WO2019104324A1

WO2019104324A1 - Ghsr1a antagonist for prader-willi syndrome treatment

Info

Publication number: WO2019104324A1
Application number: PCT/US2018/062611
Authority: WO
Inventors: Theodore Kamenecka; Hua Lin; Roy Smith; Patrick R. GRIFFIN; Cristina GRANDE; Andras Kern
Original assignee: Scripps Research Institute
Current assignee: Scripps Research Institute
Priority date: 2017-11-27
Filing date: 2018-11-27
Publication date: 2019-05-31
Anticipated expiration: 2020-05-27

Abstract

We have developed of Class B GHSR1a antagonists useful for the treatment of the genetic disease Prader-Willi syndrome, which is associated with Type 2 diabetes and obesity. Compounds that can be used in the practice of the invention include SR16281 and YIL781.

Description

GHSRIa Antagonist for Prader-Willi Syndrome Treatment

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority of U.S. provisional application Serial Number 62/590,736, filed Nov. 27, 2017, the disclosure of which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant number AG019230 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Obesity and Type-2 diabetes have reached epidemic proportions. Therapeutic agents that are designed based on new mechanisms are urgently needed. Dopamine (DA) and ghrelin play important roles in regulating food intake and glucose homeostasis.

Attenuated D2R signaling and insulin resistance are characteristic of familial and diet- induced obesity. Increasing dopamine-2 receptor (D2R) signaling by administration of D2R agonists suppresses food intake, and blocking endogenous ghrelin action improves glucose stimulated insulin secretion (GSIS) and insulin sensitivity.

Prader-Willi syndrome is a rare genetic disorder occurring in approximately 1 in 10,000 births and is associated with parent of origin imprinting. In normal subjects, maternal 15q1 1.2 is imprinted, but the paternal chromosome has a working copy of 15q1 1.2. Patients with PWS lack a working copy because of deletion or lack of expression of this region on the paternal chromosome. The resulting phenotype includes short stature, low muscle tone, uncontrollable appetite, incomplete sexual development and impaired cognition. The behavioral characteristics fall into the spectrum of autism spectrum disorders. In narrowing down the DNA region responsible for PWS it was found that individuals with paternal deletion of the SNORD116 gene cluster had all the major characteristic features of PWS [3] and with a few exceptions, the PWS cluster is well conserved between human and mouse [6] While GH replacement is frequently used to treat stunted growth and hypotonia in PWS patients, currently there is no treatment for the most serious issue, insatiable appetite. In PWS, hyperphagia begins early in childhood and is associated with a lack of satiety. Patients typically display abnormal eating behaviors including obsessive food seeking, food storage, foraging and hoarding that represent a lifelong source of distress for them and their families. Patients live a dependent life requiring continuous care and supervision and hyperphagia is associated with significant morbidity and mortality. Prader-Willi syndrome (PWS) is associated with loss or inactivation of paternal chromosome region 15q1 1 q13. In normal subjects paternal 15q1 1-q 13 is active and the maternal allele is silenced. Within this region individuals having paternal deletion of the SNORD116 gene cluster have the major PWS characteristics [3] SNORD116 encodes a small non-translated nuclear RNA expressed abundantly in areas of the hypothalamus that controls feeding. Hence, the absence of a working copy of SNORD116 is linked to the uncontrollable voracious appetite associated with PWS. Indeed without constant supervision, PWS patients will eat themselves to death! Although these patients produce high levels of the orexigenic hormone ghrelin [4, 5] the mechanisms and identity of genes causing the hyperphagia are unknown. Importantly, the PWS cluster is well conserved between human and mouse [6] and the Snord116+/- mouse model of PWS exhibits the major features of the syndrome including hyperphagia, postnatal growth retardation, high ghrelin, delayed sexual maturation, anxiety, and motor learning deficits. Dopamine (DA) and ghrelin play important roles in regulating food intake and increasing dopamine receptor D2 (DRD2 or D2R) signaling by administration of DRD2 agonists suppresses food intake [7]

SUMMARY

The growth hormone secretagogue receptor (GHSR), or ghrelin receptor, is a G protein-coupled receptor that binds ghrelin and plays a role in energy homeostasis and regulation of body weight. Development of Class B (see definition below) GHSRI a antagonists can provide new information on regulatory pathways controlling appetite that are lacking in PWS so these may be targeted for therapeutic intervention.

