A Comparison of the Central versus Peripheral Gastrointestinal Prokinetic Activity of Two Novel Ghrelin Mimetics.

The gastrointestinal (GI) prokinetic effects of ghrelin occur through direct peripheral effects on ghrelin receptors within the enteric nervous system and via the ghrelin receptor on the vagus nerve, which activate a centrally mediated mechanism. However, the relative contribution of peripheral versus central effects to the overall prokinetic effect of ghrelin agonists requires further investigation. Here, we investigated the central versus peripheral prokinetic effect of ghrelin by using two novel ghrelin agonists: HM01 (N'-[(1S)-1-(2,3-dichloro-4-methoxyphenyl)ethyl]-N-methyl-N-[1,3,3-trimethyl-(4R)-piperidyl]-urea HCL) with high brain penetration compared with HM02 (N'-[(1S)-1-(2,3-dichloro-4-methoxyphenyl)ethyl]-N-hydroxy-N-(1-methyl-4-piperidinyl)-urea), a more peripherally acting ghrelin agonist. The pharmacokinetic profiles of both ghrelin agonists were evaluated after intravenous and oral administration in rats. The efficacy of HM01 and HM02 was assessed in a rat model of postoperative ileus (POI) induced by abdominal surgery and in a rodent defecation assay. Pharmacokinetic results in our models confirmed that HM01, but not HM02, was a brain-penetrant ghrelin agonist. Administration of either HM01 or HM02 reversed the delayed upper and lower gastrointestinal transit induced by abdominal surgery to levels resembling the non-POI controls. In the defecation test, HM01, but not HM02, significantly increased the weight of fecal pellets. Our findings suggest that, in a rodent model of POI, synthetic ghrelin agonists stimulate GI transit through a peripheral site of action. However, in the defecation assay, our data suggest that a ghrelin-mediated mechanism is located at a central site. Taken together, a ghrelin agonist with both central and peripheral prokinetic activity may show therapeutic potential to treat delayed GI transit disorders.

Ghrelin receptor inverse agonists as a novel therapeutic approach against obesity-related metabolic disease.

AIMS: Ghrelin is implicated in the control of energy balance and glucose homeostasis. The ghrelin receptor exhibits ligand-independent constitutive activity, which can be pharmacologically exploited to induce inverse ghrelin actions. Because ghrelin receptor inverse agonists (GHSR-IA) might be effective for the treatment of obesity-related metabolic disease, we tested 2 novel synthetic compounds GHSR-IA1 and GHSR-IA2. MATERIALS AND METHODS: In functional cell assays, electrophysiogical and immunohistochemical experiments, we demonstrated inverse agonist activity for GHSR-IA1 and GHSR-IA2. We used healthy mice, Zucker diabetic fatty (ZDF) rats and diet-induced obese (DIO) mice to explore effects on food intake (FI), body weight (BW), conditioned taste aversion (CTA), oral glucose tolerance (OGT), pancreatic islet morphology, hepatic steatosis (HS), and blood lipids. RESULTS: Both compounds acutely reduced FI in mice without inducing CTA. Chronic GHSR-IA1 increased metabolic rate in chow-fed mice, suppressed FI, and improved OGT in ZDF rats. Moreover, the progression of islet hyperplasia to fibrosis in ZDF rats slowed down. GHSR-IA2 reduced FI and BW in DIO mice, and reduced fasting and stimulated glucose levels compared with pair-fed and vehicle-treated mice. GHSR-IA2-treated DIO mice showed decreased blood lipids. GHSR-IA1 treatment markedly decreased HS in DIO mice. CONCLUSIONS: Our study demonstrates therapeutic actions of novel ghrelin receptor inverse agonists, suggesting a potential to treat obesity-related metabolic disorders including diabetes mellitus.

Oral Treatment with the Ghrelin Receptor Agonist HM01 Attenuates Cachexia in Mice Bearing Colon-26 (C26) Tumors.

