Precision peptide theranostics: developing N- to C-terminus optimized theranostics targeting cholecystokinin-2 receptor.

Peptides are ideal for theranostic development as they afford rapid target accumulation, fast clearance from background tissue, and exhibit good tissue penetration. Previously, we developed a novel series of peptides that presented discreet folding propensity leading to an optimal candidate [68Ga]Ga-DOTA-GA1 ([D-Glu]6-Ala-Tyr-NMeGly-Trp-NMeNle-Asp-Nal-NH2) with 50 pM binding affinity against cholecystokinin-2 receptors (CCK2R). However, we were confronted with challenges of unfavorably high renal uptake. Methods: A structure activity relationship study was undertaken of the lead theranostic candidate. Prudent structural modifications were made to the peptide scaffold to evaluate the contributions of specific N-terminal residues to the overall biological activity. Optimal candidates were then evaluated in nude mice bearing transfected A431-CCK2 tumors, and their biodistribution was quantitated ex vivo. Results: We identified and confirmed that D-Glu3 to D-Ala3 substitution produced 2 optimal candidates, [68Ga]Ga-DOTA-GA12 and [68Ga]Ga-DOTA-GA13. These radiopeptides presented with high target/background ratios, enhanced tumor retention, excellent metabolic stability in plasma and mice organ homogenates, and a 4-fold reduction in renal uptake, significantly outperforming their non-alanine counterparts. Conclusions: Our study identified novel radiopharmaceutical candidates that target the CCK2R. Their high tumor uptake and reduced renal accumulation warrant clinical translation.

Implicating the cholecystokinin B receptor in liver stem cell oncogenesis.

Hepatocellular carcinoma (HCC) is the fastest-growing cause of cancer-related deaths worldwide. Chronic inflammation and fibrosis are the greatest risk factors for the development of HCC. Although the cell of origin for HCC is uncertain, many theories believe this cancer may arise from liver progenitor cells or stem cells. Here, we describe the activation of hepatic stem cells that overexpress the cholecystokinin-B receptor (CCK-BR) after liver injury with either a DDC diet (0.1% 3, 5-diethoxy-carbonyl 1,4-dihydrocollidine) or a NASH-inducing CDE diet (choline-deficient ethionine) in murine models. Pharmacologic blockade of the CCK-BR with a receptor antagonist proglumide or knockout of the CCK-BR in genetically engineered mice during the injury diet reduces the expression of hepatic stem cells and prevents the formation of three-dimensional tumorspheres in culture. RNA sequencing of livers from DDC-fed mice treated with proglumide or DDC-fed CCK-BR knockout mice showed downregulation of differentially expressed genes involved in cell proliferation and oncogenesis and upregulation of tumor suppressor genes compared with controls. Inhibition of the CCK-BR decreases hepatic transaminases, fibrosis, cytokine expression, and alters the hepatic immune cell signature rendering the liver microenvironment less oncogenic. Furthermore, proglumide hastened recovery after liver injury by reversing fibrosis and improving markers of synthetic function. Proglumide is an older drug that is orally bioavailable and being repurposed for liver conditions. These findings support a promising therapeutic intervention applicable to patients to prevent the development of HCC and decrease hepatic fibrosis.NEW & NOTEWORTHY This investigation identified a novel pathway involving the activation of hepatic stem cells and liver oncogenesis. Receptor blockade or genetic disruption of the cholecystokinin-B receptor (CCK-BR) signaling pathway decreased the activation and proliferation of hepatic stem cells after liver injury without eliminating the regenerative capacity of healthy hepatocytes.

Preclinical Evaluation of Minigastrin Analogs and Proof-of-Concept [68Ga]Ga-DOTA-CCK-66 PET/CT in 2 Patients with Medullary Thyroid Cancer.

