Tung M. Fong

[18F]MK-9470, a positron emission tomography (PET) tracer for in vivo human PET brain imaging of the cannabinoid-1 receptor

[18F]MK-9470 is a selective, high-affinity, inverse agonist (human IC50, 0.7 nM) for the cannabinoid CB1 receptor (CB1R) that has been developed for use in human brain imaging. Autoradiographic studies in rhesus monkey brain showed that [18F]MK-9470 binding is aligned with the reported distribution of CB1 receptors with high specific binding in the cerebral cortex, cerebellum, caudate/putamen, globus pallidus, substantia nigra, and hippocampus. Positron emission tomography (PET) imaging studies in rhesus monkeys showed high brain uptake and a distribution pattern generally consistent with that seen in the autoradiographic studies. Uptake was blocked by pretreatment with a potent CB1 inverse agonist, MK-0364. The ratio of total to nonspecific binding in putamen was 4–5:1, indicative of a strong specific signal that was confirmed to be reversible via displacement studies with MK-0364. Baseline PET imaging studies in human research subject demonstrated behavior of [18F]MK-9470 very similar to that seen in monkeys, with very good test–retest variability (7%). Proof of concept studies in healthy young male human subjects showed that MK-0364, given orally, produced a dose-related reduction in [18F]MK-9470 binding reflecting CB1R receptor occupancy by the drug. Thus, [18F]MK-9470 has the potential to be a valuable, noninvasive research tool for the in vivo study of CB1R biology and pharmacology in a variety of neuropsychiatric disorders in humans. In addition, it allows demonstration of target engagement and noninvasive dose-occupancy studies to aid in dose selection for clinical trials of CB1R inverse agonists.

The acyclic CB1R inverse agonist taranabant mediates weight loss by increasing energy expenditure and decreasing caloric intake

Cannabinoid 1 receptor (CB1R) inverse agonists are emerging as a potential obesity therapy. However, the physiological mechanisms by which these agents modulate human energy balance are incompletely elucidated. Here, we describe a comprehensive clinical research study of taranabant, a structurally novel acyclic CB1R inverse agonist. Positron emission tomography imaging using the selective CB1R tracer [(18)F]MK-9470 confirmed central nervous system receptor occupancy levels ( approximately 10%-40%) associated with energy balance/weight-loss effects in animals. In a 12-week weight-loss study, taranabant induced statistically significant weight loss compared to placebo in obese subjects over the entire range of evaluated doses (0.5, 2, 4, and 6 mg once per day) (p < 0.001). Taranabant treatment was associated with dose-related increased incidence of clinical adverse events, including mild to moderate gastrointestinal and psychiatric effects. Mechanism-of-action studies suggest that engagement of the CB1R by taranabant leads to weight loss by reducing food intake and increasing energy expenditure and fat oxidation.

The role of melanocortins in body weight regulation: opportunities for the treatment of obesity

Five G-protein-coupled melanocortin receptors (MC(1)-MC(5)) are expressed in mammalian tissues. The melanocortin receptors support diverse physiological functions, including the regulation of hair color, adrenal function, energy homeostasis, feed efficiency, sebaceous gland lipid production and immune and sexual function. The melanocortins (adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH), beta-MSH and gamma-MSH) are agonist peptide ligands for the melanocortin receptors and these peptides are processed from the pre-prohormone proopiomelanocortin (POMC). Peptide antagonists for the melanocortin MC(1), MC(3) and MC(4) receptors include agouti-related protein (AgRP) and agouti. Diverse lines of evidence, including genetic and pharmacological data obtained in rodents and humans, support a role for the melanocortin MC(3) and MC(4) receptors in the regulation of energy homeostasis. Recent advances in the development of potent and selective peptide and non-peptide melanocortin receptor ligands are anticipated to help unravel the roles for the melanocortin receptors in humans and to accelerate the clinical use of small molecule melanocortin mimetics.

