AIM:Glucagon-like peptide-1 (GLP-1) receptor agonists for the treatment of type 2 diabetes are administered by daily injection because of short plasma half-lives, which result partly from the biochemical instability of these peptides. There is a medical need for GLP-1 analogues that can be administered less frequently for patient convenience. METHODS:We synthesized a series of human GLP-1 (hGLP-1(7-36)NH(2) ) derivatives containing α-aminoisobutyric acid (Aib) substitutions, analysed their enzymatic stabilities and evaluated their secondary structures using circular dichroism (CD) and nuclear magnetic resonance (NMR). RESULTS:Plasma stability experiments showed that only the analogue containing Aib substitutions in both the N-terminus (position 8) and the C-terminus (position 35), [Aib⁸(,)³⁵]hGLP-1(7-36)NH₂ (BIM-51077), was fully resistant to enzymatic cleavage. Incubation with human plasma kallikrein or plasmin confirmed that the Aib substitution at position 35 prevented protease cleavage around this residue, which contributes to the significantly enhanced plasma stability and increased plasma half-life. CD revealed increased C-terminal α-helicity in Aib³⁵-substituted analogues compared with both hGLP-1(7-36)NH₂ and analogues containing only Aib⁸ substitutions. Based on NMR studies, the secondary structure of BIM-51077 is similar to hGLP-1(7-36)NH₂ with a slight increase in α-helicity in the C-terminus. Compared with hGLP-1(7-36)NH₂, BIM-51077 had similar binding affinity for the human GLP-1 receptor and activated this receptor with similar potency. CONCLUSIONS:We have discovered an Aib⁸(,)³⁵-substituted analogue of native hGLP-1(7-36)NH₂ (BIM-51077) that retains the structure of the native peptide, and has similar activity and enhanced stability. A sustained-release formulation of this molecule (taspoglutide) is in phase-3 clinical development.

Taspoglutide is a novel analog of human glucagon-like peptide-1 [hGLP-1(7-36)NH2] in clinical development for the treatment of type 2 diabetes. Taspoglutide contains alpha-aminoisobutyric acid substitutions replacing Ala(8) and Gly(35) of hGLP-1(7-36)NH2. The binding affinity [radioligand binding assay using [(125)I]hGLP-1(7-36)NH2], potency (cAMP production in CHO cells stably overexpressing hGLP-1 receptor), and in vitro plasma stability of taspoglutide compared with hGLP-1(7-36)NH2 have been evaluated. Effects on basal and glucose-stimulated insulin secretion were determined in vitro in INS-1E cells and in vivo in normal rats. Taspoglutide has comparable affinity (affinity constant 1.1 +/- 0.2 nm) to the natural ligand (affinity constant 1.5 +/- 0.3 nm) for the hGLP-1 receptor and exhibits comparable potency in stimulating cAMP production (EC(50) Taspo 0.06 nm and EC(50) hGLP-1(7-36)NH2 0.08 nm). Taspoglutide exerts insulinotropic action in vitro and in vivo and retains the glucoincretin property of hGLP-1(7-36)NH2. Stimulation of insulin secretion is concentration dependent and evident in the presence of high-glucose concentrations (16.7 mm) with a taspoglutide concentration as low as 0.001 nm. Taspoglutide is fully resistant to dipeptidyl peptidase-4 cleavage (during 1 h incubation at room temperature with purified enzyme) and has an extended in vitro plasma half-life relative to hGLP-1(7-36)NH2 (9.8 h vs. 50 min). In vitro, taspoglutide does not inhibit dipeptidyl peptidase-4 activity. This study provides the biochemical and pharmacological basis for the sustained plasma drug levels and prolonged therapeutic activity seen in early clinical trials of taspoglutide. Excellent stability and potency with substantial glucoincretin effects position taspoglutide as a promising new agent for treatment of type 2 diabetes.