Because of the need for radiolabeled theranostics for the detection and treatment of medullary thyroid cancer (MTC), and the yet unresolved stability issues of minigastrin analogs targeting the cholecystokinin-2 receptor (CCK-2R), our aim was to address in vivo stability, our motivation being to develop and evaluate DOTA-CCK-66 (DOTA-γ-glu-PEG3-Trp-(N-Me)Nle-Asp-1-Nal-NH2, PEG: polyethylene glycol) and DOTA-CCK-66.2 (DOTA-glu-PEG3-Trp-(N-Me)Nle-Asp-1-Nal-NH2), both derived from DOTA-MGS5 (DOTA-glu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1-Nal-NH2), and clinically translate [68Ga]Ga-DOTA-CCK-66. Methods: 64Cu and 67Ga labeling of DOTA-CCK-66, DOTA-CCK-66.2, and DOTA-MGS5 was performed at 90°C within 15 min (1.0 M NaOAc buffer, pH 5.5, and 2.5 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, respectively). 177Lu labeling of these 3 compounds was performed at 90°C within 15 min (1.0 M NaOAc buffer, pH 5.5, 0.1 M sodium ascorbate). CCK-2R affinity of natGa/natCu/natLu-labeled DOTA-CCK-66, DOTA-CCK-66.2, and DOTA-MGS5 was examined on AR42J cells. The in vivo stability of 177Lu-labeled DOTA-CCK-66 and DOTA-MGS5 was examined at 30 min after injection in CB17-SCID mice. Biodistribution studies at 1 h ([67Ga]Ga-DOTA-CCK-66) and 24 h ([177Lu]Lu-DOTA-CCK-66/DOTA-MGS5) after injection were performed on AR42J tumor-bearing CB17-SCID mice. In a translation to the human setting, [68Ga]Ga-DOTA-CCK-66 was administered and whole-body PET/CT was acquired at 120 min after injection in 2 MTC patients. Results: Irrespective of the metal or radiometal used (copper, gallium, lutetium), high CCK-2R affinity (half-maximal inhibitory concentration, 3.6-6.0 nM) and favorable lipophilicity were determined. In vivo, increased numbers of intact peptide were found for [177Lu]Lu-DOTA-CCK-66 compared with [177Lu]Lu-DOTA-MGS5 in murine urine (23.7% ± 9.2% vs. 77.8% ± 2.3%). Overall tumor-to-background ratios were similar for both 177Lu-labeled analogs. [67Ga]Ga-DOTA-CCK-66 exhibited accumulation (percentage injected dose per gram) that was high in tumor (19.4 ± 3.5) and low in off-target areas (blood, 0.61 ± 0.07; liver, 0.31 ± 0.02; pancreas, 0.23 ± 0.07; stomach, 1.81 ± 0.19; kidney, 2.51 ± 0.49) at 1 h after injection. PET/CT examination in 2 MTC patients applying [68Ga]Ga-DOTA-CCK-66 confirmed multiple metastases. Conclusion: Because of the high in vivo stability and favorable overall preclinical performance of [nat/67Ga]Ga-/[nat/177Lu]Lu-DOTA-CCK-66, a proof-of-concept clinical investigation of [68Ga]Ga-DOTA-CCK-66 was completed. As several lesions could be identified and excellent biodistribution patterns were observed, further patient studies applying [68Ga]Ga- and [177Lu]Lu-DOTA-CCK-66 are warranted.

Elevated Serum Gastrin Is Associated with Melanoma Progression: Putative Role in Increased Migration and Invasion of Melanoma Cells.

