Ghrelin Receptor Inverse Agonist Research Articles

LEAP2, a ghrelin receptor inverse agonist, and its effect on alcohol-related responses in rodents

The underlying neurobiology of alcohol use disorder (AUD) is complex and needs further unraveling, with one of the key mechanisms being the gut-brain peptide ghrelin and its receptor (GHSR). However, additional substrates of the ghrelin pathway, such as liver-expressed antimicrobial peptide 2 (LEAP2), an endogenous GHSR inverse agonist, may contribute to this neurobiological framework. While LEAP2 modulates feeding and reward through central mechanisms, its effects on alcohol responses are unknown. The aim of the present study was therefore to identify the impact of central LEAP2 on the ability of alcohol to activate the mesolimbic dopamine system and to define its ability to control alcohol intake. These experiments revealed that central LEAP2 (i.e. into the third ventricle) prevented the ability of alcohol to cause locomotor stimulation in male mice, suppressed the memory of alcohol reward and attenuated the dopamine release in the nucleus accumbens caused by alcohol. Moreover, central LEAP2 reduced alcohol consumption in both male and female rats exposed to alcohol for 6 weeks before treatment. However, the serum levels of LEAP2 were similar between high- and low- alcohol-consuming (male) rats. Furthermore, central LEAP2 lowered the food intake in the alcohol-consuming male rats and reduced the body weight in the females. Collectively, the present study revealed that central LEAP2 mitigates alcohol-related responses in rodents, contributing to our understanding of the ghrelin pathway’s role in alcohol effects.

Decoding the influence of central LEAP2 on food intake and its effect on accumbal dopamine release

The gut-brain peptide ghrelin and its receptor are established as a regulator of hunger and reward-processing. However, the recently recognized ghrelin receptor inverse agonist, liver-expressed antimicrobial peptide 2 (LEAP2), is less characterized. The present study aimed to elucidate LEAP2s central effect on reward-related behaviors through feeding and its mechanism. LEAP2 was administrated centrally in mice and effectively reduced feeding and intake of palatable foods. Strikingly, LEAP2s effect on feeding was correlated to the preference of the palatable food. Further, LEAP2 reduced the rewarding memory of high preference foods, and attenuated the accumbal dopamine release associated with palatable food exposure and eating. Interestingly, LEAP2 was widely expressed in the brain, and particularly in reward-related brain areas such as the laterodorsal tegmental area (LDTg). This expression was markedly altered when allowed free access to palatable foods. Accordingly, infusion of LEAP2 into LDTg was sufficient to transiently reduce acute palatable food intake. Taken together, the present results show that central LEAP2 has a profound effect on dopaminergic reward signaling associated with food and affects several aspects of feeding. The present study highlights LEAP2s effect on reward, which may have applications for obesity and other reward-related psychiatric and neurological disorders.

Differential impact of a ghrelin receptor antagonist or inverse agonist in the electrical kindling model of epilepsy

Ghrelin is a peptide, which has been shown to affect seizures. However, there is not a consensus about its real impact on the control of seizure severity. We assessed the influence of intra-amygdala injections of a ghrelin receptor (GHSR) antagonist, as well as a GHSR inverse agonist on the electrical kindling-induced seizures. Two unipolar electrodes and a tripolar electrode twisted with a guide cannula were implanted in the skull surface or the basolateral amygdala of adult male rats, respectively. A rapid electrical kindling protocol was applied for kindling epileptogenesis. The stimulations were applied until rats showed three consecutive stage five seizures. Each rat was considered as its control. D-Lys-3-GHRP-6 (1, 12.5, and 25 μg/rat) or [D-Arg, D-phe, D-Trp, heu] substance P (D-SP) (50, 500 and 5000 ng/rat) as the GHSR antagonist or inverse agonist were injected into the basolateral amygdala. Seizure parameters including after-discharge duration (ADD), stage five duration (S5D), and seizure stage (SS) were documented thirty minutes following administration of the drugs or saline. Antagonism of the GHSR in the amygdala, significantly increased seizure induction in the kindled rats, in a dose-dependent manner, and induced spontaneous seizures leading to status epilepticus. Conversely, D-SP had a dose-dependent anticonvulsant activity, indicated by decreased ADD and S5D. The results show that GHSR inverse agonism suppressed seizure severity in the rat amygdala kindling model, whereas GHSR antagonism made seizures more severe. Therefore, when considering the ghrelin system to modulate seizures, it is crucial to note the differential impact of various GHSR ligands.