The gastrointestinal hormone ghrelin reduces energy expenditure and stimulates food intake. Ghrelin analogs are a possible treatment against cancer anorexia-cachexia syndrome (CACS). This study aimed to investigate whether oral treatment with the non-peptidergic ghrelin receptor agonist HM01 counteracts CACS in colon-26 (C26) tumor-bearing mice. The C26 tumor model is characterized by pronounced body weight (BW) loss and muscle wasting in the absence of severe anorexia. We analyzed the time course of BW loss, body composition, muscle mass, bone mineral density, and the cytokines interleukin-6 (IL-6) and macrophage-inhibitory cytokine-1 (MIC-1). Moreover, we measured the expression of the muscle degradation markers muscle RING-finger-protein-1 (MuRF-1) and muscle atrophy F-box (MAFbx). After tumor inoculation, MIC-1 levels increased earlier than IL-6 and both cytokines were elevated before MuRF-1/MAFbx expression increased. Oral HM01 treatment increased BW, fat mass, and neuronal hypothalamic activity in healthy mice. In tumor-bearing mice, HM01 increased food intake, BW, fat mass, muscle mass, and bone mineral density while it decreased energy expenditure. These effects appeared to be independent of IL-6, MIC-1, MuRF-1 or MAFbx, which were not affected by HM01. Therefore, HM01 counteracts cachectic body weight loss under inflammatory conditions and is a promising compound for the treatment of cancer cachexia in the absence of severe anorexia.

The ghrelin receptor agonist HM01 mimics the neuronal effects of ghrelin in the arcuate nucleus and attenuates anorexia-cachexia syndrome in tumor-bearing rats.

The gastric hormone ghrelin positively affects energy balance by increasing food intake and reducing energy expenditure. Ghrelin mimetics are a possible treatment against cancer anorexia-cachexia syndrome (CACS). This study aimed to characterize the action of the nonpeptidergic ghrelin receptor agonist HM01 on neuronal function, energy homeostasis and muscle mass in healthy rats and to evaluate its possible usefulness for the treatment of CACS in a rat tumor model. Using extracellular single-unit recordings, we tested whether HM01 mimics the effects of ghrelin on neuronal activity in the arcuate nucleus (Arc). Furthermore, we assessed the effect of chronic HM01 treatment on food intake (FI), body weight (BW), lean and fat volumes, and muscle mass in healthy rats. Using a hepatoma model, we investigated the possible beneficial effects of HM01 on tumor-induced anorexia, BW loss, muscle wasting, and metabolic rate. HM01 (10(-7)-10(-6) M) mimicked the effect of ghrelin (10(-8) M) by increasing the firing rate in 76% of Arc neurons. HM01 delivered chronically for 12 days via osmotic minipumps (50 µg/h) increased FI in healthy rats by 24%, paralleled by increased BW, higher fat and lean volumes, and higher muscle mass. Tumor-bearing rats treated with HM01 had 30% higher FI than tumor-bearing controls and were protected against BW loss. HM01 treatment resulted in higher muscle mass and fat mass. Moreover, tumor-bearing rats reduced their metabolic rate following HM01 treatment. Our studies substantiate the possible therapeutic usefulness of ghrelin receptor agonists like HM01 for the treatment of CACS and possibly other forms of disease-related anorexia and cachexia.

Synergistic effect of 5-hydroxytryptamine 3 and neurokinin 1 receptor antagonism in rodent models of somatic and visceral pain.

Synergistic activity has been observed between serotonergic 5-hydroxytryptamine 3 (5-HT3) and tachykinergic neurokinin 1 (NK1) receptor-mediated responses. This study investigated the efficacy of a 5-HT3 antagonist, palonosetron, and a NK1 antagonist, netupitant, alone or in combination in rodent models of somatic and visceral colonic hypersensitivity. In a rat model of experimental neuropathic pain, somatic hypersensitivity was quantified by the number of ipsilateral paw withdrawals to a von Frey filament (6g). Electrophysiologic responses were recorded in the dorsal horn neurons after mechanical or thermal stimuli. Acute colonic hypersensitivity was induced experimentally in rats by infusing dilute acetic acid (0.6%) directly into the colon. Colonic sensitivity was assessed by a visceromotor behavioral response quantified as the number of abdominal contractions in response to graded isobaric pressures (0-60 mm Hg) of colorectal distension. Palonosetron or netupitant was administered alone or in combination via oral gavage. When dosed alone, both significantly reduced somatic sensitivity, decreased the evoked response of spinal dorsal horn neurons to mechanical or thermal stimulation, and caused significant (P < 0.05) inhibition of colonic hypersensitivity in a dose-dependent manner. The combined administration of palonosetron and netupitant at doses that were ineffective alone significantly reduced both somatic and visceral sensitivity and decreased the evoked response of spinal dorsal horn neurons to mechanical or thermal stimulation. In summary, the combination of palonosetron with a NK1 receptor antagonist showed synergistic analgesic activity in rodent models of somatic and visceral hypersensitivity, and may prove to be a useful therapeutic approach to treat pain associated with irritable bowel syndrome.