Micro-environmental factors, including stromal and immune cells, cytokines, and circulating hormones are well recognized to determine cancer progression. Melanoma cell growth was recently shown to be suppressed by cholecystokinin/gastrin (CCK) receptor antagonists, and our preliminary data suggested that melanoma patients with Helicobacter gastritis (which is associated with elevated serum gastrin) might have an increased risk of cancer progression. Therefore, in the present study, we examined how gastrin may act on melanoma cells. In 89 melanoma patients, we found a statistically significant association between circulating gastrin concentrations and melanoma thickness and metastasis, which are known risk factors of melanoma progression and prognosis. Immunocytochemistry using a validated antibody confirmed weak to moderate CCK2R expression in both primary malignant melanoma cells and the melanoma cell lines SK-MEL-2 and G361. Furthermore, among the 219 tumors in the Skin Cutaneous Melanoma TCGA Pan-Cancer dataset showing gastrin receptor (CCKBR) expression, significantly higher CCKBR mRNA levels were linked to stage III-IV than stage I-II melanomas. In both cell lines, gastrin increased intracellular calcium levels and stimulated cell migration and invasion through mechanisms inhibited by a CCK2 receptor antagonist. Proteomic studies identified increased MMP-2 and reduced TIMP-3 levels in response to gastrin that were likely to contribute to the increased migration of both cell lines. However, the effects of gastrin on tumor cell invasion were relatively weak in the presence of the extracellular matrix. Nevertheless, dermal fibroblasts/myofibroblasts, known also to express CCK2R, increased gastrin-induced cancer cell invasion. Our data suggest that in a subset of melanoma patients, an elevated serum gastrin concentration is a risk factor for melanoma tumor progression, and that gastrin may act on both melanoma and adjacent stromal cells through CCK2 receptors to promote mechanisms of tumor migration and invasion.

Development of Highly Potent Clinical Candidates for Theranostic Applications against Cholecystokinin-2 Receptor Positive Cancers.

Peptide receptor radionuclide therapy (PRRT) is a promising form of systemic radiation therapy designed to eradicate cancer. Cholecystokinin-2 receptor (CCK2R) is an important molecular target that is highly expressed in a range of cancers. This study describes the synthesis and in vivo characterization of a novel series of 177Lu-labeled peptides ([177Lu]Lu-2b-4b) in comparison with the reference CCK2R-targeting peptide CP04 ([177Lu]Lu-1b). [177Lu]Lu-1b-4b showed high chemical purity (HPLC ≥ 94%), low Log D7.4 (-4.09 to -4.55) with strong binding affinity to CCK2R (KD 0.097-1.61 nM), and relatively high protein binding (55.6-80.2%) and internalization (40-67%). Biodistribution studies of the novel 177Lu-labeled peptides in tumors (AR42J and A431-CCK2R) showed uptake one- to eight-fold greater than the reference compound CP04 at 1, 24, and 48 h. Rapid clearance and high tumor uptake and retention were established for [177Lu]Lu-2b-4b, making these compounds excellent candidates for theranostic applications against CCK2R-expressing tumors.

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.

[111In]In-CP04 as a novel cholecystokinin-2 receptor ligand with theranostic potential in patients with progressive or metastatic medullary thyroid cancer: final results of a GRAN-T-MTC Phase I clinical trial.

INTRODUCTION: Medullary thyroid cancer (MTC) is a rare malignant tumour of the parafollicular C-cells with an unpredictable clinical course and currently suboptimal diagnostic and therapeutic options, in particular in advanced disease. Overexpression of cholecystokinin-2 receptors (CCK2R) represents a promising avenue to diagnostic imaging and targeted therapy, ideally through a theranostic approach. MATERIALS AND METHODS: A translational study (GRAN-T-MTC) conducted through a Phase I multicentre clinical trial of the indium-111 labelled CP04 ([111In]In-CP04), a CCK2R-seeking ligand was initiated with the goal of developing a theranostic compound. Patients with proven advanced/metastatic MTC or short calcitonin doubling time were enrolled. A two-step concept was developed through the use of low- and high-peptide mass (10 and 50 µg, respectively) for safety assessment, with the higher peptide mass considered appropriate for therapeutic application. Gelofusine was co-infused in a randomized fashion in the second step for the evaluation of potential reduction of the absorbed dose to the kidneys. Imaging for the purpose of biodistribution, dosimetry evaluation, and diagnostic assessment were performed as well as pre-, peri-, and postprocedural clinical and biochemical assessment. RESULTS: Sixteen patients were enrolled. No serious adverse events after application of the compound at both peptide amounts were witnessed; transient tachycardia and flushing were observed in two patients. No changes in biochemistry and clinical status were observed on follow-up. Preliminary dosimetry assessment revealed the highest dose to urinary bladder, followed by the kidneys and stomach wall. The effective dose for 200 MBq of [111In]In-CP04 was estimated at 7±3 mSv and 7±1 mSv for 10 µg and 50 µg CP04, respectively. Administration of Gelofusine reduced the dose to the kidneys by 53%, resulting in the organ absorbed dose of 0.044±0.019 mSv/MBq. Projected absorbed dose to the kidneys with the use of [177Lu]Lu-CP04 was estimated at 0.9±0.4 Gy/7.4 GBq. [111In]In-CP04 scintigraphy was positive in 13 patients (detection rate of 81%) with superior diagnostic performance over conventional imaging. CONCLUSION: In the present study, [111In]In-CP04 was shown to be a safe and effective radiopharmaceutical with promising theranostic characteristics for patients with advanced MTC.

