Somatostatin (SST) and dopamine (DA) inhibit growth hormone (GH) secretion and proliferation of GH-secreting pituitary adenomas (GHomas) through binding to SSTR2 and D2R receptors. Chimeric SST-DA compounds (Dopastatins) display increased potency in inhibiting GH secretion, as compared with individual SST or DA analogs (alone or combined). To assess the efficacy of a second-generation dopastatin, TBR-065, in suppressing GH secretion from human GH- and GH/prolactin(PRL)-omas. We compared the ability of TBR-065 to inhibit GH secretion from primary cultures of human GH- or GH/PRLoma cells to that of the first generation dopastatin, TBR-760 (formerly BIM-23A760), octreotide (OCT) and cabergoline (CAB), the later either alone or combined. We investigated whether there was any impact of BIM-133, the metabolite of TBR-065, on the ability of TBR-065 to inhibit GH in these cultures. 17 GH- and GH/PRLomas were included in this study. Inhibition of GH secretion by TBR-065, TBR-760, OCT and CAB (0.1 pM to 0.1 µM) was assessed over a period of 8 h. All tumors expressed SSTR2 and D2R mRNAs. GH suppression was higher with TBR-065 as compared with TBR-760 (Emax = 57 ± 5.6% vs. 41.1 ± 12.5%, respectively, p < 0.001) or with OCT + CAB (Emax = 56.8 ± 7.2% vs. 44.4 ± 9.4%, p < 0.001). BIM-133 did not have any impact on the activity of TBR-065. TBR-065 has significantly improved efficacy in suppressing GH secretion as compared to current available therapies and may represent a new promising option for the treatment of acromegaly.

Abstract TBR-760 (formerly BIM-23A760) is a chimeric dopamine (DA)-somatostatin (SST) compound with potent agonist activity at both DA type 2 (D2R) and SST type 2 (SSTR2) receptors. Studies have shown that chimeric DA-SST compounds are more efficacious than individual DA and/or SST analogues, either alone or combined, in inhibiting secretion from primary cultures of human somatotroph and lactotroph tumor cells. Nonfunctioning pituitary adenomas (NFPAs) express both D2R and SSTR2 and, consequently, may respond to TBR-760. We used a mouse model with the pro-opiomelanocortin (POMC) gene knocked out that spontaneously develops aggressive NFPAs. Genomic microarray and DA and SST receptor messenger RNA expression analysis indicate that POMC KO mouse tumors and human NFPAs have similar expression profiles, despite arising from different cell lineages, establishing POMC KO mice as a model for study of NFPAs. Treatment with TBR-760 for 8 weeks resulted in nearly complete inhibition of established tumor growth, whereas tumors from vehicle-treated mice increased in size by 890 ± 0.7%. Comparing TBR-760 with its individual DA and SST components, TBR-760 arrested tumor growth. Treatment with equimolar or 10×-higher doses of the individual SST or DA agonists, either alone or in combination, had no significant effect. One exception was the lower dose of DA agonist that induced modest suppression of tumor growth. Only the chimeric compound TBR-760 arrested tumor growth in this mouse model of NFPA. Further, significant tumor shrinkage was observed in 20% of the mice treated with TBR-760. These results support the development of TBR-760 as a therapy for patients with NFPA.

Abstract TBR-760 is a chimeric dopamine (DA)-somatostatin (SST) compound with potent agonist activity at both DA type 2 (D2R; EC50 0.064nM) and SST type 2 (SSTR2; EC50 1.2nM) receptors. Prior studies have demonstrated that the chimeric DA-SST compounds are more potent and effective than either individual or combinations of individual DA and/or SST analogs in inhibiting secretion from pituitary adenomas. Non-functioning pituitary adenomas (NFPA) express high levels of D2R as well as lower levels of SSTRs, including the type 2 receptor (1), and thus have an appropriate receptor profile to respond to TBR-760. The present study examines the ability of TBR-760 to inhibit tumor growth in a mouse model of aggressive NFPA. Heterozygous and null mutant mice lacking one or both copies, respectively, of the pro-opiomelanocortin (POMC) gene (POMC-KO mice)(2) spontaneously develop aggressive, non-secreting pituitary adenomas (3). The POMC-KO mouse tumors have been shown to express D2R and SSTR2 at a similar level as human NFPAs (4). In addition, merging of microarray data (Affymetrix, U133 plus_2.0 and Mouse Genome 430 2.0 arrays), reveals 154 common gene signatures between human NFPAs and the POMC-KO mouse tumors. In an initial study, heterozygous POMC-KO mice with an established pituitary tumor of approx. 10mm3 (mean volume 8.9±0.3), as determined by MRI, were treated with a range of TBR-760 doses (0.125 to 12.5mg/kg, sc, QD) for 60 days. During that time, tumors in vehicle-treated mice increased in size by 890±0.7%, whereas all doses of TBR-760 tested resulted in a nearly complete inhibition of tumor growth from treatment initiation. We then compared the effect of the TBR-760 chimera with that of its individual SST agonist (SSTA) and DA agonist (DAA) components on tumor growth in the POMC-KO mice. As in the earlier study, TBR-760 treatment (1mg/kg, sc, QD), initiated when the mice had an established tumor of approx. 10mm3, completely arrested tumor growth during the 8 weeks of treatment (final mean tumor volume of 8.5±1.3mm3 vs. 54.61±10.6mm3 in vehicle-treated mice). Treatment with equimolar or 10x-higher doses of the individual SSTA or DAA, either alone or in combination, had no significant effect on tumor growth, except in the lower dose DAA group where a modest suppression of tumor growth was observed. These data demonstrate that only the dual DA-SST chimeric compound, TBR-760, completely arrested tumor growth in the POMC-KO mouse model of NFPA. Further, despite the highly aggressive nature of the POMC-KO tumors, significant tumor shrinkage was observed in 20% of the mice treated with TBR-760. These results support the development of TBR-760 as a medical therapy to prevent or arrest the growth of NFPAs and, potentially, to induce NFPA shrinkage. References: (1) Florio et al., 2008 Endocr Relat Cancer; 15: 583-596. (2) Yaswen et al., 1999 Nat Med; 9:1066-70 (3) Karpac J et al., 2006 Cell Mol Biol; 52: 47-52.

