CCK Receptor Research Articles

Boar sperm motility is modulated by CCK at a low concentration of bicarbonate under capacitation conditions.

In a previous study, our group detected the cholecystokinin (CCK) protein in the porcine oviduct. This fact, together with the involvement of CCK in the regulation of sperm protein tyrosine phosphorylation by the modulation of HCO3 - uptake (in mice and humans) suggests a role for CCK during sperm capacitation. Therefore, on the one hand, the expression of CCK receptors (CCK1R and CCK2R) on boar testes has been investigated and probed; on the other hand, boar spermatozoa (from seminal doses of 1-day and 5-day storage) were exposed to different concentrations of CCK (0-control, 25 or 50 μM) in a medium supporting capacitation supplemented with 0, 5 or 25 mmol/L of HCO3 - for 1 h at 38.5°C. Sperm motion (total and progressive motility), kinetic parameters, viability, acrosome status, and mitochondrial activity were determined. No differences between groups (0, 25 or 50 μM of CCK) were observed when HCO3 - was absent in the media (p > .05). However, the results showed that when the media was supplemented with 5 mmol/L HCO3 - in 1-day seminal dose storage, the linearity index (LIN, %), straightness index (STR, %) and oscillation index (WOB, %) (sperm kinetics parameters) increased in the presence of CCK regardless the concentration (p < .05). Nevertheless, CCK in sperm from 5-day storage only increased the WOB parameter in comparison to the control (p < .05). Furthermore, the average amplitude of the lateral displacement of the sperm head (ALH, μm) and curvilinear velocity (VCL, μm/s) decreased when CCK was present, depending on its concentration and sperm aging (1-day vs. 5-days) (p < .05). In the case of the media supporting capacitation supplemented with 25 mmol/L HCO3 - , any differences were observed except for sperm viability in the 5-day seminal doses, which increased in the 50 μM-CCK group compared to the control (p < .05). In conclusion, these data suggest an implication of CCK protein during sperm capacitation under low bicarbonate concentration increasing the sperm linear trajectory.

Reduced Striatal Dopamine Transporter Availability and Heightened Response to Natural and Pharmacological Stimulation in CCK-1R-Deficient Obese Rats.

Alterations in dopamine neurotransmission are associated with obesity and food preferences. Otsuka Long-Evans Tokushima Fatty (OLETF) rats that lack functional cholecystokinin receptor type-1 (CCK-1R), due to a natural mutation, exhibit impaired satiation, are hyperphagic, and become obese. In addition, compared to lean control Long-Evans Tokushima (LETO) rats, OLETF rats have pronounced avidity for over-consuming palatable sweet solutions, have greater dopamine release to psychostimulants, reduced dopamine 2 receptor (D2R) binding, and exhibit increased sensitivity to sucrose reward. This supports altered dopamine function in this strain and its general preference for palatable solutions such as sucrose. In this study, we examined the relationship between OLETF's hyperphagic behavior and striatal dopamine signaling by investigating basal and amphetamine stimulated motor activity in prediabetic OLETF rats before and after access to sucrose solution (0.3 M) compared to non-mutant control LETO rats, as well as availability of dopamine transporter (DAT) using autoradiography. In the sucrose tests, one group of OLETF rats received ad libitum access to sucrose while the other group received an amount of sucrose equal to that consumed by the LETO. OLETFs with ad libitum access consumed significantly more sucrose than LETOs. Sucrose exerted a biphasic effect on basal activity in both strains, i.e., reduced activity for 1 week followed by increased activity in weeks 2 and 3. Basal locomotor activity was reduced (-17%) in OLETFs prior to sucrose, compared to LETOs. Withdrawal of sucrose resulted in increased locomotor activity in both strains. The magnitude of this effect was greater in OLETFs and the activity was increased in restricted compared to ad-libitum-access OLETFs. Sucrose access augmented AMPH-responses in both strains with a greater sensitization to AMPH during week 1, an effect that was a function of the amount of sucrose consumed. One week of sucrose withdrawal sensitized AMPH-induced ambulatory activity in both strains. In OLETF with restricted access to sucrose, withdrawal resulted in no further sensitization to AMPH. DAT availability in the nucleus accumbens shell was significantly reduced in OLETF compared with aged-matched LETO. Together, these findings show that OLETF rats have reduced basal DA transmission and a heightened response to natural and pharmacological stimulation.