Background: Ghrelin, was observed to have treatment-potential for severe chronic heart failure (CHF) and cardiac cachexia based on anti-cachectic and cardio-protective effects.Methods: We performed two studies to assess the effects of human ghrelin on food intake, body weight and body composition, as well as heart function in a rat model of CHF. Study-1 (50 or 500 nmole/kg/d ghrelin by pump infusion) was focused on food intake and body composition, study-2 (50 or 100 nmole/kg/d ghrelin by subcutaneous injection (3-times daily) was focused on heart function due to a lack of cardiac effects observed in study-1. In both studies, myocardial infarction was induced by LAD ligation. On day 28 after surgery, rats were randomized and treated with ghrelin or placebo for 4 weeks. Food intake (study-1), body composition (NMR) cardiac function (echocardiography and invasive hemodynamics (study-2 only) were assessed.Results: In study-1, CHF rats treated with high dose ghrelin showed an increase in body weight (+25%, p<0.001), lean mass (+16%, p<0.01) and fat mass (+17%, p=0.001) vs placebo. In study-2, CHF rats treated with both low-and high dose ghrelin showed an increase in body weight (both + 18%, p=0.001), lean mass (both + 25%, p<0.001) and fat mass (50 nmole/kg/d: + 43%, p<0.05; 100 nmole/kg/d: +45%, p<0.01) vs placebo. However, no beneficial effect of ghrelin treatment on left ventricular ejection fraction or change of LV diameters was observed in either study.Conclusion: Ghrelin treatment results in dose-dependent beneficial effects on body weight and body composition, but does not improve cardiac function. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Dopamine D2 and somatostatin receptors (sstrs) were reported to affect non-functioning pituitary adenoma (NFPA) proliferation in vitro. However, the reported results differ according to the experimental conditions used. We established an experimental protocol allowing reproducible evaluation of NFPA cell proliferation in vitro, to test and compare the antiproliferative effects of dopamine and somatostatin analogs (alone or in combination) with the activity of the dopamine-somatostatin chimeric molecule BIM-23A760. The protocol was utilized by four independent laboratories, studying 38 fibroblast-deprived NFPA cell cultures. Cells were characterized for GH, POMC, sstr1-sstr5, total dopamine D2 receptor (D2R) (in all cases), and D2 receptor long and short isoforms (in 15 out of 38 cases) mRNA expression and for alpha-subunit, LH, and FSH release. D2R, sstr3, and sstr2 mRNAs were consistently observed, with the dominant expression of D2R (2.9+/-2.6 copy/copy beta-glucuronidase; mean+/-s.e.m.), when compared with sstr3 and sstr2 (0.6+/-1.0 and 0.3+/-0.6 respectively). BIM-23A760, a molecule with high affinity for D2R and sstr2, significantly inhibited [3H]thymidine incorporation in 23 out of 38 (60%) NFPA cultures (EC50=1.2 pM and Emax=-33.6+/-3.7%). BIM-23A760 effects were similar to those induced by the selective D2R agonist cabergoline that showed a statistically significant inhibition in 18 out of 27 tumors (compared with a significant inhibition obtained in 17 out of 27 tumors using BIM-23A760, in the same subgroup of adenomas analyzed), while octreotide was effective in 13 out of 27 cases. In conclusion, superimposable data generated in four independent laboratories using a standardized protocol demonstrate that, in vitro, chimeric dopamine/sstr agonists are effective in inhibiting cell proliferation in two-thirds of NFPAs.

