ABSTRACT: BACKGROUND: Cannabinoid 1 (CB1) receptor antagonists exhibit pharmacological properties favorable for the treatment of obesity and other related metabolic disorders. CE-178253 (1-[7-(2-Chlorophenyl)-8-(4-chlorophenyl)-2-methylpyrazolo[1,5-a]-[1,3,5]triazin-4-yl]-3-ethylaminoazetidine-3-carboxylic acid hydrochloride) is a recently discovered selective CB1 receptor antagonist. Despite a large body of knowledge on cannabinoid receptor antagonists little data exist on the quantitative pharmacology of this therapeutic class of drugs. The purpose of the current studies was to evaluate the quantitative pharmacology and concentration/effect relationships of CE-178253 based on unbound plasma concentration and in vitro pharmacology data in different in vivo preclinical models of FI and energy expenditure. RESULTS: In vitro, CE-178253 exhibits sub-nanomolar potency at human CB1 receptors in both binding (Ki = 0.33 nM) and functional assays (Ki = 0.07 nM). CE-178253 has low affinity (Ki >10,000 nM) for human CB2 receptors. In vivo, CE-178253 exhibits concentration-dependent anorectic activity in both fast-induced re-feeding and spontaneous nocturnal feeding FI models. As measured by indirect calorimetry, CE-178253 acutely stimulates energy expenditure by greater than 30% in rats and shifts substrate oxidation from carbohydrate to fat as indicated by a decrease the respiratory quotient from 0.85 to 0.75. Determination of the concentration-effect relationships and ex vivo receptor occupancy in efficacy models of energy intake and expenditure suggest that a greater than a 2-fold coverage of the Ki (50-75% receptor occupancy) is required for maximum efficacy. Finally, in two preclinical models of obesity, CE-178253 dose-dependently promotes weight loss in diet-induced obese rats and mice. CONCLUSIONS: We have combined quantitative pharmacology and ex vivo CB1 receptor occupancy data to assess concentration/effect relationships in food intake, energy expenditure and weight loss studies. Quantitative pharmacology studies provide a strong a foundation for establishing and improving confidence in mechanism as well as aiding in the progression of compounds from preclinical pharmacology to clinical development.

Cannabinoid CB(1) receptor antagonists exhibit pharmacologic properties favorable for the treatment of metabolic disease. CP-945,598 (1-[9-(4-chlorophenyl)-8-(2-chlorophenyl)-9H-purin-6-yl]-4-ethylamino piperidine-4-carboxylic acid amide hydrochloride) is a recently discovered selective, high affinity, competitive CB(1) receptor antagonist that inhibits both basal and cannabinoid agonist-mediated CB(1) receptor signaling in vitro and in vivo. CP-945,598 exhibits sub-nanomolar potency at human CB(1) receptors in both binding (K(i)=0.7 nM) and functional assays (K(i)=0.2 nM). The compound has low affinity (K(i)=7600 nM) for human CB(2) receptors. In vivo, CP-945,598 reverses four cannabinoid agonist-mediated CNS-driven responses (hypo-locomotion, hypothermia, analgesia, and catalepsy) to a synthetic cannabinoid receptor agonist. CP-945,598 exhibits dose and concentration-dependent anorectic activity in two models of acute food intake in rodents, fast-induced re-feeding and spontaneous, nocturnal feeding. CP-945,598 also acutely stimulates energy expenditure in rats and decreases the respiratory quotient indicating a metabolic switch to increased fat oxidation. CP-945,598 at 10 mg/kg promoted a 9%, vehicle adjusted weight loss in a 10 day weight loss study in diet-induced obese mice. Concentration/effect relationships combined with ex vivo brain CB(1) receptor occupancy data were used to evaluate efficacy in behavioral, food intake, and energy expenditure studies. Together, these in vitro, ex vivo, and in vivo data indicate that CP-945,598 is a novel CB(1) receptor competitive antagonist that may further our understanding of the endocannabinoid system.

We report the design, synthesis, and structure-activity relationships of novel bicyclic lactam-based cannabinoid type 1 (CB(1)) receptor antagonists. Members of these series are potent, selective antagonists in in vitro/in vivo efficacy models of CB(1) antagonism and exhibit robust oral activity in rodent models of food intake. These efforts led to the identification of 19d, which has been advanced to human clinical trials for weight management.

