CCK Receptor Research Articles

Association between allelic variants in the glucagon-like peptide 1 and cholecystokinin receptor genes with gastric emptying and glucose tolerance.

Nutrient-mediated release of cholecystokinin and glucagon-like peptide-1 (GLP-1) regulates gastric emptying (GE) via duodenogastric feedback mechanisms; GLP-1 also regulates postprandial insulin secretion. Some patients with functional upper gastrointestinal symptoms have impaired glucose tolerance during enteral dextrose infusion. Our hypothesis was that variants in CCK, GLP-1, and TCF7L2 (transcription factor 7-like 2 locus), which is associated with greatest genetic risk for development of type 2 diabetes mellitus, are associated with GE and independently with glucose tolerance. Our aims were to evaluate the associations between these GE, glucose tolerance, and these single nucleotide polymorphisms (SNPs). Genetic variants, scintigraphic GE of solids, plasma glucose, insulin, and GLP-1 during enteral dextrose infusion (75gm over 2hours) were measured. GE and enteral dextrose infusion were, respectively, evaluated in 44 (27 controls and 17 patients with functional dyspepsia or nausea) and 42 (28 controls, 14 patients) participants; of these, 51 participants consented to assessment of SNPs. Four functional SNPs were studied: rs6923761 and rs1042044 at GLP-1 receptor, rs7903146 (TCF7L2), and rs1800857 (CCK receptor). Gastric emptying was normal in 38, rapid in 4, and delayed in two participants; 38 had normal, and four had impaired glucose tolerance. The T allele at rs7903146 (TCF7L2) was non-significantly associated (P=.14) with faster GE. The associations between SNPs and demographic variables, GE thalf , glucose tolerance and plasma GLP1 levels were not significant. There is a trend toward an association between faster GE and the diabetes-associated allele at rs7903146 in TCF7L2. However, these SNPs were not associated with plasma glucose or GLP1 concentrations during enteral dextrose infusion.

Ser-Tyr and Asn-Ala, vasorelaxing dipeptides found by comprehensive screening, reduce blood pressure via different age-dependent mechanisms.

To understand the changes in physiological responses due to aging, a number of bioactive probes based on different signal transduction pathways are necessary. In this study, we comprehensively and systematically investigated changes in blood vessel function with age using a 336-dipeptide library.In the early stage of hypertension, the most potent vasorelaxant dipeptide was Ser-Tyr (SY) in the mesenteric artery isolated from spontaneously hypertensive rats (SHR). SY-induced vasorelaxation and anti-hypertensive effects were blocked by L-NAME, an inhibitor of nitric oxide synthase (NOS), suggesting that SY activates the NO system. On the other hand, the patterns of dipeptides with vasorelaxation activity in early and advanced stages of hypertension were different. In the advanced stage, the most potent vasorelaxing dipeptide was Asn-Ala (NA). Orally administered NA (1.5 mg/kg) reduced the blood pressure in the advanced stage, at which drugs were sometimes less effective, and the anti-hypertensive effects lasted for 6 hr. The NA-induced vasorelaxation and anti-hypertensive activity was blocked by lorglumide, an antagonist of the cholecystokinin CCK1 receptor, suggesting that NA activated the CCK system.Taken together, in the early and advanced stages of hypertension, SY and NA exhibited vasorelaxing and anti-hypertensive effects via the NO and CCK systems, respectively.

Volume changes of the pancreatic head remnant after distal pancreatectomy

BackgroundLittle is known about pancreatic regeneration in humans after surgical resection. We examined pancreatic head volume changes after distal pancreatectomy. MethodsUsing computed tomography or magnetic resonance imaging volumetry, we assessed volume changes of the pancreatic head remnant in 67 patients at defined time points (3, 6, 9, and 12 months) after distal pancreatectomy. A volume increase of >1 cm³ was defined as hypertrophy, a decrease of >1 cm³ as atrophy, and alterations of ±1 cm³ were considered as unchanged. Volumetry results were correlated with clinical patient data, histology, and immunohistochemistry for the pancreatic regeneration markers Pax4, Ghrelin, cholecystokinin receptor A, and cholecystokinin receptor B of the resection margin. ResultsOf 67 patients, 33 patients (49%) exhibited a hypertrophy of the pancreatic head remnant with a median increase of 5.08 cm³, 26 patients (39%) showed an atrophy, and in 8 patients (12%) pancreatic volume remained unchanged. No correlation of preoperative, postoperative, and new-onset diabetes with hypertrophy or atrophy was found. In patients with ductal adenocarcinoma, hypertrophy occurred less frequently compared to patients with other pathologies (38% vs 63%; P = .04). In patients with ductal adenocarcinoma, hypertrophy was associated with significantly shorter survival. Patients with a postoperative hypertrophy that did not suffer from ductal adenocarcinoma displayed significantly less fibrosis at the resection margin compared to patients with a postoperative atrophy and pancreatic ductal adenocarcinoma patients. Immunohistochemical staining revealed no differential expression of the tested regeneration markers in hypertrophy versus atrophy. ConclusionThis study demonstrates volume changes of the pancreatic head remnant after distal pancreatectomy. Clinical and functional significance and underlying molecular mechanisms in humans remain unclear.