Cholecystokinin Receptor Antagonist Induces Pancreatic Stellate Cell Plasticity Rendering the Tumor Microenvironment Less Oncogenic.

CCK receptors are expressed on pancreatic cancer epithelial cells, and blockade with receptor antagonists decreases tumor growth. Activated pancreatic stellate cells or myofibroblasts have also been described to express CCK receptors, but the contribution of this novel pathway in fibrosis of the pancreatic cancer microenvironment has not been studied. We examined the effects of the nonselective CCK receptor antagonist proglumide on the activation, proliferation, collagen deposition, differential expression of genes, and migration in both murine and human PSCs. CCK receptor expression was examined using western blot analysis. Collagen production using activated PSCs was analyzed by mass spectroscopy and western blot. Migration of activated PSCs was prevented in vitro by proglumide and the CCK-B receptor antagonist, L365,260, but not by the CCK-A receptor antagonist L365,718. Proglumide effectively decreased the expression of extracellular matrix-associated genes and collagen-associated proteins in both mouse and human PSCs. Components of fibrosis, including hydroxyproline and proline levels, were significantly reduced in PSC treated with proglumide compared to controls. CCK peptide stimulated mouse and human PSC proliferation, and this effect was blocked by proglumide. These investigations demonstrate that targeting the CCK-B receptor signaling pathway with proglumide may alter the plasticity of PSC, rendering them more quiescent and leading to a decrease in fibrosis in the pancreatic cancer microenvironment.

Looking ahead to potential incretin combination therapies for nonalcoholic steatohepatitis in patients with diabetes

ABSTRACT Introduction There are no drugs approved by regulatory agencies for the treatment of nonalcoholic fatty liver disease (NAFLD); incretin combination therapies are being developed for treatment of type 2 diabetes and research has moved to test their usefulness in NAFLD. Areas covered We reviewed the literature on the effectiveness of dual and triple peptides combining receptor agonists of the glucagon-like peptide 1, the glucose-dependent insulinotropic peptide, and glucagon to treat NAFLD and its associated metabolic diseases, and/or the cardiovascular risk intimately connected with the cluster of the metabolic syndrome. Other combination peptides involved the glucagon-like peptide 2 receptor, the fibroblast growth factor 21, the cholecystokinin receptor 2, and the amylin receptor. Expert opinion Both dual and triple agonists are promising, based on animal, pharmacokinetic and proof-of concept studies, showing effectiveness both in the presence and the absence of diabetes on a few validated surrogate NAFLD biomarkers, but the majority of studies are still in progress. Considering the long natural history of NAFLD, final proof of their efficacy on primary clinical liver outcomes might be also derived from the analysis of large databases of National Healthcare Systems or Insurance companies, when used in diabetes for improving glycemic control, after careful propensity-score matching.