Regulation of tissue-specific glucocorticoid action by inhibiting 11 beta-HSD1 activity is regarded as a potential viable treatment for metabolic and cardiovascular diseases. Our effort to find an alternative to the highly favoured adamantyl moiety found in many potent 11 beta-HSD1 inhibitors led to the synthesis and SAR study of a series of phenyl ethanone thiadiazole derivatives. Potent compounds were identified with IC50 values in the range 100-300 nM in biological evaluation on an HEK293 cell line stably transfected with the human HSD11B1 gene. These compounds are selective with no activity against human 11 beta-HSD2. An SAR study revealed the favoured combination of phenyl substitution and the linker system being meta-methoxy with a sulphide linker (18) or a para-trifluoromethyl with sulphoxide linker (27). Docking of 3 into a crystal structure of the enzyme shows how the substituted phenyl group of this new series might mimic the adamantane motif. Potent inhibitors 18 and 27 are new templates for further optimization.

The modulation of 11β-HSD1 activity with selective inhibitors has beneficial effects on various metabolic disorders including insulin resistance, dyslipidemia and obesity. Here we report the discovery of a series of novel adamantyl carboxamide and acetamide derivatives as selective inhibitors of human 11β-HSD1 in HEK-293 cells transfected with the HSD11B1 gene. Optimization based on an initially identified 11β-HSD1 inhibitor (3) led to the discovery of potent inhibitors with IC(50) values in the 100 nM range. These compounds are also highly selective 11β-HSD1 inhibitors with no activity against 11β-HSD2 and 17β-HSD1. Compound 15 (IC(50)=114 nM) with weak inhibitory activity against the key human cytochrome P450 enzymes and moderate stability in incubation with human liver microsomes is worthy of further development. Importantly, compound 41 (IC(50)=280 nM) provides a new lead that incorporates an adamantyl group surrogate and should enable further series diversification.

11β‐Hydroxysteroid dehydrogenases (11β‐HSDs) are key enzymes regulating the pre‐receptor metabolism of glucocorticoid hormones. The modulation of 11β‐HSD type 1 activity with selective inhibitors has beneficial effects on various conditions including insulin resistance, dyslipidemia and obesity. Inhibition of tissue‐specific glucocorticoid action by regulating 11β‐HSD1 constitutes a promising treatment for metabolic and cardiovascular diseases. A series of novel adamantyl ethanone compounds was identified as potent inhibitors of human 11β‐HSD1. The most active compounds identified (52, 62, 72, 92, 103 and 104) display potent inhibition of 11β‐HSD1 with IC50 values in the 50–70 nM range. Compound 72 also proved to be metabolically stable when incubated with human liver microsomes. Furthermore, compound 72 showed very weak inhibitory activity for human cytochrome P450 enzymes and is therefore a candidate for in vivo studies. Comparison of the publicly available X‐ray crystal structures of human 11β‐HSD1 led to docking studies of the potent compounds, revealing how these molecules may interact with the enzyme and cofactor.