Citrus aurantium flavonoid extract improves concentrate efficiency, animal behavior, and reduces rumen inflammation of Holstein bulls fed high-concentrate diets

One hundred fourty-four bulls (164.8 ± 5.91 kg BW and 135 ± 7.2 d of age) were randomly allocated to one of 8 pens and assigned to control (C) or citrus flavonoid (BF) treatments (Citrus aurantium, 0.4 kg per ton of concentrate of Bioflavex CA, > 20% naringin; BF). Each pen had one drinker, one separate five-space straw feeder, and one separate three-space feeder where mash concentrate containing mostly corn, barley, DDG and wheat was offered. Concentrate intake was recorded daily, whilst BW and animal behavior were recorded fortnightly. Animals were slaughtered after 168 d of study (12 periods of 14 d), and HCW and carcass quality were recorded, and rumen papillae samples were collected. Final BW (437.9 ± 1.85 kg), HCW (238.7 ± 2.02 kg), and concentrate intake (7.1 ± 0.13 kg/d) were not affected by treatment. Concentrate feed conversion ratio (kg of concentrate/ kg of BW) tended (P < 0.10) to be lesser in BF than in C bulls (5.11 vs. 5.36 ± 0.108 kg/kg). Percentage of animals eating concentrate during visual scan was greater (P < 0.01) in BF compared with C bulls (10.02% vs. 7.97% ± 0.512). Oral non-nutritive behaviors, agonistic interactions (fighting, butting, and chasing) and sexual behaviors (flehmen, attempted and complete mounts) were greater (P < 0.01) in C than in BF bulls. In the rumen epithelium, gene expression of bitter taste receptor 7, bitter taste receptor 16, bitter taste receptor 38 and bitter taste receptor 39 was greater (P < 0.05) in C compared with BF bulls, as well as was gene expression of free fatty acid receptor 2, pancreatic polypeptide receptor 1, cholecystokinin receptor 4, cytokine IL-25, Toll-like receptor-4 and β-defensin1. In conclusion, supplementation with flavonoids extracted from Citrus aurantium in bulls fed high-concentrate diets tends to improve efficiency, and reduces oral non-nutritive behaviors, agonistic interactions and sexual behavior. Moreover, flavonoid supplementation modifies the expression of genes in the rumen epithelium that could be related with eating and animal behavior regulation.

Cholecystokinin Receptor Antagonist Therapy Decreases Inflammation and Fibrosis in Chronic Pancreatitis.

Chronic pancreatitis is associated with recurrent inflammation, pain, fibrosis, and loss of exocrine and endocrine pancreatic function and risk of cancer. We hypothesized that activation of the CCK receptor contributes to pancreatitis and blockade of this pathway would improve chronic pancreatitis. Two murine models were used to determine whether CCK receptor blockade with proglumide could prevent and reverse histologic and biochemical features of chronic pancreatitis: the 6-week repetitive chronic cerulein injection model and the modified 75% choline-deficient ethionine (CDE) diet. In the CDE-fed model, half the mice received water supplemented with proglumide, for 18weeks. After chronic pancreatitis was established in the cerulein model, half the mice were treated with proglumide and half with water. Histology was scored in a blinded fashion for inflammation, fibrosis and acinar ductal metaplasia (ADM) and serum lipase levels were measured. RNA was extracted and examined for differentially expressed fibrosis genes. Proglumide therapy decreased pancreatic weight in the CDE diet study and the cerulein-induced chronic pancreatitis model. Fibrosis, inflammation, and ADM scores were significantly reduced in both models. Lipase values improved with proglumide but not in controls in both models. Proglumide decreased pancreas mRNA expression of amylase, collagen-4, and TGFβR2 gene expression by 44, 38, and 25%, respectively, compared to control mice. New strategies are needed to decreased inflammation and reduce fibrosis in chronic pancreatitis. CCK receptor antagonist therapy may improve chronic pancreatitis by reversing fibrosis and inflammation. The decrease in ADM may reduce the risk of the development of pancreatic cancer.