BRAIN CHOLECYSTOKININ SYSTEM IN NEUROTIC PATHOLOGY

Violation and discoordination of mental processes leads to changes in the ratio of the main neurotransmitters content and further to mental disorders. A significant role in such processes is played by neuropeptides, in particular anti-anxiety ones, which include cholecystokinin (CCK) and its receptors. It has been established that stimulation of CCK receptors modulates the mesolimbic system of reward, anxiety and satiety. CCK-1 (the intestine) and CCK-2 type receptors (the CNS and stomach) are isolated. In the central nervous system, the short O-sulfated form of CCK-8 and the non-sulfated form of CCK-5 predominate. It was shown that the effect of CCK on the corresponding receptors inhibits GABA-ergic receptors in the subcortical nuclei, which mediates the transmission of nerve signals from the endocannabinoid system. A significant number of CCK receptors, their agonists and antagonists were found in the body. Potentially, blockers of these receptors can be combined with other agents for the treatment of CNS diseases. In CCK, it was shown an anxiogenic effect, which was realized through the influence of a selective CCK-2 receptors agonist. There is information that CCK-4 causes anxiogenic effects due to its influence on other neurotransmitters, in particular, due to neuromodulation of the dopaminergic system, and can interact with other neurotransmission systems. In behavioral tests, the anxiolytic effect of CCK-2 receptor blockers is shown. The participation of this system in the pathogenesis of anxiety is confirmed by the influence of non-selective agonists of CCK receptors and agonists of CCK-2 receptors in the "elevated cross-shaped maze" test. At the same time, CCK-2 receptor blockers reduced anxiety in this test. In clinical studies, they suppressed panic when administered as a bolus. However, the problem of bioavailability of compounds remains unsolved. The significant diversity of CCK receptors and their expression in all parts of the brain also complicates their application greatly. So, the CCK system of the brain is involved in the development of the main manifestations of anxiety pathology, but there are prospects for the medical use of substances that regulate the functional activity of CCK receptors.

Cholecystokinin receptors in GtoPdb v.2023.1

Cholecystokinin receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on CCK receptors [90]) 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 [64, 124], 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.

D81. Gene Expression as Biomarkers of Tissue Viability in Ex Vivo Normothermic Limb Perfusion (EVNLP)

PURPOSE: Muscle viability remains the primary focus of ex vivo limb preservation. We hypothesized that genes associated with tissue injury pathophysiology are differentially expressed during EVNLP and static cold storage (SCS). METHODS: Bilateral porcine forelimbs were procured and randomly allocated to EVNLP or SCS. Skeletal muscle specimens were collected before limb procurement and during EVNLP or SCS at 0, 12, and 24h. Analysis of differential gene expression was performed with reverse transcriptase PCR to obtain fold changes. Pathway analysis was performed using Panther Classification System.The dream method from R package variance Partition (version 1.24.0) was used for differential gene expression analysis. Plots were made using log2-transformed normalized counts per million mapped reads. Statistical analyses were conducted using Bonferroni correction; the critical P value (α) was determined by the number of comparisons. RESULTS: A significantly different gene expression pattern, between EVNLP and SCS, was detected in pathways involved in inflammation, response to hypoxia, apoptosis, muscle and tissue regulation, coagulation, and other cellular signaling. At 12h, genes related to inflammation were transcribed at a higher rate in the EVNLP group, aside from macrophage inhibiting factor (MIF) which was downregulated at 12h and EVNLP endpoint. IL-10 was downregulated in both groups.Genes in pathways for coagulation, plasminogen activating cascade, presenilin pathway, cholecystokinin receptor signaling, and nicotine degradation were differentially expressed in SCS and EVNLP limbs at 12h. CONCLUSION: We identified genes differentially expressed in EVNLP compared to SCS. Increased transcription during EVNLP aligned with preserved metabolic activity.

Compounds Interacting with Cholecystokinin as Potential Drugs Against Excessive Weight Gain and Obesity

Cholecystokinin (CCK) is a peptide family that functions pancreatic enzyme secretion, gallbladder contraction, intestinal motility, satiety, and stomach acid secretion. The CCK peptides are derived from pro-CCK with a specific amino acid sequence (Asp, Tyr, Met, Gly, Trp, Met, Asp, and Phe) at their C-terminus, where the Tyr residue is sulfated. The CCK peptides are ligands of the CCK receptors 1 (CCK1) and 2 (CCK2). In this study, we propose an approach for treating overweight and obese patients, preventing or reducing CCK1 activation using compounds that specifically interact with CCK peptides to regulate their interactions with CCK1. The compounds were selected by molecular docking using a chemical library with the selected residues (Asp, Tyr, Met, Gly, Trp, Met, Asp, and Phe) in CCK. Their effects were evaluated in a diet-induced obese mice model. We show that compounds K2 and K5 decreased and maintained the body weight of the diet-induced obese mice model, respectively, likely by preventing/hindering interactions between CCK peptides and CCK1, modifying its activity. This study demonstrates another approach to treating overweight and obesity by regulating CCK1 functions. The K2 and K5 compounds have properties supporting their continued development as new drugs against these risk factors.

