Cross-talk between Human Spinal Cord µ-opioid Receptor 1Y Isoform and Gastrin-releasing Peptide Receptor Mediates Opioid-induced Scratching Behavior.

BACKGROUND: Although spinal opioids are safe and effective, pruritus is common and distressing. The authors previously demonstrated in mouse spinal cord that interactions between µ-opioid receptor isoform 1D and gastrin releasing peptide receptor mediate morphine-induced scratch. The C-terminal of 1D inhibits morphine-induced scratch without affecting analgesia. The authors hypothesize that human spinal cord also contains itch-specific µ-opioid receptor isoforms which interact with gastrin releasing peptide receptor. METHODS: Reverse transcription polymerase chain reaction was performed on human spinal cord complimentary DNA from two human cadavers. Calcium responses to morphine (1 µM) were examined using calcium imaging microscopy on human cells (HEK293) coexpressing gastrin releasing peptide receptor and different human µ-opioid receptor isoforms. The authors assessed morphine-induced scratching behavior and thermal analgesia in mice following intrathecal injection of morphine (0.3 nmol) and a transactivator of transcription peptide designed from C-terminal sequences of 1Y isoform (0, 0.1, and 0.4 nmol). RESULTS: The authors demonstrated 1Y expression in the spinal cord dorsal horn. Morphine administration evoked a calcium response (mean ± SD) (57 ± 13 nM) in cells coexpressing both gastrin releasing peptide receptor and the 1Y isomer. This was blocked by 10 µM naltrexone (0.7 ± 0.4 nM; P < 0.0001), 1 µM gastrin-releasing peptide receptor antagonist (3 ± 2 nM; P < 0.0001), or 200 µM 1Y-peptide (2 + 2 nM; P < 0.0001). In mice, 0.4 nmol 1Y-peptide significantly attenuated morphine-induced scratching behaviors (scratching bouts, vehicle vs. 1Y-peptide) (92 ± 31 vs. 38 ± 29; P = 0.011; n = 6 to 7 mice per group), without affecting morphine antinociception in warm water tail immersion test (% of maximum possible effect) (70 ± 21 vs. 67 ± 22; P = 0.80; n = 6 mice per group). CONCLUSIONS: Human µ-opioid receptor 1Y isomer is a C-terminal splicing variant of Oprm1 gene identified in human spinal cord. Cross-talk between 1Y and gastrin releasing peptide receptor is required for mediating opioid-induced pruritus. Disrupting the cross talk may have implications for therapeutic uncoupling of desired analgesic effects from side effects of opioids.

Prospects of Targeting the Gastrin Releasing Peptide Receptor and Somatostatin Receptor 2 for Nuclear Imaging and Therapy in Metastatic Breast Cancer.

BACKGROUND: The gastrin releasing peptide receptor (GRPR) and the somatostatin receptor 2 (SSTR2) are overexpressed on primary breast cancer (BC), making them ideal candidates for receptor-mediated nuclear imaging and therapy. The aim of this study was to determine whether these receptors are also suitable targets for metastatic BC. METHODS: mRNA expression of human BC samples were studied by in vitro autoradiography and associated with radioligand binding. Next, GRPR and SSTR2 mRNA levels of 60 paired primary BCs and metastases from different sites were measured by quantitative reverse transcriptase polymerase chain reaction. Receptor mRNA expression levels were associated with clinico-pathological factors and expression levels of primary tumors and corresponding metastases were compared. RESULTS: Binding of GRPR and SSTR radioligands to tumor tissue correlated significantly with receptor mRNA expression. High GRPR and SSTR2 mRNA levels were associated with estrogen receptor (ESR1)-positive tumors (p<0.001 for both receptors). There was no significant difference in GRPR mRNA expression of primary tumors versus paired metastases. Regarding SSTR2 mRNA expression, there was also no significant difference in the majority of cases, apart from liver and ovarian metastases which showed a significantly lower expression compared to the corresponding primary tumors (p = 0.02 and p = 0.03, respectively). CONCLUSION: Targeting the GRPR and SSTR2 for nuclear imaging and/or treatment has the potential to improve BC care in primary as well as metastatic disease.