Effects of dietary energy level on appetite and central adenosine monophosphate-activated protein kinase (AMPK) in broilers.

Adenosine monophosphate-activated protein kinase (AMPK) acts as a sensor of cellular energy changes and is involved in the control of food intake. A total of 216 1-d-old broilers were randomly allotted into 3 treatments with 6 replicates per treatment and 12 broilers in each cage. The dietary treatments included 1) high-energy (HE) diet (3,500 kcal/kg), 2) normal-energy (NE) diet (3,200 kcal/kg), and 3) low-energy (LE) diet (2,900 kcal/kg). The present study was conducted to investigate the effects of dietary energy level on appetite and the central AMPK signal pathway. The results showed that a HE diet increased average daily gain (ADG), whereas a LE diet had the opposite effect (P < 0.05, N = 6). The average daily feed intake (ADFI) of the chickens fed the LE diet was significantly higher than that of the control (P < 0.05, N = 6). Overall, the feed conversion rate gradually decreased with increasing dietary energy level (P < 0.05, N = 6). Moreover, the chickens fed the LE and HE diets demonstrated markedly improved urea content compared with the control group (P < 0.0001, N = 8). The triglyceride (TG) content in the LE group was obviously higher than that in the HE group but showed no change compared with the control (P = 0.0678, N = 8). The abdominal fat rate gradually increased with increased dietary energy level (P = 0.0927, N = 8). The HE group showed downregulated gene expression levels of liver kinase B1 (LKB1), neuropeptide Y (NPY), cholecystokinin (CCK), and glucocorticoid receptor (GR) in the hypothalamus compared with the control group (P < 0.05, N = 8). However, LE treatment significantly increased the mRNA level of AMP-activated protein kinase α2 (AMPKα2) compared with other groups (P = 0.0110, N = 8). In conclusion, a HE diet inhibited appetite and central AMPK signaling. In contrast, a LE diet activated central AMPK and appetite. Overall, the central AMPK signal pathway and appetite were modulated in accordance with the energy level in the diet to regulate nutritional status and maintain energy homeostasis in birds.

Gastrin vaccine improves response to immune checkpoint antibody in murine pancreatic cancer by altering the tumor microenvironment.

Pancreatic cancer has been termed a 'recalcitrant cancer' due to its relative resistance to chemotherapy and immunotherapy. This resistance is thought to be due in part to the dense fibrotic tumor microenvironment and lack of tumor infiltrating CD8 + T cells. The gastrointestinal peptide, gastrin, has been shown to stimulate growth of pancreatic cancer by both a paracrine and autocrine mechanism. Interruption of gastrin at the CCK receptor may reduce tumor-associated fibrosis and alter tumor immune cells. Polyclonal Ab Stimulator (PAS) is a vaccine that targets gastrin and has been shown to prolong survival of patients with pancreatic cancer. Here, we report that PAS vaccination monotherapy elicits both a humoral and cellular immune response when used in immune competent mice-bearing pancreatic tumors and that PAS monotherapy produced a marked T-cell activation and influx of CD8 + lymphocytes into pancreatic tumors. Isolated peripheral lymphocytes elicited cytokine release upon re-stimulation with gastrin in vitro demonstrating specificity of immune activation for the target peptide. Combination therapy with PAS and PD-1 Ab activated CD4 -/CD8 - TEMRA cells important in T-cell-mediated tumor death and memory. Tumors of mice treated with PAS (250μg) or PAS (100 and 250μg) in combination with a PD-1 Ab were significantly smaller compared to tumors from PBS or PD-1 Ab-treated mice. When PAS was given in combination with PD-1 Ab, tumors had less fibrosis, fewer inhibitory Treg lymphocytes, and fewer tumor-associated macrophages. These findings reveal a novel approach to improve treatment strategies for pancreatic cancer.

Changes in plasma levels of cholecystokinin, neurotensin, VIP and PYY in gastric and colorectal cancer – Preliminary results

Physiological roles of enterohormones such as secretion, absorption and digestion were supported by clinical data. Overexpression of cholecystokinin (CCK), neurotensin (NT) and vasoactive intestinal peptide (VIP) receptors occur in gastrointestinal (GI) malignancies. The aim of the paper was to compare plasma levels of CCK, peptide YY (PYY), VIP and NT in patients with gastrointestinal malignancies and healthy controls. The study included 80 patients (37 men and 43 women) with GI malignancies (20 with gastric and 60 with colorectal cancers). Median age of the patients was 62.9 years (range: 40–85 years). Control group was comprised of 30 healthy persons with median age 59.8 years (range: 40–82 years). Fasting plasma concentrations of CKK, PYY, NT, and VIP were determined at rest, using ELISA kits for automated systems. Comparative analysis of enterohormone levels in patients with various types of gastrointestinal malignancies demonstrated presence of some cancer-specific alterations. Patients with gastric cancers presented with lower plasma concentrations of CCK than healthy controls and individuals from colorectal cancers (p = 0.02). The highest plasma concentrations of neurotensin was found in colorectal cancer patients in comparison to gastric (p = 0.02). The plasma levels of VIP observed in gastric cancer group were lower than in colorectal cancer patients (p = 0.01). Patients with GI malignancies may present with tumor-specific alterations in plasma enterohormone levels.

