Fatty acid amide hydrolase inhibitors. 3: tetra-substituted azetidine ureas with in vivo activity.

We describe here our attempts to optimise the human fatty acid amide hydrolase (FAAH) inhibition and physicochemical properties of our previously reported tetrasubstituted azetidine urea FAAH inhibitor, VER-156084. We describe the SAR of a series of analogues and conclude with the demonstration of in vivo dose-dependant FAAH inhibition in an anandamide-loading study in rats.

Discovery and functional evaluation of diverse novel human CB(1) receptor ligands.

Ligand-based virtual screening with a 3D pharmacophore led to the discovery of 30 novel, diverse and drug-like ligands of the human cannabinoid receptor 1 (hCB(1)). The pharmacophore was validated with a hit rate of 16%, binding selectivity versus hCB(2), and expected functional profiles. The discovered compounds provide new tools for exploring cannabinoid pharmacology.

Fatty acid amide hydrolase inhibitors. Surprising selectivity of chiral azetidine ureas.

We report the discovery of a novel, chiral azetidine urea inhibitor of Fatty Acid Amide Hydrolase (FAAH,) and describe the surprising species selectivity of VER-156084 versus rat and human FAAH and also hCB1.

Discovery and optimization of potent and selective functional antagonists of the human adenosine A2B receptor.

We herein report the discovery of a novel class of antagonists of the human adenosine A2B receptor. This low molecular weight scaffold has been optimized to offer derivatives with potential utility for the alleviation of conditions associated with this receptor subtype, such as nociception, diabetes, asthma and COPD. Furthermore, preliminary pharmacokinetic analysis has revealed compounds with profiles suitable for either inhaled or systemic routes of administration.

Discovery of a novel class of selective human CB1 inverse agonists.

Ligand-based virtual screening led to the discovery of a new class of potent inverse agonists of the human cannabinoid receptor 1, hCB(1), which are selective versus hCB(2). These CB(1) ligands present intriguing departures from a classical CB(1) antagonist pharmacophore. Elements of SAR are discussed in this context.

A comparison of the effects of the CB(1) receptor antagonist SR141716A, pre-feeding and changed palatability on the microstructure of ingestive behaviour.

RATIONALE: The cannabinoid CB(1) receptor inverse agonist SR141716A (rimonabant) is known to cause hypophagia and this study uses microstructural data to elucidate the relevant behavioural mechanisms. OBJECTIVES: The aim of these studies was to determine the behavioural changes induced by SR141716A in animals consuming either a fat or carbohydrate solution. These behavioural changes were directly compared with those induced by behavioural manipulations that modify motivational state and palatability. METHODS: Male hooded Lister rats drank a highly palatable fat emulsion (10% Intralipid) or a carbohydrate solution (10% sucrose) during 30-min test sessions. Microstructural analyses of licking patterns were made after either administration of SR141716A (0, 0.3, 1 and 3 mg/kg, ip) or one of the after behavioural manipulations: pre-feeding, addition of quinine to the fat solution or changes in sucrose concentration. RESULTS: Intake of the fat solution was decreased after both the drug treatment and the behavioural manipulations of pre-feeding and addition of quinine. Pre-feeding and SR141716A-induced reductions were mediated via changes in bout number whereas addition of quinine caused a decrease in bout size. Although sucrose drinking was also decreased by pre-feeding, reduced sucrose concentration and SR141716A, the drug did not significantly alter the microstructure of intake. CONCLUSIONS: The effects of SR141716A on consumption of Intralipid solutions are likely to reflect changes in motivational state rather than modified hedonic impact.

The cannabinoid CB1 receptor inverse agonist, rimonabant, modifies body weight and adiponectin function in diet-induced obese rats as a consequence of reduced food intake.

The cannabinoid CB1 receptor inverse agonist rimonabant induces hypophagia and body weight loss. Reduced body weight may potentially be due to decreased food intake or to direct metabolic effects of drug administration on energy expenditure. This study uses a paired-feeding protocol to quantify the contributions of energy intake to rimonabant-induced body weight loss. Diet-induced obese (DIO) rats were dosed with rimonabant (3, 10 mg/kg PO once daily) and matched with pair-fed controls. Food intake and body weight were measured daily. Blood samples and adipose tissue were collected on day 15 for measurement of plasma adiponectin and adiponectin mRNA levels. DIO rats treated with rimonabant and pair-fed controls showed very similar changes in body weight. Although tolerance developed to the anorectic effect of rimonabant, total food intake was significantly decreased over the 14-day study period and fully accounted for the observed reductions in body weight. Adiponectin mRNA and plasma adiponectin were elevated in vehicle-treated chow-fed animals compared to obese controls, and did not differ between rimonabant-treated and pair-fed animals. The similarities between rimonabant-treated and pair-fed animals in body weight loss and the absence of differences in measures of adiponectin activity between drug-treated and pair-fed animals suggest that the outcomes of this experiment were solely mediated by the drug-induced reduction in food intake.

Reduced hypophagic effects of d-fenfluramine and the 5-HT2C receptor agonist mCPP in 5-HT1B receptor knockout mice.