Ca2+ signals in pancreatic acinar cells in response to physiological stimulation in vivo.

The exocrine pancreas secretes fluid and digestive enzymes in response to parasympathetic release of acetylcholine (ACh) via the vagus nerve and the gut hormone cholecystokinin (CCK). Both secretion of fluid and exocytosis of secretory granules containing enzymes and zymogens are dependent on an increase in the cytosolic [Ca2+ ] in acinar cells. It is thought that the specific spatiotemporal characteristics of the Ca2+ signals are fundamental for appropriate secretion and that these properties are disrupted in disease states in the pancreas. While extensive research has been performed to characterize Ca2+ signalling in acinar cells, this has exclusively been achieved in ex vivo preparations of exocrine cells, where it is difficult to mimic physiological conditions. Here we have developed a method to optically observe pancreatic acinar Ca2+ signals in vivo using a genetically expressed Ca2+ indicator and imaged with multi-photon microscopy in live animals. In vivo, acinar cells exhibited baseline activity in fasted animals, which was dependent on CCK1 receptors (CCK1Rs). Both stimulation of intrinsic nervous input and administration of systemic CCK induced oscillatory activity in a proportion of the cells, but the maximum frequencies were vastly different. Upon feeding, oscillatory activity was also observed, which was dependent on CCK1Rs. No evidence of a vago-vagal reflex mediating the effects of CCK was observed. Our in vivo method revealed the spatial and temporal profile of physiologically evoked Ca2+ signals, which will provide new insights into future studies of the mechanisms underlying exocrine physiology and that are disrupted in pathological conditions. KEY POINTS: In the exocrine pancreas, the spatiotemporal properties of Ca2+ signals are fundamentally important for the appropriate stimulation of secretion by the neurotransmitter acetylcholine and gut hormone cholecystokinin. These characteristics were previously defined in ex vivo studies. Here we report the spatiotemporal characteristics of Ca2+ signals in vivo in response to physiological stimulation in a mouse engineered to express a Ca2+ indicator in acinar cells. Specific Ca2+ 'signatures' probably important for stimulating secretion are evoked in vivo in fasted animals, by feeding, neural stimulation and cholecystokinin administration. The Ca2+ signals are probably the result of the direct action of ACh and CCK on acinar cells and not indirectly through a vago-vagal reflex.

Cholecystokinin receptor antagonist induces pancreatic stellate cell plasticity rendering the tumor microenvironment less oncogenic

Cholecystokinin receptor antagonist induces pancreatic stellate cell plasticity rendering the tumor microenvironment less oncogenic

Heterodimerization of cholecystokinin 1 and cholecystokinin 2 receptors in gallbladder cancer: a new mechanism for carcinogenesis.

Cholecystokinin is present in abundance in gallbladder tissue and mediates function through two structurally related receptors, CCK1R and CCK2R. Heterodimerization of these receptors is known to impact cell growth in vitro. However, the significance of these heterodimers in gallbladder carcinogenesis is relatively unknown. Therefore, we evaluated the expression and the dimerization status of CCK1 and CCK2 receptors in human gallbladder carcinoma cell line (GBC-SD) and resected gallbladder tissue from normal (n = 10), cholelithiasis (n = 25) and gallbladder cancer (n = 25) by immunofluorescence/immunohistochemistry and western blot. The dimerization status of CCK1R and CCK2R was evaluated by co-immunoprecipitation. To understand the effect of heterodimerization of these receptors on growth-related signaling pathways, the expression of p-AKT, rictor, raptor and p-ERK was evaluated by western blot. We demonstrated the expression and heterodimerization of CCK1 and CCK2 receptor in GBC-SD gall bladder carcinoma cell line. Knockdown of CCK1R and CCK2R in the cell line led to significant reduction in p-AKT (P = 0.005; P = 0.0001) and rictor (P < 0.001; P < 0.001) levels. In tissue samples, significantly higher expression of CCK1R and CCK2R was observed in gallbladder cancer when compared to other groups both by immunohistochemistry (P = 0.008 and P = 0.013) and western blot (P = 0.009 and P = 0.003). An increase in heterodimer formation of CCK1R with CCK2R was observed in gallbladder cancer when compared to normal and cholelithiasis tissues. No significant difference in the expression of p-AKT and p-ERK was observed between the three groups. Our results provide the first evidence of heterodimerization of CCK1R and CCK2R in gallbladder tissue, and its association with development of gallbladder cancer. This finding has potential clinical and therapeutic significance.