We have developed an agent that enhances D2R signaling selectively in appetite regulating neurons. Class B GSHRI a antagonists enhance D1 R activity and non-canonical signaling via GHSR1 a:D1 R heteromers, and perhaps enhancement of DA activation via GHSR1 a:D2R. The role of GHSR1 a:D1 R on appetite is unclear; therefore, GHSRI a antagonists that enhance GHSR1 a:D1 R signaling alone and those that enhance

GHSR1 a:D2R alone have been tested for appetite suppression in mice. These studies can facilitate development of antiobesity agents. More importantly, the class B GSHRI a antagonist SR16281 reduced food intake in a genetic mouse model of Prader Willi

Syndrome. This mouse model contains a deletion of a region of chromosome 15 mimicking the deletion detected in human patients and the phenotype of the mouse demonstrates increased hyperphasia as well as cognitive and behavioral defects, reminiscent of human subjects with the syndrome.

A probe study tested the dose-dependent effects of SR16281 on food intake (normal chow) in the hyperphagic Prader-Willi mouse (Snordl 16+/-) and its wild type littermate. The Snord116+/- exhibits extreme hyperphagia, and when housed at thermoneutral temperature is morbidly obese. At 20 mg/kg, food intake was markedly reduced in wild type mice (p<0.01). SR16281 dose-dependently reduced food intake in the Snord116+/- mice.

Suppression of food consumption was sustained in Snord116+/- mice by SR16281 (10 mg/kg) during the 12 h light cycle, although not in the WT counterparts at this dose.

Intriguingly, the hyperphagic Snord116+/- mice eat more during the light cycle compared to WT mice. SR16281 at 10 mg/kg normalizes food intake in Snord116+/- mice to match that of WT mice regardless of whether the mice were dosed at the beginning of the light or dark cycle. These differences indicate a greater window for suppressing hyperphagia in the Prader-Willi mice compared to WT mice.

Accordingly, the invention provides, in various embodiments, a method of treatment of Prader Willi syndrome, comprising administering to a patient afflicted therewith an effective dose of a compound of formula (I)

(I),

wherein X is (C1-C4)alkyl or halo. For instance, X can be methyl. For instance, X can be fluoro.

For a compound of formula (I), when X is methyl, the compound is termed SR16281 :

. In various embodiments, the invention also can provide a method of treatment of type 2 diabetes, comprising administering to a patient afflicted therewith an effective dose of a compound of formula (I)

(I),

In various embodiments, the invention also so provide a method of treatment of obesity, comprising administering to a patient afflicted therewith an effective dose of a compound of formula (I)

(I),

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. The effect of structurally distinct GHSRI a antagonists on [Ca²⁺], mobilization in HEK293 cells expressing GHSR1 a:DRD2 heteromers.

Figure 2. Opposite effects of structurally distinct GHSRI a antagonists (YIL781 and

JMV2959) on GHSR1 a:DRD1 ; A. Dopamine induced [Ca²⁺], mobilization; B. BRET analysis Nluc-GHSR1 a + SNAP-DRD1 in the presence of vehicle, JMV2959, YIL781 illustrating modification of allosteric interactions according to GHSRI a antagonist structure.

Figure 3. Compound progression scheme for GHSRI a-selective antagonists.

Figure 4. GHSRI a antagonist YIL781 (YIL) suppresses food intake (as described by Esler et al. 2007) and enhances cabergoline (Cab) induced suppression of food intake.

Figure 5. Selected GHSRI a antagonists (functional ICso s) from the primary or patent literature. Figure 6. Dose response curves illustrating potencies of 14 new Class B GHSRI a antagonists compared to YIL781 that enhance DA (100 nM)-induced Ca²⁺ mobilization in HEK293 cells expressing GHSRI a (left), GHSR!a:D2R (middle) and GHSR1 a:D1 R (right). Figure 7. Improved potency of SR16281 compared to YIL781 on augmenting DA-induced Ca²⁺ mobilization via GHSR1 a:D2R.