Cholecystokinin receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Cholecystokinin receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on CCK receptors [89]) are activated by the endogenous peptides cholecystokinin-8 (CCK-8), CCK-33, CCK-58 and gastrin (gastrin-17). There are only two distinct subtypes of CCK receptors, CCK1 and CCK2 receptors [63, 123], with some alternatively spliced forms most often identified in neoplastic cells. The CCK receptor subtypes are distinguished by their peptide selectivity, with the CCK1 receptor requiring the carboxyl-terminal heptapeptide-amide that includes a sulfated tyrosine for high affinity and potency, while the CCK2 receptor requires only the carboxyl-terminal tetrapeptide shared by each CCK and gastrin peptides. These receptors have characteristic and distinct distributions, with both present in both the central nervous system and peripheral tissues.

Blockade of the cholecystokinin CCK-2 receptor prevents the normalization of anxiety levels in the rat

Cholecystokinin (CCK), through the CCK-2 receptor, exerts complex effects on anxiety. While CCK agonists are panicogenic, CCK-2 antagonists fail to alleviate human anxiety. Preclinical studies with CCK-2 antagonists are also inconsistent because their anxiolytic effects largely depend on the behavioral paradigm and antecedent stress. The controversy might be accounted by the neuromodulatory role for CCK in anxiety which is ill-defined. If this is its actual role, blocking CCK-2 will have carry-over effects on the anxiety baseline over time. To test this hypothesis, the consequences of acute administration of the CCK-2 antagonist Ly225.910 (0.1 mg Kg−1) was evaluated in the temporal expression of aversion toward exploration-conflicting tasks. Ly225.910 effects were evaluated in rats exposed to the elevated plus-maze (EPM) twice, an approach-avoidance anxiety-like test. While LY225.910-treated rats had less anxiety than vehicle-treated rats, the difference was reversed during the EPM retest 24 h later without drug. Moreover, Ly225.910 effects in stress-induced cognitive impairment was measured giving the novel-object discrimination (NOD) test to rats not habituated to the exploration apparatus to elicit neophobia. After a first encounter with objects (“old”), Ly225.910-treated rats did not recognize the “novel” object introduced 6 h later. Ly225.910-exposed rats did not discriminate the new location of the “novel object” when it was repositioned in the arena 24 h later. Ly225.910-treated rats also failed to explore objects. In line with its neuromodulatory role, aversive carry-over effects of Ly225.910 suggest that CCK-2 activation by endogenous CCK, rather than triggering anxiety, may return the anxiety state to its normal level.

Update on the Molecular Mechanisms Underlying the Effect of Cholecystokinin and Cholecystokinin-1 Receptor on the Formation of Cholesterol Gallstones.

Cholecystokinin (CCK) is an important neuro-intestinal peptide hormone produced by the enteroendocrine I-cells in the upper part of small intestine. Protein- and fat-enriched food plays an important role in triggering CCK secretion from the intestine. Carbohydrates stimulate only small amounts of CCK release. The CCK-1 receptor (CCK-1R) is largely localized in the gallbladder, sphincter of Oddi, pancreas, small intestine, gastric mucosa, and pyloric sphincter, where it is responsible for CCK to regulate multiple digestive processes including gallbladder contraction, pancreatic secretion, small intestinal transit, and gastric emptying. Accumulated evidence clearly demonstrates that CCK regulates gallbladder and small intestinal motility through CCK-1R signaling cascade and the effect of CCK-1R on small intestinal transit is a physiological response for regulating intestinal cholesterol absorption. Disruption of the Cck or the Cck-1r gene in mice significantly increases the formation of cholesterol gallstones by disrupting gallbladder emptying and biliary cholesterol metabolism, as well as promoting intestinal absorption of cholesterol. Abnormalities in gallbladder motility function in response to exogenously administered CCK are found primarily in patients with cholesterol gallstones. Patients with pigment gallstones display an intermediate degree of gallbladder motility defect without gallbladder inflammation and enlarged fasting gallbladder. Dysfunctional gallbladder contractility has been found under several conditions such as pregnancy, obesity, diabetes, celiac disease, and total parenteral nutrition although gallstones are not observed. The gallbladder-specific CCK-1R-selective agonist may lead to an efficacious novel way for preventing gallstone formation by promoting gallbladder emptying, particularly for pregnant women and subjects with dysfunctional gallbladder motility function such as celiac patients, as well as patients with total parenteral nutrition.

