Modification of receptor selectivity and functional activity in cholecystokinin peptoid ligands.

Hybrid analogs of the cholecystokinin A (CCK-A) receptor selective tetrapeptide agonist Boc-Trp-Lys(Tac)-Asp-MePhe-NH2 (1,A-71623) and the CCK-B receptor selective antagonists PD-135118 (2) and CI-988 (3) were prepared. Incorporation of the Lys(Tac) side chain into 2 produced a moderately potent antagonist of CCK-8 in the isolated guinea pig gallbladder (GPGB). Incorporation of the Lys(Tac) side chain into 3 produced the novel agonist analog 7 (EC50 = 28 nM in the GPGB) with excellent affinity for both human CCK-A (IC50 = 12 nM) and CCK-B (IC50 = 17 nM) receptors. Analog 7 was a full agonist (EC50 = 3.5 nM) for calcium mobilization on CHO-K1 cells expressing hCCK-A receptors but a partial agonist on CHO-K1 cells expressing hCCK-B receptors, eliciting a weak agonist response (EC50 = 2800 nM) and antagonizing CCK-8-induced calcium mobilization (KB = 20 nM). Despite this unusual in vitro profile, analog 7 was a potent anorectic agent in rats (ED50 = 30 nmol/kg) following intraperitoneal administration.

3-[2-(N-phenylacetamide)]-1,5-benzodiazepines: orally active, binding selective CCK-A agonists.

A series of modifications were made to the C-3 substituent of the 1,5-benzodiazepine CCK-A agonist 1. Replacement of the inner urea NH and addition of a methyl group to generate a C-3 quaternary carbon resulted in acetamide 6, which showed CCK-A receptor binding selectivity and sub-micromolar agonist activity in vitro. Benzodiazepine 6 was active in an in vivo mouse gallbladder emptying assay and represents a novel orally active, binding selective CCK-A agonist.

Discovery of 1,5-benzodiazepines with peripheral cholecystokinin (CCK-A) receptor agonist activity (II): Optimization of the C3 amino substituent.

Analogs of the previously reported 1,5-benzodiazepine peripheral cholecystokinin (CCK-A) receptor agonist 1 were prepared which explore substitution and/or replacement of the C-3 phenyl urea moiety. Agonist efficacy on the isolated guinea pig gallbladder (GPGB) was retained with a variety of substituted ureas and amide analogs. Three compounds were identified which were orally active in the mouse gallbladder emptying assay (MGBE). The 2-indolamide (52) and N-(carboxymethyl)-2-indolamide (54) derivatives had improved affinity for the human CCK-A receptor but reduced agonist efficacy on the GPGB. Neither indolamide was orally active in a rat feeding assay. In contrast, the (3-carboxyphenyl)urea derivative (29, GW7854) had moderately increased affinity for the human CCK-B receptor but was a potent full agonist on the GPGB and was orally active in both the MGBE and rat feeding assays. GW7854 was a full agonist (EC50 = 60 nM) for calcium mobilization on CHO K1 cells expressing hCCK-A receptors and a potent antagonist of CCK-8 (pA2 = 9.1) on CHO K1 cells expressing hCCK-B receptors. GW7854 is a potent mixed CCK-A agonist/CCK-B antagonist which is orally active in two in vivo models of CCK-A-mediated agonist activity.

Discovery of 1,5-benzodiazepines with peripheral cholecystokinin (CCK-A) receptor agonist activity. 1. Optimization of the agonist "trigger".

Directed screening of compounds selected from the Glaxo registry file for contractile activity on the isolated guinea pig gallbladder (GPGB) identified a series of 1,5-benzodiazepines with peripheral cholecystokinin (CCK) receptor agonist activity. Agonist efficacy within this series was modulated by variation of substituents on the N1-anilinoacetamide moiety. Remarkably, a single methyl group confers agonist activity, with an N-isopropyl substituent providing optimal efficacy. Hydrophilic substituents on the anilino nitrogen abolish agonist activity or produce antagonists of CCK. In contrast, hydrophilic electron-donating groups at the para-position of the anilino ring enhance or maintain in vitro and in vivo agonist activity. Despite decreased affinity for the human CCK-A receptor, relative to CCK-8, some of these compounds are equipotent to CCK as anorectic agents in rats following intraperitoneal administration.

Optimization of 3-(1H-indazol-3-ylmethyl)-1,5-benzodiazepines as potent, orally active CCK-A agonists.

