Mauro A. M. Carai

Differential distribution of functional cannabinoid CB1 receptors in the mouse gastroenteric tract.

Recently, the gastrointestinal pharmacology of cannabinoid CB(1) receptors has been extensively explored. We employed western blotting and immunohistochemistry techniques to study the distribution of the cannabinoid CB(1) receptor protein in the mouse gastroenteric tract. The cannabinoid CB(1) receptor peptide was detected by western blotting only in its glycosylated form (63 kDa) with a significant differential distribution. The highest levels of expression were detected in the stomach and in the colon, while the pyloric valve was devoid of any cannabinoid CB(1) receptor protein. The immunohistochemical study showed intense cannabinoid CB(1) receptor immunoreactivity in ganglia subadjacent to the gastric epithelium and in the smooth muscle layers of both the small and large intestine. Only the small intestine showed (-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)-phenyl]-4-(3-hydroxylpropyl) cyclohexan-1-ol) ([3H]CP 55,940) specific binding (27%). These receptors mediated pharmacologically significant effects since the cannabinoid CB(1) receptor agonist R(-)-7-hydroxy-delta-6-tetra-hydrocannabinol-dimethylheptyl (HU 210) dose dependently inhibited gastrointestinal transit up to 70%, while the cannabinoid CB(1) receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide (SR 141716A) increased gastrointestinal transit. Moreover, the dose of 0.3 microg/kg of HU 210, devoid per se of any activity on mouse intestinal propulsion, blocked the increased gastroenteric transit induced by the cannabinoid CB(1) antagonist SR 141716A.

Tricyclic Pyrazoles. Part 1: Synthesis and Biological Evaluation of Novel 1,4-Dihydroindeno[1,2-c]pyrazol-based Ligands for CB1and CB2 Cannabinoid Receptors

Cannabinoids receptors, cellular elements of the endocannabinoid system, have been the focus of extensive studies because of their potential functional role in several important physiological and pathological processes. To further evaluate the properties of CB receptors, especially CB(1) and CB(2) subtypes, we have designed, using SR141716A as a benchmark, a new series of rigid 1-aryl-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamides. Compounds 1 were synthesized from substituted 1-aryl-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxylic acids and requisite amines. The various analogues were assayed for binding both to the brain and peripheral cannabinoid receptors (CB(1) and CB(2)). Seven of the new compounds displayed very high in vitro CB(2) binding affinities, especially 1a, 1b, 1c, 1e, 1g, 1h and 1j which showed K(i) values of 0.34, 0.225, 0.27, 0.23, 0.385, 0.037 and 0.9 nM, respectively. Compounds 1a, 1b, 1c and 1h showed the highest selectivity for CB(2) receptor with K(i)(CB(1)) to K(i)(CB(2)) ratios of 6029, 5635, 5814 and 9810, respectively. Noticeably, 1h exhibited the highest affinity and selectivity for CB(2) receptors.

Voluntary alcohol drinking enhances proopiomelanocortin gene expression in nucleus accumbens shell and hypothalamus of Sardinian alcohol-preferring rats.

BACKGROUND Evidence obtained in humans and rodents indicates that beta-endorphin (encoded by the proopiomelanocortin [POMC] gene) is critical in the regulation of alcohol drinking behavior. However, the alcohol effect on POMC gene expression has not been studied in rodent mesolimbic regions, such as the nucleus accumbens (NAc). METHODS In this study, we first utilized POMC-enhanced green fluorescent protein (EGFP) transgenic mice to visualize POMC neurons and found that POMC-EGFP cells were modestly distributed throughout the NAc shell and core, in addition to the hypothalamic arcuate nucleus. POMC mRNA expression in the NAc of mice and rats was confirmed using reverse transcriptase-polymerase chain reaction and solution hybridization assays. We then investigated whether there are genetically determined differences in basal mRNA levels of POMC and mu opioid receptor (MOP-r) between selectively bred Sardinian alcohol-preferring (sP) and nonpreferring (sNP) rats, and whether these mRNA levels are altered in sP rats after alcohol drinking (10%, unlimited access) for 17 days. RESULTS Alcohol-naïve sP rats had higher basal POMC mRNA levels than sNP rats only in hypothalamus. Alcohol drinking increased POMC mRNA levels in both the NAc shell (by 100%) and the hypothalamus (by 50%) of sP rats. Although sP rats had lower basal levels of MOP-r mRNA and GTPγS binding in NAc shell than sNP rats, voluntary alcohol consumption had no effect on MOP-r mRNA levels in the NAc shell. CONCLUSIONS Our results define the distribution of POMC-expressing neurons in the NAc of mice and rats. Higher POMC expression at basal levels in sP rats (genetically determined), along with increases after drinking (alcohol-induced) in the NAc shell and hypothalamus, suggests that the POMC systems play a role in high alcohol preference and consumption.