Isatoic Anhydride: A Fascinating and Basic Molecule for the Synthesis of Substituted Quinazolinones and Benzo di/triazepines

This review article is focused on the synthesis of compounds with quinazolinones and benzo di/triazepine scaffolds. These invaluable derivatives are of great interest in medicinal and pharmaceutical studies because of their important biological properties. Quinazolinones have diverse applications due to their antibacterial, analgesic, antiinflammatory, antifungal, antimalarial, antihypertensive, CNS depressant, anticonvulsant, antihistaminic, antiparkinsonism, antiviraland and anticancer activities. On the other hand, pharmacological properties of benzodiazepines include antianxiety, anticancer, anticonvulsant, antagonists of cholecystokinin receptors (CCK), antileishmanial, sleep-inducing muscle relaxant and several other useful and interesting properties. As an example, three main categories of drugs, namely anxiolytics, sedative hypnotics (sleep inducers) and anticonvulsants are constructed by 1,4-benzodiazepines. Finally, benzotriazepines are believed to possess various pharmacological properties such as antipsychotic and antitumor activities. Hence, this review is divided into three major sections, considering quinazolinones, benzodiazepines and benzotriazepines. In the first section, we take a brief look at various approaches towards synthesis of substituted quinazolin-4(3H)-ones and 2,3-dihydroquinazolin-4(1H)-ones. Also in this section, we try to give an overview of the synthetic routes and strategies recently reported for the generation of various classes of substituted 4(3H)-quinazolinones and 2,3-dihydroquinazolin-4(1H)-ones. Accordingly, quinazolin-4(3H)-ones, were subdivided into three major classes: 2-substituted, 3-substituted and 2,3-disubstituted-quinazolinones. 2,3- dihydroquinazolin-4(1H)-ones also were subdivided into six sub-categories: 2-monosubstituted, 2,2- disubstituted, 2,3-disubstituted, 1,2,3-trisubstituted, 2,2,3-trisubstituted 2,3-dihydroquinazolin-4(1H)-ones and boron-containing quinazoline-4(1H)-ones. In the other two sections, we cover the literature related to synthesis of benzo di/triazepine. The most recent developments are highlighted with a special emphasis on new synthetic routes based on isatoic anhydride as starting material.

Targeting cholecystokinin-2 receptor for pancreatic cancer chemoprevention.

Gastrin signaling mediated through cholecystokinin-2 receptor (CCK2R) and its downstream molecules is altered in pancreatic cancer. CCK2R antagonists, YF476 (netazepide) and JNJ-26070109, were tested systematically for their effect on pancreatic intraepithelial neoplasia (PanIN) progression to pancreatic ductal adenocarcinoma (PDAC) in KrasG12D mice. After dose selection using wild-type mice, six-week-old p48Cre/+ -LSL-KrasG12D (22-24 per group) genetically engineered mice (GEM) were fed AIN-76A diets containing 0, 250, or 500 ppm JNJ-26070109 or YF-476 for 38 weeks. At termination, pancreata were collected, weighed, and evaluated for PanINs and PDAC. Results demonstrated that control-diet-fed mice showed 69% (males) and 33% (females) incidence of PDAC. Administration of low and high dose JNJ-26070109 inhibited the incidence of PDAC by 88% and 71% (P < .004) in male mice and by 100% and 24% (P > .05) in female mice, respectively. Low and high dose YF476 inhibited the incidence of PDAC by 74% (P < .02) and 69% (P < .02) in male mice and by 45% and 33% (P > .05) in female mice, respectively. Further, transcriptome analysis showed downregulation of Cldn1, Sstr1, Apod, Gkn1, Siglech, Cyp2c44, Bnc1, Fmo2, 623169, Kcne4, Slc27a6, Cma1, Rho GTPase activating protein 18, and Gpr85 genes in JNJ-26070109-treated mice compared with untreated mice. YF476-treated mouse pancreas showed downregulation of Riks, Zpbp, Ntf3, Lrrn4, Aass, Skint3, Kcnb1, Dgkb, Ddx60, and Aspn gene expressions compared with untreated mouse pancreas. Overall, JNJ-26070109 showed better chemopreventive efficacy than YF476. However, caution is recommended when selecting doses, as the agents appeared to exhibit gender-specific effects.

