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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2016 Mar 17;173(9):1452–1464. doi: 10.1111/bph.13439

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

J Kong 1, J Chuddy 1, I A Stock 1, P M Loria 1, S V Straub 1, C Vage 1, K O Cameron 1, S K Bhattacharya 1, K Lapham 1, K F McClure 1, Y Zhang 1, V M Jackson 1,✉
PMCID: PMC4831304  PMID: 26784385

Abstract

Background and Purpose

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.

Experimental Approach

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.

Key Results

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 K d 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 K i 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.

Conclusions and Implications

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.

Abbreviations

CI

confidence interval

[d‐Lys3]‐GHRP‐6

[d‐Lys3]‐growth hormone releasing peptide‐6

Eu‐GTP

europium‐labelled GTP

GLP‐1

glucagon‐like peptide‐1

[3H]‐IP

tritium labelled inositol phosphate

PYY3–36

peptide YY

SPA

scintillation proximity assay

Tables of Links

TARGETS
Ghrelin receptor
LIGANDS
Ghrelin GLP‐1 Insulin
Glibenclamide GTP PYY3–36
GHRP‐6 GTPγS
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These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015).

Introduction

Ghrelin is an endogenous 28 amino acid acylated peptide synthesized within the stomach fundus that has been associated with modulating growth hormone secretion, gastric motility, gastric acid secretion and hunger. Acylated ghrelin is the only peripheral hunger‐stimulating hormone that is elevated preprandially in plasma, suggesting a role in meal initiation in humans (Cummings et al., 2001). Circulating endogenous baseline and pulsatile patterns of total ghrelin are inhibited in obese subjects following gastric bypass surgery (Cummings et al., 2002; Roth et al., 2008), whereas endogenous levels of acylated ghrelin are reported to be decreased with increased body mass index (Homaee et al., 2011; Moradi et al., 2011; Dardzińska et al., 2014; van Name et al., 2015), elevated in obese type 2 diabetics (Rodríguez et al., 2009) and inversely correlated with insulin sensitivity (Pagotto et al., 2002; Barazzoni et al., 2007). Conflicting data may be attributed to differences in patient populations used, assays or plasma sample handling (Blatnik and Soderstrom, 2011; Blatnik et al., 2012). Exogenous administration of acylated ghrelin increases blood glucose and decreases insulin levels in both rodents and humans (Broglio et al., 2001, 2002, 2003a, 2003b, 2004; Arosia et al., 2003; Dezaki et al., 2004; Sun et al., 2006), and also inhibits glucose‐stimulated insulin secretion (Reimer et al., 2003; Dezaki et al., 2007; Tong et al., 2010) and peripheral insulin sensitivity (Gauna et al., 2004; Lucidi et al., 2005; Damjanovic et al., 2006; Vestergaard et al., 2007, 2008a, 2008b).

Acylated ghrelin is the endogenous ligand for the growth hormone secretagogue receptor 1a (ghrelin receptor; Alexander et al., 2003) of which des‐acylated ghrelin has no activity. The ghrelin receptor is unique in that it has been shown to be constitutively active, suggesting that blocking ghrelin receptor tone rather than blocking endogenous acylated ghrelin binding may be beneficial (Mear et al., 2013). The metabolic effects of acylated ghrelin were originally considered to be acting on central ghrelin receptors as ghrelin crosses the blood brain barrier (Banks et al., 2002); however, it is now evident that many of ghrelin's actions are mediated peripherally as the brain‐impaired selective ghrelin receptor agonist capromorelin caused insulin resistance in humans (White et al., 2009), and ghrelin receptors are located on islets and vagal afferent soma (Gnanapavan et al., 2002; Volante et al., 2002; Wierup et al., 2002; Sakata et al., 2003; Kageyama et al., 2005; Wierup and Sundler, 2005; Burdyga et al., 2006; Dezaki et al., 2007; Sato et al., 2007; Jia et al., 2008; Damdindorj et al., 2012; Grabauskas et al., 2015). Ghrelin‐induced food intake is blocked by vagotomy in rodents and humans (Date et al., 2002; le Roux et al., 2005), and ghrelin (i.v.) suppresses vagal afferent firing (Asakawa et al., 2001; Date et al., 2002, 2012; Inui et al., 2004). Selective blockade of the gastric vagus nerve either by capsaicin (afferent only) or surgical differentiation (afferent and efferent pathways) blocks c‐Fos expression within the arcuate nucleus in response to ghrelin‐induced feeding. Vagotomy also eliminates ghrelin‐induced growth hormone secretion, indicating a novel vagus‐mediated growth hormone secretion pathway (Date et al., 2002), in addition to the known vagus‐mediated efferent regulation of gastrointestinal motility, gastric acid secretion and nausea (Yakabi et al., 2008; Sanger et al., 2013; Swartz et al., 2014).

Whilst the effect of ghrelin and small‐molecule agonists have been well characterized pharmacologically and profiled in man, there have been a paucity of reports on the translational pharmacology of ghrelin receptor antagonists comparing potency and efficacy in preclinical models and no published clinical data. Antagonists routinely used preclinically to evaluate ghrelin receptor activity, such as [d‐Arg1,d‐Phe5,d‐Trp7,9,Leu11]‐substance P and [d‐Lys3]‐GHRP‐6, are hampered by the fact that they are peptidic or peptidomimetic antagonists with limited potency (>100 nM), and selectivity. For example, [d‐Arg1,d‐Phe5,d‐Trp7,9,Leu11]‐substance P is a potent bombesin antagonist and [d‐Lys3]‐GHRP‐6 can activate 5‐HT receptors and antagonize CCR4/CCR5 receptors, hence limiting their use in translational ex vivo and in vivo experimentation (Woll and Rozengurt, 1988; Depoortere et al., 2006; Patel et al., 2012a, 2012b).

