting schedule. Twenty hours before behavioral testing, food was removed from the cages. Water remained available constantly. L-364,718, g i f t kindly supplied by Merck Sharp and Dohme Research Laboratories, West Point, PA, was suspended in 1% Emulphor EL-620 (GAF Corporation, New York, NY) and sonicated for intraperitoneal injection in an volume of 5 ml/kg. Sulfated cholecystokinin 26-33 (Bachem Co., Torrance, CA) was dissolved in saline for intraperitoneal injection of 5 ~g/kg in a volume of 5 ml/kg. L-364,718 100 ng/kg, 500 ng/kg, 2 ~g/kg, 10 ug/kg, 50 ug/kg, 200 ~g/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, or vehicle was injected into the l e f t side of the abdominal region, five minutes before CCK or saline was injected into the right side of the abdominal region. Immediately after the second injection, each mouse was individually placed in an 18 x 28 x 12 cm polypropylene cage, containing a square of fringed cardboard taped to one side. As previously described (7), exploratory behaviors were scored by a human observer uninformed of the treatment condition, over a five minute session. The parameter which produced the most consistent results was the cumulative duration of pauses, in which the total time spent in the pause mode was t a l l i e d . A pause was defined as total immobility for a minimum of three seconds duration. Immediately after the five minute exploration test, each mouse was placed in another clean cage containing a plastic weighing dish with a wet mash of one vanilla wafer cookie (Nilla Wafers, Nabisco Company, East Hanover, NJ) and three m i l l i l i t e r s of d i s t i l l e d water. Access to the palatable food source was available for a t h i r t y minute period, The dish containing the wet mash was weighed before and after the t h i r t y minute food consumption test by an observer uninformed of the treatment condition. Each mouse was used only once. Eight mice were used for each dose of each treatment. Time course analysis was performed by injecting L-364,718 or vehicle intraperitoneally O, 10, 20, 30, 60, 90, 120, or 180 minutes before intraperitoneal injection of CCK, 5 ~g/kg, and the beginning of the behavioral testing. Each mouse was used only once. Four mice were used for each dose of each treatment. Food intake and cumulative pause duration values were analyzed for significance of treatment and time effects by Analysis of Variance, followed by Newman-Keuls test for significance of individual means. Results L-364,718 significantly blocked the a b i l i t y of CCK to reduce food consumption (Figure 1). At doses from 10 ~g/kg - 10 mg/kg, L-364,718 significantly increased the amount of food consumed after CCK administration,
Vol. 42, No. 2, 1988
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155
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FIG. 1 L-364,718 or vehicle (1% emulphor in 0.9 % physiological saline) was administered intraperitoneally, five minutes before cholecystokinin octapeptide sulfate (CCK), 5 ug/kg, or saline, to male Swiss-Webster mice, previously fasted overnight. Mice were individually exposed for t h i r t y minutes to a wet cookie mash, which was weighed before and after the test session. CCK reduced food consumption to approximately 30% of control values. L-364,718 antagonized the CCK-induced reduction in food intake, while having no effect alone on food intake. Data are presented as mean + standard error of the mean. N=8 for each dose of each treatment. *p < .05, **p < .01, as compared to vehicle + CCK, by Newman-Keuls analysis following significant ANOVA. as compared to vehicle + CCK (ANOVA F10.137 = 5.13, p < .01; Newman-Keuls p < .05 for 10 ug/kg, 200 ~g/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg; p < .01 for 50 ~g/kg). L-364,718 had no significant effect when given in combination with saline on food consumption at doses from 100 ng/kg - 10 mg/kg (ANOVA F7,63 = 1.31, NS). L-364,718 significantly blocked the a b i l i t y of CCK to induce pauses of behavioral i n a c t i v i t y (Figure 2). At doses from 500 ng/kg - 10 mg/kg, L-364,718 significantly decreased the cumulative duration of pauses after CCK administration, as compared to vehicle + CCK (ANOVA F10,125 = 10.52, p < .01; Nev~nan-Keuls p < .01 for 500 ng/kg, 2 ~g/kg, 10 ug/kg, 50 ~g/kg, 200 ug/kg, 1 mg/kg, 5 mg/kg and 10 mg/kg). L-364,718 had no significant effect when given in combination with saline on cumulative
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duration of pauses at doses from 100 ng/kg - 10 mg/kg (ANOVA F7,63 = 0.37, NS). The time course of action of L-364,718 was in the range of 0 - 90 minutes (Figure 3). L-364,718, 10 ug/kg i . p . , s i g n i f i c a n t l y blocked the a b i l i t y of CCK, 5 pg/kg i . p . , to reduce food intake when given 0 - 60 minutes before CCK (ANOVA F8,41 = 3.08, p < .01; Newman-Keuls p < .05 for t = O, 10, 30 and 60 minutes, as compared to vehicle + CCK at t = 0 minutes and to vehicle + CCK at t = 60 minutes). L-364,718, 10 ~g/kg i . p . , s i g n i f i c a n t l y blocked the a b i l i t y of CCK, 5 ~g/kg i . p . , to induce pauses of behavioral i n a c t i v i t y when given 0 - 90 minutes before CCK (ANOVA F9 46 = 4.68, p < .01; Newman-Keuls p < .01 for t = O, 10 and 20"minutes, p < .05 for t = 30, 60 and 90 minutes, as compared to vehicle + CCK at t = 0 minutes and to vehicle + CCK at t = 60 minutes).