A Cholecystokinin Receptor Antagonist Halts Nonalcoholic Steatohepatitis and Prevents Hepatocellular Carcinoma

Background and AimsNonalcoholic steatohepatitis (NASH) is a common inflammatory liver condition that may lead to cirrhosis and hepatocellular carcinoma (HCC). Risk factors for NASH include a saturated fat diet, altered lipid metabolism, and genetic and epigenetic factors, including microRNAs. Serum levels of cholecystokinin (CCK) are elevated in mice and humans that consume a high-saturated fat diet. CCK receptors (CCK-Rs) have been reported on fibroblasts which when activated can induce fibrosis; however, their role in hepatic fibrosis remains unknown. We hypothesized that elevated levels of CCK acting on the CCK-Rs play a role in the development of NASH and in NASH-associated HCC.MethodsWe performed a NASH Prevention study and Reversal study in mice fed a saturated fat 75% choline-deficient–ethionine-supplemented (CDE) diet for 12 or 18 weeks. In each study, half of the mice received untreated drinking water, while the other half received water supplemented with the CCK-R antagonist proglumide. CCK-R expression was evaluated in mouse liver and murine HCC cells.ResultsCCK receptor antagonist treatment not only prevented NASH but also reversed hepatic inflammation, fibrosis, and steatosis and normalized hepatic transaminases after NASH was established. Thirty-five percent of the mice on the CDE diet developed HCC compared with none in the proglumide-treated group. We found that CCK-BR expression was markedly upregulated in mouse CDE liver and HCC cells compared with normal hepatic parenchymal cells, and this expression was epigenetically regulated by microRNA-148a.ConclusionThese results support the novel role of CCK receptors in the pathogenesis of NASH and HCC.

Anxiety-induced hyperalgesia in female rats is mediated by cholecystokinin 2 receptor in rostral ventromedial medulla and spinal 5-hydroxytryptamine 2B receptor.

BackgroundPreoperative anxiety is associated with postoperative hyperalgesia; however, few studies have investigated the mechanism underlying this association in female surgical patients. Research has suggested that ON cells in the rostral ventromedial medulla (RVM) receive nerve impulses via cholecystokinin 2 (CCK2) receptors, facilitating hyperalgesia. Additionally, the downstream serotonergic projection system from the RVM to the spinal cord has a dual regulating effect on pain responses, and the 5-hydoxytryptophan 2B (5-HT2B) receptor in spinal dorsal horn neurons is critically involved in mechanical allodynia.MethodsOvariectomized rats were treated with estrogen replacement, single prolonged stress (SPS), and plantar incision. Various receptor agonists and antagonists were then administered into the RVM and spinal cord to study the mechanism underlying postoperative hyperalgesia caused by preoperative anxiety in female rats.ResultsBehavioral testing revealed that preoperative SPS induced postoperative hyperalgesia, as well as the expression of the CCK2 receptor in the RVM and the expression of the 5-HT2B receptor, protein kinase Cγ (PKCγ), and phosphorylation of the N-methyl-d-aspartate receptor1 (p-NR1) in the spinal cord increased confirmed by Western blot. RVM microinjection of the CCK2 receptor agonist CCK-8 and intrathecal injection of the 5-HT2B receptor agonist BW723C86 both produced hyperalgesia in female rats after plantar incision, whereas the CCK2 receptor antagonist YM022, the 5-HT2B receptor antagonist RS127445, and the PKCγ inhibitor C37H65N9O13 decreased the rats’ sensitivity to the same stimulus. Additionally, electrophysiological analysis suggested that activation of the 5-HT2B receptor increased the whole-cell current (IBa) in superficial dorsal horn neurons through the PKCγ pathway.ConclusionOur study demonstrated that preoperative anxiety-induced postoperative hyperalgesia in female rats is associated with descending pain pathways. The CCK2 receptor in the RVM and spinal 5-HT2B receptor may play a role in this hyperalgesic effect.