Pharmacotherapy in detrusor underactivity: A new challenge for urologists and pharmacologists (from lab to clinic).

Higher incidence of functional urinary bladder dysfunction (detrusor overactivity - DO and detrusor underactivity - DU) occurs in elderly people. Effective therapy is widely used in patients with DO, in contrast DU seems to be a serious burden for the older population due to the lack of successful treatment. The aim of the study was to review the potential pharmacological targets in DU treatment in the animal model. This review is based on systemic literature research. The Medline/Pubmed, Scopus, Embase, and Web of Science databases were searched in order to identify original and review articles, as well as editorials relating to underactive bladder, detrusor underactivity. The following Medical Subject Headings (MeSH) terms were used to ensure the sensitivity of the searches: urinary bladder, animal models, humans and therapy. 19 papers met the criteria and were included for this review. 19 papers met the criteria and were included for this review. The pathophysiology of DU and its animal models were described. Moreover, the potential pharmacological targets in DU therapy were discussed, such as bombesin receptors, prostaglandin-, ATP-, NO-, CGRP-, SP-, Dopamine-, NGF-, M2-, and agrin-dependent pathways. In conclusion, due to the lack of effective treatment strategies in DU, further research is necessary. Close cooperation between urologists and pharmacologists should be maintained for optimal research on DU pharmacotherapy.

Clinical Relevance of Targeting the Gastrin-Releasing Peptide Receptor, Somatostatin Receptor 2, or Chemokine C-X-C Motif Receptor 4 in Breast Cancer for Imaging and Therapy.

UNLABELLED: Imaging and therapy using radioligands targeting receptors overexpressed on tumor cells is successfully applied in neuroendocrine tumor patients. Because expression of the gastrin-releasing peptide receptor (GRPR), somatostatin receptor 2 (SSTR2), and chemokine C-X-C motif receptor 4 (CXCR4) has been demonstrated in breast cancer, targeting these receptors using radioligands might offer new imaging and therapeutic opportunities for breast cancer patients. The aim of this study was to correlate messenger RNA (mRNA) expression of GRPR, SSTR2, and CXCR4 with clinicopathologic and biologic factors, and with prognosis and prediction to therapy response, in order to identify specific breast cancer patient groups suited for the application of radioligands targeting these receptors. METHODS: First, we studied GRPR and SSTR2 expression in 13 clinical breast cancer specimens by in vitro autoradiography and correlated this with corresponding mRNA levels to investigate whether mRNA levels reliably represent cell surface expression. Next, GRPR, SSTR2, and CXCR4 mRNA levels were measured by quantitative reverse transcriptase polymerase chain reaction in 915 primary breast cancer tissues and correlated with known clinicopathologic and biologic factors, disease-free survival, distant metastasis-free survival, and overall survival (DFS, MFS, and OS, respectively). In 224 adjuvant hormonal treatment-naïve estrogen receptor (ER, ESR1)-positive patients who received tamoxifen as first-line therapy for recurrent or metastatic disease, the expression levels of the receptors were correlated with progression-free survival. RESULTS: Our results showed a significant positive correlation between GRPR and SSTR2 expression analyzed by in vitro autoradiography and by quantitative reverse transcriptase polymerase chain reaction (Spearman's rank correlation coefficient [Rs]=0.94, P<0.001, and Rs=0.73, P=0.0042, respectively). Furthermore, high GRPR and SSTR2 mRNA levels were observed more frequently in ESR1-positive specimens, whereas high CXCR4 expression was associated with ESR1-negative specimens. Also, high mRNA expression of CXCR4 was associated with a prolonged DFS, MFS, and OS (multivariate hazard ratio MFS=0.76 [95% confidence interval, 0.64-0.90], P=0.001), whereas high mRNA levels of GRPR were associated with a prolonged progression-free survival after the start of first-line tamoxifen treatment (multivariate hazard ratio=0.68 [95% confidence interval, 0.48-0.97], P=0.031). CONCLUSION: Our data indicate that imaging and therapy using GRPR or SSTR2 radioligands might especially be beneficial for ESR1-positive breast cancer and CXCR4 radioligands for ESR1-negative breast cancer.