Due to the potential therapeutic utility of ghrelin receptor antagonists, the search for potent selective small‐molecule antagonists has been pursued. These molecules have been discovered using recombinant cell lines to evaluate binding and antagonizing ghrelin‐induced activation of Gq‐coupled pathways known to underlie growth hormone secretion in the pituitary (Pasternak et al., 2009; Perdonà et al., 2011; Mihalic et al., 2012; Puleo et al., 2012; Alexander et al., 2003; Moulin et al., 2013; www.iuphar.org/). However, the signal transduction mechanisms mediating the effect of the ghrelin receptor in pancreatic beta cells are distinct from those utilized in GH‐releasing and/or ghrelin receptor‐expressing cells as demonstrated by the use of pertussis toxin to inhibit the ghrelin‐induced inhibition of insulin secretion, suggesting Gi‐mediated pathways maybe more relevant to insulin secretion (Dezaki et al., 2007, 2008; Damdindorj et al., 2012). In addition, the ghrelin receptor has been reported to be constitutively active using recombinant in vitro systems, suggesting an inverse agonist rather than a neutral antagonist may be therapeutically relevant; however, this remains to be demonstrated in native tissue or in vivo physiological systems.

The current study reports a potent and selective small‐molecule ghrelin receptor competitive antagonist with inverse agonism that is equally potent and efficacious within native physiological systems underlying food intake and insulin secretion ex vivo. A high‐throughput screening of the Pfizer corporate compound file led to the identification of a spiro‐azetidino piperidine series of analogues (Cameron et al., 2014). Medicinal chemistry optimization of initial leads in this series for ghrelin receptor activity while balancing absorption, distribution, metabolism, and excretion and off‐target selectivity provided PF‐05190457 (2‐(2‐methylimidazo[2,1‐b][1,3]thiazol‐6‐yl)‐1‐{2‐[(1R)‐5‐(6‐methylpyrimidin‐4‐yl)‐2,3‐dihydro‐1 H‐inden‐1‐yl]‐2,7‐diazaspiro[3.5]non‐7‐yl}ethanone) (Bhattacharya et al., 2014). Based on its promising pharmacological and safety profile, PF‐05190457 has advanced into human clinical trials.

Methods

In vitro radioligand binding, direct and indirect quantitative kinetics

The in vitro potency of PF‐05190457 (Bhattacharya et al., 2014) against the human ghrelin receptor was determined by radioligand binding in two different assay formats: filter plate and scintillation proximity assay (SPA). Both assays utilized membranes prepared from HEK293 cells which had been stably transfected with a tet‐inducible construct expressing the human ghrelin receptor. The stable cell lines were grown under Zeocin/blasticidin selection in T‐Rex‐293 cells (Life Technologies) and receptor expression was induced with doxycycline. Clonal cell lines (human, rat and mouse) and stable cell pools (dog and primate) were evaluated for receptor expression via saturation binding to ensure appropriate receptor expression. B max and K d were determined for individual membrane preparations. Control membranes were also prepared from parental T‐Rex‐293 cells and were negative for [125I]‐ghrelin binding. Membranes were incubated in the presence of PF‐05190457 (1 × 10–10 − 1 × 10–5 M) and a single concentration (~50 pM) of human [125I]‐ghrelin (Perkin Elmer Life Sciences) for either 90 min (filter plate) or 8 h (SPA) and a K i value was determined. The binding potency for PF‐05190457 against the ghrelin receptor for dog, primate, rat and mouse was also determined by a filter binding assay, using membranes prepared from HEK293 cells which had been transfected with a tet‐inducible construct expressing dog, primate, rat or mouse ghrelin receptor. Binding data were analysed using internal software. An IC50 value was determined using a four‐parameter fit algorithm, and the K i value was generated using the Cheng–Prusoff equation: K i = IC50/(1 + ([L]/K d), where [L] was the actual radioligand concentration in the assay and K d was the equilibrium dissociation constant specific for the batch of receptor membrane used in the experiment. The K i values are reported as the geometric mean with 95% confidence interval (CI) for the number (n) of determinations.

The binding kinetics of PF‐05190457 was evaluated using both an indirect Motulsky and Mahan method (Motulsky and Mahan, 1984) and by directly radiolabelling PF‐05190457 ([3H]‐PF‐05190457; 3 × 10–9 − 1 × 10–8 M) with a specific activity of 4.9 Ci⋅mmol−1 and >98% radiochemical purity, provided by Groton Radiochemistry Laboratory (see modelling methodology in Supporting Information 2S). Using the indirect method, [125I]‐ghrelin 0.25 nM was profiled with and without PF‐05190457 (1 × 10–9 − 1 × 10–7 M) over a 7 h period to evaluate the association and dissociation rate for PF‐05190457. A saturation binding was also performed under these conditions to obtain the K d of PF‐05190457.