Synthesis of PF-6870961, a major hydroxy metabolite of the novel ghrelin receptor inverse agonist PF-5190457

Preclinical and human studies have indicated involvement of the ghrelin system in alcohol-related behaviors illuminating the possibility of using ghrelin receptor blockers as a pharmacological intervention for alcohol use disorder (AUD). Preliminary data from a recently conducted phase 1b human study with a ghrelin receptor inverse agonist, PF-5190457 (2-(2-methylimidazo[2,1-b][1,3thiazol-6-yl)-1-{2-(1R)-5-(6-methylpyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl]-2,7-diazaspiro[3.5]non-7-ylethanone), provided evidence on the safety and tolerability of this compound when co-administered with alcohol. Furthermore, the study revealed important information on the biotransformation pathways for this compound and prompted the discovery and then synthesis of a newly identified major metabolite, PF-6870961 ((R)-1-(2-(5-(2-hydroxy-6-methylpyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)-2,7-diazaspiro[3.5]nonan-7-yl)-2-(2-methylimidazo[2,1-b]thiazol-6-yl)ethan-1-one). The metabolite was synthesized and fully characterized through a design that enabled it to be prepared in useful quantities. The synthesis provided direct access to the recently discovered PF-6870961 and is allowing researchers to conduct additional and deeper evaluation of its in vitro and in vivo properties.

Development of a ghrelin receptor inverse agonist for positron emission tomography.

Imaging of Ghrelin receptors in vivo provides unique potential to gain deeper understanding on Ghrelin and its receptors in health and disease, in particular, in cancer. Ghrelin, an octanoylated 28-mer peptide hormone activates the constitutively active growth hormone secretagogue receptor type 1a (GHS-R1a) with nanomolar activity. We developed novel compounds, derived from the potent inverse agonist K-(D-1-Nal)-FwLL-NH2 but structurally varied by lysine conjugation with 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), palmitic acid and/or diethylene glycol (PEG2) to allow radiolabeling and improve pharmacokinetics, respectively. All compounds were tested for receptor binding, potency and efficacy in vitro, for biodistribution and -kinetics in rats and in preclinical prostate cancer models on mice. Radiolabeling with Cu-64 and Ga-68 was successfully achieved. The Cu-64- or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiotracer were specifically accumulated by the GHS-R1a in xenotransplanted human prostate tumor models (PC-3, DU-145) in mice. The tumors were clearly delineated by PET. The radiotracer uptake was also partially blocked by K-(D-1-Nal)-FwLL-NH2 in stomach and thyroid. The presence of the GHS-R1a was also confirmed by immunohistology. In the arterial rat blood plasma, only the original compounds were found. The Cu-64 or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH2 radiolabeled inverse agonists turned out to be potent and safe. Due to their easy synthesis, high affinity, medium potency, metabolic stability, and the suitable pharmacokinetic profiles, they are excellent tools for imaging and quantitation of GHS-R1a expression in normal and cancer tissues by PET. These compounds can be used as novel biomarkers of the Ghrelin system in precision medicine.

A Population Pharmacokinetic Analysis of PF-5190457, a Novel Ghrelin Receptor Inverse Agonist in Healthy Volunteers and in Heavy Alcohol Drinkers.