Synthesis and characterization of a high-affinity NOTA-conjugated bombesin antagonist for GRPR-targeted tumor imaging.

The gastrin-releasing peptide receptor (GRPR/BB2) is a molecular target for the visualization of prostate cancer. This work focused on the development of high-affinity, hydrophilic, antagonistic, bombesin-based imaging agents for PET and SPECT. The bombesin antagonist analog d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 ([d-Phe(6),Sta(13),Leu(14)]bombesin[6-14]) was synthesized and conjugated to 1,4,7-triazacyclononane-N,N',N″-triacetic acid (NOTA) via a diethylene glycol (PEG2) linker. The resulting conjugate, NOTA-PEG2-[d-Phe(6),Sta(13),Leu(14)]bombesin[6-14] (NOTA-P2-RM26), was labeled with (68)Ga (T1/2 = 68 min, positron emitter) and (111)In (T1/2 = 2.8 days, gamma emitter). The labeling stability, specificity, inhibition efficiency (IC50), and dissociation constant (KD) of both labeled compounds as well as their cellular retention and internalization were investigated. The pharmacokinetics of the dual isotope ((111)In/(68)Ga)-labeled peptide in both normal NMRI mice and PC-3 tumor-bearing Balb/c nu/nu mice was also studied. NOTA-P2-RM26 was labeled with (111)In and (68)Ga at a radiochemical yield of >98%. Both conjugates were shown to have high specificity and binding affinity for GRPR. The KD value was determined to be 23 ± 13 pM for the (111)In-labeled compound in a saturation binding experiment. In addition, (nat)In- and (nat)Ga-NOTA-P2-RM26 showed low nanomolar binding inhibition concentrations (IC50 = 1.24 ± 0.29 nM and 0.91 ± 0.19 nM, respectively) in a competitive binding assay. The internalization rate of the radiolabeled conjugates was slow. The radiometal-labeled tracers demonstrated rapid blood clearance via the kidney and GRPR-specific uptake in the pancreas in normal mice. Tumor targeting and biodistribution studies in mice bearing PC-3 xenografts displayed high and specific uptake in tumors (8.1 ± 0.4%ID/g for (68)Ga and 5.7 ± 0.3%ID/g for (111)In) and high tumor-to-background ratios (tumor/blood: 12 ± 1 for (68)Ga and 10 ± 1 for (111)In) after only 1 h p.i. of 45 pmol of peptide. The xenografts were visualized by gamma and microPET cameras shortly after injection. In conclusion, the antagonistic bombesin analog NOTA-PEG2-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (NOTA-P2-RM26) is a promisindg candidate for prostate cancer imaging using PET and SPECT/CT.

Autocrine effects of neuromedin B stimulate the proliferation of rat primary osteoblasts.

Neuromedin B (NMB) is a mammalian bombesin-like peptide that regulates exocrine/endocrine secretion, smooth muscle contraction, body temperature, and the proliferation of some cell types. Here, we show that mRNA encoding Nmb and its receptor (Nmbr) are expressed in rat bone tissue. Immunohistochemical analysis demonstrated that NMB and NMBR colocalize in osteoblasts, epiphyseal chondrocytes, and proliferative chondrocytes of growth plates from mouse hind limbs. Then, we investigated the effect of NMB on the proliferation of rat primary cultured osteoblasts. Proliferation assays and 5-bromo-2'-deoxyuridine incorporation assays demonstrated that NMB augments the cell number and enhances DNA synthesis in osteoblasts. Pretreatment with the NMBR antagonist BIM23127 inhibited NMB-induced cell proliferation and DNA synthesis. Western blot analysis showed that NMB activates ERK1/2 MAPK signaling in osteoblasts. Pretreatment with the MAPK/ERK kinase inhibitor U0126 attenuated NMB-induced cell proliferation and DNA synthesis. We also investigated the effects of molecules that contribute to osteoblast proliferation and differentiation on Nmb expression in osteoblasts. Real-time PCR analysis demonstrated that 17ß-estradiol (E2) and transforming growth factor ß1 increase and decrease Nmb mRNA expression levels respectively. Finally, proliferation assays revealed that the NMBR antagonist BIM23127 suppresses E2-induced osteoblast proliferation. These results suggest that NMB/NMBR signaling plays an autocrine or paracrine role in osteoblast proliferation and contributes to the regulation of bone formation.