Somatostatin is an inhibitor of hormone secretion through specific receptors (sst(1-5)). The aim of this study was to investigate the putative regulatory role of somatostatin analogues on the secretion of insulin and glucagon by rat pancreatic islets.After 48 h exposure only the non-selective agonists (somatostatin, octreotide and SOM-230) inhibited insulin accumulation. The inhibition of insulin secretion was accompanied by increased islet insulin contents. None of the analogues showed a consistent effect on the glucagon accumulation in the medium after 48 h.Since we observed a difference in the regulatory effect between the non-selective and selective analogues, combinations of selective analogues were studied. Combination of sst(2) + sst(5) agonists inhibited the medium insulin accumulation, while combination of sst(1) + sst(2) analogues caused a decrease in glucagon accumulation.After removal of somatostatin a rebound effect with increased insulin secretion were observed. This effect was reversed after 6 h. For SOM-230 insulin secretion continued to be suppressed even after the analogue was removed and returned to control values after 3 h. As for glucagon secretion there was an initial decline after culture with octreotide, while the other substances failed to induce any changes.In summary, non-selective somatostatin analogues or combinations of receptor selective analogues may cause inhibition of hormone secretion from rat pancreatic islets. For insulin and glucagon, combinations of sst(2) + sst(5) and sst(1) + sst(2), respectively may exert this effects. Thus, our data suggest that more than one sst must be involved to down-regulate islet glucagon and insulin secretion. (c) 2006 Elsevier B.V. All rights reserved.

Glucocorticoids are important immunosuppressive hormones; these steroids also inhibit somatic growth by decreased growth hormone (GH) secretion and induced protein catabolism. The ability of ghrelin, the endogenous ligand for the GHS-1a receptor, to increase body weight is attributed to a combination of enhanced food intake, increased gastric emptying and increased food assimilation, coupled with potent GH releasing activity. The aim of the present study was to evaluate the ability of a full-length, metabolically stabilized ghrelin agonist, BIM-28125, to reverse the dexamethasone-induced decrease of growth rate of prepubertal Sprague–Dawley male rats. Twenty-one days old rats were randomly assigned to two treatment groups. Beginning on day 23 of age, 16 animals were treated ip either with saline or DEX (40 μg/kg/day). On day 33 after birth, these two groups were further subdivided and treated sc with either vehicle or BIM-28125 (80 nmol/kg, t.i.d.). On day 47 after birth, rats were killed and trunk blood was collected for hormone determinations. DEX significantly reduced final body weight and nose-anal length; BIM-28125 increased linear growth in saline-treated rats and reversed growth inhibition in DEX-treated rats. The inhibitory effects of DEX on somatic growth was paralleled by decreased 24 h food intake (FI), decreased food efficiency (FE) and lower plasma IGF-1 levels versus vehicle-treated rats. BIM-28125 induced an increase of FI, FE and plasma IGF-1 in saline-treated rats, and reversed the inhibitory effects of DEX. These preclinical results leads to the conclusion that BIM-28125 may represent a good tool to reverse the catabolic effects induced by glucocorticoids.

Chronic kidney disease (CKD) is associated with an increase in inflammatory cytokines and can result in cachexia with loss of muscle and fat stores. We previously demonstrated the efficacy of treating a model of cancer cachexia with ghrelin and a ghrelin receptor agonist. Currently, we examine a surgical model of CKD in rats, resulting in uremia and decreased accrual of lean body mass. Treatment with ghrelin and two ghrelin receptor agonists (BIM-28125 and BIM-28131) resulted in increased food intake and an improvement in lean body mass accrual that was related in part to a decrease in muscle protein degradation as assessed by muscle levels of the 14-kDa actin fragment resulting from cleaved actomyosin. Additionally, there was a decrease in circulating inflammatory cytokines in nephrectomized animals treated with ghrelin relative to saline treatment. Ghrelin-treated animals also had a decrease in the expression of IL-1 receptor in the brainstem and a decrease in expression of prohormone convertase-2, an enzyme involved in the processing of proopiomelanocortin to the anorexigenic peptide alpha-MSH. We conclude that ghrelin treatment in uremia results in improved lean mass accrual in part due to suppressed muscle proteolysis and possibly related to antiinflammatory effects.