Europium‐labelled GTP functional in vitro assay

The functional activity of PF‐05190457 was determined using a Eu‐GTP assay on membranes prepared from HEK293 cells stably transfected with the human ghrelin receptor. Membranes were incubated in the presence PF‐05190457 (3 × 10–11 − 1 × 10–4 M) with and without an 80% maximal effective concentration of human acylated ghrelin (1.5 nM; AnaSpec) for 114 min. PF‐05190457 was profiled as an agonist, partial agonist or inverse agonist in the absence of ghrelin, whereas the antagonist mode measured the ability of PF‐05190457 to block the ghrelin‐stimulated changes. To evaluate the competitive antagonist nature of PF‐05190457, membranes were incubated in the presence human acylated ghrelin (1 × 10–11 − 3 × 10–6 M) with or without a single concentration of PF‐05190457 (1 × 10–9 − 1 × 10–5 M) pre‐incubated for 2 h. The relative fluorescence unit of membrane‐bound Eu‐GTP was determined by an EnVision Mutilabel Reader (PerkinElmer). To generate a Schild Plot, a graph with log of antagonist concentration on the x‐axis and log of ‘concentration ratio −1’ on the y‐axis was plotted by using the Graphpad Prism programme (GraphPad Software, Inc).

Rat stomach–gastric vagus preparation and unit action potential recording ex vivo

Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015). Male Sprague Dawley rats (Charles River, ~150–250 g/2‐ to 3‐months‐old; group housed), with ad libitum access to normal chow and water, were anaesthetized with an i.p. injection of Nembutal sodium (0.4–0.6 mL of 50 mg⋅mL−1 Nembutal sodium solution) prior to decapitation. All experiments were performed in accordance with Institutional Animal Care and Use Committee guidelines and regulations at Pfizer Inc. (Groton, CT). Rats were used as the pharmacological model of choice as (i) there are no known species differences in rodent physiology and (ii) to align with toxicology species as PF‐05190457 is a clinical candidate. To ensure replacement, refinement or reduction of animals, tissue harvesting was shared amongst groups, and preclinical statistics was involved in experimental design in addition to analysis of all ex vivo studies. Experiments were conducted on an isolated rat stomach gastric vagus nerve ex vivo preparation, as previously described (Wei and Wang, 2000). The esophagus and stomach were pinned to a Sylgard (Dow Corning)‐filled dissection dish, and the tissue was rinsed several times with ice cold recording buffer that contained 134.7 mM NaCl, 16.3 mM NaHCO3, 3.4 mM KCl, 1.3 mM KH2PO4, 2.8 mM CaCl2, 0.6 mM MgSO4 and 7.7 mM glucose and continuously bubbled with 95% O2/5% CO2. The vagal nerves and surrounding connective tissue were carefully dissected free from the oesophagus and pinned out. A piece of connective tissue was attached to the left platinum electrode wire (0.25 mm diameter; World Precision Instruments), and several nerve fibres were wrapped around the right electrode wire. The platinum wires were connected to an NL‐100 headstage (NeuroLog) which was connected to an NL‐104 pre‐amplifier. The signal was filtered through an NL 125/126 filter which was set at 200 Hz and 3 kHz. The signal from the NeuroLog was passed through a HumBug filter (Quest Scientific) and acquired using labchart software (version 7.0.1, AD Instruments) via a PowerLab 4/30 A‐D interface (AD Instruments) sampling at 20 kHz. The recording buffer was perfused at a rate of ~10 mL⋅min−1 via a peristaltic pump, and heated to 35°C via an electronically controlled in‐line solution heater (TC344B, Warner Instruments). To ensure that the level of spontaneous afferent nerve activity was stable, spontaneous activity was recorded for ~20 min at the beginning of each experiment prior to intragastric arterial infusion of increasing concentrations of PF‐05190457 (1 × 10–10 − 1 × 10–6 M) or the neutral antagonist compound 2 (1 × 10–10 − 1 × 10–6 M) (Supporting Information Figure 1S). Each drug concentration‐effect was allowed to plateau prior to addition of a higher drug concentration (circa 10–15 min). Nerve recordings were analysed using labchart software, and action potentials were detected using the simple threshold analysis detection function. Firing frequency was expressed as mean ± SD. For each individual experiment, the total nerve activity generated over a 5 min period sampled 5 min after injection of 0.3 mL of control recording solution (basal activity) was subtracted from all drug‐treated responses, and the responses were then normalized to the maximum activity elicited during the experiment. Normalized data from all experiments were averaged and plotted as a concentration–response curve, which was fit using the log [agonist] versus response function within graphpad prism to generate a concentration of PF‐05190457 required for 50% inhibition. EC50 values are reported as mean ± SEM.