We report the structure-activity relationships, design, and synthesis of the novel cannabinoid type 1 (CB1) receptor antagonist 3a (CP-945,598). Compound 3a showed subnanomolar potency at human CB1 receptors in binding (Ki = 0.7 nM) and functional assays (Ki = 0.12 nM). In vivo, compound 3a reversed cannabinoid agonist-mediated responses, reduced food intake, and increased energy expenditure and fat oxidation in rodents.

c-Jun NH(2)-terminal kinase (JNK) plays an important role in insulin resistance; however, identification of pharmacologically potent and selective small molecule JNK inhibitors has been limited. Compound A has a cell IC(50) of 102 nM and is at least 100-fold selective against related kinases and 27-fold selective against glycogen synthase kinase-3beta and cyclin-dependent kinase-2. In C57BL/6 mice, compound A reduced LPS-mediated increases in both plasma cytokine levels and phosphorylated c-Jun in adipose tissue. Treatment of mice fed a high-fat diet with compound A for 3 wk resulted in a 13.1 +/- 1% decrease in body weight and a 9.3 +/- 1.5% decrease in body fat, compared with a 6.6 +/- 2.1% increase in body weight and a 6.7 +/- 2.1% increase in body fat in vehicle-treated mice. Mice pair fed to those that received compound A exhibited a body weight decrease of 7 +/- 1% and a decrease in body fat of 1.6 +/- 1.3%, suggesting that reductions in food intake could not account solely for the reductions in adiposity observed. Compound A dosed at 30 mg/kg for 13 days in high-fat fed mice resulted in a significant decrease in phosphorylated c-Jun in adipose tissue accompanied by a decrease in weight and reductions in glucose and triglycerides and increases in insulin sensitivity to levels comparable with those in lean control mice. The ability of compound A to reduce the insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1) von Ser307 and partially reverse the free fatty acid inhibition of glucose uptake in 3T3L1 adipocytes, suggests that enhancement of insulin signaling in addition to weight loss may contribute to the effects of compound A on insulin sensitization in vivo. Pharmacological inhibition of JNK using compound A may therefore offer an effective therapy for type 2 diabetes mediated at least in part via weight reduction.

A series of conformationally constrained bicyclic derivatives derived from SR141716 was prepared and evaluated as hCB(1)-R antagonists and inverse agonists. Optimization of the structure-activity relationships around the 2,6-dihydro-pyrazolo[4,3-d]pyrimidin-7-one derivative 2a led to the identification of two compounds with oral activity in rodent feeding models (2h and 4a). Replacement of the PP group in 2h with other bicyclic groups resulted in a loss of binding affinity.

The cannabinoid-1 (CB1) receptor plays a role in the regulation of appetitive behavior. Exogenously administered cannabinoid receptor agonists stimulate food consumption in animals and humans. Endogenous cannabinoid receptor agonists are present in the brain, and the brain level of these agonists increases with greater demand of food by rodents. Specific CB1 receptor antagonist compounds have been discovered that display high affinity and selectivity for the CB1 receptor. CB1 receptor antagonists inhibit both acute and long-term food intake in rodents. Chronic treatment with CB1 antagonists results in a sustained reduction in body weight in rodents (5 weeks), and weight loss in humans (16 weeks). Patent literature indicates CB1 receptor antagonist discovery efforts at a number of pharmaceutical companies. The CB1 receptor antagonist, rimonabant (SR-141716), discovered by Sanofi-Synthélabo, is in phase III clinical trials for the treatment of obesity and has been found to decrease appetite and body weight in humans.

The serine/threonine kinase Akt/PKB plays key roles in the regulation of cell growth, survival, and metabolism. It remains unclear, however, whether the functions of individual Akt/PKB isoforms are distinct. To investigate the function of Akt2/PKBbeta, mice lacking this isoform were generated. Both male and female Akt2/PKBbeta-null mice exhibit mild growth deficiency and an age-dependent loss of adipose tissue or lipoatrophy, with all observed adipose depots dramatically reduced by 22 weeks of age. Akt2/PKBbeta-deficient mice are insulin resistant with elevated plasma triglycerides. In addition, Akt2/PKBbeta-deficient mice exhibit fed and fasting hyperglycemia, hyperinsulinemia, glucose intolerance, and impaired muscle glucose uptake. In males, insulin resistance progresses to a severe form of diabetes accompanied by pancreatic beta cell failure. In contrast, female Akt2/PKBbeta-deficient mice remain mildly hyperglycemic and hyperinsulinemic until at least one year of age. Thus, Akt2/PKBbeta-deficient mice exhibit growth deficiency similar to that reported previously for mice lacking Akt1/PKBalpha, indicating that both Akt2/PKBbeta and Akt1/PKBalpha participate in the regulation of growth. The marked hyperglycemia and loss of pancreatic beta cells and adipose tissue in Akt2/PKBbeta-deficient mice suggest that Akt2/PKBbeta plays critical roles in glucose metabolism and the development or maintenance of proper adipose tissue and islet mass for which other Akt/PKB isoforms are unable to fully compensate.