Cancer cachexia is a debilitating syndrome of anorexia and loss of lean body mass that accompanies many malignancies. Ghrelin is an orexigenic hormone with a short half-life that has been shown to improve food intake and weight gain in human and animal subjects with cancer cachexia. We used a rat model of cancer cachexia and administered human ghrelin and a synthetic ghrelin analog BIM-28131 via continuous infusion using sc osmotic minipumps. Tumor-implanted rats receiving human ghrelin or BIM-28131 exhibited a significant increase in food consumption and weight gain vs. saline-treated animals. We used dual-energy x-ray absorptiometry scans to show that the increased weight was due to maintenance of lean mass vs. a loss of lean mass in saline-treated animals. Also, BIM-28131 significantly limited the loss of fat mass normally observed in tumor-implanted rats. We further performed real-time PCR analysis of the hypothalami and brainstems and found that ghrelin-treated animals exhibited a significant increase in expression of orexigenic peptides agouti-related peptide and neuropeptide Y in the hypothalamus and a significant decrease in the expression of IL-1 receptor-I transcript in the hypothalamus and brainstem. We conclude that ghrelin and a synthetic ghrelin receptor agonist improve weight gain and lean body mass retention via effects involving orexigenic neuropeptides and antiinflammatory changes.

CONTEXT Cortistatin (CST) is a neuropeptide that shares high homology with somatostatin and binds with high affinity to all somatostatin receptor (SSTR) subtypes. Many of its endocrine and biological activities overlap with those of somatostatin. OBJECTIVE/DESIGN The objective of the study was to assess the direct in vitro effects of CST on human pituitary hormone secretion. SETTING This study was performed in the endocrine laboratory of a tertiary academic medical center. MATERIALS Primary cell cultures of human fetal (21-25 wk gestation) pituitary tissues and cultured hormone-secreting adenoma cells were used in this study. INTERVENTIONS Cell cultures were incubated with CST-14 or CST-17, somatostatin, GHRH, SSTR analogs, and ghrelin analogs, and hormone secretion was analyzed. OUTCOME MEASURES GH and prolactin (PRL) medium concentrations were tested by hormone assay, and SSTR mRNA was tested by RT-PCR. RESULTS CST-14 (10 nm) inhibited GH secretion by up to 65% in all fetal pituitary specimens after 4-h incubation (P < 0.05). CST-14 or CST-17 (10 nm) inhibited basal GH secretion in six of the 13 GH-cell adenomas and two of the three GH-PRL mixed adenomas. CST-17 (100 nm) suppressed the GH response to GHRH and ghrelin analog (10 nm each) by 30-50% in adenomas (P < 0.05). Three PRL-adenomas treated with CST-17 (10 nm) showed a 20-40% inhibition of PRL release (P < 0.05), whereas in three others no suppression or mild response was achieved at this concentration. A comparable inhibition of PRL secretion was obtained with SSTR5-selective analog but significantly less with SSTR2-preferential compounds. RT-PCR revealed the expression of both SSTR2 and SSTR5 in all GH-cell and mixed adenomas studied and all PRL-secreting adenomas studied, except for two of the CST-resistant prolactinomas, in which SSTR5 was absent. CONCLUSIONS This is the first report of in vitro CST suppression of human GH and PRL in cultured pituitary tissues. The regulation of PRL release from cultured adenomas appears to be primarily mediated by SSTR5.

Bombesin/gastrin releasing peptide (BN/GRP) functions as an auto crine growth factor in small cell lung cancer (SCLC). Previously, this autocrine growth cycle was disrupted by a monoclonal antibody which binds to the carboxyl terminal of BN and neutralizes the peptide so that it is unable to interact with the BN/GRP receptor. Here a series of BN analogues were synthesized which have a reduced peptide bond near the carboxyl terminal. The analogues inhibited specific binding of I2SI-GRP to SCLC cell line NCI-H345 in a dose-dependent manner and the analogue [D-Nal6, Psi13-14, Phe'4| BN6"14was approximately 6-fold more potent than was (Psi13-14, Leu'4)BN with a 50% inhibition concentration value of 5 nivi. |oNal«,Psi'3-14,Phe'4]BN6-'4 and |Psi13-14,Leu'4|BN had no effect on the cytosolic Ca2* levels but antagonized the increase in cytosolic Ca2+caused by 10 niviBN. |Psi13-14, Leu'4]BN (1 MM)inhibited the growth of SCLC in vitro using a clonogenic assay by approximately 70% Also, injection of [Psi13-14,Leu14]BN (10 fig, s.c.) inhibited the growth of SCLC xenografts in nude mice in vivo by approximately 50%. These data suggest that the autocrine growth cycle of BN/GRP in SCLC may also be disrupted by peptide antagonists which bind to the BN receptor.