Figure 8. Dependence of the anorexigenic effect of a D2R agonist on GHSRI a in fasted mice. Effect of cabergoline administration on food intake in controls (n = 4 for cabergoline and vehicle) and Ghsr^loxP /Drd2^Cre mice (n = 4 for cabergoline and vehicle). Mice were injected i.p. with cabergoline (0.5 mg/kg) in 100 pi of physiological saline or with 100 pi saline alone (vehicle). Food intake was measured at 1 , 2, 4, 6, 20, and 24 hr after injections. *p < 0.05. **p < 0.01 versus control vehicle treatments. The data represent the mean ± SEM for each time point.

Figure 9. Suppression of food intake (1 hr) in wild type (WT) and Snord116+/- (Het) mice by SR16281.

Figure 10. Inhibitory effects of 10 mg/kg SR16281 ± cabergoline on food intake during 12 hr light cycle illustrating increased sensitivity in Snord116+/- compared to WT mice.

DETAILED DESCRIPTION

GHSR1 a:D2R alone have been tested for appetite suppression in mice. These studies will facilitate development of antiobesity agents. More importantly, the class B GSHRI a antagonist SR16281 reduced food intake in a genetic mouse model of Prader Willi

Prader-Willi syndrome (PWS) is associated with loss or inactivation of paternal chromosome region 15q1 1 q13. In normal subjects paternal 15q1 1-q 13 is active and the maternal allele is silenced. Within this region individuals having paternal deletion of the SNORD116 gene cluster have the major PWS characteristics [3] SNORD116 encodes a small non-translated nuclear RNA expressed abundantly in areas of the hypothalamus that controls feeding. Hence, the absence of a working copy of SNORD116 is linked to the uncontrollable voracious appetite associated with PWS. Indeed without constant supervision, PWS patients will eat themselves to death! Although these patients produce high levels of the orexigenic hormone ghrelin [4, 5] the mechanisms and identity of genes causing the hyperphagia are unknown. Importantly, the PWS cluster is well conserved between human and mouse [6] and the Snord116+/- mouse model of PWS exhibits the major features of the syndrome including hyperphagia, postnatal growth retardation, high ghrelin, delayed sexual maturation, anxiety, and motor learning deficits. Dopamine (DA) and ghrelin play important roles in regulating food intake and increasing dopamine receptor D2 (DRD2 or D2R) signaling by administration of DRD2 agonists suppresses food intake [7] A ghrelin receptor (GHSRI a) antagonist (YIL781) enhances the anorexigenic effect of the dopamine receptor-2 (DRD2) agonist cabergoline on ad libitum feeding of fasted (12h) C57BL/6 mice.

Our central hypothesis is that hyperphagia in PWS is caused by attenuated

GHSRI a-dependent hypothalamic DRD2 signaling. This hypothesis is formulated on our team’s recent discovery that the anorexigenic effect of a DRD2 agonist is dependent on allosteric interactions between two GPCRs, the GHSRI a and DRD2 receptors, that results from GHSR1 a:DRD2 heteromer formation exclusively in the hypothalamus [1 , 2] Allosteric interactions between GHSRI a and DRD2 in the heteromeric complex produce non- canonical DA/DRD2 signaling through phospholipase C and mobilization of intracellular [Ca²⁺] instead of canonical DRD2 signaling (inhibition of cAMP production) [1]

We have developed cell-based assays that allow classification of GHSRI a antagonists into two distinct functional classes: Class A, or those that inhibit, and Class B, those that augment non-canonical DA/DRD2 signal transduction via GHSR1 a:DRD2 heteromers. Using these assays, we modified the GHSRI a antagonist YIL781 to develop compounds of both Class A and Class B. In contrast to Class A GHSRI a antagonists, Class B compounds inhibit food intake in the Snord116+/- mouse.

Our studies involve the use of cell lines and the Snord116+/- mouse that inform on GHSR1 a:DRD receptor signaling and aid in compound development. While hyperphagia exhibited by young Snord116+/- mice is modest, like humans, ghrelin levels are elevated and hyperphagia becomes more evident during adulthood. Also, like PWS in humans, the Snord116+/- mouse exhibits impaired growth associated with low growth hormone (GH). More importantly, the Snord116+/- mouse exhibits the major features of PWS such as hyperphagia, postnatal growth retardation, high ghrelin, delayed sexual maturation, anxiety, and motor learning deficits. At thermoneutrality, Snord116+/- mice exhibit significant weight gain versus WT litter mates.