Rat dispersed islets and single cell calcium imaging

Male Sprague Dawley rats (~275 g/~3 months old; group housed) were killed by CO2 and cervical dislocation, the pancreas was removed and islet cells were isolated as previously described (Pakhtusova et al., 2003). All experiments were performed in accordance with Institutional Animal Care and Use Committee guidelines and regulations at Pfizer Inc. (Groton, CT). Dispersed cells were placed in Ca2 + imaging buffer containing 129 mM NaCl, 5 mM NaHCO3, 4.7 mM KCl, 1.2 mM KH2PO4, 2 mM CaCl2, 1.2 mM MgSO4, and 10 mM HEPES and pH adjusted to 7.4. Depending on the experiment, either 3 or 9 mM glucose was added. For imaging, cells were loaded with the Ca2 +‐sensitive fluorescent indicator, Fura‐2AM for 35 min at room temperature and perfused (1–2 mL⋅min−1) with imaging buffer (35°C) for at least 5 min prior to imaging. Cells were excited at wavelengths of 340 and 380 nm every 5–8 s, and emission values were detected above 510 nm. A single concentration of PF‐05190457 or Compound 2 (1 × 10–10 − 1 × 10–6 M) was applied until the calcium response plateaued (5–15 min) followed by a washout until the calcium response returned to baseline prior to the addition of the sulphonylurea glibenclamide (100 μM). In all experiments, a concentration of 9 mM glucose imaging buffer was used except when testing the glucose dependence of PF‐05190457. In these experimental runs, 3 mM glucose Ca2 + imaging buffer was used. To test the additive effects of PF‐05190457 and glucagon‐like peptide‐1 (GLP‐1), the EC50 of each were combined and administered at the same time in the perfusion system to the cells (Moens et al., 1996). An average trace was generated per experiment, and the initial baseline value was recorded and compared with the peak response of PF‐05190457 as well as to the peak response of glibenclamide. The average compound response was then reported as a percentage of the glibenclamide response. Normalized data from all experiments were averaged and plotted as a concentration–response curve (n = 3 rats statistically relevant for a 10‐point concentration–response curve), which was fit using the log [agonist] versus response function within graphpad prism to generate an EC50. Data are expressed as mean ± SEM.

Data and statistical analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). To assess the uncertainty associated with the estimates of association rate constant and time to achieve equilibrium, the Delta method was applied to approximate the standard errors of on‐rate and time to achieve equilibrium using the estimates of binding kinetic parameters and their standard errors based on the direct [3H]‐PF‐05190457 and indirect Motulsky modelling implemented in NONMEM version V (University of California at San Francisco, CA).

A paired t‐test was used to determine the significance of the effect of PF‐05190457 on vagal afferent firing (two tailed). One‐way ANOVA was applied to analyse the change in intracellular Ca2 + in rat dispersed islets to determine whether PF‐05190457 has an additive effect with GLP‐1 on intracellular Ca2 + and peak calcium response (%) to test whether the co‐administration of an EC50 of PF‐05190457 (10 nM) and EC50 of GLP‐1 will result in a maximal increase in intracellular Ca2 +, where the two‐sided P‐values adjusted by the Dunnett's test were provided. P < 0.05 was used as the level of probability deemed to constitute the threshold for statistical significance when comparing groups.

Results

PF‐05190457 is a potent ghrelin receptor ligand across species

The binding kinetics of PF‐05190457 where approximated in the radioligand filter assay by determining K i values after a 90 min and 24 h incubation. Potency was similar over time with a K i of 2.49 nM CI 2.18–2.85 nM at 90 min versus 1.42 nM CI 1.21–1.6 nM at 24 h (Figure 1A). The human potency for PF‐05190457 was comparable using SPA (K i 4.4 nM CI 2.1–9.1 nM, n = 10) and no species difference was detected (dog 3.6 nM CI 2.9–4.4 nM, n = 9; primate 1.8 nM CI 1.6–2.2 nM, n = 8; rat 3.2 nM C.I. 2.8–3.6, n = 49 and mouse 7.7 nM C.I. 5.9–10.1 nM, n = 15; Figure 1B). To determine PF‐05190457 K on and K off, a quantitative Motulsky and Mahan indirect method was applied (Motulsky and Mahan, 1984). PF‐05190457 had an estimated K on of 109275 M−1*s−1, K off of 0.000628 s−1 (Table 1A) and required 1.2 h to achieve equilibrium (Figure 1C). The human K d for PF‐05190457 at equilibrium was 5.75 nM (n = 8). PF‐0590457 was also radiolabelled to evaluate direct receptor kinetics. [3H]PF‐05190457 had an estimated K on of 65534 M−1*s−1, K off of 0.0002 s−1 (Table 1B) and required 3.8 h to achieve equilibrium (Figure 1D). The human K d for [3H]‐PF‐05190457 was 3.09 nM (n = 7).

Figure 1.

Figure 1

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(A) The in vitro potency of PF‐05190457 against the human ghrelin receptor using filter binding at 90 min and 24 h. The concentration–response curves represent the average ± SD for % inhibition at each concentration tested where n = 86–103 replicates over 52 (90 min) or 44 experiments (24 h). (B) The quantitative association and dissociation kinetics of PF‐05190457 using [125I]ghrelin binding to human ghrelin receptor membranes in the presence or absence of 1, 10 or 100 nM PF‐05190457. The specific binding plot represents the arithmetic mean ± SEM at each time point tested over eight experiments. (C) The in vitro potency of PF‐051907457 against the ghrelin receptor across species. The concentration–response curves represent the mean ± SD for the % inhibitation values at each concentration tested where n = 16–103 replicates over 52 (human), 9 (dog), 8 (primate), 49 (rat) or 15 experiments (mouse). (D) The association and dissociation kinetics of [3H]PF‐05190457 a (3 and 10 nM) over 7 h. Each specific binding point represents the arithmetic mean ± SEM at each time point tested over seven experiments.

Table 1A.

Indirect quantitative (Motulsky and Mahan) method parameter summary of PF‐05190457

Table 1A Indirect method (Motulsky and Mahan)
Parameter Estimate IOV (CV%) RSE (CV%)
B max (M) 1.21E‐11* 108.0** 7.2
K d L (M) 1.23E‐10, fixed* 37.0, fixed
K off L (s−1) 0.0013 9.1
K off D, PF‐05190457 (s−1) 0.000628 14.4
K d D, PF‐05190457 (M) 5.75E‐09 30.9 12.0
K on D, PF‐05190457 (M−1 s−1) 109275 10.7
Equilibrum time (s) 4423
Additive residual error 0.1, fixed
Proportional residual error (CV%) 33
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Data represent mean, interocassion variability (IOV), and relative standard error (RSE). Maximum binding (B max), tracer equilibrium dissociation constant (K d L), tracer dissociation rate constant (K off L), tracer association rate constant (K on L), drug equilibrium dissociation constant (K d D), drug dissociation rate constant (K off D), drug association rate constant (K on D) estimates are summarized in this table. B max was estimated for each experiment (n = 9 experiments). K d L was estimated separately.