The ghrelin receptor (GHS-R1a) is a potential target for alcohol use disorders. PF-5190457 is the first inverse agonist of GHS-R1a to progress to clinical development with potential to treat alcohol use disorder. We present a population pharmacokinetic model for PF-5190457 in non-heavy (alcohol consumption status = 0) and heavy alcohol drinkers (alcohol consumption status = 1), and identify relevant factors that can influence its pharmacokinetics. Plasma concentration-time data from non-heavy (n = 35) and heavy drinkers (n = 12) were pooled for the population pharmacokinetic model development. The influence of various covariates including alcohol consumption status was evaluated. The accuracy, precision, and robustness of the model were also evaluated using bootstrapping and visual predictive checks. A two-compartment model best described the pharmacokinetics of PF-5190457. The apparent volume of distribution of 44.5 L, apparent clearance of 72.0 L/h, apparent peripheral volume of distribution of 271 L, apparent distributional clearance of 28.7 L/h, and first-order absorption rate constant of 0.27/h were accurate and precise. The apparent volume of distribution was 3.8-fold higher (169 L) in heavy drinkers, and correlated with a lower maximum plasma concentration in heavy drinkers compared with non-heavy drinkers at the same dose; and a corresponding reduced incidence of somnolence in heavy drinkers at doses > 50mg. This work provides an accurate, precise, and robust two-compartment model that describes the pharmacokinetics of PF-5190457 and suggests a possible link of PF-5190457 pharmacokinetics with somnolence. ClinicalTrials.gov identifier numbers NCT01247896 and NCT02039349.

Tryptophan Analogues with Fixed Side-Chain Orientation: Expanding the Scope.

A generalized synthetic strategy is proposed here for the synthesis of asymmetric β‐indoylated amino acids by 8‐aminoquinoline (8AQ)‐directed C(sp3)‐H functionalization of suitably protected precursors. Peptides containing one of the four stereoisomers of (indol‐3‐yl)‐3‐phenylalanine at position 2 of the parent peptide KwFwLL‐NH2 (w=d‐Trp) cover a wide range of activities as ghrelin receptor inverse agonists, among them the most active described until now. This application exemplarily shows how β‐indoylated amino acids can be used for the systematic variation of the position of an indole group in a bioactive peptide.

Development and validation of an assay for a novel ghrelin receptor inverse agonist PF-5190457 and its major hydroxy metabolite (PF-6870961) by LC-MS/MS in human plasma

PF-5190457 is a selective and potent ghrelin receptor inverse agonist presently undergoing clinical trials to treat alcohol use disorder (AUD). We describe the development and validation of a selective and sensitive liquid chromatography-tandem mass spectrometry-based method for quantification of PF-5190457 and its recently discovered hydroxy metabolite PF-6870961 in human plasma. Analytes were extracted after simple protein precipitation using methanol (2.5 ng mL−1 tacrine as an internal standard). A gradient liquid chromatography method was used to separate the analytes on an Acquity UPLC BEH C18 analytical column. The separation was achieved at a flow rate of 0.25 mL min−1 and the total chromatographic runtime was 11.30 min. Positive electrospray ionization and multiple reaction monitoring mode were used for the quantification of all the analytes. The calibration curves from six validation runs were linear with a correlation coefficient of ≥0.996 for the concentration range of 1–1000 ng mL−1 and 2–250 ng mL−1 for PF-5190457 and PF-6870961, respectively. The retention time for PF-5190457, PF-6870961 and tacrine were 4.4, 3.8, and 4.6 min, respectively. The lower limit of quantification for PF-5190457 and PF-6870961 was 1 and 2 ng mL−1, respectively. The inter-assay precision and accuracy results obtained were within the Food and Drug Administration recommended ±15% limit of nominal values. All the analytes were found to be stable under varied stability conditions. The recovery of PF-5190457 and PF-6870961 ranged from 95 to 103%. Further, the application of the method was demonstrated by measuring the concentration of PF-5190457 and its hydroxy metabolite in patient plasma samples from 100 mg dose.

Endocrine effects of the novel ghrelin receptor inverse agonist PF-5190457: Results from a placebo-controlled human laboratory alcohol co-administration study in heavy drinkers

Both animal and human work suggests that the ghrelin system may be involved in the mechanisms that regulate the development and maintenance of alcohol use disorder. Previously, in a Phase 1b study, we tested pharmacological blockade of the growth hormone secretagogue receptor 1a (GHS-R1a, also known as the ghrelin receptor), in heavy drinking individuals with PF-5190457, an orally bioavailable, potent and selective GHS-R1a inverse agonist. We report here the effects of PF-5190457 on endocrine blood concentrations of amylin, gastric inhibitory polypeptide, glucagon-like peptide 1, insulin, leptin, pancreatic polypeptide, peptide YY, thyroid stimulating hormone, free triiodothyronine (T3), thyroxine (T4), cortisol, prolactin, and glucose during PF-5190457 dosing, as compared to placebo, in absence of alcohol as well as during an alcohol challenge when PF-5190457 was on steady-state. Blood hormone levels were largely unaffected by PF-5190457, both during dosing and in the context of alcohol challenge. The safety-related relevance of these findings to further develop PF-5190547 in alcohol use disorder is discussed. Clinicaltrials.govNCT02039349.This article is part of the special issue on ‘Neuropeptides’.