Despite attempts to make correlations between phenotype and changes in hormones that regulate appetite in PWS, none have yet emerged [10, 1 1] Hence, knowledge of factors and gene regulatory pathways contributing towards obsessive overeating in PWS is lacking. A consistent finding is that levels of the orexigenic hormone ghrelin are elevated [4, 5, 1 1]; nevertheless, the relationship between ghrelin and overeating is unclear. We have discovered that the anorexigenic activity of DRD2 agonists is dependent on allosteric interactions with GHSRI a [1] Formation of GHSR1 a:DRD2 heteromers in cells results in mobilization of intracellular [Ca²⁺] by non-canonical dopamine signaling; this same signaling occurs in hypothalamic neurons. In mice, administration of ghrelin increases food intake and cabergoline inhibits food intake; thus, it is possible that cabergoline inhibits feeding by interfering with ghrelin signaling. To investigate this link further, food intake was measured in ghrelin-/-, ghsr-/- mice and WT controls treated with cabergoline. Interestingly, food intake was suppressed in ghrelin-/- and WT mice, but the ghsr-/- mice were completely refractory to the anorexigenic effects of cabergoline supporting the notion of GHSR-dependency on DRD2 agonists effects on appetite [1] Similar results were obtained in mice where ghsr was selectively inactivated in DRD2 expressing neurons. To test whether the allosteric interaction between DRD2 and GHSRI a could be targeted pharmacologically, mice were pretreated with the highly selective Class A GHSRI a antagonist JMV2959 and then administered cabergoline. As predicted by our hypothesis, JMV2959 blocked cabergoline-induced anorexia [1] The demonstration that JMV2959 treatment of WT mice recapitulates the phenotype observed in ghsr-/- mice indicates that the resistance of ghsr-/- mice to cabergoline treatment is further evidence of an allosteric interaction between GHSRI a and DRD2 for DRD2-mediated inhibition of food intake. This result also argues against possible developmental changes caused by ghsr ablation as an explanation of the resistance of ghsr- /- mice to cabergoline. These collective results provide additional evidence that cabergoline- induced anorexia is dependent upon allosteric interactions between GHSRIa and DRD2.

Our findings are of fundamental importance because they support the notion that in subsets of neurons co-expressing GHSRI a and DRD2, allosteric modulation by GHSRI a can modify the action of endogenous dopamine.

JMV2959 and YIL781 exhibit opposing actions on non-canonical DA signaling via

GHSR1 a:DRD:

We have categorized GHSRI a antagonists into two distinct functional classes: Class A or those that inhibit DA/DRD signaling, and Class B, those that augment non-canonical DA/DRD signal transduction via GHSR1 a:DRD receptors. Our preliminary animal studies suggest that JMV2959 is a Class A antagonist and YIL781 is a Class B antagonist. To confirm this, and to determine if the different effects of these two structurally distinct GHSRI a antagonists on feeding behavior could be predicted from in vitro assays, GHSRI a and DRD2 were co-expressed in HEK293 cells. In the absence of ghrelin, dopamine (DA) dose dependently activated GHSR1 a:DRD2 heteromers to induce [Ca²⁺], mobilization [1] The GHSRI a antagonist JMV2959, and the peptide GHSRI a inverse agonist (Subst P derivative) dose-dependently inhibited DA (100 nM) induced [Ca²⁺], mobilization (Fig. 1A). By contrast the structurally distinct antagonist YIL781 (discussed below) increased DA- induced [Ca²⁺]i mobilization (Fig. 1 B), and like JMV2959 and the Subst P derivative, YIL781 dose-dependently inhibited ghrelin (10 nM) induced mobilization of [Ca²⁺], (Fig. 1C). Neither compound binds to or activates the DRD receptors directly. These results confirm that JMV2959 is a Class A GHSRI a antagonist and YIL781 belongs to Class B. Importantly, other GHSRI a antagonists that failed to suppress food intake in our studies also

demonstrated Class A behavior in these cell assays.