Activation of the GABAB Receptor Prevents Nicotine-Induced Locomotor Stimulation in Mice

Recent studies demonstrated that activation of the GABAB receptor, either by means of orthosteric agonists or positive allosteric modulators (PAMs), inhibited different nicotine-related behaviors, including intravenous self-administration and conditioned place preference, in rodents. The present study investigated whether the anti-nicotine effects of the GABAB receptor agonist, baclofen, and GABAB PAMs, CGP7930, and GS39783, extend to nicotine stimulant effects. To this end, CD1 mice were initially treated with baclofen (0, 1.25, and 2.5 mg/kg, i.p.), CGP7930 (0, 25, and 50 mg/kg, i.g.), or GS39783 (0, 25, and 50 mg/kg, i.g.), then treated with nicotine (0 and 0.05 mg/kg, s.c.), and finally exposed to an automated apparatus for recording of locomotor activity. Pretreatment with doses of baclofen, CGP7930, or GS39783 that did not alter locomotor activity when given with nicotine vehicle fully prevented hyperlocomotion induced by 0.05 mg/kg nicotine. These data extend to nicotine stimulant effects the capacity of baclofen and GABAB PAMs to block the reinforcing, motivational, and rewarding properties of nicotine. These data strengthen the hypothesis that activation of the GABAB receptor may represent a potentially useful, anti-smoking therapeutic strategy.

Anti-alcohol and anxiolytic properties of a new chemical entity, GET73

N-[(4-trifluoromethyl)benzyl]4-methoxybutyramide (GET73) is a newly synthesized compound structurally related to the clinically used, alcohol-substituting agent, gamma-hydroxybutyric acid (GHB). The present study was designed to assess whether GET73 may share with GHB the capacity to reduce alcohol intake in rats. Additionally, the effect of treatment with GET73 on anxiety-related behaviors and cognitive tasks in rats was investigated. A series of in vitro binding assays investigated the capacity of GET73 to bind to the GHB binding site and multiple other receptors. GET73 (10−9–10−3 M) failed to inhibit [3H]GHB binding at both high- and low-affinity GHB recognition sites in rat cortical membranes. GET73 displayed minimal, if any, binding at dopamine, serotonin, GABA, and glutamate receptors in membranes from different rat brain areas. Acute treatment with low-to-moderate, non-sedative doses of GET73 (5–50 mg/kg, i.g. or i.p.) (a) reduced alcohol intake and suppressed “alcohol deprivation effect” (a model of alcohol relapse) in selectively bred, Sardinian alcohol-preferring (sP) rats, (b) exerted anxiolytic effects in Sprague-Dawley (SD) and sP rats exposed to the Elevated Plus Maze test, and (c) tended to induce promnestic effects in SD rats exposed to a modified water version of the Hebb–Williams maze test. Although the mechanism of GET73 action is currently unknown, the results of the present study suggest that GET73 has a multifaceted pharmacological profile, including the capacity to reduce alcohol drinking and anxiety-related behaviors in rats.

Synthesis, cannabinoid receptor affinity, molecular modeling studies and in vivo pharmacological evaluation of new substituted 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides. 2. Effect of the 3-carboxamide substituent on the affinity and selectivity profile.

New substituted 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides were synthesized by replacing the 2,4-dichlorobenzyl and cyclohexyl moieties at the 3-carboxamide nitrogen of the previously reported CB(1) receptor antagonists/inverse agonists 4 and 5. Several ligands showed potent affinity for the hCB(1) receptor, with K(i) concentrations comparable to the reference compounds 1, 4 and 5, and exhibited CB(1) selectivity comparable to 1 and 2. Docking experiments and molecular dynamics (MD) simulations explained the potent hCB(1) binding affinity of compounds 31 and 37. According to our previous studies, 31 and 37 formed a H-bond with K3.28(192), which accounted for the high affinity for the receptor inactive state and the inverse agonist activity. The finding of inhibition of food intake following their acute administration to rats, supported the concept that the CB(1) selective compounds 4 and 52 act as antagonists/inverse agonists.