Role of neurokinin 1 receptors in dextran sulfate-induced colitis: studies with gene-deleted mice and the selective receptor antagonist netupitant.

OBJECTIVE AND DESIGN: The function of the neurokinin 1 (NK1) receptor was investigated in the DSS-induced mouse colitis model using NK1 receptor-deficient mice and the selective antagonist netupitant. SUBJECTS: Colitis was induced by oral administration of 20 mg/ml DSS solution for 7 days in C57BL/6 and Tacr1 KO animals (n = 5-7). TREATMENT: During the induction, one-half of the C57BL/6 and Tacr1 KO group received one daily dose of 6 mg/kg netupitant, administered intraperitoneally, the other half of the group received saline, respectively. METHODS: Disease activity index (DAI), on the basis of stool consistency, blood and weight loss, was determined over 7 days. Histological evaluation, myeloperoxidase (MPO) measurement, cytokine concentrations and receptor expression analysis were performed on the colon samples. RESULTS: NK1 receptors are up-regulated in the colon in response to DSS treatment. DSS increased DAI, histopathological scores, BLC, sICAM-1, IFN-γ, IL-16 and JE in wildtype mice, which were significantly reduced in NK1 receptor-deficient ones. NK1 receptor antagonism with netupitant significantly diminished DAI, inflammatory histopathological alterations, BLC, IFN-γ, IL-13 and IL-16 in wildtype mice, but not in the NK1-deficient ones. MPO was similarly elevated and netupitant significantly decreased its activity in both groups. CONCLUSIONS: NK1 receptor antagonism could be beneficial for colitis via inhibiting different inflammatory mechanisms.

Activation of neurokinin-1 receptors increases the excitability of guinea pig dorsal root ganglion cells.

The suppression of overactive bladder symptoms in patients and overactive bladder reflexes in animal models by neurokinin (NK)-1 receptor antagonists raises the possibility that these drugs target sensory neurons. This mechanism was evaluated by examining the interactions between a specific NK-1 agonist, [Sar(9),Met(O(2))(11)]-substance P (Sar-Met-SP), and a potent NK-1 antagonist, netupitant (NTP), on small size (20-30 µm) dissociated L6 and S1 dorsal root ganglion (DRG) neurons from female guinea pigs. Current-clamp recording revealed that Sar-Met-SP (1 µM) elicited membrane depolarization (average 8.05 ± 1.38 mV) in 27% (18 of 65) of DRG neurons. In 74% of the remaining neurons (35 of 47) Sar-Met-SP decreased the rheobase for action potential (AP) generation and increased the response to a suprathreshold stimulus (3 times rheobase) without changing the membrane potential. Sar-Met-SP also induced changes in the action potential (AP) wave form, including 1) an increase in overshoot (average 5 mV, n = 35 neurons), 2) a prolongation of AP duration (from 4.64 to 5.29 ms, n = 34), and 3) a reduction in the maximal rate of AP repolarization. NTP (200 nM) reversed the Sar-Met-SP-induced changes. Ca(2+) imaging showed that application of Sar-Met-SP (1 µM) decreased the tachyphylaxis induced by repeated application of capsaicin (0.5 µM), an effect blocked by pretreatment with NTP (200 nM). These results raise the possibility that activation of NK-1 receptors in primary sensory neurons plays a role in the generation of overactive bladder and that block of NK-1 receptors in these neurons may contribute to efficacy of NK-1 antagonists in the treatment of overactive bladder symptoms.

Study of the antilymphoma activity of pracinostat reveals different sensitivities of DLBCL cells to HDAC inhibitors.