*

median estimate

**

posthoc calculation

Table 1B.

Direct quantitative method parameter summary of PF‐05190457

Table 1B Direct method
Parameter Estimate IOV (CV%) RSE (CV%)
B max – Study (M) 7.12E‐10 — 2.0
K off D, PF‐05190457 (s−1) 0.0002 — 4.7
K d D, PF‐05190457 (M) 3.09E‐9 — 3.9
K on D, PF‐05190457 (M−1 s−1) 65534 — 4.3
Equilibrium time (s) 13689
Additive residual error (CV%) 0, fixed
Proportional residual error for saturation binding data (CV%) 5
Proportional residual error for association data (CV%) 4
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Data represent mean, interocassion variability (IOV) and relative standard error (RSE). Maximum binding (B max), drug equilibrium dissociation constant (K d D), drug dissociation rate constant (K off D), drug association rate constant (K on D) estimates are summarized in this table.

PF‐05190457 is a competitive antagonist with inverse agonist activity

To evaluate functional activity, PF‐05190457 was profiled as an agonist, inverse agonist and as an antagonist. As shown in Figure 2A, the concentration–response curve generated in the agonist mode produced a decreasing curve that dipped below the 0% effect line (basal activity), indicating inverse agonist activity (IC50 4.9 nM CI 3.5–6.7 nM, n = 16). PF‐05190457 also antagonized ghrelin‐stimulated changes with a K i 6.6 nM C.I. 5–8.6 nM (n = 16; Figure 2B). PF‐05190457 was a potent competitive antagonist with a slope of 0.9 ± 0.1 and a K b of 1.8 ± 0.2 nM, n = 10 (Figure 2C).

Figure 2.

Figure 2

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(A) The in vitro functional activity of PF‐05190457 against the human ghrelin receptor as measured by europium‐labelled guanosine‐5′‐triphosphate (Eu‐GTP). The agonist mode measured the ability of PF‐05190457 to act as an agonist, partial agonist or inverse agonist receptor, and the observed negative % effect values were characteristic of an inverse agonist. The antagonist mode measured the ability of increasing concentrations of PF‐05190457 to block ghrelin‐stimulated changes (B). The concentration–response plot is a representative experiment from n = 16 with 1–2 replicates per concentration and plotted as the mean % effect ± SD. (C) is the K b potency of PF‐05190457. Data represent the mean ± SEM at each concentration of PF‐05190457 tested. Concentration ratio = EC50 of ghrelin with PF‐05190457/EC50 of ghrelin without PF‐05190457 (n = 10 experiments).

PF‐05190457 increases gastric vagal afferent firing in rat ex vivo

Ghrelin has been shown to increase food intake via the gastric vagal afferents in vivo, an effect that is abolished in rats with gastric vagotomy (Date et al., 2002). In addition, ghrelin inhibits neuronal firing of mouse vagal afferent nerve fibres ex vivo (Page et al., 2007) and in rats (Supporting Information Figure 3S). The effects of PF‐05190457 on gastric vagal nerve activity were assessed through electrophysiological recordings of gastric afferent nerve activity in a rat ex vivo stomach/vagal preparation. Intragastric arterial administration of PF‐05190457 elicited a concentration‐dependent increase in gastric afferent vagal nerve activity (baseline 9.2 ± 2.1 Hz; PF‐05190457 E max 16.8 ± 4.8 Hz; n = 5; P < 0.05) with an EC50 of 10.7 ± 0.5 nM (Figure 3A and B). The neutral antagonist Compound 2 with no inverse agonism also elicited a concentration‐dependent increase in vagal firing (n = 4; Figure 3C).

Figure 3.

Figure 3

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(A) An example trace of PF‐05190457 E max 10−6 M increasing vagal afferent firing. (B) and (C) Concentration–response curves of PF‐05190457 and compound 2 respectively, on rat gastric vagal afferent firing ex vivo. Data represent mean ± SEM (PF‐05190457 0.1–1 nM n = 4; 10–1000 nM n = 5; compound 2 n = 4).