Discovery of N-[(1S,2S)-3-(4-Chlorophenyl)-2- (3-cyanophenyl)-1-methylpropyl]-2-methyl-2- {[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide (MK-0364), a novel, acyclic cannabinoid-1 receptor inverse agonist for the treatment of obesity.

The discovery of novel acyclic amide cannabinoid-1 receptor inverse agonists is described. They are potent, selective, orally bioavailable, and active in rodent models of food intake and body weight reduction. A major focus of the optimization process was to increase in vivo efficacy and to reduce the potential for formation of reactive metabolites. These efforts led to the identification of compound 48 for development as a clinical candidate for the treatment of obesity.

Antiobesity Efficacy of a Novel Cannabinoid-1 Receptor Inverse Agonist, N-[(1S,2S)-3-(4-Chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-{[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide (MK-0364), in Rodents

The cannabinoid-1 receptor (CB1R) has been implicated in the control of energy balance. To explore the pharmacological utility of CB1R inhibition for the treatment of obesity, we evaluated the efficacy of N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-{[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide (MK-0364) and determined the relationship between efficacy and brain CB1R occupancy in rodents. MK-0364 was shown to be a highly potent CB1R inverse agonist that inhibited the binding and functional activity of various agonists with a binding Ki of 0.13 nM for the human CB1R in vitro. MK-0364 dose-dependently inhibited food intake and weight gain, with an acute minimum effective dose of 1 mg/kg in diet-induced obese (DIO) rats. CB1R mechanism-based effect was demonstrated for MK-0364 by its lack of efficacy in CB1R-deficient mice. Chronic treatment of DIO rats with MK-0364 dose-dependently led to significant weight loss with a minimum effective dose of 0.3 mg/kg (p.o.), or a plasma Cmax of 87 nM. Weight loss was accompanied by the loss of fat mass. Partial occupancy (30–40%) of brain CB1R by MK-0364 was sufficient to reduce body weight. The magnitude of weight loss was correlated with brain CB1R occupancy. The partial receptor occupancy requirement for efficacy was also consistent with the reduced food intake of the heterozygous mice carrying one disrupted allele of CB1R gene compared with the wild-type mice. These studies demonstrated that MK-0364 is a highly potent and selective CB1R inverse agonist and that it is orally active in rodent models of obesity.

Anti-obesity efficacy of a novel cannabinoid-1 receptor inverse agonist MK-0364 in rodents

The cannabinoid-1 receptor (CB1R) has been implicated in the control of energy balance. To explore the pharmacological utility of CB1R inhibition for the treatment of obesity, we evaluated the efficacy of N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-{[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide (MK-0364) and determined the relationship between efficacy and brain CB1R occupancy in rodents. MK-0364 was shown to be a highly potent CB1R inverse agonist that inhibited the binding and functional activity of various agonists with a binding Ki of 0.13 nM for the human CB1R in vitro. MK-0364 dose-dependently inhibited food intake and weight gain, with an acute minimum effective dose of 1 mg/kg in diet-induced obese (DIO) rats. CB1R mechanism-based effect was demonstrated for MK-0364 by its lack of efficacy in CB1R-deficient mice. Chronic treatment of DIO rats with MK-0364 dose-dependently led to significant weight loss with a minimum effective dose of 0.3 mg/kg (p.o.), or a plasma Cmax of 87 nM. Weight loss was accompanied by the loss of fat mass. Partial occupancy (30–40%) of brain CB1R by MK-0364 was sufficient to reduce body weight. The magnitude of weight loss was correlated with brain CB1R occupancy. The partial receptor occupancy requirement for efficacy was also consistent with the reduced food intake of the heterozygous mice carrying one disrupted allele of CB1R gene compared with the wild-type mice. These studies demonstrated that MK-0364 is a highly potent and selective CB1R inverse agonist and that it is orally active in rodent models of obesity.

Synergistic effects of cannabinoid inverse agonist AM251 and opioid antagonist nalmefene on food intake in mice.