Furthermore, these results recapitulate the effects of Class A and B GHSR1 a antagonists on DRD2 agonist-induced [Ca²⁺] mobilization observed in primary hypothalamic neurons [1] Similar to that observed with DRD2, DA activated non-canonical signaling and [Ca²⁺] mobilization in GHSR1 a:DRD1 heteromers, which was also inhibited by JMV2959. YIL781 enhanced [Ca²⁺] mobilization induced by DA via GHSR1a:DRD1 heteromers. In fact, YIL781 was more potent at enhancing DA induced [Ca²⁺] mobilization through

GHSR1 a:DRD1 than through GHSR1 a:DRD2 (compare Fig. 2A and Fig. 1 B). To determine whether the opposing effects of YIL781 and JMV2959 on DA-induced mobilization of [Ca²⁺] are explained by allosteric interactions between the two receptors, we performed

bioluminescence resonance energy transfer (BRET) analysis. Nluc-GHSR1a and SNAP- DRD1 were co-transfected into HEK293 cells in the presence and absence of the GHSRI a antagonists (Fig. 2B). Compared to vehicle, BRET titration in the presence of JMV2959 produced a significant decrease in the BRETmax (p<0.05), whereas YIL781 increased the BRETmax (p<0.05, Fig. 2B). BRETso was unaffected showing that the GHSRI a antagonists acted by altering allosteric interactions between GHSRI a and DRD1.

Since DRD2 anorexigenic activity is dependent on signaling through GHSR1 a:DRD2, the response to a DRD2 agonist is dependent on the numbers of these receptors on the surface of the hypothalamic neurons. How does high ghrelin affect hyperphagia? Does ghrelin increase GHSRI a concentrations resulting in hyperstimulation, or reduce the numbers of GHSR1a:DRD2 heteromers on the cell surface via internalization thereby reducing anorexigenic signaling? These possibilities can be tested. Internalization of GHSRI a by endogenous ghrelin is preventable by treating mice with a GHSRI a antagonist. In the case of GHSRI a antagonist YIL781 , the anorexigenic action of cabergoline is augmented (Fig. 4). Our studies also provide important information on whether combination therapy using an approved drug, cabergoline, co-administered with a Class B GHSRI a antagonist, could be an effective approach for treating PWS associated hyperphagia.

Mechanisms and gene expression pathways that cause life-threatening hyperphagia in PWS are unknown. Members of our team have shown that the anorexigenic effects of DRD2 agonists are dependent upon allosteric interactions between GHSRIa and DRD2 as a likely result of formation of GHSR1 a:DRD2 heteromers [1] Indeed, ghsr-/- mice are completely resistant to the anorexigenic effects of cabergoline, a D2 agonist. Importantly, the allosteric interactions result in non-canonical DRD2 signaling. D2 monomers or homomers suppress cAMP in response to dopamine or D2 agonists; however, activation of

GHSR1 a:DRD2 by dopamine results in signaling through [Ca²⁺] rather than cAMP. Hence, our approach only affects dopamine signaling in neurons expressing both GHSRI a and DRD2, and co-localization of these two receptors is limited and enriched in the

hypothalamus. This specific allosteric interaction is a new discovery that can be exploited pharmacologically to manipulate function. A GHSRIa ligand modifies signaling of DRD2 by an allosteric mechanism. To our knowledge it is the first example of targeting this system to suppress food intake by augmenting dopamine signaling through an allosteric mechanism. A GHSRI a antagonist, dictated by its chemical structure, will either inhibit or enhance DRD2 agonist signaling via GHSR1 a:DRD2. For example, two GHSRI a antagonists JMV2959 and YIL781 both inhibit ghrelin binding and activation of GHSRI a [12, 13]; however, JMV2959, blocks the suppressive effect of cabergoline on food intake [1] In contrast, food intake was suppressed by each YIL781 and cabergoline alone, but the combination augments the anorexigenic activity (Fig. 4, p<0.05).