RATIONALE: The possible role of compensatory changes in 5-HT2C receptors in the reduced hypophagic action of d-fenfluramine in 5-HT1B knockout (KO) mice was assessed by comparing their response to d-fenfluramine and the 5-HT2C receptor agonist mCPP. In addition we measured 5-HT(2C/A) receptor binding in 5-HT1B KO and wild-type (WT) mice and examined the effects of 5-HT1B receptor antagonists on d-fenfluramine-induced hypophagia in WT mice. METHODS: Hypophagic responses to d-fenfluramine (1-30 mg/kg) and mCPP (1-5.6 mg/kg) were measured using a behavioural satiety sequence paradigm. The effects of the 5-HT1B receptor antagonists GR 127,935 and SB 224289 in opposing the hypophagic action of d-fenfluramine were evaluated in WT mice. The binding of [3H]-mesulergine was compared in the brains of both mouse strains. RESULTS: The hypophagic effects of moderate doses of d-fenfluramine and mCPP were attenuated in 5-HT1B KO mice. Pretreatment of WT mice with the 5-HT(1B/1D) receptor antagonist GR 127,935, or food-deprived WT mice with the 5-HT1B receptor antagonist SB 224289, did not reproduce the reduction in sensitivity to the effects of d-fenfluramine on feeding behaviour observed in 5-HT1B KO mice. Estimates of 5-HT2C receptor binding were similar in 5-HT1B KO and WT mice. CONCLUSIONS: The hypophagic effect of d-fenfluramine in mice is unlikely to be mediated by the 5-HT1B receptor. Instead, the evidence suggests that an adaptive change in 5-HT2C receptor function occurs in 5-HT1B receptor KO mice and contributes to their reduced response to d-fenfluramine.

5-HT1B receptors modulate components of satiety in the rat: behavioural and pharmacological analyses of the selective serotonin1B agonist CP-94,253.

RATIONALE: 5-HT(1B) receptors are thought to be one of the receptor subtypes that mediate the inhibitory control of serotonin on food intake and satiety. OBJECTIVE: To use the selective 5-HT(1B) receptor agonist, CP-94,253 as a probe of 5-HT(1B) receptor function in feeding behaviour, and to confirm the pharmacological selectivity of CP-94,253-induced hypophagia with a range of antagonists. METHODS: Dose-response functions for CP-94,253 (0, 1.25, 2.5, 5.0 mg/kg; IP) were determined in animals consuming wet mash in a 40-min test session during which time-sampled behavioural observations were collected to evaluate satiety sequences. A meal patterning study was carried out in a separate group of rats. The 5-HT(1A) antagonist WAY 100,635 (0, 1.0, 3.0 mg/kg; SC), the 5-HT(1B/1D)antagonist GR 127,935 (0, 3 mg/kg; IP), and the 5-HT(1B) antagonist SB 224289 (0, 2.5, 5.0 mg/kg; IP) were used to confirm that 5-HT(1B) receptor subtypes were responsible for the action of CP-94,253 on feeding behaviour. RESULTS: CP-94,253 (2.5 mg/kg) reduced food intake and preserved the satiety sequence in animals consuming a diet of mash. GR 127,935 (3.0 mg/kg) and SB 224289 (2.5 mg/kg), but not WAY 100,635, attenuated the hypophagic effect of the 5-HT(1B) agonist, and returned the changes in satiety sequence to control patterns. Meal patterning analyses indicated that CP-94,253 (2.5 mg/kg) reduced food intake through a decrease in meal size and duration in the absence of any alteration in the rate of eating. A hypodipsic action of CP-94,253 was also observed (2.5 and 5.0 mg/kg). CONCLUSION: These findings imply that 5-HT(1B) receptors regulate discrete elements of satiety. We discuss the potential role of 5-HT(1B) agonists for the treatment of obesity.

Pharmacological characterisation of the agonist radioligand binding site of 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptors.

In the present study we compared the affinity of various drugs for the high affinity "agonist-preferring" binding site of human recombinant 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptors stably expressed in monoclonal mammalian cell lines. To ensure that the "agonist-preferring" conformation of the receptor was preferentially labelled in competition binding experiments, saturation analysis was conducted using antagonist and agonist radiolabels at each receptor. Antagonist radiolabels ([3H]-ketanserin for 5-HT(2A) receptor and [3H]-mesulergine for 5-HT(2B) and 5-HT(2C) receptor) bound to a larger population of receptors in each preparation than the corresponding agonist radiolabel ([125I]-DOI for 5-HT(2A) receptor binding and [3H]-5-HT for 5-HT(2B) and 5-HT(2C) receptor binding). Competition experiments were subsequently conducted against appropriate concentrations of the agonist radiolabels bound to the "agonist-preferring" subset of receptors in each preparation. These studies confirmed that there are a number of highly selective antagonists available to investigate 5-HT2 receptor subtype function (for example, MDL 100907, RS-127445 and RS-102221 for 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptors respectively). There remains, however, a lack of highly selective agonists. (-)DOI is potent and moderately selective for 5-HT(2A) receptors, BW723C86 has poor selectivity for human 5-HT(2B) receptors, while Org 37684 and VER-3323 display some selectivity for the 5-HT(2C) receptor. We report for the first time in a single study, the selectivity of numerous serotonergic drugs for 5-HT2 receptors from the same species, in mammalian cell lines and using, exclusively, agonist radiolabels. The results indicate the importance of defining the selectivity of pharmacological tools, which may have been over-estimated in the past, and highlights the need to find more selective agonists to investigate 5-HT2 receptor pharmacology.