PF‐05190457 increases intracellular Ca2+ in rat dispersed islets ex vivo

As ghrelin receptors are expressed locally within the pancreatic islet and ghrelin inhibits intracellular Ca2 + within single cells (Gnanapavan et al., 2002; Volante et al., 2002; Wierup et al., 2002; Kageyama et al., 2005; Wierup and Sundler, 2005; Dezaki et al., 2007; Damdindorj et al., 2012), changes in intracellular Ca2 + in rat dispersed islets ex vivo after treatment with PF‐05190457 were measured to (i) assess intracellular Ca2 + increases in the presence of 3 mM (low) and 9 mM (high) glucose; (ii) evaluate the ex vivo potency of PF‐05190457 on intracellular Ca2 +; and (iii) determine whether PF‐05190457 has an additive effect with GLP‐1 on intracellular Ca2 +. A high concentration of PF‐05190457 (100 nM) failed to evoke a change in intracellular Ca2 + in the presence of 3 mM glucose but evoked a maximal increase in intracellular Ca2 + in 9 mM glucose (Figure 4A), which was comparable with the insulin secretagogue glibenclamide (100 nM), confirming efficacy and glucose dependence of PF‐05190457 (n = 4; Figure 4C). In contrast, the neutral antagonist Compound 2 which lacks inverse agonism had no effect (n = 3; Figure 4B and D). PF‐05190457 induced a concentration‐dependent increase in intracellular Ca2 + in 9 mM glucose with an EC50 9.3 ± 1 nM, n = 3–7 (Figure 4E). In contrast to Compound 2 which had no effect, co‐administration of an EC50 of PF‐05190457 (10 nM) and EC50 of GLP‐1 (1 nM; Moens et al., 1996) resulted in a maximal increase in intracellular Ca2 + (n = 6; P < 0.05; Figure 4F).

Figure 4.

Figure 4

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(A) Example raw trace of PF‐5109457 (1 × 10–7 M) in the presence of 9 mM glucose on intracellular Ca2 + in rat dispersed islets (A: 9 mM glucose buffer baseline; B: 1 × 10–7 M PF‐05190457 in 9 mM glucose buffer; C: 9 mM glucose buffer wash; D: unrecorded wash to prevent bleaching and E: 1 × 10–7 M Glibenclamide in 9 mM glucose wash). (B) The same as (A) but with compound 2. (C) The average glucose‐dependent effect of PF‐05190457 on intracellular Ca2 + in rat dispersed islets in the presence of 3 and 9 mM glucose. All data are normalized against the positive control glibenclamide. Data represent the mean ± SEM (n = 4). Comparison of compound 2 to glibenclamide in the presence of 9 mM glucose (n = 3; D). Concentration–response curve of PF‐05190457 showing the mean ± SEM, where n = 3–7 pending concentration (E). (F) Additive effect of GLP‐1 EC50 and PF‐05190457 IC50 on intracellular calcium in rat dispersed islets in the presence of 9 mM glucose. All data are normalized against glibenclamide. Data represent the mean ± SEM (n = 6; ***P < 0.05 two‐tailed t‐test with Dunnett adjustment).

Discussion

Here, we report a novel potent small‐molecule ghrelin receptor competitive antagonist that has (i) equipotent inverse agonism activity; (ii) moderately fast on and fast off kinetics requiring 4 h to achieve equilibrium; (iii) increases rat vagal afferent firing ex vivo, (iv) increases intracellular calcium in rat dispersed islets that is comparable with sulphonylureas but in a glucose‐dependent manner; and (v) has the potential to have additive efficacy on top of approved GLP‐1 receptor agonist therapy.

Since the discovery of ghrelin, various functions of ghrelin, including growth hormone release, gastric acid secretion, gastrointestinal motility, feeding behaviour, glucose metabolism, memory and also antidepressant effects, have been studied (Sakata and Sakai, 2010). Since the first reports of ghrelin as the only peripheral hunger‐stimulating hormone more than a decade ago, much attention has been focused on developing a novel antagonist approach to treat obesity and more recently improve insulin secretion and insulin sensitivity in humans (Broglio et al., 2001, 2002, 2003a, 2003b, 2004; Arosia et al., 2003; Reimer et al., 2003; Dezaki et al., 2004, 2007; Gauna et al., 2004; Lucidi et al., 2005; Damjanovic et al., 2006; Sun et al., 2006; Vestergaard et al., 2007, 2008a, 2008b; Tong et al., 2010). Unfortunately, the field has been hampered due to lack of non‐peptidic antagonists, poor potency, lack of selectivity, limited efficacy and toleration issues which have ultimately prevented testing the hypothesis in humans (Schellekens et al., 2010). As a consequence, there has been a demand for novel strategic approaches to ensure a deeper understanding of the pharmacology of ghrelin receptor antagonists underlying ghrelin‐mediated pathways controlling energy homeostasis and insulin secretion necessary for a unique therapeutic intervention.