Abstract 2728: Cholecystokinin receptor antagonist proglumide reverses nonalcoholic steatohepatitis and prevents hepatocellular carcinoma

Abstract Background: The fastest growing cause of cancer related death is hepatocellular carcinoma (HCC), in part due to the rising prevalence of nonalcoholic steatohepatitis (NASH). HCC cases are expected to increase during 2015-2030. Cholecystokinin (CCK), blood levels are increased in those consuming high fat diets and CCK receptors have been identified in normal liver, fibroblasts, and immune cells. Hypothesis: We hypothesize that CCK receptor blockade could decrease NASH and prevent HCC in a mouse model.Methods: We examined whether CCK receptor blockade with proglumide could decrease NASH in the choline deficient ethionine supplemented (CDE)-diet mouse model. Two cohorts of Female C57BL/6 mice (N=65) mice were examined: (1) a preventive study in which mice were treated for 12 and 18 weeks to determine if proglumide could prevent NASH and HCC and (2) a ‘reversal study’ in which histologic and biochemical NASH was induced on a 75% CDE high fat diet for 12 weeks followed by treatment with proglumide for 6 weeks (until week 18) while remaining on the CDE diet to determine if CCK-receptor blockade could reverse NASH once established. Control mice received the same high fat diet with choline and half received proglumide therapy. Results: Alanine aminotransferase, aspartate aminotransferases, and bilirubin were increased in mice on the CDE/Regular (untreated water) diet and these values returned to near normal with proglumide therapy in the prevention study and also reversed after NASH was established. Histologic scoring showed significant decreased inflammation, fibrosis and steatosis in mice on the high fat CDE diet when treated with proglumide. 35% of the mice (N=23) at week 18 developed HCC or dysplastic nodules on the CDE diet while none of the mice in the CDE/proglumide prevention (N=15) or reversal therapy groups (N=15) had HCC. Mice on the lard-control diet had fatty livers at week 18 but no evidence of NASH histologically or biochemically and no HCC. Fibrosis was decreased in mice treated with proglumide as evidence by Masson’s trichrome stain and Western blot for FAP. Gene expression of the liver’s collagen-4, TGFβR11, PDGH-Rβ, and collagen-1α1 as determined by qRT-PCR decreased with proglumide treatment. Control diet fed mice had normal serum tests and fibrosis biomarkers. Conclusion: This data supports our novel hypothesis that CCK receptors play a role in NASH-associated HCC. CCK receptor blockade decreased histologic and biochemical evidence of NASH and prevented HCC. Since proglumide has already been tested in human subjects for peptic ulcer disease decades ago and is known to be orally bioavailable, results of our study could easily be translated into a clinical trial in human subjects to improve NASH to prevent HCC. Supported by AGA Research Foundation, Ruesch Foundation, and a grant from NIH, TL1TR001431 Citation Format: Martha D. Gay, Victor Ciofoaia, Sandeep Nadella, Hong Cao, Matthew Huber, Robin D. Tucker, Narayan Shivapurkar, Bhaskar Kallakury, Alexander H. Kroemer, Jill P. Smith. Cholecystokinin receptor antagonist proglumide reverses nonalcoholic steatohepatitis and prevents hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2728.

OP#40 - [99mTc]Tc-HYNIC-MGS5 and [68Ga]Ga-DOTA-MGS5: two novel imaging probes for cholecystokinin-2 receptor targeting

OP#40 - [99mTc]Tc-HYNIC-MGS5 and [68Ga]Ga-DOTA-MGS5: two novel imaging probes for cholecystokinin-2 receptor targeting

Abstract 5079: Targeting cholecystokinin-2 receptor (CCK2R) for pancreatic ductal adenocarcinoma prevention in p48Cre/+-LSL-KrasG12D/+ mice model