Spinal CCK1 Receptors Contribute to Somatic Pain Hypersensitivity Induced by Malocclusion via a Reciprocal Neuron-Glial Signaling Cascade.

Recent studies have shown that the incidence of chronic primary pain including temporomandibular disorders (TMD) and fibromyalgia syndrome (FMS) often exhibit comorbidities. We recently reported that central sensitization and descending facilitation system contributed to the development of somatic pain hypersensitivity induced by orofacial inflammation combined with stress. The purpose of this study was to explore whether TMD caused by unilateral anterior crossbite (UAC) can induce somatic pain hypersensitivity, and whether the cholecystokinin (CCK) receptor-mediated descending facilitation system promotes hypersensitivity through neuron-glia cell signaling cascade. UAC evoked thermal and mechanical pain hypersensitivity of the hind paws from day 5 to 70 that peaked at week 4 post UAC. The expression levels of CCK1 receptors, interleukin-18 (IL-18) and IL-18 receptors (IL-18R) were significantly up-regulated in the L4 to L5 spinal dorsal horn at 4 weeks post UAC. Intrathecal injection of CCK1 and IL-18 receptor antagonists blocked somatic pain hypersensitivity. IL-18 mainly co-localized with microglia, while IL-18R mainly co-localized with astrocytes and to a lesser extent with neurons. These findings indicate that the signaling transduction between neurons and glia at the spinal cord level contributes to the descending pain facilitation through CCK1 receptors during the development of the comorbidity of TMD and FMS. PERSPECTIVE: CCK1 receptor-dependent descending facilitation may mediate central mechanisms underlying the development of widespread somatic pain via a reciprocal neuron-glial signaling cascade, providing novel therapeutic targets for the clinical treatment of TMD and FMS comorbidities.

Intestinal Gastrin/CCKBR (Cholecystokinin B Receptor) Ameliorates Salt-Sensitive Hypertension by Inhibiting Intestinal Na+/H+ Exchanger 3 Activity Through a PKC (Protein Kinase C)-Mediated NHERF1 and NHERF2 Pathway.

BACKGROUND: The present study directly tested the crucial role of intestinal gastrin/CCKBR (cholecystokinin B receptor) in the treatment of salt-sensitive hypertension. METHODS: Adult intestine-specific Cckbr-knockout mice (Cckbrfl/fl villin-Cre) and Dahl salt-sensitive rats were studied on the effect of high salt intake (8% NaCl, 6-7 weeks) on intestinal Na+/H+ exchanger 3 expression, urine sodium concentration, and blood pressure. High-salt diet increased urine sodium concentration and systolic blood pressure to a greater extent in Cckbrfl/fl villin-Cre mice and Dahl salt-sensitive rats than their respective controls, Cckbrfl/fl villin mice and SS13BN rats. We constructed gastrin-SiO2 microspheres to enable gastrin to stimulate specifically and selectively intestinal CCKBR without its absorption into the circulation. RESULTS: Gastrin-SiO2 microspheres treatment prevented the high salt-induced hypertension and increase in urine Na concentration by inhibiting intestinal Na+/H+ exchanger 3 trafficking and activity, increasing stool sodium without inducing diarrhea. Gastrin-mediated inhibition of intestinal Na+/H+ exchanger 3 activity, related to a PKC (protein kinase C)-mediated activation of NHERF1 and NHERF2. CONCLUSIONS: These results support a crucial role of intestinal gastrin/CCKBR in decreasing intestinal sodium absorption and keeping the blood pressure in the normal range. The gastrointestinal administration of gastrin-SiO2 microspheres is a promising and safe strategy to treat salt-sensitive hypertension without side effects.