Intrathecal bombesin is sympathoexcitatory and pressor in rat.

Bombesin, a 14 amino-acid peptide, is pressor when administered intravenously in rat and pressor and sympathoexcitatory when applied intracerebroventricularly. To determine the spinal effects of bombesin, the peptide was administered acutely in the intrathecal space at around thoracic spinal cord level six of urethane-anesthetized, paralyzed, and bilaterally vagotomized rats. Blood pressure, heart rate, splanchnic sympathetic nerve activity (sSNA), phrenic nerve activity, and end-tidal CO(2) were monitored to evaluate changes in the cardiorespiratory systems. Bombesin elicited a long-lasting excitation of sSNA associated with an increase in blood pressure and tachycardia. There was a mean increase in arterial blood pressure of 52 ± 5 mmHg (300 µM; P < 0.01). Heart rate and sSNA also increased by 40 ± 4 beats/min (P < 0.01) and 162 ± 33% (P < 0.01), respectively. Phrenic nerve amplitude (PNamp, 73 ± 8%, P < 0.01) and phrenic expiratory period (+0.16 ± 0.02 s, P < 0.05) increased following 300 µM bombesin. The gain of the sympathetic baroreflex increased from -2.8 ± 0.7 to -5.4 ± 0.9% (P < 0.01), whereas the sSNA range was increased by 99 ± 26% (P < 0.01). During hyperoxic hypercapnia (10% CO(2) in O(2), 90 s), bombesin potentiated the responses in heart rate (-25 ± 5 beats/min, P < 0.01) and sSNA (+136 ± 29%, P < 0.001) but reduced PNamp (from 58 ± 6 to 39 ± 7%, P < 0.05). Finally, ICI-216,140 (1 mM), an in vivo antagonist for the bombesin receptor 2, attenuated the effects of 300 µM bombesin on blood pressure (21 ± 7 mmHg, P < 0.01). We conclude that bombesin is sympathoexcitatory at thoracic spinal segments. The effect on phrenic nerve activity may the result of spinobulbar pathways and activation of local motoneuronal pools.

Effects of intracerebral ventricular administration of gastrin-releasing peptide and its receptor antagonist RC-3095 on learned fear responses in the rat.

Several lines of evidence have implicated bombesin and its mammalian analogue, gastrin-releasing peptide (GRP), in the mediation and/or modulation of the stress response. However, the physiological role of GRP in mediating conditioned fear responses remains to be elucidated. The objective of the present study was to characterize the role(s) of GRP and its receptor antagonist (D-Tpi6, Leu13 psi[CH2NH]-Leu14) BB((6-14)) (RC-3095) in fear-related responses using two animal models of conditioned fear. To this end, the effects of intracerebroventricular (i.c.v.) administration of GRP (0.062, 0.30, 3.0 nmol) and RC-3095 (0.3, 3.0 and 9.0 nmol) were assessed in the conditioned emotional response (CER) and the fear-potentiated startle (FPS) paradigms. In the CER paradigm, i.c.v. administration of GRP dose-dependently (all doses) attenuated the expression of both contextual and cued fear as reflected by a reduction in freezing behavior to both the context (cage where shock was received) and cue (tone paired with shock). Conversely, pretreatment with RC-3095 (high dose), blocked the reduction of contextual and cued fear normally observed over time. Further, in the FPS paradigm, i.c.v. administration of GRP significantly attenuated the fear-potentiated startle response at medium and high doses without affecting basal startle amplitude. In contrast, pretreatment with RC-3095 at the highest dose (9.0 nmol) significantly increased the basal startle amplitude without affecting fear-potentiation, suggesting elevated fear at the onset of testing. These data provide further evidence that GRP is involved in conditioned fear responses.

Anticancer activity of a peptide combination in gastrointestinal cancers targeting multiple neuropeptide receptors.