Effect of N-Terminal Peptide Modifications on In Vitro and In Vivo Properties of 177Lu-Labeled Peptide Analogs Targeting CCK2R

The therapeutic potential of minigastrin (MG) analogs for the treatment of cholecystokinin-2 receptor (CCK2R)-expressing cancers is limited by poor in vivo stability or unfavorable accumulation in non-target tissues. Increased stability against metabolic degradation was achieved by modifying the C-terminal receptor-specific region. This modification led to significantly improved tumor targeting properties. In this study, further N-terminal peptide modifications were investigated. Two novel MG analogs were designed starting from the amino acid sequence of DOTA-MGS5 (DOTA-DGlu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1Nal-NH2). Introduction of a penta-DGlu moiety and replacement of the four N-terminal amino acids by a non-charged hydrophilic linker was investigated. Retained receptor binding was confirmed using two CCK2R-expressing cell lines. The effect on metabolic degradation of the new 177Lu-labeled peptides was studied in human serum in vitro, as well as in BALB/c mice in vivo. The tumor targeting properties of the radiolabeled peptides were assessed using BALB/c nude mice bearing receptor-positive and receptor-negative tumor xenografts. Both novel MG analogs were found to have strong receptor binding, enhanced stability, and high tumor uptake. Replacement of the four N-terminal amino acids by a non-charged hydrophilic linker lowered the absorption in the dose-limiting organs, whereas introduction of the penta-DGlu moiety increased uptake in renal tissue.

A Novel CCK Receptor GPR173 Mediates Potentiation of GABAergic Inhibition.

Cholecystokinin (CCK) enables excitatory circuit long-term potentiation (LTP). Here, we investigated its involvement in the enhancement of inhibitory synapses. Activation of GABA neurons suppressed neuronal responses in the neocortex to a forthcoming auditory stimulus in mice of both sexes. High-frequency laser stimulation (HFLS) of GABAergic neurons potentiated this suppression. HFLS of CCK interneurons could induce the LTP of their inhibition toward pyramidal neurons. This potentiation was abolished in CCK knock-out mice but intact in mice with both CCK1R and 2R knockout of both sexes. Next, we combined bioinformatics analysis, multiple unbiased cell-based assays, and histology examinations to identify a novel CCK receptor, GPR173. We propose GPR173 as CCK3R, which mediates the relationship between cortical CCK interneuron signaling and inhibitory LTP in the mice of either sex. Thus, GPR173 might represent a promising therapeutic target for brain disorders related to excitation and inhibition imbalance in the cortex.SIGNIFICANCE STATEMENT CCK, the most abundant and widely distributed neuropeptide in the CNS, colocalizes with many neurotransmitters and modulators. GABA is one of the important inhibitory neurotransmitters, and much evidence shows that CCK may be involved in modulating GABA signaling in many brain areas. However, the role of CCK-GABA neurons in the cortical microcircuits is still unclear. We identified a novel CCK receptor, GPR173, localized in the CCK-GABA synapses and mediated the enhancement of the GABA inhibition effect, which might represent a promising therapeutic target for brain disorders related to excitation and inhibition imbalance in the cortex.

Cholecystokinin receptor antagonist challenge elicits brain-wide functional connectome modulation with micronetwork hippocampal decreased calcium transients.