This putative mechanism explains why some GHSRI a antagonists tested as antiobesity agents increased appetite while others decreased appetite. As a result of this disconnect, most if not all discovery programs were abandoned based on insufficient understanding of GHSRI a signaling. We hypothesize that in PWS the concentration of GHSRI a, and hence GHSR1 a:DRD2 heteromers, on the surface of hypothalamic neurons is low because of sustained high levels of circulating ghrelin. Low levels of these heteromers on the cell surface will result in unopposed orexigenic activity. This situation is reversible by blocking ghrelin-mediated GHSRI a internalization with YIL781. Our breakthrough was the demonstration that by modifying the chemical structure of YIL781 , we identified compounds such as SR16281 that enhance non-canonical DRD2 signaling (Class B), and others that block it (Class A). In contrast to compounds that block, those that enhance DRD2 signaling also inhibit food intake in the Snord116+/- mouse. To guide medicinal chemistry efforts we developed cell-based assays to test which GHSRI a antagonists inhibit (Class A) and which enhance (Class B) DA/DRD2 induction of [Ca²⁺] transients via GHSR1 a:DRD2. These effects of GHSRI a antagonists on DRD2 agonist-induced signal transduction with dependence on GHSRI a were confirmed in hypothalamic slices and neurons from wild type and Ghsr-/- mice [1] On this basis, we selected Class B antagonists for structural optimization, followed by pharmacokinetics and testing in mice for inhibition of food intake. In testing our central hypothesis we plan to design additional Class B GHSRI a antagonists that augment DA activation of GHSR1 a:DRD2 selectively over GHSR1 a:DRD1. However, it is possible that selectivity over DRD1 is not required. The result of our studies will inform on the need for or avoiding augmentation of GHSR1 a:DRD1 signaling.

We have in hand lead GHSRI a antagonists (YIL781 and SR16281) that demonstrate non-canonical signaling through both GHSR1a:DRD1 and GHSR1 a:DRD2 receptors or heteromers (Fig. 6). Our novel lead molecules have good plasma and brain exposure following intraperitoneal (IP) administration in mice (Table 2). SR16281 decreases food intake in Snord116+/- mice in vivo (Figs. 9-10).

Compound Synthesis: There are more than a dozen chemically distinct GHSRIa antagonists or inverse agonists identified by pharmaceutical companies published in the primary and patent literature [13, 22-38] For many of these analogs, there is no in vivo data, but for several of them, effects on feeding or food intake have been published. Some of these compounds have been reported to increase food intake when dosed to rats (cmpd 1 , Fig. 5)

[30, 31].

With no guidance on where to initiate SAR investigations based on GHSR1 a:DR signaling, we started with the structural series related to YIL781 (cmpd 4, Fig. 5), which produced the desired pharmacological effect of reducing food intake in mice. Scientists at Bayer reported this class of potent and selective small molecule GHSRI a antagonists typified by YIL781. Several analogs from this scaffold were shown to suppress food intake and promote body weight loss through loss of fat mass. However, there was not a clear correlation between drug brain concentrations and the reduction in food intake. Based on results described above in the significance section with JMV2959 and YIL781 , the variability likely reflects their ability to impact signaling through GHSR1 a:DRD heteromers. Although YIL781 augments DA-induced [Ca²⁺], mobilization and enhances the anorexigenic effect of cabergoline, its effects are short-lived (Fig. 4). YIL781 is also considerably more potent on GHSR1 a:DRD1 than on GHSR1 a:DRD2; however, its PK characteristics (short half-life) might explain the short duration of action.

Based on the structure of YIL781 , several new GHSRI a antagonists were synthesized (Table 1 , below). Synthesis of this series is highlighted in Scheme 1 [24]

Conditions: a) K₂C0₃; b) CH₃C(OCH₃)₃; c) TFA; d) (CH₃)₂CHI, K₂C0₃; e) R-OH, Cs₂C0₃, Cul

The chemistry is routine, following standard protocols as described in the primary and patent literature [24, 39] All synthetic analogs demonstrate antagonism of ghrelin- induced [Ca²⁺], mobilization in cells expressing GHSRI a (Fig. 6 left panel), but in cells expressing GHSR1 a:DRD2 (Fig. 6 center), or GHSR1 a:DRD1 (Fig. 6 right panel), they enhance DA-induced [Ca²⁺], mobilization. Although potencies vary, some selectivity is noted. YIL781 activates both GHSR1 a:DRD1 and GHSR1 a:DRD2, with greater activity on