We determined the effect of a cannabinoid CB1 receptor antagonist (AM-251; N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) on food intake, body weight and adipose tissue mass in Western diet-induced obese (DIO) mice using a chronic, interrupted, oral dosing paradigm. The dosing paradigm was 2 weeks on treatment (treatment 1), 2 weeks off-treatment, followed by 2 weeks on treatment (treatment 2). During treatment 1 and treatment 2, food intake and body weight were reduced after a single dose. At 30 mg/kg/day, anorectic efficacy was maintained through 12 days (treatment 1) and 7 days (treatment 2). Body weight of AM-251-treated mice remained less than vehicle-treated mice throughout treatment 1 and treatment 2. Administration of AM-251 reduced inguinal subcutaneous, retroperitoneal and mesenteric adipose tissue mass. Antiobesity effects of AM-251 were lost during the off-treatment period, and hyperphagia was observed in treated animals. With re-initiation of AM-251 treatment, mice again responded to the effects of the compound. These results support the hypothesis that chronic treatment of obese individuals with cannabinoid CB1 receptor antagonists is a viable pharmacologic approach to sustained weight loss.

INTRODUCTION: Obesity is a significant public health concern with considerable academic and industrial research effort underway to discover novel drugs to treat this disease. The aim of this study was to validate a recently developed high-resolution X-ray computed tomography (micro CT) system capable of measuring murine adipose tissue depot mass in situ. METHODS: The micro CT was used to generate a series of cross-sectional X-ray images from which individual adipose tissue depot mass was quantified. Four individual adipose tissue depots were studied: inguinal subcutaneous, epididymal, retroperitoneal, and mesenteric. The relationship between micro CT-derived adipose tissue mass and adipose mass measured gravimetrically was determined. The effect of strain (C57/Bl6, C3H/HeNCR1BR, and db/db) and age (49 vs. 99 days) on adipose tissue depot mass was studied. RESULTS: Validation studies in which adipose tissue depot mass was determined by micro CT and by gravimetry were conducted in the three strains of mice at 49 and 99 days of age. The correlation of micro CT and gravimetric measures of adipose tissue mass exceeded 90% in all strains at 99 days, and in the C57/Bl6 and C3H/HeNCR1BR strains at 49 days. At 49 days, the correlation in the db/db strain was 82%. Micro CT methodology distinguished both age and strain differences in the adipose tissue depots studied (P<.0001, in all cases). DISCUSSION: Micro CT is a valid method to quantify the mass of individual adipose tissue depots in mice. This method of determining adipose tissue mass is not a terminal procedure; thus, this methodology may be particularly useful for the longitudinal assessment of the effects of drug intervention on adipose tissue depot mass.

The cardioprotective efficacy of the pyrazolinone-piperidine dipeptide growth hormone secretagogue (GHS) CP-424,391 was studied in an in vivo rabbit model of ischemia and reperfusion. CP-424,391 was administered at 25 mg/kg p.o. x 7 days. Ischemia was induced by left coronary artery occlusion for 30 min, after which the heart was reperfused for 2 h. At the end of reperfusion, animals were euthanized and the infarct size was determined. The area at risk of infarct was not different between the control (45.8+/-3.7%, n=6) and CP-424,391-treated groups (36.9+/-4.3%, n=11). The infarct size of the control animals was 49.5+/-7.1% and was significantly (P<0.05) lower in the CP-424,391-treated group (infarct size=17.3+/-3.0). There was a trend, albeit not significant, for the left ventricular function to recover to a greater extent in CP-424,391-treated rabbits. Thus, the treatment of rabbits for 7 days with CP-424,391 was cardioprotective against ischemia/reperfusion injury.

This review discusses the pharmacology of regional myocardial ischemia and reperfusion (I/R) injury and the utilization of in vivo animal models in the preclinical development of novel therapeutic compounds. The manuscript aims to provide an overview of a number of different cardioprotective strategies that have been successful from a preclinical perspective and to also present where possible results of clinical trials of the respective compounds. Myocardial ischemia reperfusion injury may be manifested as myocardial stunning, ventricular arrhythmias, coronary vascular dysfunction, or the development of a myocardial infarct. This review is principally concerned with preclinical studies related to reduction of infarct size. The pathophysiology of the reperfusion injury process is complex, including primarily cellular and humoral components of inflammation, as well as myocellular ionic and metabolic disturbances. This review will discuss strategies directed at oxygen-derived free radicals, neutrophils, adenosine, and the sodium-hydrogen exchanger (NHE). The results of preclinical cardioprotective studies are influenced by the paradigm used therefore methodological considerations will also be presented where appropriate.