Discovery of once-weekly, peptide-based selective GLP-1 and cholecystokinin 2 receptors co-agonizts.

A group of long-acting, peptide-based, and selective GLP-1R/CCK-2R dual agonizts were identified by rational design. Guided by sequence analysis, structural elements of the CCK-2R agonist moiety were engineered into the GLP-1R agonist Xenopus GLP-1, resulting in hybrid peptides with potent GLP-1R/CCK-2R dual activity. Further modifications with fatty acids resulted in novel metabolically stable peptides, among which 3d and 3 h showed potent GLP-1R and CCK-2R activation potencies and comparable stability to semaglutide. In food intake tests, 3d and 3 h also showed a potent reduction in food intake, superior to that of semaglutide. Moreover, the acute hypoglycemic and insulinotropic activities of 3d and 3 h were better than that of semaglutide and ZP3022. Importantly, the limited pica response following 3d and 3 h administration in SD rats preliminarily indicated that the food intake reduction effects of 3d and 3 h are independent of nausea/vomiting. In a 35-day study in db/db mice, every two days administration of 3d and 3 h increased islet areas and numbers, insulin contents, ß-cell area, ß-cell proliferation, as well as improved glucose tolerance, and decreased HbA1c, to a greater extent than ZP3022 and semaglutide. In a 21-day study in DIO mice, once-weekly administration of 3d and 3 h significantly induced body weight loss, improved glucose tolerance, and normalized lipid metabolism, to a greater extent than semaglutide. The current study showed the antidiabetic and antiobesity potentials of GLP-1R/CCK-2R dual agonizts that warrant further investigation.

Unique membranous gastrin receptor expression of parietal cells and its distribution pattern in the gastric oxyntic mucosa and fundic gland polyps.

The aim of this study was to clarify the correlation between gastrin receptor (GR) expression in the gastric oxyntic mucosa and fundic gland polyps (FGPs) and the histological and immunohistochemical findings of the mucosa as well as the history of proton pump inhibitor (PPI) administration. The unique membranous linear positivity of GR in parietal cells was reproducibly observed by immunohistochemistry, which was also validated by immunofluorescence. Further histological and immunohistochemical examination of 34 oxyntic mucosae and 43 FGPs revealed the following: 1) parietal cells (PCs) with membranous linear GR expression (mGR) were observed to be limited to the isthmus-neck region in the normal state; 2) appearance of PCs with mGR in the deep oxyntic gland regions was significantly related to the PPI medication history; 3) PCs with mGR were more frequently observed in the deep oxyntic gland regions when the oxyntic mucosa showed derangement of mucosal component cell compartmentalization revealed by MUC5AC and MUC6 immunohistochemistry, which was also significantly related to the PPI use; and 4) PCs with intense membranous linear positivity of GR were observed to be diffusely distributed in all of the cases of FGPs. In conclusion, the distribution of unique GR membranous linear expression in PCs of the oxyntic mucosa under PPI medication and FGPs could reflect the pathologic mucosal state characterized by derangement of the compartmentalization of mucosal component cells, which could be another basis for evaluating physiologic and/or pathophysiologic conditions of the gastric mucosa.

Automated Synthesis of 68Ga-Labeled DOTA-MGS8 and Preclinical Characterization of Cholecystokinin-2 Receptor Targeting.