A novel peptide combination consisting of four synthetic neuropeptide analogs of Vasoactive Intestinal Peptide (VIP), Bombesin, Substance P and Somatostatin has been found to have potent anticancer activity in vitro and in vivo. The receptors of these four neuropeptides are known to be over expressed in various cancers. We have found the presence of native neuropeptides in the culture supernatant of the primary tumor cells of human colon adenocarcinomas. It was further demonstrated by receptor-ligand assays that not only do these tumor cells synthesize and secrete four peptide hormones but also possess specific high affinity receptors on their surface. Screening a large panel of analogs to the four peptide hormones on tumor cell proliferation led to the identification of four cytotoxic analogs, the combination of which was code-named DRF7295. The design and synthesis of the peptide analogs have been described in this paper. In vitro anticancer activity of DRF7295 was studied in a large panel of human tumor cells. Gastrointestinal tumor cells of the colon, pancreas and duodenum were found to be most sensitive to DRF7295 with moderate activity seen in glioblastoma, prostate, leukemia and those of oral cancer cells. Efficacy studies in xenograft models of colon and duodenum resulted in T/C% of less than 40%, which is indicative of strong tumor regressing potential of DRF7295 in gastrointestinal cancers. Acute and long-term toxicity studies as well as safety pharmacology studies conducted indicate the safety of the drug upon systemic administration with no significant adverse pharmacological effects.

In vitro and in vivo antitumor effects of cytotoxic camptothecin-bombesin conjugates are mediated by specific interaction with cellular bombesin receptors.

Most human tumors overexpress or ectopically express peptide hormone/neurotransmitter receptors, which are being increasingly studied as a means to selectively deliver cytotoxic agents. Although a number of peptide ligand-constructs demonstrate tumor cytotoxicity, the role of specific tumoral receptor interaction in its mediation is unclear. To address this question, we synthesized camptothecin (CPT) bombesin (Bn) analogs, in which CPT was coupled via a novel carbamate linker, L2 [N-(N-methyl-amino-ethyl)-glycine carbamate], that were chemically similar but differed markedly in their potency/affinity for human Bn receptors. We then examined their ability to interact with Bn receptors and cause in vitro and in vivo tumor cytotoxicity. CPT-L2-[D-Tyr(6),beta-Ala(11),D-Phe(13),Nle(14)] Bn (6-14) (BA3) bound with high affinity and had high potency for all three human Bn receptor subtypes, whereas CPT-L2-[D-Tyr(6),beta-Ala(11), D-Phe(13),Nle(14)] Bn (6-14) [D-Phe-CPT-L2-BA3] had >1400-fold lower affinity/potency. (125)I-CPT-L2-BA3 but not (125)I-D-Phe-CPT-L2-BA3 was internalized by Bn receptor subtype-containing cells. CPT-L2-BA3 displayed significantly more cytotoxicity than D-Phe-CPT-L2-BA3 toward NCI-H1299 lung cancer cells in both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide and clonogenic assays and more potently inhibited H1299 xenograft growth in nude mice. CPT-L2-BA3 was also metabolically more stable than its parent peptide and inhibited growth of a number of other tumor cell lines in vitro and in vivo. These results demonstrate that specific tumoral receptor interaction is important in mediating the ability of peptide ligand-cytotoxic constructs to cause cytotoxicity. Because many tumors overexpress Bn receptors, these results also demonstrate that CPT-L2-BA3 will be a useful agent for delivering receptor-mediated cytotoxicity to many different human tumors.

Targeting gastrin-releasing peptide receptors on small cell lung cancer cells with a bispecific molecule that activates polyclonal T lymphocytes.