PF‐05190457 is the first ghrelin receptor antagonist to advance into human clinical studies (NCT01247896). PF‐05190457 is approximately 10‐fold more potent than previously identified small‐molecule antagonists (Perdonà et al., 2011; Mihalic et al., 2012; Moulin et al., 2013; Cameron et al., 2014), and also has greater than 100‐fold potency and selectivity compared with peptide antagonists. To date, little attention has been spent looking at the kinetics of peptide or non‐peptide ghrelin receptor antagonists to optimize the potency and temporal requirements for efficacy studies. Perdonà et al. (2011 revealed that GSK1614343 and YIL‐781 showed higher potency in FLIPR assays than in [3H]‐IP assays suggesting the hemi‐equilibria conditions were not optimal to determine compound behaviour or potency. Likewise, [d‐Arg1,d‐Phe5,d‐Trp7,9,Leu11]‐substance P is reported to be <100‐fold more potent as an inverse agonist than as an antagonist, yet the temporal conditions of the assays were not reported (Holst et al., 2003). The present study addresses for the first time ghrelin receptor antagonist kinetics. A good correlation was observed between the Motulsky and Mahan methodology and direct radiolabelling of PF‐05190457 which highlighted PF‐05190457 as a moderately fast on and fast off ligand that requires 4 h to achieve equilibrium. To compare kinetics, the D2 receptor antagonists have been most studied and provide the most diverse range in K on and K off reported. The receptor kinetics of D2 receptor antagonist antipsychotic drugs are well studied and range from 3.3E−5 to 5E− 2 s−1 in K off, and 1.8E8 to 2.8E9 M−1 s−1 in K on (Kapur and Seeman, 2000). Their binding affinity and the functional activity has been shown to correlate with K off, highlighting the importance of this measure. Compared with mid‐range binding affinity antipsychotic drugs (sertindole, chlorpromazine and raclopride), PF‐05190457 K off is similar (within twofold), and K on is ~20 000‐fold slower (Kapur and Seeman, 2000). PF‐05190457 K off at the ghrelin receptor is similar (within twofold) to risperidone, paliperidone, aripiprazole, ziprasidone and haloperidol; ~10‐fold slower than olanzipine and dopamine; and ~100‐fold slower than quetiapine, raclopride and IBZM at D2 receptor (Richelson and Souder, 2000). PF‐05190457 K on at ghrelin receptor is approximately twofold slower compared with risperidone, paliperidone, aripiprazole, ziprasidone, haloperidol, olanzipine, dopamine, quetiapine, raclopride and IBZM at the D2 receptor. A summary of PF‐05190457 potency versus time for different assay conditions is provided in Figure 5 and clearly shows how potency increases with longer incubation of PF‐05190457 with the ghrelin receptor and plateaus once equilibrium has been achieved. This effect is important to consider when directly comparing and referring to concentration‐dependent effects across a range of in vitro recombinant and ex vivo preparations which are limited by the temporal conditions necessary for each assay. A full understanding of receptor kinetics is also important when designing complex in vivo or clinical studies when full blockade of the receptor is required over acute or chronic efficacy studies in order to aid in the interpretation of pharmacokinetic/pharmacodynamic relationships and evaluation of adverse events.

Figure 5.

Figure 5

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In vitro and ex vivo potency of PF‐05190457 across all assays against time. Good correlation in potency of PF‐05190457 is observed using assays with the same exposure time, with an observed increase in potency with longer incubation times as equilibrium is achieved around 4 h.

The use of ghrelin receptor antagonists in vivo has been confusing to date; BIM28163 and GSK1614343 surprisingly increased food intake and body wt, whereas two chemically distinct small‐molecule antagonists (compound 14f and YIL‐781) decreased food intake (Halem et al., 2005; Esler et al., 2007; Costantini et al., 2011; Puleo et al., 2012). Whilst the pharmacokinetic properties and effects of central versus peripheral mechanism of actions is unknown, the dilemma raises many potential questions regarding the nature of the antagonist, such as (i) selectivity and off target pharmacology; (ii) does the neutral antagonist act like a partial agonist at the target sites of action depending on receptor densities; (iii) what functional cell‐based screens should be used to elucidate the functional characteristics of the antagonist; and (iv) how would the ghrelin receptor antagonist behave at the site of action underlying ghrelin's orexigenic effects. The authors did a convincing job demonstrating that GSK1614343 effects are not driven by off target pharmacology as the increase in food intake and body wt were not observed in the ghrelin receptor knockout mouse. One notable difference between the antagonists that increased food intake versus those that decreased was the functional assay used to define the antagonist pharmacology. Agents that increased food intake were profiled by blocking ghrelin‐induced increases in calcium. A Ca2 + mobilization assay has been traditionally used because the ghrelin‐induced increases in growth hormone effects via the ghrelin receptor in the pituitary were shown to be Gq‐coupled. However, the ghrelin receptor underlying insulin secretion or food intake via the islet and vagus afferents are unlikely to be Gq mediated. Studies by Dezaki et al. (2007, 2011) and Damdindorj et al. (2012) have shown that exogenous ghrelin stimulates a pertussis–toxin‐sensitive pathway within rodent islets that attenuates cAMP‐PKA signalling, activates Kv2.1 channels inhibiting glucose‐dependent intracellular Ca2 + and insulin secretion. Peng et al. (2012) also demonstrated that ghrelin inhibits insulin release by regulating the expression of inwardly rectifying potassium channel 6.2 in islets. Whilst the ghrelin signalling pathway within vagal afferent nerve terminals has not been reported and technically challenging due to neuronal viability with an overnight toxin treatment, ghrelin does modulate vagal afferent cell bodies excitability by activating KATP conductance via a Gαi‐PI3K‐Erk1/2‐KATP pathway (Grabauskas et al., 2015). Other gastrointestinal anorexigenic agents known to excite gastric vagal afferents, for example, cholecystokinin (CCK), PYY3–36, GLP‐1 and CB1 receptor antagonists, are typically associated with increasing intracellular Ca2 + or cAMP for efficacy (http://www.guidetopharmacology.org; Kakei et al., 2002; Lankisch et al., 2002; Burdyga et al., 2008, 2010; Alexander et al., 2003). Therefore, blocking elevations of intracellular Ca2 + via calcium entry into beta cells or vagal afferents would decrease insulin secretion and vagal afferent firing. The non‐calcium mobilization assays such as forskolin‐stimulated luciferase and [35S]‐GTPγS used in identification of antagonists that decrease food intake may have identified alternative bias antagonists. Here, a non‐traditional screening approach within the ghrelin receptor antagonist competitive space was applied. Using a non‐biased GTPγS screen, the in vitro pharmacology was confirmed in putative native tissues underlying metabolic efficacy such as food intake and insulin secretion once potency, kinetics, selectivity, antagonism and inverse agonism was fully characterized. This approach simply builds the basic translation understanding of the pharmacology and mechanism of action prior to complex in vivo studies which have proved challenging to interpret alone.