1-Aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide: An effective scaffold for the design of either CB1 or CB2 receptor ligands

New 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides were synthesized as cannabinoid (CB) receptor ligands. Compound 11 (CB(1)K(i) = 2.3 nM, CB(1) SI = 163.6) showed CB(1) receptor affinity and selectivity superior to Rimonabant and AM251. Acute administration of 2mg/kg 11 reduced sucrose, but not regular food, intake in rats. On the other hand, compound 23 (CB(2)K(i) = 0.51 nM, CB(2) SI = 30.0) showed significant affinity and selectivity for the CB(2) receptor. The results presented here show that the 1-aryl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide may serve as an effective scaffold for the design of either CB(1) or CB(2) receptor ligands.

Reducing effect of a combination of Phaseolus vulgaris and Cynara scolymus extracts on operant self-administration of a chocolate-flavoured beverage in rats.

Treatment with a rational combination of standardized extracts of Phaseolus vulgaris and Cynara scolymus reduced food intake and glycemia in rats. The present study was designed to assess the effect of this extract combination and of each single extract in an experimental model of food craving, made up of rats displaying exaggerated seeking and taking behaviors for a chocolate‐flavoured beverage. After training to lever‐respond for the chocolate‐flavoured beverage, rats were treated with vehicle, Phaseolus vulgaris extract alone (200 mg/kg), Cynara scolymus extract alone (400 mg/kg), or combination of Phaseolus vulgaris (200 mg/kg) and Cynara scolymus (400 mg/kg) extracts. The Phaseolus vulgaris extract and the extract combination exerted similar and substantial decrements in the number of lever‐responses and amount of self‐administered chocolate‐flavoured beverage; conversely, the Cynara scolymus extract was totally ineffective. These results suggest that (i) the capacity of the extract combination to reduce the self‐administration of the chocolate‐flavoured beverage entirely relied on the Phaseolus vulgaris extract, (ii) Phaseolus vulgaris extract may interfere with the mechanisms regulating food‐related addictive‐like behaviors, and (iii) combinations of Phaseolus vulgaris and Cynara scolymus extracts may possess a broad spectrum of activities, from treatment of metabolic syndrome to overweight, obesity, and possibly food‐related addictive disorders. Copyright

Reducing Effect of a Combination of Phaseolus vulgaris and Cynara scolymus Extracts on Food Intake and Glycemia in Rats

Extracts from Phaseolus vulgaris and Cynara scolymus may reduce food intake and/or postprandial glycemia. This study investigated the effect of standardized extracts of P. vulgaris and C. scolymus and their combination on food intake and glycemia in rats. P. vulgaris and C. scolymus extracts, and their 1:2 combination, were administered acutely to rats (a) given access to regular food and water, (b) given access to regular food, water, and a chocolate‐flavored beverage, or (c) infused with a starch bolus. P. vulgaris extract and the combination produced comparable reductions in intake of regular food and chocolate‐flavored beverage; conversely, C. scolymus extract was ineffective on both parameters. P. vulgaris and C. scolymus extracts additively contributed to the reducing effect of the combination on glycemic rise. These results suggest that a mixture of P. vulgaris and C. scolymus extracts is preferable over each single extract, as it combines the anorectic effect of the P. vulgaris extract with the hypoglycemic effect of both extracts. These data support the recent clinical use of the combination of P. vulgaris and C. scolymus extracts in the control of appetite, food intake, and postprandial glycemia and represent a successful example of translational research in the nutraceutical field. Copyright

Reducing effect of an extract of Phaseolus vulgaris on food intake in mice--focus on highly palatable foods.

Different lines of experimental evidence indicate that treatment with extracts from and derivatives of Phaseolus vulgaris reduces intake of food, including highly palatable foods and beverages, in rats. The present study was designed to extend to mice these lines of evidence. To this end, CD1 mice were treated acutely with a standardized extract of P. vulgaris and then exposed to unlimited access to regular food pellets (Experiment 1) or 1-hour limited access to three different palatable foods/beverages, such as butter cookies (Experiment 2), a condensed-milk beverage (Experiment 3), and a chocolate-flavored beverage (Experiment 4). Treatment with P. vulgaris extract resulted in a significant reduction in the intake of regular food pellets, that was still evident 24h later, as well as of the three palatable nourishments. Together, these results (a) extend to mice several previous findings on the capacity of P. vulgaris extracts to suppress food intake in rats, (b) suggest that P. vulgaris extracts may interfere with the central mechanisms regulating appetite, food intake, palatability, and/or the rewarding and hedonic properties of food, and (c) P. vulgaris extracts may represent a potentially effective therapy for overeating, obesity, and food craving.