The cortical distribution and functional role of cholecystokinin (CCK) are largely unknown. Here, a CCK receptor antagonist challenge paradigm was developed to assess functional connectivity and neuronal responses. Structural-functional magnetic resonance imaging and calcium imaging were undertaken in environmental enrichment (EE) and standard environment (SE) groups (naïve adult male mice, n = 59, C57BL/B6J, P = 60). Functional connectivity network-based statistics and pseudo-demarcation Voronoi tessellations to cluster calcium signals were used to derive region of interest metrics based on calcium transients, firing rate, and location. The CCK challenge elicited robust changes to structural-functional networks, decreased neuronal calcium transients, and max firing rate (5s) of dorsal hippocampus in SE mice. However, the functional changes were not observed in EE mice, while the decreased neuronal calcium transients and max firing rate (5s) were similar to SE mice. Decreased gray matter alterations were observed in multiple brain regions in the SE group due to CCK challenge, while no effect was observed in the EE group. The networks most affected by CCK challenge in SE included within isocortex, isocortex to olfactory, isocortex to striatum, olfactory to midbrain, and olfactory to thalamus. The EE group did not experience network changes in functional connectivity due to CCK challenge. Interestingly, calcium imaging revealed a significant decrease in transients and max firing rate (5s) in the dorsal CA1 hippocampus subregion after CCK challenge in EE. Overall, CCK receptor antagonists affected brain-wide structural-functional connectivity within the isocortex, in addition to eliciting decreased neuronal calcium transients and max firing rate (5s) in CA1 of the hippocampus. Future studies should investigate the CCK functional networks and how these processes affect isocortex modulation. Significance Statement Cholecystokinin is a neuropeptide predominately found in the gastrointestinal system. Albeit abundantly expressed in neurons, the role and distribution of cholecystokinin are largely unknown. Here, we demonstrate cholecystokinin affects brain-wide structural-functional networks within the isocortex. In the hippocampus, the cholecystokinin receptor antagonist challenge decreases neuronal calcium transients and max firing rate (5s) in CA1. We further demonstrate that mice in environmental enrichment do not experience functional network changes to the CCK receptor antagonist challenge. Environmental enrichment may afford protection to the alterations observed in control mice due to CCK. Our results suggest that cholecystokinin is distributed throughout the brain, interacts in the isocortex, and demonstrates an unexpected functional network stability for enriched mice.

Effects of Side Chain and Peptide Bond Modifications on the Targeting Properties of Stabilized Minigastrin Analogs.

Different attempts have been made in the past two decades to develop radiolabeled peptide conjugates with enhanced pharmacokinetic properties in order to improve the application for tumor imaging and peptide receptor radionuclide therapy (PRRT), which targets the cholecystokinin-2 receptor (CCK2R). In this paper, the influence of different side chain and peptide bond modifications has been explored for the minigastrin analog DOTA-DGlu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1Nal-NH2 (DOTA-MGS5). Based on this lead structure, five new derivatives were synthesized for radiolabeling with trivalent radiometals. Different chemical and biological properties of the new derivatives were analyzed. Receptor interaction of the peptide derivatives and cell internalization of the radiolabeled peptides were studied in A431-CCK2R cells. The stability of the radiolabeled peptides in vivo was investigated using BALB/c mice. Tumor targeting of all 111In-labeled peptide conjugates, and of a selected compound radiolabeled with gallium-68 and lutetium-177, was evaluated in BALB/c nude mice xenografted with A431-CCK2R and A431-mock cells. All 111In-labeled conjugates, except [111In]In-DOTA-[Phe8]MGS5, showed a high resistance against enzymatic degradation. A high receptor affinity with IC50 values in the low nanomolar range was confirmed for most of the peptide derivatives. The specific cell internalization over time was 35.3-47.3% for all radiopeptides 4 h after incubation. Only [111In]In-DOTA-MGS5[NHCH3] exhibited a lower cell internalization of 6.6 ± 2.8%. An overall improved resistance against enzymatic degradation was confirmed in vivo. Of the radiopeptides studied, [111In]In-DOTA-[(N-Me)1Nal8]MGS5 showed the most promising targeting properties, with significantly increased accumulation of radioactivity in A431-CCK2R xenografts (48.1 ± 9.2% IA/g) and reduced accumulation of radioactivity in stomach (4.2 ± 0.5% IA/g). However, in comparison with DOTA-MGS5, a higher influence on the targeting properties was observed for the change of radiometal, resulting in a tumor uptake of 15.67 ± 2.21% IA/g for [68Ga]Ga-DOTA-[(N-Me)1Nal8]MGS5 and 35.13 ± 6.32% IA/g for [177Lu]Lu-DOTA-[(N-Me)1Nal8]MGS5.