PURPOSE: Gastrin-releasing peptide (GRP) is a growth factor for small cell lung cancer (SCLC). GRP belongs to the bombesin peptide family and has significant homology to bombesin. We constructed a bispecific molecule, OKT3xAntag2, by conjugating a monoclonal antibody OKT3 (anti-CD3) with a bombesin/GRP antagonist (Antag2) and evaluated cytotoxicity against SCLC cells. EXPERIMENTAL DESIGN: We tested binding of the bispecific molecule to SCLC cell lines and T cells by flow cytometry, antibody-dependent cellular cytotoxicity (ADCC) of SCLC cells in vitro and in a murine SCLC xenograft model. We studied SCLC apoptosis and necrosis during ADCC and the activity and cleavage of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP). RESULTS: The bispecific molecule functions as a cross-linker between T cells and SCLC cells, induces T cell activation, and mediates ADCC of SCLC cells; 40% to 80% growth inhibition of SCLC cells mediated by the bispecific molecule at low effector to target cell ratios was achieved. Activation of T cells by the bispecific molecule resulted in significant increases in IFNgamma production and apoptosis and necrosis of SCLC cells associated with cleavage of PARP and caspase-3. Targeted immunotherapy with the bispecific molecule-armed human T cells significantly reduced SCLC tumor burdens in a mouse model. CONCLUSION: The bispecific molecule OKT3xAntag2 mediates growth inhibition and apoptosis of SCLC cells by activated T cells through activation and cleavage of caspase-3 and PARP in vitro and in vivo. Clinical trials of this bispecific molecule through adoptive transfer of ex vivo activated T cells in GRP receptor-positive tumors, such as SCLC, are warranted.

Gastrin-releasing peptide receptor as a molecular target for psychiatric and neurological disorders.

The mammalian bombesin (BB)-like peptide gastrin-releasing peptide (GRP) stimulates cell proliferation, displays a range of neuroendocrine activities, and acts as a growth factor in the pathogenesis of several types of human cancer. Several lines of evidence have indicated that GRP and its receptor (GRPR) might also be involved in the neurochemical alterations associated with psychiatric and neurological disorders. GRP and GRPR are distributed throughout the mammalian central nervous system (CNS). Altered levels of BB-like peptides have been found in the CNS of patients with schizophrenia and Parkinson's disease. Dysfunctions in GRPR-induced cellular calcium signaling have been reported in fibroblasts from patients with Alzheimer's disease. A translocation in the GRPR gene has been associated with autism. Pharmacological and genetic studies in rodents have shown that GRPRs in brain areas such as the dorsal hippocampus and amygdala are importantly involved in regulating synaptic plasticity and aspects of behavior that might be altered in disorders such as anxiety, schizophrenia, depression, autism and dementia. Behaviors modulated by the GRPR in rodents include grooming, food intake, stereotypy, social behavior, and emotionally-motivated learning and memory. Together, these findings support the view that the GRPR should be considered a therapeutic target for a subset of CNS diseases.

Targeted chemotherapy with cytotoxic bombesin analogue AN-215 inhibits growth of experimental human prostate cancers.

We developed a powerful cytotoxic analogue of bombesin AN-215, in which the bombesin (BN)-like carrier peptide is conjugated to 2-pyrrolino doxorubicin (AN-201). Human prostate cancers express high levels of receptors for BN/gastrin releasing peptide (GRP) that can be used for targeted chemotherapy. The effects of targeted chemotherapy with cytotoxic BN analogue AN-215 were evaluated in nude mice bearing subcutaneous xenografts of DU-145, LuCaP-35, MDA-PCa-2b and intraosseous implants of C4-2 human prostate cancers. Intraosseous growth of C4-2 tumors was monitored by serum PSA. BN/GRP receptors were evaluated by 125I-[Tyr4]BN binding assays and RT-PCR. The effects of AN-215 on apoptosis and cell proliferation were followed by histology, and the expression of Bcl-2 and Bax protein was determined by Western blot analysis. Targeted analog AN-215 significantly inhibited growth of subcutaneously implanted DU-145, LuCaP-35 and MDA-PCa-2b prostate cancers by 81% to 91% compared to controls, while cytotoxic radical AN-201 was less effective and more toxic. Serum PSA levels of mice bearing intraosseous C4-2 prostate tumors were significantly reduced. In LuCaP-35 tumors administration of BN antagonist RC-3095 prior to AN-215 blocked the receptors for BN/GRP and inhibited the effects of AN-215. High affinity receptors for BN/GRP and their m-RNA were detected on membranes of all 4 tumor models. Therapy with AN-215, but not with AN-201, decreased the ratio of Bcl-2/Bax in DU-145 and the expression of antiapoptotic Bcl-2 in LuCaP-35 tumors. The presence of BN/GRP receptors on primary and metastatic prostate cancers makes possible targeted chemotherapy with AN-215 for the treatment of this malignancy.