GHSR1 a:DRD1. The p-tolyl analog SR16281 was >100-fold more potent on GHSR1 a:DRD2 compared to YIL781 (Fig. 7). An increase in the size of the substituent at the para-position of the phenol ring in YIL781 leads to a remarkable shift in potency and selectivity for

GHSR1 a:DRD2 over GHSR1 a:DRD1. This trend is observable, as methyl (SR16281) is better than chlorine (SR16279), which is better than fluorine (YIL781) (Figs. 6 and 7). Good correlation is observed as increasing the size of the phenoxy group (SR18279, SR18280, SR18282) leads to additional analogs with enhanced signaling through GHSR1 a:DRD2, whereas compounds lacking para-substitution lose potency and efficacy (SR18283, SR18284). YIL781 potently increases signaling through GHSR1 a:DRD1 heteromers while retaining moderate activity at GHSR1 a:DRD2 (Fig. 6). SR16281 is a marked improvement over YIL781 in augmenting signaling through GHSR1 a:DRD2, but it is also potent on GHSR1 a:DRD1 heteromers (Fig. 6), representing a potent dual modulator of DRD1 and DRD2. The goal is to modify YIL781 to generate analogs that are selective towards either DRD1 or DRD2 heteromers. With such compounds in-hand, we can interrogate the function of each heteromer complex in vivo with regards to effects on hyperphagia in Snord116+/- and WT mice.

The aryloxy position of the molecule appears to be a pivot point for selectivity between GHSR1 a:DRD1 and GHSR1 a:DRD2 receptors. Two compounds from the quinazolone series, SR16279 and SR16281 were profiled for in vivo exposure in mice

(Table 2). SR16281 had excellent CNS exposure following 30 mg/kg IP injection. SR16279 also demonstrated high brain penetration with a 10 mg/kg ip dose and excellent

pharmacokinetic parameters in mice following iv dosing. Hence, compounds from this particular series are likely good candidates for probing the central effects of GHSR1 a:DRD2 signaling and are likely not substrates for P-glycoprotein.

Mice where GHSRI a is selectively inactivated in DRD2 neurons (Ghsr^loxP/Drd2^Cre) are resistant to cabergoline-induced inhibition of food intake (Fig. 8). The dependence on Ghsr for DRD2 agonist-induced suppression of food intake in global Ghsr-/- mice has been reported [1 ] To confirm the requirement for coexpression of GHSRI a and DRD2 in the same neuron, Ghsr was selectively inactivated in DRD2 expressing neurons using ^loxPGhsr^plox and Drd2^Cre mice to generate Ghsr^loxP/Drd2^Cre mice. Mice were fasted overnight and then injected i.p. with cabergoline (0.2 mg/kg) in 100 pi of physiological saline or with 100 pi saline alone (vehicle). Food intake was measured at 1 , 2, 4, 6, and 24 hr after injections. Food intake was markedly suppressed by cabergoline in control mice, but in mice where Ghsr was selectively inactivated in DRD2 expressing neurons, food intake was unaffected by cabergoline, supporting the hypothesis that suppression of food intake by a DRD2 agonist is dependent on co-expression of GHSRI a with DRD2 in the same cell. This correlates with previous data wherein Ghsr-/- mice are resistant to cabergoline-induced suppression of food intake [1 ] Although JMV2959 blocks DRD2 signaling through

GHSR1 a:DRD2 in hypothalamic neurons, DRD2 activity in the striatum would be unaffected, because there is no evidence for formation of GHSR1 a:DRD2 heteromers in the striatum. YIL781 enhances the suppressive effect of the dopamine receptor-2 (DRD2) agonist cabergoline on food intake whereas_JMV2959 does not prevent ghrelin enhanced food intake [63] Additionally, in diet-induced obese (DIO) mice YIL781 improved glucose tolerance, reduced food intake, and weight loss; pair-feeding experiments indicated weight loss was associated with lower food intake [12]

A probe study tested the dose-dependent effects of the novel analog SR16281 on food intake (normal chow) in the hyperphagic Prader-Willi mouse (Snordl 16+/-) and its wild type littermate. The Snordl 16+/- exhibits extreme hyperphagia, and when housed at thermoneutral temperature is morbidly obese. At a dose of 20 mg/kg (IP), food intake was markedly reduced in wild type mice (p<0.01) and dose dependently reduced in the