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer that is difficult to treat. Hence, developing strategies that delay/inhibit/prevent the progression of pancreatic intraepithelial neoplasms (PanINs) to PDAC are of utmost importance. Gastrin signaling mediated through gastrin/cholecystokinin-2 receptor (CCK2R) and its down-stream signaling molecules are overexpressed in both human and KrasG12D mouse PDACs. Particularly, CCK2R is highly expressed in PDAC stellate cells and is involved in fibrosis and PDAC progression. Thus, targeting CCK2R for pancreatic chemoprevention is a valid approach for PDAC prevention. Two CCK2R antagonists, netazepide (YF476) and JNJ-26070109, were tested for their effect on PanINs and their progression to PDAC in p48Cre/+-LSL-KrasG12D/+ mice. Six-week-old p48Cre/+-LSL-KrasG12D/+ (22-24/group) mice were fed (AIN-76A) diets containing 0, 250 or 500 ppm netazepide or JNJ-26070109 for 38 weeks. After sacrifice at 44 weeks of age, pancreata were collected, weighed, and evaluated histopathologically for PanINs and PDAC. To understand the molecular mechanisms, levels of proliferation and apoptosis (PCNA, p21, and caspase3) were analyzed by Immunohistochemistry (IHC). Results demonstrated that control diet fed mice showed 69% and 33% incidence of PDAC in male and female mice. Dietary administration of low dose and high dose JNJ-26070109 inhibited the incidence of PDAC by 88% (p&amp;lt;0.003) and 71% (p&amp;lt;0.004) in male mice and by 100% and 24 % (not significant, p&amp;gt;0.05) in female mice respectively. Dietary administration of low dose and high dose netazepide inhibited the incidence of PDAC by 74% (p&amp;lt;0.018) and 69% (p&amp;lt;0.02) in male mice and by 45% and 33 % (not significant, p&amp;gt;0.05) in female mice, respectively. Both agents reduced PanIN 3 (carcinoma in-situ) multiplicity compared to untreated mice in both genders, with JNJ-26070109 showing greater inhibitory effect compared to netazepide. Both agents showed a decrease in caspase-3, p21, and PCNA expressions. Transcriptome analysis showed downregulation of Cldn1, Sstr1, Apod, Gkn1, Siglech, Cyp2c44, Bnc1, Fmo2, 623169, Kcne4, Slc27a6, Cma1, Rho GTPase activating protein 18 and Gpr85 genes in JNJ-26070109-treated mice compared to untreated mice. Netazepide-treated mouse pancreas specifically showed down regulation of Riks, Zpbp, Ntf3, Lrrn4, Aass, Skint3, Kcnb1, Dgkb, Ddx60, and Aspn genes compared to untreated mice pancreas. In summary, targeting the CCK2R pathway by netazepide and JNJ-26070109 showed significant chemopreventive effects in suppression of PDAC and PanIN3. Overall, JNJ-26070109 showed better chemopreventive efficacy on PDAC incidence and PanIN 3 multiplicity as compared to netazepide. However, caution is recommended while selecting the doses as the agents appeared to exhibit gender-specific effects. [Supported by NCICN25001-26} Citation Format: Altaf Mohammed, Naveena B. Janakiram, Chen Suen, Nicole Stratton, Stanley Lightfoot, Anil Singh, Gopal Pathuri, Rebekah Ritchie, Venkateshwar Madka, Chinthalapally V. Rao. Targeting cholecystokinin-2 receptor (CCK2R) for pancreatic ductal adenocarcinoma prevention in p48Cre/+-LSL-KrasG12D/+ mice model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5079.

QSAR Modeling of CCK2 Receptor Antagonists Utilizing Computed Structural Indices

In the present case study, a QSAR model has been developed to identify CCK2 receptor antagonists. The earlier reported 3D pharmacophore modeling of these molecules involved commercial software. Since the reduction in the cost involved in the drug discovery phase is very crucial, in the present study, QSAR models based on the structural indices including 1D, 2D and 3D indices computed from the structures of CCK2 receptor antagonists has been developed utilizing NanoBRIDGES software which is openly accessible (http://nanobridges.eu/software/). This QSAR model is not only comparable to the earlier reported model, but it also reasonably predicts the external set of nine compounds including the drug benzotript and the most active 6a described in earlier work on structure-based homology model CCK2 receptor antagonists as antiulcer agents and thus may be useful in virtual screening for the identification of new CCK2 receptor antagonists.

The cholecystokinin receptor agonist, CCK-8, induces adiponectin production in rat white adipose tissue.

A cholecystokinin (CCK) system has been identified in white adipose tissue (WAT). Nevertheless, the endocrine actions of CCK on WAT remain unknown. Our goal was to investigate the role of CCK in regulating the production of adiponectin, an adipokine expressed in WAT, which is pivotal in preserving energy homeostasis. The effect of the bioactive CCK fragment CCK-8 on adiponectin production was studied both in vivo and in vitro. CCK-8 effects were characterized in rats treated with selective CCK1 and CCK2 receptor antagonists as well as in pre-adipocytes carrying the selective silencing of either CCK1 or CCK2 receptors. The influence of insulin on CCK-8 responses was also analysed. In WAT, CCK-8 increased plasma adiponectin levels and the expression of the adiponectin gene (Adipoq). In pre-adipocytes, CCK-8 up-regulated adiponectin production. CCK-8 effects were abolished by L-365,260, a selective CCK2 receptor antagonist. CCK2 receptor knockdown also abolished the effects of CCK-8 in pre-adipocytes. Moreover, in vitro CCK-8 effects were blocked by triciribine, a specific inhibitor of protein kinase B (Akt) and by the PPARγ antagonist T0070907. Silencing the expression of the insulin receptor inhibited CCK-8-induced Adipoq expression in pre-adipocytes. Furthermore, insulin potentiated the effect of CCK-8. CCK-8 stimulates adiponectin production in WAT by acting on CCK2 receptors, through a mechanism involving both Akt and PPARγ. Moreover, CCK-8 actions are only observed in the presence of insulin. Our results could have translational value in the design of new insulin-sensitizing therapies.