New chelation strategy allows for quick and clean 99mTc-labeling of synthetic peptides.

Analogues of bombesin have been synthesized in which a N2S2 (bis-mercaptoacetyl functionalized diaminopropionic acid) or a N3S (mercaptoacetyl-Gly-Gly-Gly) radiometal-chelating center has been incorporated that allows radiolabeling of these peptides with 99mTc without the need for conjugation or harsh reaction conditions. A mild radiolabeling is possible by using an acetyl-moiety as sulfur protecting group, which can be removed by mild hydroxylamine-treatment at room temperature before radiolabeling. Retained receptor binding is demonstrated in competitive binding experiments with 99mTc-radiolabeled peptides and PC-3 cells with bombesin receptors.

Preclinical evaluation of therapeutic effects of targeted cytotoxic analogs of somatostatin and bombesin on human gastric carcinomas.

BACKGROUND: New modalities are necessary for the treatment of patients with unresectable gastric carcinoma. The authors investigated whether receptors for somatostatin and bombesin were present in human gastric carcinoma lines and tested the antitumor effects of cytotoxic somatostatin analog AN-238 and cytotoxic bombesin conjugate AN-215. METHODS: Nude mice bearing AGS, Hs 746T, and NCI-N87 human gastric carcinomas were treated with AN-238, AN-215, or their cytotoxic moiety 2-pyrrolinodoxorubicin (AN-201). Tumor growth reduction and tumor doubling times were calculated, and histologic characteristics of cell proliferation and apoptosis were examined. The expression of mRNA for somatostatin and bombesin receptors in tumors was investigated by reverse transcriptase-polymerase chain reaction. Subtypes 2 and 5 of somatostatin receptor proteins (sst2 and sst5, respectively) were analyzed using immunohistochemistry and immunoblotting. Binding assays were performed with radiolabeled somatostatin and bombesin analogs. RESULTS: Cytotoxic bombesin analog AN-215 powerfully inhibited the growth of AGS carcinomas that expressed high-affinity subtype 1 bombesin receptors. All three carcinomas expressed high-affinity sst2 and sst5 receptors. Cytotoxic somatostatin analog AN-238 exerted a strong inhibitory effect on NCI-N87 and Hs 746T carcinomas, which exhibited high concentrations of somatostatin receptors, but had a weaker effect on AGS tumors, which expressed the lowest receptor levels. AN-201 had only nonsignificant effects. CONCLUSIONS: Experimental human gastric carcinomas that expressed high-affinity subtype 1 bombesin receptors were inhibited by cytotoxic bombesin analog AN-215, and tumors with high concentrations of sst2 or sst5 somatostatin receptors were suppressed by cytotoxic somatostatin analog AN-238. These findings suggest that this class of targeted compounds should be considered for the therapy of patients with advanced gastric carcinoma.

Molecular basis of the pharmacological difference between rat and human bombesin receptor subtype-3 (BRS-3).

We cloned the gene and cDNA for rat bombesin receptor subtype-3 (BRS-3) and characterized its mRNA expression pattern and pharmacological properties. Despite the high degree of sequence similarity (80% identical), rat and human BRS-3 differ markedly in their pharmacological properties. Although the natural ligand for BRS-3 is still unknown, a synthetic peptide, dY-Q-W-A-V-(beta-A)-H-F-Nle-amide (dY-bombesin), activates human BRS-3 with an EC(50) of 1.2 nM. In contrast, dY-bombesin had a very poor potency for rat BRS-3 (EC(50) = 2 microM). To understand the molecular basis of this pharmacological difference, we constructed chimeric receptors in which individual extracellular loops of rat BRS-3 were replaced with the corresponding human sequences. Switching the N-terminal region or the second extracellular loop did not significantly change receptor properties. However, switching the third extracellular loop (E3) in the rat BRS-3 resulted in a chimeric receptor (RB3-E3) that behaved almost identically to human BRS-3. RB3-E3 bound dY-bombesin with high affinity (K(i) = 1.2 +/- 0.7 nM), and was activated by dY-bombesin with high potency (EC(50) = 1.8 +/- 0.5 nM). Within the E3 loop, mutation of Y(298)E(299)S(300) to S(298)Q(299)T(300) (RB3-SQT) or of D(306)V(307)P(308) to A(306)M(307)H(308) (RB3-AMH) only partially mimicked the effect of switching the entire E3 loop, and mutation of A(302)E(303) to V(302)D(303) or of V(310)V(311) to I(310)F(311) had little effect on the dY-bombesin potency. These results indicate that the sequence variation in the E3 loop is responsible for the species difference between rat and human BRS-3, and multiple residues in the E3 loop are involved in interactions with the agonist dY-bombesin.