We previously described a series of 3-(1H-indazol-3-ylmethyl)-1,5-benzodiazepine CCK-A agonists exemplified by compound 1 (GW 5823), which is the first reported binding selective CCK-A full agonist demonstrating oral efficacy in a rat feeding model. In this report we describe analogs of compound 1 designed to explore changes to the C3 and N1 pharmacophores and their effect on agonist activity and receptor selectivity. Agonist efficacy in this series was affected by stereoelectronic factors within the C3 moiety. Binding affinity for the CCK-A vs CCK-B receptor showed little dependence on the structure of the C3 moiety but was affected by the nature of the second substituent at C3. Structure-activity relationships at the N1-anilidoacetamide "trigger" moiety within the C3 indazole series were also investigated. Both agonist efficacy and binding affinity within this series were modulated by variation of substituents on the N1-anilidoacetamide moiety. Evaluation of several analogs in an vivo mouse gallbladder emptying assay revealed compound 1 to be the most potent and efficacious of all the analogs tested. The pharmacokinetic and pharmacodynamic profile of 1 in rats is also discussed.

Relationship between native and recombinant cholecystokinin receptors: role of differential glycosylation.

In an attempt to establish the relationship between the protein encoded by the recently cloned type A cholecystokinin (CCK) receptor cDNA and the two distinct plasmalemmal proteins on the rat pancreatic acinar cell that were previously described as candidates to represent this receptor, we have established a Chinese hamster ovary (CHO) cell line stably expressing large amounts of this recombinant protein and have used biochemical methods to characterize it directly. Upon affinity labeling, this protein migrated faster on a sodium dodecyl sulfate-polyacrylamide gel than the M(r) 85,000-95,000 molecule previously felt to represent the best candidate. However, deglycosylation with endoglycosidase F demonstrated that it had the same size core protein as that candidate, and this identification was further supported by protease peptide mapping. We postulated that the structural differences between the recombinant and the native proteins related to differences in glycosylation. Consistent with this, lectin-binding experiments demonstrated that both represented complex glycoproteins but that only the native receptor-bound Ulex europeus agglutinin I. Since this lectin binds to fucose residues that are added late in glycoprotein biosynthesis, it is possible that the distinct processing observed affected only that step. In spite of this structural difference, the type A CCK receptor-bearing CHO cell CCK receptor was functionally indistinguishable from the native acinar cell receptor. This included its ability to initiate signaling cascades, its sensitivity to stable GTP analogues, and its binding affinities for agonists and antagonists. The fidelity of this receptor expression system, while representing a 25-fold increase in receptor density over the native pancreatic acinar cell, should provide an ideal substrate for the examination of structure-function relationships within this molecule.

Dominant lethal assay of chlordecone and its distribution in the male reproductive tissues of the rat.

Male rats received 3.6 or 11.4 mg/kg/day of chlordecone orally for 5 days. Some statistically significant events were seen in the reproductive data of females mated to males receiving chlordecone. However, these events did not follow a consistent pattern and do not suggest the conclusion that chlordecone causes dominant lethal effects. Male rats received a single oral dose (40 mg/kg) of chlordecone and were killed at 1, 2, 3, 5, 7, 14 or 21 days. Chlordecone was distributed throughout the reproductive tract. The descending order of concentration was seminal vesicular fluid greater than prostate greater than vas deferens greater than seminal vesicle greater than unwashed sperm greater than washed sperm. It is concluded that chlordecone is well distributed throughout the reproductive tract of the male rat, appears in the ejaculate, and does not appear to produce dominant lethal effects.

Changes in motility and ph in the digestive tract of experimentally overfed sheep.

Motility of the digestive tract of 4 sheep was studied with radiotelemetric equipment. After base line records were made, each sheep was overfed with 70 g of grain per kilogram of body weight. The ruminoreticulum did not become static until the ingesta pH was less than 5. The cecum had the same patterns of motility and pH as did the ruminoreticulum, but these returned to normal more quickly in surviving sheep. The motility patterns of the abomasum and the small intestine were more erratic. Results indicated that considerable quantities of grain (substrate for microbial growth) reached the cecum before ruminoreticular motility was inhibited.

Intestinal infusion of endotoxin in adrenalectomized calves.

