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Cholecystokinin-JMV-180 (JMV-180) is an analogue of cholecystokinin C-terminal octapeptide (CCK-8), which has been shown to be an agonist at the proposed CCK pancreatic high-affinity site and a functional antagonist at the pancreatic low-affinity site in rats and to have agonist activity at both high- and low-affinity sites in the mouse. In this study we used JMV-180 to evaluate the potential participation of these two CCK-A sites in the satiety effect of CCK-8 in rats and mice. When tested at doses that ranged from 0.01 to 9.2 mumol/kg, JMV-180 did not reliably affect food intake of solid or liquid test diets in rats. When combined with CCK-8 (3.2 or 8.5 nmol/kg) JMV-180 dose dependently reversed the satiety effect of CCK-8. In contrast to these results in the rat, both JMV-180 (3.7-14.8 mumol/kg) and CCK-8 (1.7-6.8 nmol/kg) dose dependently reduced the intake of 20% sucrose in mice. Both CCK-8- and JMV-180-induced suppression of food intake were attenuated by the CCK-A antagonist MK-329 (24.8 nmol/kg). The results of these studies suggest that agonist activity at sites pharmacologically similar to the CCK pancreatic high-affinity site is not sufficient for expression of CCK satiety, whereas agonist activity at low-affinity-like sites is necessary to reduce food intake. Thus the anorexic activity of CCK appears to be mediated through an interaction with a receptor site pharmacologically similar to the pancreatic low-affinity CCK receptor site.

Cholecystokinin represents a family of gut hormones which among other activities, have been proposed to participate in satiety signaling. Ac-CCK-7[Ac-Tyr(SO3H)-Met-Gly-Trp30-Met-Asp-Phe-NH2 (2)] possesses the full spectrum of activity and potency of the intact hormone; thus analogs of 2 may be useful as anorectic agents. A series of derivatives has been prepared in which the tryptophan indole moiety of 2 has been modified. The new compounds were assayed in CCK binding assays using homogenated rat pancreatic membranes and bovine striatum as a source of CCK-A and CCK-B receptors respectively and in vivo in rats for anorectic activity. Although previous studies have concluded that the indole ring of Trp30 is a critical pharmacophore for the interaction of CCK with both its A and B type receptors, we find 2-Nal30-Ac-CCK-7 (20) to be nearly equipotent to 2 in both CCK binding and as an anorectic agent sensitive to blockade by the Merck CCK-A receptor antagonist MK-329. The extreme structural sensitivity of this anorectic activity is illustrated by the 1-naphthylalanine30 (19) and (benzo[b]thien-2-yl)alanine30 (21) analogs which are 30 and 100 times less potent than 2 respectively. Other mono- and bicyclic Trp30 replacements, including substituted phenylalanines, 3-quinolinylalanine, and 2-(5,6,7,8-tetrahydro)naphthylalanine, gave inactive compounds.

Previous work indicates that both the C-terminal phenylalanine amide and the tryptophan moieties of cholecystokinin (CCK) are critical pharmacophores for interaction with either the A or B receptor subtypes. We have examined a series of analogs of Ac-CCK-7 [Ac-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe33-NH2] (2) in which the phenyl ring of the C-terminal Phe-NH2 has been modified. Compounds were assessed in binding assays using homogenated rat pancreatic membranes and bovine striatum as the source of CCK-A and CCK-B receptors respectively and for anorectic activity after intraperitoneal administration to rats. Substitution of a number of cycloalkyl or bicyclic aryl moieties for the phenyl ring of phenylalanine33 including cyclopentyl (20), cyclohexyl (21), cyclooctyl (23), 2-(5,6,7,8-tetrahydro)naphthyl (26), 2-naphthyl (27), and 1-naphthyl (29) led to analogs with 10-70 times the anorectic potency of 2. The anorectic activity of 21 was blocked by the specific CCK-A receptor antagonist MK-329. Other bulky aliphatic groups in place of the phenylalanine33 aromatic ring such as isopropyl, 2-adamantyl and cyclohexylmethyl gave derivatives similar to 2 in potency. While most of the new compounds were comparable to CCK in binding assays, 23, 26, 27 and 29 were exceptionally potent with IC50s 10(-11)-10(-14) M in the pancreas. Compounds 23 and 29 were further evaluated for their ability to stimulate amylase secretion and found to have potencies similar to that of CCK. The dissociation between potency in the binding and amylase secretion assays suggests that they may interact with a high affinity binding site which is not coupled to amylase secretion. We conclude that CCK receptors possess a generous hydrophobic pocket capable of accommodating large alkyl groups in place of the side chain of phenylalanine33 and that the pharmacological profile of CCK analogs can be tailored by appropriate exploitation of this finding.

A series of analogues of the satiety-inducing peptide cholecystokinin (CCK-8) was prepared in which the sulfated tyrosine required for activation of peripheral receptors was replaced with a carboxy(alkyl)- or tetrazolyl(alkyl)-phenylalanine to investigate whether an organic acid could serve the role of the sulfate group at the receptor. The necessary intermediates were prepared by previously reported procedures or by alkylation of carboxy(alkyl)- or tetrazolyl(alkyl)phenylmethyl bromides with a glycine-derived anion followed by protecting-group manipulations, and these were incorporated into derivatives of acetyl-CCK-7 using solid-phase synthesis. Peptide analogues were evaluated in a CCK-binding assay for affinity for either peripheral (CCK-A) receptors using homogenated rat pancreatic membranes as the receptor source or for central (CCK-B) receptors using bovine striatum as the receptor source. They were further evaluated for effects on food intake in rats after intraperitoneal (ip) injection. A number of the compounds reported are active in the CCK-A receptor binding assay although less potent than acetyl-CCK-7 and decrease food intake with comparable potency to acetyl-CCK-7. In a meal feeding model designed to assess appetite suppressant activity, acetyl-CCK-7 has an ED50 of 7 nmol/kg ip, while the ED50s of Ac-Phe(4-CH2CO2H)-Met-Gly-Trp-Met-Asp-Phe-NH2 (28) and Ac-Phe[4-(tetrazol-5-yl)]-Met-Gly-Trp-Met-Asp-Phe-NH2 (34) were 9 and 11 nmol/kg ip, respectively. An analogue of 28 lacking the N-terminal acetamido group, 3-[4-(carboxymethyl)-phenyl]propanoyl-Met-Gly-Trp-Met-Asp-Phe-NH2 (50), was also active in the meal feeding assay with an ED50 of 3 nmol/kg ip. Its anorexic effect was blocked by simultaneous administration of the CCK-A receptor antagonist MK 329, indicating that the observed anorexic activity is mediated by CCK-A receptors. We conclude from this work that the requirement for a negative charge at the CCK-A receptor provided in the natural substrate by a sulfate group can be satisfied by organic acids.