Inhibition of proliferation of human small cell lung cancer cells expressing an autocrine system for gastrin releasing peptide by antisense oligodeoxynucleotides to gastrin releasing peptide receptor.

The objectives of this study were to investigate the effect of antisense (AS) oligodeoxynucleotides (ODNs) directed against gastrin releasing peptide (GRP) receptor mRNA on proliferation of human small cell lung cancer (SCLC) NCI-H345 cells which express the autocrine system for GRP. The methods used were to expose human SCLC cell lines to antisense ODNs or sense ODNs and to measure their proliferation by spectrophotometric assay or viable cell counts. Our results demonstrated that the single or combined AS ODNs against GRP receptor inhibited proliferation of human SCLC NCI-H345 cells significantly by 37% (P<0.01), but did not inhibit proliferation of either human bronchial epithelial BEAS 2B cells or human SCLC NCI-N417 cells, neither of which express the GRP autocrine system. The sense controls did not significantly inhibit proliferation compared with no treatment controls. Specificity was also demonstrated by the observation that cells exposed to AS ODNs had a decrease in GRP receptor expression as measured by specific binding of 34% (P<0.01), and when all three AS ODNs were used, binding was decreased by 60% (P<0.03). Furthermore, AS ODNs decreased by 75% the maximum percentage of cells responding to GRP in an intracellular calcium release assay. Our conclusions are that antisense ODNs directed against a GRP receptor which is involved in an autocrine loop in human SCLC cells inhibited proliferation of these cells by their impact on reducing GRP receptor expression. Further development of means of increasing AS ODN specificity and effectiveness in human SCLC cell is warranted.

Identification and characterisation of functional bombesin receptors in human astrocytes.

Reverse transcription polymerase chain reaction (RT-PCR) demonstrated the presence of bombesin BB2 receptor mRNA but not bombesin BB1 receptor or bombesin BB3 receptor mRNA in cultured human astrocytes. Neuromedin C hyperpolarised human astrocytes in whole-cell current and voltage clamp recordings and increased the intracellular free Ca(2+) ion concentration ([Ca(2+)](i)) in single astrocytes. Treatment with neuromedin C caused larger and more frequent increases in [Ca(2+)](i) than those triggered by neuromedin B, with 96% and 78% of cells responding, respectively. The stimulatory effects of neuromedin C were inhibited significantly by treatment with U73122 or the bombesin BB2 receptor antagonist [D-Phe(6), des-Met(14)]bombesin-(6-14) ethylester. A Fluorometric Imaging Plate Reader (FLIPR) was used to measure [Ca(2+)](i) in cell populations. Neuromedin C was approximately 50-fold more potent than neuromedin B in elevating [Ca(2+)](i) in astrocytes and Chinese hamster ovary (CHO) cells expressing human bombesin BB2 receptors (hBB2-CHO). However, in CHO cells expressing the bombesin BB1 receptor hBB1-CHO, neuromedin B was 32-fold more potent than neuromedin C. [D-Phe(6), des-Met(14)]bombesin-(6-14) ethylester was a partial agonist in hBB1-CHO cells (E(max)=55%) but was a noncompetitive antagonist in both hBB2-CHO cells and astrocytes. These studies report the first identification of functional bombesin receptors on cultured human astrocytes and have demonstrated that the bombesin BB2 receptor contributes significantly to astrocyte physiology.