The new minigastrin analog DOTA-MGS8 targeting the cholecystokinin-2 receptor (CCK2R) used in this study displays the combination of two site-specific modifications within the C-terminal receptor binding sequence together with an additional N-terminal amino acid substitution preventing fast metabolic degradation. Within this study, the preparation of 68Ga-labeled DOTA-MGS8 was validated using an automated synthesis module, describing the specifications and analytical methods for quality control for possible clinical use. In addition, preclinical studies were carried out to characterize the targeting potential. [68Ga]Ga-DOTA-MGS8 showed a high receptor-specific cell internalization into AR42J rat pancreatic cells (~40%) with physiological expression of rat CCK2R as well as A431-CCK2R cells transfected to stably express human CCK2R (~47%). A favorable biodistribution profile was observed in BALB/c nude mice xenografted with A431-CCK2R cells and mock-transfected A431 cells as control. The high tumor uptake of ~27% IA/g together with low background activity and limited uptake in non-target tissue confirms the potential for high-sensitivity positron emission tomography of stabilized MG analogs in patients with MTC and other CCK2R-related malignancies.

Effects of galantamine on social interaction impairments in cholecystokinin receptor-2 overexpression mice.

We examined whether galantamine (GAL), a cholinesterase inhibitor and allosteric potentiating ligand for α7 nicotinic acetylcholine receptor (nAChR), had an impact on emotional abnormalities in forebrain-specific cholecystokinin receptor-2 overexpressed transgenic mice. Treatment with GAL (1 mg/kg, s.c.) attenuated the decrease of social interaction time, but failed to attenuate anxiety-like behavior in the elevated plus-maze test. The effect of GAL was blocked by an α7 nAChR antagonist, methyllycaconitine (3 mg/kg, i.p.). These results suggest that GAL improved social interaction impairments via α7 nAChR and could be useful to treat sociability-related emotional abnormalities.

Downregulation of CCKBR Expression Inhibits the Proliferation of Gastric Cancer Cells, Revealing a Potential Target for Immunotoxin Therapy.

BACKGROUND: Increased CCKBR expression density or frequency has been reported in many neoplasms. OBJECTIVE: We aimed to investigate whether CCKBR drives the growth of gastric cancer (GC) and its potential as a therapeutic target of immunotoxins. METHODS: A lentiviral interference system was used to generate CCKBR-knockdown gastric cancer cells. Cell Counting Kit-8 and clonogenic assays were used to evaluate cell proliferation. Woundhealing and cell invasion assays were performed to evaluate cell mobility. Cell cycle was analyzed by flow cytometry. Tumor growth in vivo was investigated using a heterologous tumor transplantation model in nude mice. In addition, we generated the immunotoxin FQ17P and evaluated the combining capacity and tumor cytotoxicity of FQ17P in vitro. RESULTS: Stable downregulation of CCKBR expression resulted in reduced proliferation, migration and invasion of BGC-823 and SGC-7901 cells. The impact of CCKBR on gastric cancer cells was further verified through CCKBR overexpression studies. Downregulation of CCKBR expression also inhibited the growth of gastric tumors in vivo. Furthermore, FQ17P killed CCKBR-overexpressing GC cells by specifically binding to CCKBR on the tumor cell surface. CONCLUSION: The CCKBR protein drives the growth, migration, and invasion of gastric cancer cells, and it might be a promising target for immunotoxin therapy based on its aberrant expression, functional binding interactions with gastrin, and subsequent internalization.

Inhibition of Geranylgeranylacetone on cholecystokinin-B receptor, BDNF and dopamine D1 receptor induced by morphine.

Morphine is the pain releasing and abusing drug. Morphine leads to addiction by activating dopaminergic rewarding system consisted of the ventral tegmental area (VTA) and nucleus accumbens (NAc). Cholecystokinin (CCK) is a gut-brain neuropeptide and involved in morphine dependence. Brain-derived neurotrophic factor (BDNF) is a neurotrophin and plays roles in regulating addiction. Geranylgeranylacetone (GGA) is a medicine of protecting gastric mucosal injury and protecting neurons. Our previous study showed that GGA blocked morphine-induced withdrawal and relapse through inducing thioredoxin 1(Trx1). In this study, we investigated that whether cholecystokinin-B receptor (CCKB receptor) and BDNF were related to GGA inhibition on morphine addiction. At first, we made conditioned place preference (CPP) model and confirmed again that GGA blocked the expression of morphine-CPP in present study. Then, our results showed that morphine increased the expressions of dopamine D1 receptor, tyrosine hydroxylase (TH), CCKB receptor and BDNF in the VTA and NAc in mice, which was inhibited by GGA. These results suggest that CCK and BDNF in dopaminergic systems are associated with the role of GGA blocking morphine-CPP.