Ghrelin: From a Gut Hormone to a Potential Therapeutic Target for Alcohol Addiction

Alcohol use disorder (AUD) is a chronic relapsing disease with significant medical, psychosocial, and economic burden. Pharmacological treatments for AUD are, however, limited in number and efficacy. Beyond classic neurotransmitter systems, there has been a growing interest in understanding the role of peripheral pathways (e.g., gut‐brain communications) in AUD, with the ultimate goal of identifying novel therapeutic targets. Ghrelin is an orexigenic peptide synthesized by enteroendocrine cells primarily located in the gastric mucosa. Ghrelin is known as the ‘hunger hormone’, given its role in stimulating both homeostatic and hedonic food intake. Ghrelin has also been shown to modulate alcohol seeking and consummatory behaviors, possibly through interactions with reward and stress pathways. Here we investigated whether and how pharmacological manipulation of the ghrelin system may affect biobehavioral correlates of alcohol use in heavy‐drinking alcohol‐dependent individuals. First, in a randomized, double‐blind, placebo‐controlled, human laboratory study, intravenous (IV) ghrelin/placebo was administered and two experiments were conducted: progressive‐ratio IV alcohol self‐administration (IV‐ASA) and brain functional magnetic resonance imaging (fMRI). Results showed that IV ghrelin, compared to placebo, significantly increased the number of alcohol infusions self‐administered (p=0.04) and reduced the time to initiate alcohol self‐administration (p=0.03). In addition, ghrelin increased the alcohol‐related signal in the amygdala (p=0.01) and modulated the food‐related signal in the medial orbitofrontal cortex (p=0.01) and nucleus accumbens (p=0.08). In the second study, we examined the effects of a novel ghrelin receptor inverse agonist (PF‐5190457) co‐administered with alcohol in both rodents and humans. No pharmacological interactions between alcohol and PF‐5190457 were found. PF‐5190457 did not interact with the effects of alcohol on locomotor activity or loss‐of‐righting reflex. In humans, PF‐5190457 was well tolerated, did not cause any significant side effect, and did not alter alcohol pharmacokinetics or alcohol‐induced subjective effects. As a secondary outcome, a cue‐reactivity procedure was also conducted in a bar‐like laboratory setting. Results showed that PF‐5190457, compared to placebo, reduced cue‐elicited craving for alcohol (p=0.05), as well as attention to alcohol cues (p=0.02). In conclusion, the ghrelin system seems to regulate both behavioral and neurobiological correlates of alcohol consumption and may represent a potential target for developing novel medications to treat AUD.Support or Funding InformationThis work was supported by A) NIH intramural funding ZIA‐AA000218 jointly supported by NIAAA and NIDA, and B) NCATS grant UH2/UH3‐TR000963. Development of the Computerized Alcohol Infusion System software was supported by the NIAAA‐funded Indiana Alcohol Research Center (AA007611). Pfizer provided the PF‐5190457 drug under the NCATS grant UH2/UH3‐TR000963 grant.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Differential functional selectivity and downstream signaling bias of ghrelin receptor antagonists and inverse agonists.