Treatment with a Cholecystokinin Receptor Antagonist, Proglumide, Improves Efficacy of Immune Checkpoint Antibodies in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-associated deaths worldwide. Treatment with immune checkpoint antibodies has shown promise in advanced HCC, but the response is only 15-20%. We discovered a potential target for the treatment of HCC, the cholecystokinin-B receptor (CCK-BR). This receptor is overexpressed in murine and human HCC and not in normal liver tissue. Mice bearing syngeneic RIL-175 HCC tumors were treated with phosphate buffer saline (PBS; control), proglumide (a CCK-receptor antagonist), an antibody to programmed cell death protein 1 (PD-1Ab), or the combination of proglumide and the PD-1Ab. In vitro, RNA was extracted from untreated or proglumide-treated murine Dt81Hepa1-6 HCC cells and analyzed for expression of fibrosis-associated genes. RNA was also extracted from human HepG2 HCC cells or HepG2 cells treated with proglumide and subjected to RNA sequencing. Results showed that proglumide decreased fibrosis in the tumor microenvironment and increased the number of intratumoral CD8+ T cells in RIL-175 tumors. When proglumide was given in combination with the PD-1Ab, there was a further significant increase in intratumoral CD8+ T cells, improved survival, and alterations in genes regulating tumoral fibrosis and epithelial-to-mesenchymal transition. RNAseq results from human HepG2 HCC cells treated with proglumide showed significant changes in differentially expressed genes involved in tumorigenesis, fibrosis, and the tumor microenvironment. The use of the CCK receptor antagonist may improve efficacy of immune checkpoint antibodies and survival in those with advanced HCC.

Peripheral administration of neuropeptide W inhibits gastric emptying in rats: The role of small diameter afferent fibers and cholecystokinin receptors

Neuropeptide W (NPW), a novel hypothalamic peptide, contributes to the central regulation of food intake and energy balance, and suppresses feeding behavior when administered centrally. The aim of our study was to investigate the role of peripherally administered NPW in the modulation of gastric emptying, and to evaluate the participation of afferent fibers, cholecystokinin (CCK) receptors and gastric smooth muscle contractility in the regulatory effects of NPW on gastric motility. In Sprague-Dawley male rats equipped with gastric fistula, gastric emptying rate of the saline and peptone solutions was measured following subcutaneous administration of NPW (0.1 or 5 μg/kg) preceded by subcutaneous injections of saline, CCK-1 or CCK-2 receptor antagonists. Another group of rats with cannulas were injected subcutaneously with capsaicin for afferent denervation before commencing emptying trials. The effect of NPW on carbachol-induced gastric contractility and the role of CCK receptors in gastric smooth muscle contractility were also assessed in gastric strips. Peripheral injection of NPW delayed gastric emptying rate of both caloric and non-caloric liquid test meals, while administration of CCK-1 or CCK-2 receptor antagonists or denervation of small diameter afferents reversed NPW–induced delay in gastric emptying. Moreover, NPW inhibited antrum contractility in the organ bath. Our results revealed that peripherally administered NPW delayed liquid emptying from the stomach via the involvement of small diameter afferent neurons and CCK receptors, and thereby this regulatory role may contribute to its central regulatory role in controlling food intake and energy balance.