Another potential difference to explain observed in vivo differences between antagonists may be whether the small molecules are solely neutral antagonists or also have inverse agonist properties. Because the ghrelin receptor has been shown in vitro to be constitutively active, it is debatable whether a ghrelin receptor inverse agonist or a neutral antagonist would be beneficial for obesity or metabolic disorders (Holst et al., 2003). To date, the majority of nonpeptide compounds have claimed to be antagonists, with the exception of Pasternak et al. (2009) who identified an inverse agonist with poor p.o. bioavailability (Rudolph et al., 2007; Perdonà et al., 2011; Sabbatini et al., 2011; Mihalic et al., 2012; Puleo et al., 2012; Moulin et al., 2013). PF‐05190457 is an equipotent inverse agonist and neutral competitive antagonist working independently of ghrelin tone with the added advantage of not switching to a partial agonist.

Whilst the ghrelin receptor is constitutively active when used in vitro recombinant assays, the translation to inverse agonist activity in native tissues remains uncharacterized. In whole islets and perfused pancreas, a non‐selective peptide ghrelin receptor antagonist and ghrelin anti‐serum have been shown to increase intracellular glucose‐dependent insulin secretion, suggesting an endogenous ghrelin tone (Egido et al., 2002; Colombo et al., 2003; Reimer et al., 2003; Dezaki et al., 2004, 2007, 2008). Here, we report that the ghrelin receptor in rat dispersed islet cells is constitutively active, as a minimal increase in intracellular calcium was observed with a neutral antagonist lacking inverse agonist activity, whereas in contrast PF‐05190457 induced a profound increase that was equally efficacious to glibenclamide at pharmacologically relevant concentrations. Interestingly, the effect of PF‐05190457 was glucose‐dependent which is critical for a type 2 diabetes therapy to prevent complications of hypoglycaemia associated with glucose‐independent treatments like sulphonylureas. The only glucose‐dependent insulin secretagogues approved to date are GLP‐1 receptor agonist analogues and dipeptidyl peptidase IV inhibitors that elevate endogenous GLP‐1 levels. Despite superior efficacy, GLP‐1 receptor agonists are considered third‐line therapy because of dose limiting side effects (nausea) and their mode of administration (injectable). Interestingly, Damdindorj et al. (2012 has shown that ghrelin counteracts GLP‐1 action to stimulate cAMP signalling and insulin secretion in islets. In support of these data, the present study shows for the first time that a ghrelin receptor inverse agonist can have an additive effect with GLP‐1 receptor agonism and could therefore be used in combination with approved GLP‐1 receptor agonists.

In summary, PF‐05190457 is a novel potent ghrelin receptor competitive antagonist with inverse agonist activity in vitro that translates to putative native target tissues of interest involved in energy homeostasis and insulin secretion. The ghrelin receptor is constitutively active in dispersed islets and is the key receptor mediating the inhibitory effects of endogenous ghrelin secreted from the stomach on gastric vagal afferents to modulate gut brain neuronal signalling. PF‐05190457 is a pharmacologically validated clinical candidate with potential to be a first in class treatment for excessive eating disorders, that is, Prader–Willi syndrome or type 2 diabetes, as a monotherapy or in combination with GLP‐1.

Author contributions

J.K. performed in vitro binding and functional experiments (Motulsky/Schild). J.C. was in‐charge in disperse islet calcium imaging experiments. I.S. performed in vitro binding and functional experiments. P.L. participated in the design and interpretation of in vitro pharmacology. S.S. participated in vagal afferent firing experiments. C.V. performed Motulsky analysis and pharmacology temporal analysis. K.C., S.B. and K.M. participated in the design and synthesis of PF‐05190457. K.L. participated in pharmadynamic and PF‐05190457 free concentration study design. Y.Z. was the statistician on all in vitro and ex vivo assay design and analysis. M.J. participated in the design and interpretation of all in vitro and ex vivo studies.

Conflict of interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Supporting information

Figure 3SA is an example electrophysiological trace highlighting ghrelin‐induced inhibition of a rat gastric vagal afferent firing. Capsaicin was used as a positive inhibitor control to confirm afferent neuron activity. 3SB, Same as A but the average inhibition across 3–5 preparations.

Supporting info item

Click here for additional data file. (453.2KB, pdf)

Supporting info item

Click here for additional data file. (147.2KB, pdf)

Acknowledgements

We would like to acknowledge Professor Gino Saccone (Flinders) and his laboratory for vagal afferent extracellular recording training and consultancy. We also acknowledge Jeffrey Koup, Pharm.D., A2PG consultancy for the indirect method analysis of PF‐05190457 receptor kinetics.

Kong, J. , Chuddy, J. , Stock, I. A. , Loria, P. M. , Straub, S. V. , Vage, C. , Cameron, K. O. , Bhattacharya, S. K. , Lapham, K. , McClure, K. F. , Zhang, Y. , and Jackson, V. M. (2016) 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 . British Journal of Pharmacology, 173: 1452–1464. doi: 10.1111/bph.13439.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure 3SA is an example electrophysiological trace highlighting ghrelin‐induced inhibition of a rat gastric vagal afferent firing. Capsaicin was used as a positive inhibitor control to confirm afferent neuron activity. 3SB, Same as A but the average inhibition across 3–5 preparations.

Supporting info item

Click here for additional data file. (453.2KB, pdf)

Supporting info item

Click here for additional data file. (147.2KB, pdf)

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