The ghrelin receptor [growth hormone secretagogue receptor (GHSR)-1a] represents a promising pharmacologic target for the treatment of metabolic disorders, including obesity and cachexia, via central appetite modulation. The GHSR-1a has a complex pharmacology, highlighted by G-protein-dependent and -independent downstream signaling pathways and high basal constitutive activity. The functional selectivity and signaling bias of many GHSR-1a-specific ligands has not been fully characterized. In this study, we investigated the pharmacologic properties of ghrelin, MK-0677, L692,585, and [d-Lys3]-growth hormone-releasing peptide-6 (Dlys), JMV2959, and [d-Arg(1),d-Phe(5),d-Trp(7, 9),Leu(11)]-substance P (SP-analog). We investigated their effect on basal GHSR-1a constitutive signaling, ligand-directed downstream GHSR-1a signaling, functional selectivity, and signaling bias. Dlys behaved as a partial antagonist with a strong bias toward GHSR-1a-β-arrestin signaling, whereas JMV2959 acted as a full unbiased GHSR-1a antagonist. Moreover, the SP-analog behaved as an inverse agonist increasing G-protein-dependent signaling, but only at high concentrations, whereas, at low concentrations, the SP-analog attenuated β-arrestin-dependent signaling. Considering the limited success in the clinical development of GHSR-1a-targeted drugs so far, these findings provide a novel insight into the pharmacologic characteristics of GHSR-1a ligands and their signaling bias, which has important implications in the design of novel, more selective GHSR-1a ligands with predictable functional outcome and selectivity for preclinical and clinical drug development.-Ramirez, V. T., van Oeffelen, W. E. P. A., Torres-Fuentes, C., Chruścicka, B., Druelle, C., Golubeva, A. V., van de Wouw, M., Dinan, T. G., Cryan, J. F., Schellekens, H. Differential functional selectivity and downstream signaling bias of ghrelin receptor antagonists and inverse agonists.

Pharmacological manipulation of the ghrelin system and alcohol hangover symptoms in heavy drinking individuals: Is there a link?

Ghrelin, an orexigenic peptide synthesized in the stomach, is a key player in the gut-brain axis. In addition to its role in regulating food intake and energy homeostasis, ghrelin has been shown to modulate alcohol-related behaviors. Alcohol consumption frequently results in hangover, an underexplored phenomenon with considerable medical, psychological, and socioeconomic consequences. While the pathophysiology of hangover is not clear, contributions of mechanisms such as alcohol-induced metabolic/endocrine changes, inflammatory/immune response, oxidative stress, and gut dysbiosis have been reported. Interestingly, these mechanisms considerably overlap with ghrelin's physiological functions. Here, we investigated whether pharmacological manipulation of the ghrelin system may affect alcohol hangover symptoms. Data were obtained from two placebo-controlled laboratory studies. The first study tested the effects of intravenous (IV) ghrelin and consisted of two experiments: a progressive-ratio IV alcohol self-administration (IV-ASA) and a fixed-dose IV alcohol clamp. The second study tested the effects of an oral ghrelin receptor inverse agonist (PF-5190457) and included a fixed-dose oral alcohol administration experiment. Alcohol hangover data were collected the morning after each alcohol administration experiment using the Acute Hangover Scale (AHS). IV ghrelin, compared to placebo, significantly reduced alcohol hangover after IV-ASA (p = 0.04) and alcohol clamp (p = 0.04); PF-5190457 had no significant effect on AHS scores. Females reported significantly higher hangover symptoms than males following the IV-ASA experiment (p = 0.04), but no gender × drug condition (ghrelin vs. placebo) effect was found. AHS total scores were positively correlated with peak subjective responses, including ‘stimulation’ (p = 0.08), ‘sedation’ (p = 0.009), ‘feel high’ (p = 0.05), and ‘feel intoxicated’ (p = 0.03) during the IV-ASA. IV ghrelin blunted the positive association between alcohol sedation and hangover as shown by trend-level drug × sedation effect (p = 0.08). This is the first study showing that exogenous ghrelin administration, but not ghrelin receptor inverse agonism, affects hangover symptoms. Future research should investigate the potential mechanism(s) underlying this effect.

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

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. 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. 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. Our study demonstrates therapeutic actions of novel ghrelin receptor inverse agonists, suggesting a potential to treat obesity-related metabolic disorders including diabetes mellitus.

Pharmacokinetics and pharmacodynamics of PF-05190457: The first oral ghrelin receptor inverse agonist to be profiled in healthy subjects.