5-HT(2C) receptor activation inhibits appetitive and consummatory components of feeding and increases brain c-fos immunoreactivity in mice.

5-Hydroxytryptamine (5-HT)(2C) and 5-HT(1B) receptors are implicated in the inhibitory modulation of feeding behaviour. However, their respective, and possibly different, roles have not been clearly identified because of a lack of selective 5-HT(2C) receptor agonists. Here, using the putative, selective 5-HT(2C) receptor agonist VER23779, we show that its effects on feeding are fully reversed by pretreatment with a selective 5-HT(2C) receptor antagonist, but unaffected by pretreatment with either a 5-HT(1B) or a 5-HT(2A) receptor antagonist. In mice eating a palatable mash, feeding ends earlier, inactivity is increased but the behavioural satiety sequence is preserved. In a second-order schedule of reinforcement with an initial, non-food-reinforced appetitive phase, VER23779 produces a much greater relative reduction in appetitive responding than the 5-HT(1B) receptor agonist CP-94,253. Increased c-fos immunoreactivity patterns following VER23779 also differ from those described for CP-94,253, in particular showing strong activation of the basolateral amygdala. The different behavioural consequences of 5-HT(2C) and 5-HT(1B) receptor activation may relate to the patterns of c-fos immunoreactivity. In particular, the basolateral amygdala may have a role in maintaining response in the appetitive phase of the second-order schedule and also be susceptible to serotonergic modulation through activation of 5-HT(2C) receptors.

Pyrrolo(iso)quinoline derivatives as 5-HT(2C) receptor agonists.

A series of 1-(1-pyrrolo(iso)quinolinyl)-2-propylamines was synthesised and evaluated as 5-HT(2C) receptor agonists for the treatment of obesity. The general methods of synthesis of the precursor indoles are described. The functional efficacy and radioligand binding data for the compounds at 5-HT(2) receptor subtypes are reported. The analogue which showed the highest 5-HT(2C) binding affinity (27, 1.6nM) was found to be successful in reducing food intake in rats.

5-HT1B receptor knockout mice show a compensatory reduction in 5-HT2C receptor function.

Although null mutant ('knockout') mice have provided valuable animal models to complement traditional approaches to psychopharmacology, such animals may also show complex adaptations to the induced mutation. Here we demonstrate that serotonin1B (5-HT1B) receptor knockout (KO) mice show adaptations in serotonin2C (5-HT2C) receptor-mediated functions. They show smaller reductions in food intake and locomotor activity in response to administration of 5-HT2C receptor agonists that are not accounted for by altered drug disposition. These effects are not mimicked by pretreatment of wildtype (WT) mice with a 5-HT1B receptor antagonist showing that they result from a longer term adaptation to the loss of 5-HT1B receptor function and not from a short-term interaction between 5-HT1B- and 5-HT2C-mediated functions. In addition, we show that 5-HT1B receptor KO mice have a lowered hypothalamic c-fos response to the administration of 5-HT2C receptor agonists. These results demonstrate that compensatory adaptations to the constitutive loss of 5-HT1B receptors may be an important determinant of the altered response of 5-HT1B KO mice to a variety of pharmacological challenges.

Tonic regulation of satiety by 5-HT receptors in the mouse: converging evidence from behavioural and c-fos immunoreactivity studies?

Activation of 5-HT(1B) receptors is thought to play an important role in the inhibitory influence of serotonin on feeding behaviour and body weight in mammals. Earlier studies have shown that 5-HT(1B)-knockout (KO) mice eat more and are heavier than wild-type (WT) controls and that the selective 5-HT(1B) receptor agonist CP-94,253 reduces food intake in food-deprived mice. Here we characterize the behavioural effects of both CP-94,253 and the selective 5-HT(1B) receptor antagonist SB224289 on feeding and other behaviours within the behavioural satiety sequence, and also report a c-fos mapping study using CP-94,253. CP-94,253 produced a dose-dependent suppression of food intake with a profile consistent with a selective effect on feeding behaviour. These effects were absent or reduced in 5-HT(1B)-KO mice and in WT mice pretreated with SB224289. SB224289 administered alone enhanced food intake consistent with impaired satiation; a similar effect was apparent in 5-HT(1B)-KO mice compared to WT. CP-94,253 induced c-fos in a range of structures previously implicated in the expression of feeding behaviour. These results suggest that the activation of 5-HT(1B) receptors is an important component of endogenous satiation mechanisms in the mouse.