Melanocortin receptor agonists act in the brain to regulate food intake and body weight and, independently of these actions, affect insulin sensitivity. These experiments investigated the function of novel non-selective melanocortin receptor agonists (BIM-22493, BIM-22511) that cross the blood–brain barrier when administered peripherally. Treatment of diet induced obese C57BL/6J (B6) mice with melanocortin agonists administered peripherally improved obesity, hyperinsulinemia (∼50%) and fatty liver disease. Specificity of function was determined using B6 melanocortin-3 and melanocortin-4 receptor knockout mice (MC3RKO, MC4RKO). Chow fed MC4RKO but not MC3RKO used for these tests exhibited obesity, hyperinsulinemia and severe hepatosteatosis associated with increased expression of insulin-stimulated genes involved in lipogenesis. Reduced food intake associated with acute BIM-22493 treatment, and weight loss associated with 14 days of treatment with BIM-22511, required functional MC4R but not MC3R. However, while 14 days of treatment with BIM-22511 did not affect body weight and even increased cumulative food intake in MC4RKO, a significant reduction (∼50%) in fasting insulin was still observed. Despite lowering insulin, chronic treatment with BIM-22511 did not improve hepatosteatosis in MC4RKO, and did not affect hepatic lipogenic gene expression. Together, these results demonstrate that peripherally administered melanocortin receptor agonists regulate body weight, liver metabolism and glucose homeostasis through independent pathways. MC4R are necessary for melanocortin agonist-induced weight loss and improvements in liver metabolism, but are not required for improvements in hyperinsulinemia. Agonists with activity at MC4R improve glucose homeostasis at least partially by causing weight loss, however other melanocortin receptors may have potential for treating aberrations in glucose homeostasis associated with obesity.

Obesity and nutrient homeostasis are linked by mechanisms that are not fully elucidated. Here we describe a secreted protein, adropin, encoded by a gene, Energy Homeostasis Associated (Enho), expressed in liver and brain. Liver Enho expression is regulated by nutrition: lean C57BL/6J mice fed high-fat diet (HFD) exhibited a rapid increase, while fasting reduced expression compared to controls. However, liver Enho expression declines with diet-induced obesity (DIO) associated with 3 months of HFD or with genetically induced obesity, suggesting an association with metabolic disorders in the obese state. In DIO mice, transgenic overexpression or systemic adropin treatment attenuated hepatosteatosis and insulin resistance independently of effects on adiposity or food intake. Adropin regulated expression of hepatic lipogenic genes and adipose tissue peroxisome proliferator-activated receptor gamma, a major regulator of lipogenesis. Adropin may therefore be a factor governing glucose and lipid homeostasis, which protects against hepatosteatosis and hyperinsulinemia associated with obesity.

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.

Two anatomically and neurochemically distinct zones within the vomeronasal organ (VNO) and accessory olfactory bulb (AOB) have been identified that are responsible for the detection of pheromones. Using markers to distinguish between apical and basal neurons of the VNO neuroepithelium and rostral versus caudal AOB glomeruli, we examined immediate early gene immunoreactivity (IEG-IR) in gonadectomized, steroid-treated mice in response to pheromones of male and female conspecifics. After exposure of estradiol-treated females to soiled male bedding, more VNO neurons in the basal than the apical layer exhibited IEG-IR compared with VNO neurons of estradiol-treated males. Conversely, whereas soiled female bedding failed to induce IEG-IR in VNO neurons of estradiol-treated males or females, both apical and basal neurons were activated in testosterone-treated males. Male and female pheromones also activated mitral and granule cells in the AOBs of all subjects, but responses to different pheromones were distributed across the boundary of the rostral and caudal regions. These data show that differences in the response of males and females to the same pheromonal stimulus are found in the sensory neurons of the VNO. We propose that centrifugal, noradrenergic inputs to VNO neurons, which may differ in the two sexes and respond differently to adult sex steroids, modulate sensitivity to pheromonal stimulation.

Exposure of recently mated female mice to the urinary odours of an unfamiliar male blocks pregnancy (the Bruce effect). The absence of a pregnancy block in response to the stud male's familiar odours depends on an olfactory memory that is formed in the accessory olfactory bulb (AOB) in response to vomeronasal organ (VNO) inputs during mating. Sexually naive Balb/c female mice in pro-oestrus/oestrus were either placed onto soiled bedding ('bedding-only' females) from, or allowed to mate with, a Balb/c male ('recently mated' females). After 42 h, females were placed for 90 min onto clean bedding (controls) or onto soiled bedding from either a C57BL/6 male (unfamiliar bedding) or a Balb/c male (familiar bedding). Significant increases in Fos-immunoreactivity (Fos-IR, a marker of neuronal activation) occurred in the medial amygdala and the medial preoptic area (MPA) of 'bedding only' females exposed to either unfamiliar or familiar bedding and in 'recently mated' females exposed to unfamiliar bedding but not to familiar bedding. This suggests that a mating-induced memory prevents the later activation by the familiar stud male's odours of neurons in forebrain regions that receive inputs from the VNO--AOB. 'Bedding-only' females later exposed to either familiar or unfamiliar bedding had increased Fos-IR in the G alpha(o) protein-expressing basal zone of the VNO whereas no such effect occurred in 'recently mated' females. Familiar, as well as unfamiliar, male odours augmented Fos-IR in significantly more rostral than caudal AOB granule cells in all groups, with the effect being strongest in 'recently mated' females exposed to familiar male bedding. This outcome is consistent with the absence of odour-induced Fos-IR in forebrain regions of these females and, presumably, the absence of a pregnancy block.