To evaluate safety, tolerability and pharmacokinetics of oral PF-05190457, an oral ghrelin receptor inverse agonist, in healthy adults. Single (SAD) and multiple ascending dose (MAD) studies were randomised, placebo-controlled, double-blind studies. Thirty-five healthy men (age 38.2±10.4years; body mass index 24.8±3.1kgm-2 [mean±standard deviation]) received ≥1 dose (2, 10, 40 [divided], 50, 100, 150, and 300 [single or divided] mg) of PF-05190457 and/or placebo in the SAD. In the MAD study, 35 healthy men (age 39.7±10.1years; body mass index 25.9±3.3kgm-2 ) received ≥1 dose (2, 10, 40 and 100mg twice daily) of PF-05190457 and/or placebo daily for 2weeks. PF-05190457 absorption was rapid with a Tmax of 0.5-3hours and a half-life between 8.2-9.8hours. PF-05190457 dose-dependently blocked ghrelin (1pmolkg-1 min-1 )-induced growth hormone (GH) release with (mean [90% confidence interval]) 77% [63-85%] inhibition at 100mg. PF-05190457 (150mg) delayed gastric emptying lag time by 30% [7-58%] and half emptying time by 20% [7-35%] with a corresponding decrease in postprandial glucose by 9mgdL-1 . The most frequent adverse event reported by 30 subjects at doses ≥50mg was somnolence. PF-05190457 plasma concentrations also increased heart rate up to 13.4 [4.8-58.2] beatsmin-1 and, similar to the effect on glucose and ghrelin-induced GH, was lost within 2weeks. PF-05190457 is a well-tolerated first-in-class ghrelin receptor inverse agonist with acceptable pharmacokinetics for oral daily dosing. Blocking ghrelin receptors inhibits ghrelin-induced GH, and increases heart rate, effects that underwent tachyphylaxis with chronic dosing. PF-051940457 has the potential to treat centrally-acting disorders such as insomnia.

Pharmacological characterization of the first in class clinical candidate PF-05190457: a selective ghrelin receptor competitive antagonist with inverse agonism that increases vagal afferent firing and glucose-dependent insulin secretion ex vivo.

Ghrelin increases growth hormone secretion, gastric acid secretion, gastric motility and hunger but decreases glucose-dependent insulin secretion and insulin sensitivity in humans. Antagonizing the ghrelin receptor has potential as a therapeutic approach in the treatment of obesity and type 2 diabetes. Therefore, the aim was to pharmacologically characterize the novel small-molecule antagonist PF-05190457 and assess translational pharmacology ex vivo. Radioligand binding in filter and scintillation proximity assay formats were used to evaluate affinity, and europium-labelled GTP to assess functional activity. Rat vagal afferent firing and calcium imaging in dispersed islets were used as native tissues underlying food intake and insulin secretion respectively. PF-05190457 was a potent and selective inverse agonist on constitutively active ghrelin receptors and acted as a competitive antagonist of ghrelin action, with a human Kd of 3 nM requiring 4 h to achieve equilibrium. Potency of PF-05190457 was similar across different species. PF-05190457 increased intracellular calcium within dispersed islets and increased vagal afferent firing in a concentration-dependent manner with similar potency but was threefold less potent as compared with the in vitro Ki in recombinant overexpressing cells. The effect of PF-05190457 on rodent islets was comparable with glibenclamide, but glucose-dependent and additive with the insulin secretagogue glucagon-like peptide-1. Together, these data provide the pharmacological in vitro and ex vivo characterization of the first ghrelin receptor inverse agonist, which has advanced into clinical trials to evaluate the therapeutic potential of blocking ghrelin receptors in obesity and type 2 diabetes.

C-terminus of a hexapeptidic ghrelin receptor inverse agonist can switch peptide behavior from inverse agonism to agonism.