Cholecystokinin-B Receptor-Targeted Nanoparticle for Imaging and Detection of Precancerous Lesions in the Pancreas.

Survival from pancreatic cancer remains extremely poor, in part because this malignancy is not diagnosed in the early stages, and precancerous pancreatic intraepithelial neoplasia (PanIN) lesions are not seen on routine radiographic imaging. Since the cholecystokinin-B receptor (CCK-BR) becomes over-expressed in PanIN lesions, it may serve as a target for early detection. We developed a biodegradable fluorescent polyplex nanoparticle (NP) that selectively targets the CCK-BR. The NP was complexed to a fluorescent oligonucleotide with Alexa Fluor 647 for far-red imaging and to an oligonucleotide conjugated to Alexa Fluor 488 for localization by immunohistochemistry. Fluorescence was detected over the pancreas of five- to ten-month-old LSL-KrasG12D/+; P48-Cre (KC) mice only after the injection of the receptor target-specific NP and not after injection of untargeted NP. Ex vivo tissue imaging and selective immunohistochemistry confirmed particle localization only to PanIN lesions in the pancreas and not in other organs, supporting the tissue specificity. A human pancreas tissue microarray demonstrated immunoreactivity for the CCK-BR only in the PanIN lesions and not in normal pancreas tissue. The long-term goal would be to develop this imaging tool for screening human subjects at high risk for pancreatic cancer to enable early cancer detection.

Spinal CCK contributes to somatic hyperalgesia induced by orofacial inflammation combined with stress in adult female rats.

In some chronic primary pain conditions such as temporomandibular disorder (TMD) and fibromyalgia syndrome (FMS), mild or chronic stress enhances pain. TMD and FMS often occur together, but the underlying mechanisms are unclear. The purpose of this study was to investigate the role of cholecystokinin (CCK) in the spinal cord in somatic hyperalgesia induced by orofacial inflammation combined with stress. Somatic hyperalgesia was detected by the thermal withdrawal latency and mechanical withdrawal threshold. The expression of CCK1 receptors, CCK2 receptors, ERK1/2 and p-ERK1/2 in the spinal cord was examined by Western blot. After the stimulation of orofacial inflammation combined with 3 day forced swim, the expression of CCK2 receptors and p-ERK1/2 protein in the L4-L5 spinal dorsal horn increased significantly, while the expression of CCK1 receptors and ERK1/2 protein remained unchanged. Intrathecal injection of the CCK2 receptor antagonist YM-022 or mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor PD98059 blocked somatic hyperalgesia induced by orofacial inflammation combined with stress. Intrathecal administration of the MEK inhibitor blocked somatic sensitization caused by the CCK receptor agonist CCK8. The CCK2 receptor antagonist YM-022 significantly reduced the expression of p-ERK1/2. These data indicate that upregulation of CCK2 receptors through the MAPK pathway contributes to somatic hyperalgesia in this comorbid pain model. Thus, CCK2 receptors and MAPK pathway may be potential targets for the treatment of TMD comorbid with FMS.

Cholecystokinin acts in the dorsomedial hypothalamus of young male rats to suppress appetite in a nitric oxide-dependent manner.