Four adrenalectomized calves, 3 to 4 months old, were allowed to develop adrenocorticosteroid insufficiency and then were given 50 mg of Escherichia coli endotoxin by infusion via cannula into the duodenum. Several physiologic variables were studied to detect changes indicative of endotoxemia. It is concluded that adrenocorticosteroid insufficiency in calves is not an independent factor allowing an intestinal source of endotoxin to cause the development of clinical signs and physiologic changes characteristic of endotoxemia.

Preliminary study of appearance of endotoxin in circulatiory system of sheep and cattle after induced grain engorgement.

Edotoxin was detected, using the limulus amebocyte lysate (LAL) test, in the blood of 3 sheep and 1 steer which had been experimentally "overfed" (induced grain engorgement) with a mixture of corn and oats (2:1). The 1st postfeeding blood samples were collected 24 hours after overfeeding. In 2 sheep and 1 steer, the 24-hours blood samples were test positive. In 1 sheep which died, the 48-hour blood sample was the 1st test-positive sample. In all cases, pre-overfeeding blood samples were taken just before overfeeding.

Grain overload in cattle and sheep: changes in microbial populations in the cecum and rumen.

Samples from the rumen and cecum of cattle and sheep were cultured to determine changes in microbial populations resulting from overfeeding with grain. Before the animals were overfed, the predominant organisms from both sides were those that grew anaerobically on a relatively nonselective ruminal fluid medium and would not grow on selective mediums designed to culture lactobacilli, streptococci, coliforms, or Clostridium perfringens. By 24 hours after overfeeding, lactic acid bacteria had increased in numbers so that they were the most numerous organisms in both the rumen and the cecum. The concentrations of coliforms and C perfringens also increased after overfeeding and were generally higher in the cecum than in the rumen.

Physiologic studies of experimentally grain-engorged cattle and sheep.

Seven sheep weighing 34 to 41 kg, each, 3 steers and 1 heifer weighing 230 to 460 kg each, were experimentally, "overfed" (induced grain engorgement). The most significant changes occurred in ruminal ingesta pH, blood pH, packed cell volume (PCV), hemoglobin (Hb), carbon dioxide pressure (PCO2, total CO2 (volume %), blood D-lactic acid, blood HCO3, and base excess. There was no common denominator that was especially pathognomonic.

Dysphoria and the failure to perceive and use discounting information: implications for internalizing negative feedback.

The present study examined the proposition that dysphoric individuals make internal attributions because they do not use available discounting cues. To test this hypothesis, 23 dysphoric and 32 nondysphoric college students were either provided a discounting cue or were led to believe that an internal attribution for failure was appropriate (no discounting cue). On the primary measure of internality, nondysphoric individuals made greater external attributions when a discounting cue was available than they did when no such cue was present, but attributions made by dysphoric individuals were unaffected by the presence of a discounting cue. On the other hand, using a secondary dependent measure inserted to replicate a prior study in this area, key comparison differences were not obtained.

Cytosolic calcium regulates phorbol diester binding affinity in intact phagocytes.

The mobilization of internally sequestered stores of Ca2+ and activation of protein kinase C appear to be involved in neutrophil activation. We have examined the inter-relationship of these two pathways by investigating the effects of modulating Ca2+ activity on the binding of [3H]phorbol 12,13-dibutyrate (PDBU) to protein kinase C in intact phagocytes. Differentiated HL-60 cells were equilibrated with [3H]PDBU prior to stimulation with various agents known to alter Ca2+ homeostasis in cells. Agents that elevated cytosolic Ca2+, such as f-Met-Leu-Phe and A23187, up-regulated radioligand binding by increasing the affinity of the PDBU/protein kinase C interaction. These effects were time- and agonist concentration-dependent and temperature-sensitive. The kinetics of the up-regulation of binding by f-Met-Leu-Phe coincided with the kinetics of Ca2+ mobilization (by quin2 fluorescence measurements). The putative intracellular Ca2+ antagonist 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate alone down-regulated [3H]PDBU binding and inhibited the up-regulation of ligand binding by f-Met-Leu-Phe and A23187. Low concentrations of La3+ (0.1-10 microM) also inhibited up-regulation of radioligand binding to f-Met-Leu-Phe and A23187, whereas higher concentrations (0.1-1 mM) alone increased [3H] PDBU binding and supported further up-regulation of ligand binding by the Ca2+-mobilizing agents. These data suggest a role for Ca2+ in the regulation of phorbol diester binding to protein kinase C in intact cells.