Subtle changes in the sequence at the N-terminus and in the aromatic core of hexapeptidic ghrelin receptor inverse agonists can switch behavior from inverse agonism to agonism, but the C-terminal role of the sequence is unclear. Thus, analogs of the ghrelin receptor inverse agonist KbFwLL-NH2 (b = β-(3-benzothienyl)-d-alanine) were synthesized by solid phase peptide synthesis in order to identify the influence of aromaticity, charge, and hydrophobicity. Potency and efficacy of the hexapeptides were evaluated in inositol triphosphate turnover assays. Notably, modifications directly at the C-terminal Leu(6) could influence peptide efficacy leading to decreased constitutive activity. High hydrophobicity at the C-terminal position was of importance for elevated inverse agonist activity, the introduction of charged amino acids led to decreased potency. In contrast, structure-activity relationship studies of Leu(5) located closer to the aromatic core revealed an agonism-inducing position. These findings imply that amino acids with possible cation-π or π-π interactions and a suitable orientation at the C-terminus of the aromatic core induce agonism. Receptor binding studies showed that most peptides bind to the receptor at a concentration of 1 µM and modification directly at the C-terminus is generally more accepted than Leu(5) substitution. Interestingly, this observation is not dependent on the type of modification. These studies reveal another switch region of the short ghrelin receptor ligand pointing out the sensitivity of the ghrelin receptor binding pocket.

Orally active ghrelin receptor inverse agonists and their actions on a rat obesity model

A series of 2-alkylamino nicotinamide analogs was prepared as orally active ghrelin receptor (ghrelinR) inverse agonists. Starting from compound 1, oral bioavailability was improved by modifying metabolically unstable sites and reducing molecular weight. Brain-permeable compound 33 and compound 24 with low brain permeability were tested in rat models of obesity; 30mg/kg of compound 33 suppressed weight gain. PK/PD analysis revealed that the anti-obesity effect of ghrelinR inverse agonists depends on their brain concentrations.

Development and validation of an UPLC-MS/MS assay for quantitative analysis of the ghrelin receptor inverse agonist PF-5190457 in human or rat plasma and rat brain.

PF-5190457 is a ghrelin receptor inverse agonist that is currently undergoing clinical development for the treatment of alcoholism. Our aim was to develop and validate a simple and sensitive assay for quantitative analysis of PF-5190457 in human or rat plasma and rat brain using liquid chromatography-tandem mass spectrometry. The analyte and stable isotope internal standard were extracted from 50 μL plasma or rat brain homogenate by protein precipitation using 0.1% formic acid in acetonitrile. Chromatography was carried out on an Acquity UPLC BEH C18 (2.1 mm × 50 mm) column with 1.7 μm particle size and 130 Å pore size. The flow rate was 0.5 mL/min and total chromatographic run time was 2.2 min. The mobile phase consisted of a gradient mixture of water: acetonitrile 95:5% (v/v) containing 0.1% formic acid (solvent A) and 100% acetonitrile containing 0.1% formic acid (solvent B). Multiple reaction monitoring was carried out in positive electro-spray ionization mode using m/z 513.35 → 209.30 for PF-5190457 and m/z 518.47 → 214.43 for the internal standard. The recovery ranged from 102 to 118% with coefficient of variation (CV) less than 6% for all matrices. The calibration curves for all matrices were linear over the studied concentration range (R(2) ≥ 0.998, n = 3). The lower limit of quantification was 1 ng/mL in rat or human plasma and 0.75 ng/g in rat brain. Intra- and inter-run mean percent accuracies were between 85 and 115% and percent imprecision was ≤15%. The assays were successfully utilized to measure the concentration of PF-5190457 in pre-clinical and clinical pharmacology studies of the compound.

Rational design of dual peptides targeting ghrelin and Y2 receptors to regulate food intake and body weight.

Ghrelin and Y2 receptors play a central role in appetite regulation inducing opposite effects. The Y2 receptor induces satiety, while the ghrelin receptor promotes hunger and weight gain. However, the food regulating system is tightly controlled by interconnected pathways where redundancies can lead to poor efficacy and drug tolerance when addressing a single molecule. We developed a multitarget strategy to synthesize dual peptides simultaneously inhibiting the ghrelin receptor and stimulating the Y2 receptor. Dual peptides showed dual activity in vitro, and one compound induced a slight diminution of food intake in a rodent model of obesity. In addition, stability studies in rats revealed different behaviors between the dual peptide and its corresponding monomers. The Y2 receptor agonist was unstable in blood, while the dual peptide showed an intermediate stability compared to that of the highly stable ghrelin receptor inverse agonist.