PAUSES
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100rig 500no 2~g 10ug 50~g 2OSug I~Q L-364,718 (dose/kg)
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FIG. 2 L-364,718 or vehicle was administered f i v e minutes before CCK, 5 ~g/kg, i . p . , to mice immediately before individual exposure to a novel environment. Cumulativenumber of seconds spent in pauses of complete behavioral i n a c t i v i t y was scored by a human observer uninformed of the treatment condition, over a f i v e minute test session. L-364,718 antagonized the CCK-induced pauses of behavioral i n a c t i v i t y , while having no effect alone in this paradigm. Data are presented as mean + standard error of the mean. N=8 for each dose of each treatment. **p < .01 as compared to vehicle + CCK, by Newman-Keuls analysis following significant ANOVA.
Vol. 42, No. 2, 1988
L-364,718, Cholecystokinin Antagonist
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60 70 120 TIMEAFTERL-364.718ADMINISTRATION(rain)
I 180
FIG. 3 L-364,718, 10 ~g/kg i . p . , or vehicle was administered O, 10, 20, 30, 60, 90, 120, or 180 minutes before CCK, 5 ~g/kg i . p . , to mice for behavioral testing as described in the legends to Figures 1 and 2. (Top panel) L-364,718 significantly blocked the effects of CCK on food intake when administered O, 10, 30, and 60 minutes before CCK (*p < .05 as compared to vehicle + CCK at t = 0 or to vehicle + CCK at t = 60 minutes; Newman-Keuls analysis following significant ANOVA). (Bottom panel) L-364,718 significantly blocked the effects of CCK on cumulative duration of pauses of behavioral inactivity when administered O, 10, 20, 30, 60 and 90 minutes before CCK (*p < .05, **p < .01, as compared to vehicle + CCK at t = 0 or to vehicle + CCK at t = 60 minutes; Newman-Keuls analysis following significant ANOVA). Data are presented as mean + standard error of the mean. N=4 for each time point of each treatment.