Histone deacetylase inhibitors (HDACis) are antitumor agents with distinct efficacy in hematologic tumors. Pracinostat is a pan-HDACi with promising early clinical activity. However, similar to other HDACis, its activity as a single agent is limited. Diffuse large B-cell lymphoma (DLBCL) includes distinct molecular subsets or metabolically defined subtypes that rely in different ways on the B-cell receptor signaling pathway, oxidative phosphorylation, and glycolysis for their survival. The antitumor activity of pracinostat has not been determined in lymphomas. We performed preclinical in vitro activity screening of 60 lymphoma cell lines that included 25 DLBCLs. DLBCL cells belonging to distinct metabolic subtypes were treated with HDACis for 6 hours or 14 days followed by transcriptional profiling. DLBCL xenograft models enabled assessment of the in vivo antilymphoma activity of pracinostat. Combination treatments with pracinostat plus 10 other antilymphoma agents were performed. Western blot was used to assess acetylation levels of histone and nonhistone proteins after HDACi treatment. Robust antiproliferative activity was observed across all lymphoma histotypes represented. Focusing on DLBCL, we identified a low-sensitivity subset that almost exclusively consists of the oxidative phosphorylation (OxPhos)-DLBCL metabolic subtype. OxPhos-DLBCL cells also showed poorer sensitivity to other HDACis, including vorinostat. Transcriptomic analysis revealed fewer modulated transcripts but an enrichment of antioxidant pathway genes after HDACi treatment of OxPhos-DLBCLs compared with high-sensitivity B-cell receptor (BCR)-DLBCLs. Pharmacologic inhibition of antioxidant production rescued sensitivity of OxPhos-DLBCLs to pracinostat whereas BCR-DLBCLs were unaffected. Our study provides novel insights into the antilymphoma activity of pracinostat and identifies a differential response of DLBCL metabolic subtypes to HDACis.

The antiemetic 5-HT3 receptor antagonist Palonosetron inhibits substance P-mediated responses in vitro and in vivo.

Palonosetron is the only 5-HT(3) receptor antagonist approved for the treatment of delayed chemotherapy-induced nausea and vomiting (CINV) in moderately emetogenic chemotherapy. Accumulating evidence suggests that substance P (SP), the endogenous ligand acting preferentially on neurokinin-1 (NK-1) receptors, not serotonin (5-HT), is the dominant mediator of delayed emesis. However, palonosetron does not bind to the NK-1 receptor. Recent data have revealed cross-talk between the NK-1 and 5HT(3) receptor signaling pathways; we postulated that if palonosetron differentially inhibited NK-1/5-HT(3) cross-talk, it could help explain its efficacy profile in delayed emesis. Consequently, we evaluated the effect of palonosetron, granisetron, and ondansetron on SP-induced responses in vitro and in vivo. NG108-15 cells were preincubated with palonosetron, granisetron, or ondansetron; antagonists were removed and the effect on serotonin enhancement of SP-induced calcium release was measured. In the absence of antagonist, serotonin enhanced SP-induced calcium-ion release. After preincubation with palonosetron, but not ondansetron or granisetron, the serotonin enhancement of the SP response was inhibited. Rats were treated with cisplatin and either palonosetron, granisetron, or ondansetron. At various times after dosing, single neuronal recordings from nodose ganglia were collected after stimulation with SP; nodose ganglia neuronal responses to SP were enhanced when the animals were pretreated with cisplatin. Palonosetron, but not ondansetron or granisetron, dose-dependently inhibited the cisplatin-induced SP enhancement. The results are consistent with previous data showing that palonosetron exhibits distinct pharmacology versus the older 5-HT(3) receptor antagonists and provide a rationale for the efficacy observed with palonosetron in delayed CINV in the clinic.

Attenuation of Visceral and Somatic Nociception by Ghrelin Mimetics.

PURPOSE: The anti-nociceptive properties of ghrelin have been demonstrated in alleviating inflammatory and neuropathic pain. Whether a ghrelin receptor-mediated mechanism attenuates visceral and somatic pain in the absence of active inflammation remains to be explored. Here, we investigate the efficacy of peripherally restricted (ipamorelin) and a globally active (HM01) selective ghrelin receptor agonist in an experimental model of non-inflammatory visceral hypersensitivity and somatic mechanical allodynia. MATERIALS AND METHODS: Visceral hypersensitivity was induced by dilute acetic acid (0.6%) infusion in the colon of rats in the absence of colonic epithelial inflammation. Ghrelin mimetics HM01 and ipamorelin were administered orally or intravenously, respectively. The ghrelin receptor antagonist H0900 was administered orally. Colonic sensitivity was assessed via a visceromotor behavioral response (VMR) quantified as the number of abdominal contractions in response to graded isobaric pressures (0-60 mmHg) of colorectal distension (CRD). Somatic mechanical allodynia was quantified by the number of ipsilateral paw withdrawals in response to a calibrated von Frey filament. RESULTS: Compared to vehicle controls, ghrelin mimetics HM01 and ipamorelin significantly attenuated colonic hypersensitivity and somatic allodynia. The anti-nociceptive effects of the ghrelin mimetics were blocked after administration of the ghrelin receptor antagonist H0900. CONCLUSION: We have shown that ghrelin receptor-mediated mechanisms are involved in visceral and somatic hypersensitivity in the absence of active colonic inflammation. Furthermore, visceral and somatic hypersensitivity could be attenuated by a peripherally restricted ghrelin mimetic. These results highlight a potential novel approach for treating acute visceral and somatic pain by ghrelin mimetics.