Cholecystokinin (CCK) is an appetite-suppressing hormone that acts in the dorsomedial hypothalamus (DMH) in adult rats to suppress food intake. It remains unknown, however, whether CCK has the same affect in young animals, despite the rising prevalence of childhood obesity and drastic need for research in this area. At the synaptic level, CCK has been shown to inhibit putative orexigenic DMH neurons in young male rats by increasing GABA release onto these neurons via a CCK2 receptor and nitric oxide-dependent pathway. Whether this pathway leads to appetite suppression in young rats is not known. We therefore investigated whether intra-DMH administration of CCK, with or without inhibitors of the CCK2 receptor and nitric oxide signaling pathways, affects food intake in young, male, fasted Sprague-Dawley rats. We implanted bilateral guide cannulas into the DMH and allowed animals to recover from the surgery. Animals were then fasted for 24 h, following which they received intra-DMH microinjections of vehicle, CCK-8S, or CCK-8S combined with either LY-225910 (CCK2 receptor antagonist), L-NAME (a nitric oxide synthase inhibitor), or ODQ (a soluble guanylate cyclase inhibitor) and were given access to regular chow. Following a two hour refeeding period during which food intake, latency to feed, and body weight were measured, brains were subsequently removed to confirm cannula placement in the DMH. The effect of CCK on these parameters in rats given a high fat diet were also measured. Here we show that intra-DMH administration of CCK suppresses food intake and body weight in young rats. This effect requires activation of CCK2 receptors and nitric oxide signaling. Finally, CCK has no effect on consumption of a high fat diet when administered into the DMH. Overall, these findings demonstrate a potential pathway through which CCK might suppress appetite in young rats.

Rapid Increase of Gastrointestinal P-Glycoprotein Functional Activity in Response to Etoposide Stimulation.

We previously reported that exposure of human colon adenocarcinoma (Caco-2) cells to the bitter substance phenylthiocarbamide (PTC) rapidly enhanced the transport function of P-glycoprotein (P-gp). In this study, we investigated the short-term effect of etoposide, another bitter-tasting P-gp substrate, on P-gp transport function in the same cell line. We found that etoposide exposure significantly increased both the P-gp protein level in the plasma membrane fraction and the efflux rate of rhodamine123 (Rho123) in Caco-2 cells within 10 min. The efflux ratio (ratio of the apparent permeability coefficient in the basal-to-apical direction to that in the apical-to-basal direction) of Rho123 in etoposide-treated cells was also significantly increased compared with the control. These results indicated that etoposide rapidly enhances P-gp function in Caco-2 cells. In contrast, P-gp expression in whole cells at both the mRNA and protein level was unchanged by etoposide exposure, compared with the levels in non-treated cells. Furthermore, etoposide increased the level of phosphorylated ezrin, radixin and moesin (P-ERM) proteins in the plasma membrane fraction of Caco-2 cells within 10 min. P-gp functional changes were blocked by YM022, an inhibitor of cholecystokinin (CCK) receptor. These results suggest that etoposide induces release of CCK, causing activation of the CCK receptor followed by phosphorylation of ERM proteins, which recruit intracellular P-gp for trafficking to the gastrointestinal membrane, thereby increasing the functional activity of P-gp.

A New Turn in Peptide-Based Imaging Agents: Foldamers Afford Improved Theranostics Targeting Cholecystokinin-2 Receptor-Positive Cancer.

Proteins adopt unique folded secondary and tertiary structures that are responsible for their remarkable biological properties. This structural complexity is key in designing efficacious peptides that can mimic the three-dimensional structure needed for biological function. In this study, we employ different chemical strategies to induce and stabilize a ß-hairpin fold of peptides targeting cholecystokinin-2 receptors for theranostic application (combination of a targeted therapeutic and a diagnostic companion). The newly developed peptides exhibited enhanced folding capacity as demonstrated by circular dichroism (CD) spectroscopy, ion-mobility spectrometry-mass spectrometry, and two-dimensional (2D) NMR experiments. Enhanced folding characteristics of the peptides led to increased biological potency, affording four optimal Ga-68 labeled radiotracers ([68Ga]Ga-4b, [68Ga]Ga-11b-13b) targeting CCK-2R. In particular, [68Ga]Ga-12b and [68Ga]Ga-13b presented improved metabolic stability, enhanced cell internalization, and up to 6 fold increase in tumor uptake. These peptides hold great promise as next-generation theranostic radiopharmaceuticals.