Ghrelin, the natural ligand for the growth hormone secretagogue-1a (GHS-1a) receptor, has received a great deal of attention due to its ability to stimulate weight gain and the hope that an antagonist of the GHS-1a receptor could be a treatment for obesity. We have discovered an analog of full-length human ghrelin, BIM-28163, which fully antagonizes GHS-1a by binding to but not activating the receptor. We further demonstrate that BIM-28163 blocks ghrelin activation of the GHS-1a receptor, and inhibits ghrelin-induced GH secretion in vivo. Unexpectedly, however, BIM-28163 acts as an agonist with regard to stimulating weight gain. These results may suggest the presence of an unknown ghrelin receptor that modulates ghrelin actions on weight gain. In keeping with our results on growth hormone (GH) secretion, BIM-28163 acts as an antagonist of ghrelin-induced Fos protein immunoreactivity (Fos-IR) in the medial arcuate nucleus, an area involved in the ghrelin modulation of GH secretion. However, in the dorsal medial hypothalamus (DMH), a region associated with regulation of food intake, both ghrelin and BIM-28163 act as agonists to upregulate Fos-IR. The observation that ghrelin and BIM-28163 have different efficacies in inducing Fos-IR in the DMH, and that concomitant administration of ghrelin and an excess of BIM-28163 results in the same level of Fos-IR as BIM-28163 administered alone may demonstrate that in the DMH both ghrelin and BIM-28163 act via the same receptor. If so, it is unlikely that this receptor is GHS-1a. Collectively, our findings suggest that the action of ghrelin to stimulate increased weight gain may be mediated by a novel receptor other than GHS-1a, and further imply that GHS-1a may not be the appropriate target for anti-obesity strategies.

BIM-23627 is a synthetic peptide with “in vitro” and “in vivo” properties consistent with a pure sst2 antagonist. The aim of the present study was to evaluate the effects of long-term administration of BIM-23627 and the combined effects of BIM-23627 and dexamethasone (DEX) on the somatotropic axis, including growth, epididymal fat accumulation, glucose homeostasis and insulin activity, in young male rats. Beginning on day 23 of age, 16 animals were treated daily with saline or DEX (40 μg/kg/daily). Each group was subdivided into two paired groups and treated with either vehicle or BIM-23627 (0.5 mg/kg, t.i.d.). The treatment period lasted 31 days. The animals were killed by decapitation; trunk blood and pituitaries were collected for the determination of hormone concentrations and GH mRNA expression, respectively. Based on plasma GH and IGF-I concentrations and GH mRNA expression in the pituitary, BIM-23627 was able to counteract the inhibitory effects of DEX on the somatotropic axis; however, only a partial reversal of somatic growth inhibition was observed. DEX-treated rats remained euglycemic, but their insulin levels were significantly increased, indicating an incipient insulin resistance. Although BIM-23627 itself tended to increase insulin concentration in saline-treated rats, its administration to DEX-treated rats reduced insulin levels (saline: 25±3; DEX: 55±16*; DEX+BIM-23627: 34±5; BIM-23627: 38±7 μIU/ml; *P<0.05 vs. saline), apparently improving the degree of insulin sensitivity. DEX administration significantly reduced circulating ghrelin, whereas the sst2 antagonist had no significant effect. An inverse correlation was found between ghrelin concentrations and plasma insulin levels. Both rats receiving DEX and rats receiving BIM-23627 had decreased plasma concentration of total testosterone (P<0.05); however, the effects of DEX and BIM-23627 were not additive. In conclusion, BIM-23627 may represent a new pharmacological agent to reduce the suppression of the GH-IGF-I axis in long-term GC treated patients and enhance insulin sensitivity. Further studies are required in order to fully optimize the SSTR-2 antagonist-induced reversal of DEX-induced somatic growth inhibition.