The GLP-2 analogue elsiglutide reduces diarrhoea caused by the tyrosine kinase inhibitor lapatinib in rats.

PURPOSE: Lapatinib is a small molecule tyrosine kinase inhibitor used to treat breast cancer, often in combination with chemotherapy. Diarrhoea commonly occurs in up to 78% of patients undertaking lapatinib treatment. The mechanism of this diarrhoea is currently unknown. Elsiglutide is a GLP-2 analogue known to increase cell proliferation and reduce apoptosis in the intestine. METHODS: We used a previously developed rat model of lapatinib-induced diarrhoea to determine if co-treatment with elsiglutide was able to reduce diarrhoea caused by lapatinib. Additionally, we analysed the caecal microbiome of these rats to assess changes in the microbiome due to lapatinib. RESULTS: Rats treated with lapatinib and elsiglutide had less severe diarrhoea than rats treated with lapatinib alone. Serum lapatinib levels, blood biochemistry, myeloperoxidase levels and serum limulus amebocyte lysate levels were not significantly different between groups. Rats treated with lapatinib alone had significantly higher histopathological damage in the ileum than vehicle controls. This increase was not seen in rats also receiving elsiglutide. Rats receiving lapatinib alone had lower microbial diversity than rats who also received elsiglutide. CONCLUSIONS: Elsiglutide was able to reduce diarrhoea from lapatinib treatment. This does not appear to be via reduction in inflammation or barrier permeability, and may be due to thickening of mucosa, leading to increased surface area for fluid absorption in the distal small intestine. Microbial changes seen in this study require further research to fully elucidate their role in the development of diarrhoea.

The brain-penetrating, orally bioavailable, ghrelin receptor agonist HM01 ameliorates motion-induced emesis in Suncus murinus (house musk shrew).

BACKGROUND AND PURPOSE: HM01, a novel, orally bioavailable, brain-penetrating agonist of ghrelin receptors, ameliorates emesis in Suncus murinus. This study compared HM01's activity against motion sickness with that of the less brain-penetrating ghrelin receptor agonist, HM02. EXPERIMENTAL APPROACH: The potential of HM01 and HM02 to relax isolated mesenteric arteries and to increase feeding was investigated. Radio telemetry was used to record gastric slow waves and body temperature. Plethysmography was used to measure respiratory function. HM01 and HM02 were administered p.o. 1 hr prior to provocative motion, and c-Fos expression in brain sections was assessed. KEY RESULTS: HM01 and HM02 both relaxed precontracted arteries, yielding EC50 values of 2.5 ± 0.5 and 3.5 ± 0.4 nM respectively. HM01 increased feeding, but HM02 did not. Both compounds caused hypothermia and bradygastria. Motion induced 123 ± 24 emetic events. HM01, but not HM02, reduced motion-induced emesis by 67.6%. Motion increased c-Fos expression in the nucleus tractus solitarius (NTS), dorsal motor nucleus of the vagus (DMNV), medial vestibular nucleus (MVe), central nucleus of the amygdala, and paraventricular hypothalamic nucleus (PVH). HM01 alone increased c-Fos expression in the area postrema, NTS, DMNV, PVH, and arcuate hypothalamic nucleus; HM02 had a similar pattern except it did not increase c-Fos in the PVH. Both compounds antagonized the motion-induced increases in c-Fos expression in the MVe. CONCLUSIONS AND IMPLICATIONS: HM01 is more effective than HM02 in preventing motion-induced emesis. The difference in potency may relate to activation of ghrelin receptors in the PVH.

Behavioural characterization of ghrelin ligands, anamorelin and HM01: Appetite and reward-motivated effects in rodents.