Mechanism of Action and Structure-Activity Relationships of Tetracyclic Small Molecules Acting as Universal Positive Allosteric Modulators of the Cholecystokinin Receptor.

As part of an ongoing effort to develop a drug targeting the type 1 cholecystokinin receptor (CCK1R) to help prevent and/or treat obesity, we recently performed a high throughput screening effort of small molecules seeking candidates that enhanced the action of the natural agonist, CCK, thus acting as positive allosteric modulators without exhibiting intrinsic agonist action. Such probes would be expected to act in a temporally finite way to enhance CCK action to induce satiety during and after a meal and potentially even modulate activity at the CCK1R in a high cholesterol environment present in some obese patients. The current work focuses on the best scaffold, representing tetracyclic molecules identified through high throughput screening we previously reported. Extensive characterization of the two top "hits" from the previous effort demonstrated them to fulfill the desired pharmacologic profile. We undertook analog-by-catalog expansion of this scaffold using 65 commercially available analogs. In this effort, we were able to eliminate an off-target effect observed for this scaffold while retaining its activity as a positive allosteric modulator of CCK1R in both normal and high cholesterol membrane environments. These insights should be useful in the rational medicinal chemical enhancement of this scaffold and in the future development of candidates to advance to pre-clinical proof-of-concept and to clinical trials.

Preliminary Clinical Experience with Cholecystokinin-2 Receptor PET/CT Using the 68Ga-Labeled Minigastrin Analog DOTA-MGS5 in Patients with Medullary Thyroid Cancer.

PET/CT with the new 68Ga-labeled minigastrin analog DOTA-dGlu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1-Nal-NH2 (68Ga-DOTA-MGS5) was performed on patients with advanced medullary thyroid cancer (MTC) to evaluate cholecystokinin-2 receptor expression status. Methods: Six patients with advanced MTC underwent PET/CT with 68Ga-DOTA-MGS5. From the images acquired 1 and 2 h after injection, preliminary data on the biodistribution and tumor-targeting properties were evaluated in a retrospective analysis. Results: In total, 87 lesions with increased radiotracer uptake considered malignant were detected (2 local recurrences, 8 lymph node lesions, 27 liver lesions, and 50 bone lesions). In general, radiotracer accumulation in lesions was higher at 2 h than at 1 h after injection (mean SUVmax, 7.2 vs. 6.0, respectively; mean SUVmean, 4.4 vs. 3.6, respectively). Conclusion: The preliminary results clearly demonstrate the potential of 68Ga-DOTA-MGS5 PET/CT in detecting local recurrence and metastases in patients with advanced MTC.

Bromophenols from Symphyocladia latiuscula (Harvey) Yamada as Novel Cholecystokinin 2 Receptor Antagonists.

Cholecystokinin (CCK) is one of the most abundant peptides in the central nervous system and is believed to function as a neurotransmitter as well as a gut hormone with an inverse correlation of its level to anxiety and depression. Therefore, CCK receptors (CCKRs) could be a relevant target for novel antidepressant therapy. In silico target prediction was first employed to predict the probability of the bromophenols interacting with key protein targets based on a model trained on known bioactivity data and chemical similarity considerations. Next, we tested the functional effect of natural bromophenols from Symphyocladia latiuscula on the CCK2 receptor followed by a molecular docking simulation to predict interactions between a compound and the binding site of the target protein. Results of cell-based functional G-protein coupled receptor (GPCR) assays demonstrate that bromophenols 2,3,6-tribromo-4,5-dihydroxybenzyl alcohol (1), 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether (2), and bis-(2,3,6-tribromo-4,5-dihydroxybenzyl) ether (3) are full CCK2 antagonists. Molecular docking simulation of 1‒3 with CCK2 demonstrated strong binding by means of interaction with prime interacting residues: Arg356, Asn353, Val349, His376, Phe227, and Pro210. Simulation results predicted good binding scores and interactions with prime residues, such as the reference antagonist YM022. The results of this study suggest bromophenols 1-3 are CCK2R antagonists that could be novel therapeutic agents for CCK2R-related diseases, especially anxiety and depression.