Oral administration of the opioid antagonist nalmefene alone (up to 20 mg/kg) failed to show a significant effect on acute food intake in mice. However, combined oral dosing of nalmefene and subthreshold doses of AM251, a cannabinoid CB1 receptor inverse agonist, led to a significant reduction in food intake in both lean and diet-induced obese (DIO) mice. Furthermore, the anorectic effect of a high dose of AM251 was further enhanced when co-administered with nalmefene. The results support a synergistic interaction between opioid and cannabinoid systems in regulating feeding behavior.

Regulation of Energy Homeostasis by Bombesin Receptor Subtype-3: Selective Receptor Agonists for the Treatment of Obesity

Bombesin receptor subtype 3 (BRS-3) is a G protein coupled receptor whose natural ligand is unknown. We developed potent, selective agonist (Bag-1, Bag-2) and antagonist (Bantag-1) ligands to explore BRS-3 function. BRS-3-binding sites were identified in the hypothalamus, caudal brainstem, and several midbrain nuclei that harbor monoaminergic cell bodies. Antagonist administration increased food intake and body weight, whereas agonists increased metabolic rate and reduced food intake and body weight. Prolonged high levels of receptor occupancy increased weight loss, suggesting a lack of tachyphylaxis. BRS-3 agonist effectiveness was absent in Brs3(-/Y) (BRS-3 null) mice but was maintained in Npy(-/-)Agrp(-/-), Mc4r(-/-), Cnr1(-/-), and Lepr(db/db) mice. In addition, Brs3(-/Y) mice lost weight upon treatment with either a MC4R agonist or a CB1R inverse agonist. These results demonstrate that BRS-3 has a role in energy homeostasis that complements several well-known pathways and that BRS-3 agonists represent a potential approach to the treatment of obesity.

Pharmacological evaluation of LH-21, a newly discovered molecule that binds to cannabinoid CB1 receptor.

LH-21 (5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-hexyl-1H-1,2,4-triazole) was previously reported as a neutral antagonist at the cannabinoid CB1 receptor which, despite its reported poor ability to penetrate into the brain, suppressed food intake and body weight in rats by intraperitoneal administration. In the present study, we studied the mechanism of action of LH-21 by characterizing its in vitro pharmacological properties and in vivo efficacy. LH-21 inhibited the binding of [3H]CP55940 to cloned human and rat CB1 receptors with IC50 values of 631+/-98 nM, and 690+/-41 nM, respectively, and acted as an inverse agonist in a cAMP functional assay using cultured cells expressing human, rat or mouse CB1 receptor. The compound was shown to be brain-penetrant in rats by intravenous administration. Importantly, a single dose of LH-21 (60 mg/kg, i.p.) caused a similar suppression of overnight food intake and body weight gain in wild-type and CB1 receptor knockout mice. Our results suggest that LH-21 is a low affinity inverse agonist for the CB1 receptor and does not act on the CB1 receptor to inhibit food intake in mice.

Conformational Analysis and Receptor Docking of N-[(1S,2S)-3-(4-Chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-{[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide (Taranabant, MK-0364), a Novel, Acyclic Cannabinoid-1 Receptor Inverse Agonist

X-ray crystallographic, NMR spectroscopic, and computational studies of taranabant afforded similar low-energy conformers with a significant degree of rigidity along the C11-N13-C14-C16-C17 backbone but with more flexibility around bonds C8-C11 and C8-O7. Mutagenesis and docking studies suggested that taranabant and rimonabant shared the same general binding area of CB1R but with significant differences in detailed interactions. Similar to rimonabant, taranabant interacted with a cluster of aromatic residues (F(3.36)200, W(5.43)279, W(6.48)356, and Y(5.39)275) through the two phenyl rings and with F(2.57)170 and L(7.42)387 through the CF 3-Pyr ring. The notable distinction between taranabant and rimonabant was that taranabant was hydrogen-bonded with S(7.39)383 but not with K(3.28)192, while rimonabant was hydrogen-bonded with K(3.28)192 but not with S(7.39)383. The strong hydrogen bonding between the amide NH of taranabant and hydroxyl of S(7.39)383 was key to the superior affinity of taranabant to CB1R.