The ghrelinergic system has been steadily investigated as a therapeutic target in the treatment of metabolic disorders and modulation of appetite. While endogenous ghrelin activates the full complement of the growth hormone secretagogue receptor (GHSR-1a) pathways, synthetic GHSR-1a ligands display biased signalling and functional selectivity, which have a significant impact on the intended and indeed, unintended, therapeutic effects. The widespread expression of the GHSR-1a receptor in vivo also necessitates an imperative consideration of the biodistribution of GHSR-1a ligands. Here, we investigate anamorelin and HM01, two recently described synthetic GHSR-1a ligands which have shown promising effects on food intake in preclinical and clinical studies. We compare the downstream signalling pathways in cellular in vitro assays, including calcium mobilization, IP-one, internalization and ß-arrestin recruitment assays. We describe a novel divergent activation of central reward circuitry by anamorelin and HM01 using c-Fos immunostaining as well as behavioural effects in food intake and reward paradigms. Interestingly, we found a paradoxical reduction in reward-related behaviour for anamorelin and HM01 treated animals in our chosen paradigms. The work highlights the critical importance to consider signalling bias in relation to future ghrelin-based therapies. In addition, central access of GHSR-1a ligands, particularly to reward areas of the brain, remains a crucial factor in eliciting potent appetite-stimulating effects. The precise characterization of downstream ghrelinergic signalling and biodistribution of novel GHSR-1a ligands will be decisive in their successful development and will allow predictive modelling and design of future synthetic ligands to combat metabolic and appetite disorders involving the ghrelinergic system. This article is part of the special issue on 'Neuropeptides'.

Acyl-ghrelin Is Permissive for the Normal Counterregulatory Response to Insulin-Induced Hypoglycemia.

Insulin-induced hypoglycemia leads to far-ranging negative consequences in patients with diabetes. Components of the counterregulatory response (CRR) system that help minimize and reverse hypoglycemia and coordination between those components are well studied but not yet fully characterized. Here, we tested the hypothesis that acyl-ghrelin, a hormone that defends against hypoglycemia in a preclinical starvation model, is permissive for the normal CRR to insulin-induced hypoglycemia. Ghrelin knockout (KO) mice and wild-type (WT) littermates underwent an insulin bolus-induced hypoglycemia test and a low-dose hyperinsulinemic-hypoglycemic clamp procedure. Clamps also were performed in ghrelin-KO mice and C57BL/6N mice administered the growth hormone secretagogue receptor agonist HM01 or vehicle. Results show that hypoglycemia, as induced by an insulin bolus, was more pronounced and prolonged in ghrelin-KO mice, supporting previous studies suggesting increased insulin sensitivity upon ghrelin deletion. Furthermore, during hyperinsulinemic-hypoglycemic clamps, ghrelin-KO mice required a 10-fold higher glucose infusion rate (GIR) and exhibited less robust corticosterone and growth hormone responses. Conversely, HM01 administration, which reduced the GIR required by ghrelin-KO mice during the clamps, increased plasma corticosterone and growth hormone. Thus, our data suggest that endogenously produced acyl-ghrelin not only influences insulin sensitivity but also is permissive for the normal CRR to insulin-induced hypoglycemia.

Multiple Beneficial Effects of Ghrelin Agonist, HM01 on Homeostasis Alterations in 6-Hydroxydopamine Model of Parkinson's Disease in Male Rats.