Snordl 16+/- mice (Fig. 9). Suppression of food consumption was sustained in Snordl 16+/- mice by SR16281 (10 mg/kg) during the 12 h light cycle, although not in the WT counterparts at this dose (Fig. 10, left). Intriguingly, the hyperphagic Snordl 16+/- mice eat more during the light cycle compared to WT mice. SR16281 at 10 mg/kg normalizes food intake in Snordl 16+/- mice to match that of WT mice regardless of whether the mice were dosed at the beginning of the light or dark cycle. These differences indicate a greater window for suppressing hyperphagia in the Prader-Willi mice compared to WT mice. At a dose of 10mg/kg SR16281 alone had no effect on food intake in WT mice; however, at this same dose it was effective alone and in combination with cabergoline in Snordl 16+/- mice. Although a surprising result, it indicates that the mechanism of suppression of food intake is different in the PWS model. It also shows that the inhibitory effect on feeding is unlikely to be caused by inducing sickness; the treated mice showed no adverse effects in response to SR16281. For the Snord116+/- mice, reduced food intake was evident at 1 h (Fig. 9) and the effect was sustained for up to 12 h, no inhibitory effects were observed during the dark cycle (Fig. 10, right), which may relate to drug clearance, or more interestingly may have great biological significance (circadian) since overeating in the Snord116+/- mice occurs during the light cycle.

Testing Class B GHSRI a antagonists for food intake controlling for biological variables: A scheduled feeding paradigm is employed to synchronize metabolism and activity to the time of food presentation [64, 65] To reduce biological variables, testing is conducted in age-matched intact virgin males and females. Males and females are housed separately. No attempts is made to control for changes in sex hormones in the females. We, and others have shown that 6-10 animals/group is sufficient to detect statistical significance (p<0.05) in food intake with YIL781 and SR16281 (see Figs. 9-10) using 2-way analysis of variance and Bonferroni post hoc tests (Graphpad Prism, Prism 5.0, San Diego, CA).

Individuals analyzing food intake data are blinded as to which mice have been treated with vehicle, Class B antagonist + cabergoline. Mice are carefully observed each day by an independent observer to check for any drug related changes in activity or demeanor following injection. Adverse events are immediately reported to the PI. Should poor reproducibility be noted in females possibly as a result of differences in reproductive cycling, this will be addressed by repeating the studies in ovariectomized mice. Six month-old and 12 mo Snordl 16+/-m ice and WT littermates are housed individually for 10 days allowing access to food for 3 h during the light phase. The next morning (9:00 am) vehicle and vehicle containing three different doses of the GHSRI a antagonist + cabergoline are injected i.p.

The mice are presented with food immediately following injection, and cumulative food intake is measured. Episodic food and water intake is monitored using the BioDAQ system. Body weight and body composition (determined by NMR) are recorded before and after treatment and tested for significance by 2-way ANOVA and Bonferroni post hoc tests. Data are analyzed using GraphPad Instat Software and differences judged to be statistically significant if P<0.05. Documents Cited

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All patents and publications referred to herein are incorporated by reference herein to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.

Tables

Table 1. New synthetic GHSRla analogs

Table 2. DMPK parameters of new Class B GHSRla antagonists SR16281 and SR16279

¹ 0.5mg/kg IV; * 30mg/kg IP; ^& 10mg/kg IP

Claims

CLAIMS What is claimed is:

1. A method of treatment of Prader Willi syndrome, comprising administering to a patient afflicted therewith an effective dose of a compound of formula (I)

(I), wherein X is (C1-C4)alkyl or halo.

2. The method of claim 1 wherein X is methyl.

3. The method of claim 1 wherein X is fluoro.

4. A method of treatment of type 2 diabetes, comprising administering to a patient afflicted therewith an effective dose of a compound of formula (I)

(I), wherein X is (C1-C4)alkyl or halo.

5. The method of claim 4 wherein X is methyl.

6. The method of claim 4 wherein X is fluoro.

7. A method of treatment of obesity, comprising administering to a patient afflicted therewith an effective dose of a compound of formula (I)

(I),

wherein X is (C1-C4)alkyl or halo.

8. The method of claim 7 wherein X is methyl.

9. The method of claim 7 wherein X is fluoro.