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Discussion The data presented herein describe the efficacy and potency of a new cholecystokinin antagonist, L-364,718, in blocking the effects of peripherally administered CCK on food intake and exploration in mice. This compound had no effect alone on either behavior, over a wide range of doses. The lack of effect of L-364,718 alone raises questions about the role of endogenous CCK in feeding behaviors. However, these data support the published reports which showed that L-364,718 binds to the peripheral CCK binding site as a competitive antagonist, and blocks physiological actions of CCK, while i t s e l f being devoid of agonist properties (2,3,4). This compound was highly potent and efficacious in blocking the behavioral effects of peripherally administered CCK. The exploratory behavior effects of an intraperitoneal dose of CCK of 5 ~g/kg (approximately 5 nmol/kg) were completely blocked by an intraperitoneal dose of L-364,718 of 500 ng/kg (approximately 1.2 nmol/kg). The feeding behavior effects of CCK, 5 ~g/kg i . p . , were completely blocked by an intraperitoneal dose of L-364,718 of I0 ug/kg (approximately 25 nmol/kg). In contrast, in similar behavioral paradigms in the same strain of mice, other antagonists of the peripheral CCK binding s i t e required much higher doses for a similar level of antagonism: proglumide, I00 - 300 mg/kg [approximately 300 - go0 umol/kg ( 7 ) ] ; benzotript, 0.I mg/kg - i0 mg/kg [approximately 300 nmol/kg - 30 umol/kg ( 7 ) ] ; CR-1409, a proglumide analog, I mg/kg [approximately 3 umol/kg (12)]. Therefore, L-364,718 appears to be considerably more potent than other available antagonists of the peripheral CCK binding s i t e . The minimum effect dose of L-364,718 given intraperitoneally in the present feeding paradigm in mice was 10 ~g/kg. The ED 50 for L-364,718 given orally in rats was 321 ug/kg (5). This discrepancy may relate to a) species differences; b) route of administration; c) the higher dose of CCK employed (5 ~g/kg versus 12.4 ug/kg), or d) the time point employed (5 minutes before CCK and behavioral testing versus 1 hour and 10 minutes before CCK and 2 hours before behavioral testing). With respect to the last point, the time course of L-364,718 activity after intraperitoneal administration in an emulphor vehicle was 0 - 90 minutes in our paradigm, while the time course of L-364,718 activity after oral administration in a methocel vehicle was at least 4 hours in the previous report (5). The potency of L-364,718 is coupled with pharmacological s p e c i f i c i t y for CCK as opposed to other gut peptides, in that t h i s compound did not block motilin-induced gall bladder contractions or secretin-induced pancreatic secretion (5). These properties make L-364,718 an a t t r a c t i v e research tool for investigating the role of endogenous CCK in the regulation of feeding behaviors. In addition, considering the b i o a v a i l a b i l i t y of L-364-718 given o r a l l y , t h i s compound may also have potential for c l i n i c a l applications in gastrointestinal dysfunction. References 1. 2. 3. 4. 5.
R.S.L. CHANG, V.J. LOTTI, R.L. MONAGHAN,J. BIRNBAUM, E.O. STAPLEY, M.A. GOETZ, G. ALBERS-SCHONBERG,A.A. PATCHETT, J.M. LIESCH, O.D. HENSENS and J.P. SPRINGER, Science 230 177-179 (1985). R.S.L. CHANG and V. J. LOTTI, Proc. Natl. Acad. Sci. USA 83 4923-4926 (1986a). R.S.L. CHANG, V.J. LOTTI, T.B. CHEN and K.A. KUNKEL, Molecular Pharmacology 30 212-217 (1986b). V.-~J. LOTTI, D.L. CERINO, P.J. KLING and R.S.L. CHANG, Life Sciences 39 1631-1638 (1986). V.J. LOTTI, R.G. PENDLETON, R.J. GOULD, H.M. HANSON, R.S.L. CHANGand B.V. CLINESCHMIDT, J. Pharmacol. Exptl. Therap.~ 241:1, 103-109 (1987).
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J. ANTIN, J. GIBBS, J. HOLT, R.C. YOUNG, and G.P. SMITH, J. Comp. Phxsiol. Psychol. 89 784-790 (1975). 7. J.N. CRAWLEY, J.A. STIVERS, D.W. HOMMER,L.R. SKIRBOLL and S.M. PAUL, J. Phamacol Exptl. Therap. 236 320-330 (1986). 8. J.E. MORLEY, Life Sciences 30 479-493 (1982). 9. G.P. SMITH, J. GIBBS and P.J. KULKOSKY, The Neural Basis of Feeding and and Reward, ed. B.G. Hoebel and D. Novin, p. 149, Haer I n s t i t u t e , Brunswick, ME (1982). 10. J.N. CRAWLEY, S.E. HAYS, S.M. PAUL and F.K. GOODWIN, Physiol. Behav. 27 407-411 (1981). i i . J.N. CRAWLEY, S. ST. PIERRE and P. GAUDREAU, J. Pharmacol. Exptl. Therap. 230 438-444 (1984). 12. M.T. KALTWASSER, B. PETRACK and J.N. CRAWLEY, Neurochemistry International 10:4 547-553 (1987).