To study somatostatin/dopamine (SS/D) synergy in a human cell system constitutively expressing SS and D receptors (SSR and DR, respectively), we characterized the expression of SSR and DR subtypes in the non-small-cell lung cancer line Calu-6, and then we evaluated the effect on cell proliferation of SS/D chimeric molecules (BIM-23A387 and BIM-23A370), which bind with high affinity both sst(2) and D(2)R, and compared the results with those obtained by using SS-14 and subtype-selective SS analogs (SSA) and D agonists (DA). Because Calu-6 cells produce insulin-like growth factor (IGF) and IGF-binding protein (IGFBP) peptides, which play a role in the autocrine/paracrine control of cell growth, we also investigated the effects of chimeric compounds on secretion and expression of IGF system components. Relative high levels of sst(2) and the long isoform of the D(2)R were detected by real-time RT-PCR and Western blot in Calu-6, together with sst(5) and to a lesser extent sst(3) and D(4)R. BIM-23A387 and BIM-23A370 significantly inhibited growth of Calu-6, whereas IGF-IGFBP secretion or expression was unaffected, suggesting a direct inhibitory effect. The inhibition of cell growth, measured by both [(3)H]thymidine incorporation and cell count, was significantly lower when individual SSA and DA control peptides or subtype-specific SSA and DA were tested. BIM-23A370 was more potent than BIM-23A387 (P < 0.001). These findings show that SS/D chimeras can inhibit Calu-6 proliferation in an IGF-independent manner and suggest that this enhanced potency might be because of the induction of SSR/DR dimerization. The Calu-6 cell line, constitutively expressing SSR and DR, provides a suitable model to elucidate the mechanism of action of SSA and DA on regulation of cell growth and to characterize the interaction between SSR and DR.

Somatostatin inhibits adrenocorticotropin ( ACTH) secretion from pituitary tumor cells. To assess the contribution of somatostatin receptor subtype 5 (SST5) to somatostatin receptor subtype 2 ( SST2) action in these cells, we assessed multipathway responses to novel highly monoreceptor-selective peptide agonists and multireceptor agonists, including octreotide and somatostatin-28. Octreotide and somatostatin-28 cell membrane binding affinities correlated with their respective SST2-selective peptide ligand. Although octreotide had similar inhibiting potency (picomolar) for cAMP accumulation and ACTH secretion as an SST2-selective agonist, somatostatin-28 exhibited a higher potency (femtomolar). Baseline spontaneous calcium oscillations assessed by fluorescent confocal microscopy revealed two distinct effects: SST2 activation reduced oscillations at femtomolar concentrations reflected by high inhibiting potency of averaged normalized oscillation amplitude, whereas SST5 activation induces brief oscillation pauses and increased oscillation amplitude. Octreotide exhibits an integrated effect of both receptors; however, somatostatin-28 exhibited a complex response with two separate inhibitory potencies. SST2 internalization was visualized with SST2-selective agonist at lower concentrations than for octreotide or somatostatin-28, whereas SST5 did not internalize. Using monoreceptor-selective peptide agonists, the results indicate that, in AtT-20 cells, SST5 regulates the dominant SST2 action, attenuating SST2 effects on intracellular calcium oscillation and internalization. This may explain superior somatostatin-28 potency and provides a rationale for somatostatin ligand design to treat ACTH-secreting pituitary tumors.