Background and objective: Developing therapy for non-motor symptoms of Parkinson's disease (PD) is important for improving patients' quality of life. Previously, we reported that the ghrelin receptor agonist, HM01 normalized the decreased 4-h fecal output and levodopa-inhibited gastric emptying in 6-OHDA rats, and activated selective areas in brain and spinal cord. In this study, we evaluated whether chronic HM01 treatment influences motor functions and/or has beneficial effects on non-motor symptoms including alterations of body weight and composition, defecation, feeding and water intake in 6-OHDA rats. Methods: Male rats were microinjected unilaterally into the medial forebrain bundle with either vehicle or 6-OHDA. Three weeks later, we assessed basal body weight, and 24-h fecal output (pellets, weight, dry weight and water content), water intake and food intake (ingested and spillage). Then, HM01 (3 mg/kg) or vehicle was given per gavage daily for 10-12 days and the same parameters were re-assessed daily. Motor behavior (stepping and rotations tests), body composition were monitored before and after the HM01 treatment. Results: 6-OHDA rats showed motor deficits in rotation test induced by apomorphine and stepping test. They also displayed a significant reduction in body weight, water consumption, fecal weight and water content and an increase in food spillage compared to vehicle microinjected rats. Daily oral treatment of HM01 did not modify motor alterations compared to vehicle but significantly increased the body weight, fat mass, and 24-h fecal weight, fecal water content, food and water intake in 6-OHDA rats, while HM01 had no significant effect in vehicle microinjected rats. Fecal weight and water content were both correlated with water intake, but not with food intake. Fat mass, but not body weight, was correlated with food intake. HM01 effects were significant after 24 h and remained similar during the treatment. Conclusions: Chronic treatment with ghrelin agonist, HM01 improved several non-motor symptoms in the rat PD model induced by 6-OHDA lesion including the decrease in body weight, water consumption, fecal weight and water content, and increased food intake while not improving the motor deficits. These findings provide pre-clinical evidence of potential benefits of ghrelin agonists to alleviate non-motor symptoms in PD patients.

Corrigendum: Anti-emetic Action of the Brain-Penetrating New Ghrelin Agonist, HM01, Alone and in Combination With the 5-HT3 Antagonist, Palonosetron and With the NK1 Antagonist, Netupitant, Against Cisplatin- and Motion-Induced Emesis in Suncus murinus (House Musk Shrew).

[This corrects the article DOI: 10.3389/fphar.2018.00869.].

Anti-emetic Action of the Brain-Penetrating New Ghrelin Agonist, HM01, Alone and in Combination With the 5-HT3 Antagonist, Palonosetron and With the NK1 Antagonist, Netupitant, Against Cisplatin- and Motion-Induced Emesis in Suncus murinus (House Musk Shrew).

Ghrelin has well-known activity to stimulate appetite and weight gain. Evidence suggests that ghrelin may also have effects in reducing chemotherapy-induced emesis via growth hormone secretagogue receptors (GHS-R1A) in the brain. However, it is not known whether the stimulation of GHS-R1A has broad inhibitory anti-emetic effects. In the present studies, we used Suncus murinus to investigate the potential of the new and novel orally bioavailable brain-penetrating GHS-R1A mimetic, HM01 (1-[(1S)-1-(2,3-dichloro-4-methoxyphenyl)ethyl]-3-methyl-3-[(4R)-1-Methyl-3,3-dimethyl-4-piperidyl]urea), to reduce emesis induced by a variety of emetic challenges. HM01 (1 to 30 mg/kg, p.o.) antagonized emesis induced by cisplatin (30 mg/kg, i.p.) and by motion (4 cm horizontal displacement, 1 Hz) but was ineffective against emesis induced by nicotine (5 mg/kg, s.c.) and copper sulfate (120 mg/kg by intragastric gavage). In other experiments, HM01 (3 mg/kg, p.o.) enhanced the anti-emetic control of a regimen of palonosetron (0.01 mg/kg, p.o.) alone and palonosetron (0.01 mg/kg p.o.) plus netupitant (1 mg/kg, p.o.). HM01 (10 mg/kg, p.o.) also had positive effects in increasing feeding and drinking in nicotine-treated animals, and it shortened the latency to drink in animals treated with cisplatin. These data indicate that brain-penetrating GHS-R1A agonists may have use alone and/or in combination with standard anti-emetic regimens for the treatment of chemotherapy-induced nausea and vomiting and motion sickness. Highlights: - The novel orally bioavailable brain-penetrating GHS-R1A agonist, HM01 (1-[(1S)-1-(2,3-dichloro-4-methoxyphenyl)ethyl]-3-methyl-3-[(4R)-1-Methyl-3,3-dimethyl-4-piperidyl]urea), antagonizes motion- and cisplatin-induced emesis.- HM01 did not reduce emesis induced by nicotine or by intragastric copper sulfate.- HM01 has positive effects on food consumption after treatment with nicotine.- HM01 has synergistic effects against cisplatin when combined with palonosetron and palonosetron/netupitant regimens.- It is suggested that GHS-R1A agonists may be protective against chemotherapy-induced nausea and vomiting in combination with traditional anti-emetics and against motion-induced emesis.

Rational Design, Synthesis, and Pharmacological Characterization of Novel Ghrelin Receptor Inverse Agonists as Potential Treatment against Obesity-Related Metabolic Diseases.

A new chemotype of ghrelin inverse agonists was discovered through chimeric design based on molecular scaffolds known as growth-hormone secretagogue receptor (GHSR) modulators but with divergent pharmacodynamic and pharmacokinetic properties. The structure-activities/properties exploration led to compound 47, which displayed potent human GHSR antagonism and inverse agonism in cellular assays (IC50 = 68 nM, EC50 = 29 nM), moderate oral bioavailability, and notable brain penetration in rat ( F = 27%, B/ P ratio = 1.9). First in vivo studies demonstrated effective reduction of food intake after oral or parenteral administration to mouse (78% at 1 h and 38% at 8 h, respectively). Further preclinical studies are needed to evaluate the most suited mode of administration with the aim of promoting a first central-acting ghrelin inverse agonist molecule to development, which would represent a significant step toward therapeutic agents to treat metabolic disorders related to obesity, such as type 2 diabetes mellitus.

Ghrelin Receptor Agonist Rescues Excess Neonatal Mortality in a Prader-Willi Syndrome Mouse Model.

In the current study, we sought to determine the significance of the ghrelin system in Prader-Willi Syndrome (PWS). PWS is characterized by hypotonia and difficulty feeding in neonates and hyperphagia and obesity beginning later in childhood. Other features include low GH, neonatal hypoglycemia, hypogonadism, and accelerated mortality. Although the hyperphagia and obesity in PWS have been attributed to elevated levels of the orexigenic hormone ghrelin, this link has never been firmly established, nor have ghrelin's potentially protective actions to increase GH secretion, blood glucose, and survival been investigated in a PWS context. In the current study, we show that placing Snord116del mice modeling PWS on ghrelin-deficient or ghrelin receptor [GH secretagogue receptor (GHSR)]-deficient backgrounds does not impact their characteristically reduced body weight, lower plasma IGF-1, delayed sexual maturation, or increased mortality in the period prior to weaning. However, blood glucose was further reduced in male Snord116del pups on a ghrelin-deficient background, and percentage body weight gain and percentage fat mass were further reduced in male Snord116del pups on a GHSR-deficient background. Strikingly, 2 weeks of daily administration of the GHSR agonist HM01 to Snord116del neonates markedly improved survival, resulting in a nearly complete rescue of the excess mortality owing to loss of the paternal Snord116 gene. These data support further exploration of the therapeutic potential of GHSR agonist administration in limiting PWS mortality, especially during the period characterized by failure to thrive.

Ghrelin agonist HM01 attenuates chemotherapy-induced neurotoxicity in rodent models.

Chemotherapy-Induced Peripheral Neurotoxicity (CIPN) is often dose-limiting and impacts life quality and survival of cancer patients. Ghrelin agonists have neuroprotectant effects and may have a role in treating or preventing CIPN. We evaluated the CNS-penetrant ghrelin agonist HM01 in three experimental models of CIPN at doses of 3-30 mg/kg p.o. daily monitoring orexigenic properties, nerve conduction, mechanical allodynia, and intra-epidermal nerve fiber density (IENFD). In a cisplatin-based study, rats were dosed daily for 3 days (0.5 mg/kg i.p.) + HM01. Cisplatin treatment induced mechanical hypersensitivity which was significantly reduced by HM01. In a second study, oxaliplatin was administered to mice (6 mg/kg i.p. 3 times/week for 4 weeks) resulting in significant digital nerve conduction velocity (NCV) deficits and reduction of IENFD. Concurrent HM01 dose dependently prevented the decline in NCV and attenuated the reduction in IENFD. Pharmacokinetic studies showed HM01 accumulation in the dorsal root ganglia and sciatic nerves which reached concentrations > 10 fold that of plasma. In a third model, HM01 was tested in preventive and therapeutic paradigms in a bortezomib-based rat model (0.2 mg/kg i.v., 3 times/week for 8 weeks). In the preventive setting, HM01 blocked bortezomib-induced hyperalgesia and IENFD reduction at all doses tested. In the therapeutic setting, significant effect was observed, but only at the highest dose. Altogether, the robust peripheral nervous system penetration of HM01 and its ability to improve multiple oxaliplatin-, cisplatin-, and bortezomib-induced neurotoxicities suggest that HM01 may be a useful neuroprotective adjuvant for CIPN.