The aim of this study was to determine the effect of different administration protocols on the cardioprotective efficacy of the non-selective, irreversible caspase inhibitors N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) and bocaspartyl-(OMe)-fluoromethylketone (BocD.fmk) in a rat in vivo ischemia and reperfusion paradigm. Hearts were made ischemic for 45 min and reperfused for 180 min. Under these conditions, it was determined that zVAD.fmk was cardioprotective when administered before or after the onset of ischemia, whereas BocD.fmk was efficacious only when administered before the onset of ischemia. This is the first report of in vivo cardioprotection by a caspase inhibitor when administered after the onset of ischemia.

Apoptotic cell suicide initiated by ligation of CD95 (Fas/APO-1) occurs through recruitment, oligomerization and autocatalytic activation of the cysteine protease, caspase-8 (MACH, FLICE, Mch5). An endogenous mammalian regulator of this process, named Usurpin, has been identified (aliases for Usurpin include CASH, Casper, CLARP, FLAME-1, FLIP, I-FLICE and MRIT). This protein is ubiquitously expressed and exists as at least three isoforms arising by alternative mRNA splicing. The Usurpin gene is comprised of 13 exons and is clustered within approximately 200 Kb with the caspase-8 and -10 genes on human chromosome 2q33-34. The Usurpin polypeptide has features in common with pro-caspase-8 and -10, including tandem 'death effector domains' on the N-terminus of a large subunit/small subunit caspase-like domain, but it lacks key residues that are necessary for caspase proteolytic activity, including the His and Cys which form the catalytic substrates diad, and residues that stabilize the P1 aspartic acid in substrates. Retro-mutation of these residues to functional caspase counterparts failed to restore proteolytic activity, indicating that other determinants also ensure the absence of catalytic potential. Usurpin heterodimerized with pro-caspase-8 in vitro and precluded pro-caspase-8 recruitment by the FADD/MORT1 adapter protein. Cell death induced by CD95 (Fas/APO-1) ligation was attenuated in cells transfected with Usurpin. In vivo, a Usurpin deficit was found in cardiac infarcts where TUNEL-positive myocytes and active caspase-3 expression were prominent following ischemia/reperfusion injury. In contrast, abundant Usurpin expression (and a caspase-3 deficit) occurred in surrounding unaffected cardiac tissue, suggesting reciprocal regulation of these pro- and anti-apoptotic molecules in vivo. Usurpin thus appears to be an endogenous modulator of apoptosis sensitivity in mammalian cells, including the susceptibility of cardiac myocytes to apoptotic death following ischemia/ reperfusion injury.

Renal effects of a selective cyclooxygenase-2 (COX-2) inhibitor [MF-Tricyclic; 3-(3,4-difluorophenyl)-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone] were studied in control and volume-depleted conscious dogs. MF-Tricyclic was compared with the nonselective COX-1/COX-2 inhibitor indomethacin. Six instrumented male dogs were randomly selected to receive MF-Tricyclic or indomethacin at 10 mg/kg. Volume depletion was effected by a sodium-restricted diet (14 days) with administration of furosemide (7.5 mg/kg, i.v.) the day before the experiment. Indomethacin ablated systemic COX-1 activity (p < 0.05), whereas MF-Tricyclic did not affect this activity. Each compound achieved plasma concentrations in excess of their respective median inhibitory concentrations (IC50 values) against canine COX-2. In controls, neither compound affected mean arterial pressure (MAP), heart rate (HR), renal blood flow (RBF), fractional excretion (FE) Na+, or FE K+. In volume-depleted dogs, indomethacin reduced RBF (p < 0.05), whereas MF-Tricyclic did not affect this parameter. Indices of renal function in volume-depleted dogs were not affected. These data are consistent with the view that the effects of indomethacin on RBF are a consequence of inhibition of COX-1 activity. Furthermore, in these studies, short-term administration of a selective COX-2 inhibitor was without deleterious effects on renal function.

Cardioprotective effects of the angiotensin-converting enzyme inhibitors captopril and ramiprilat were studied in two in vivo canine models of myocardial ischemic injury: 90 min of regional ischemia with 18 h reperfusion (protocol I) and 6 h of continuous ischemia without reperfusion (protocol II). In protocol I, neither ramiprilat (50 micrograms/kg) nor captopril (5 mg/kg + 0.25 mg/kg/h) reduced infarct size after 18 h of reperfusion (vs. controls). In protocol II, drugs were administered directly into both left anterior descending coronary artery and left circumflex branch. Compared to controls, continuous intracoronary administration of ramiprilat (40 ng/kg/min) or captopril (400 ng/kg/min) did not reduce infarct size. Thus neither captopril nor ramiprilat protected the heart from injury under conditions of ischemia with reperfusion or ischemia without reperfusion.

The aim of our study was to characterize the temporal relationship of apoptosis to regional myocardial ischemia and reperfusion and we aimed to determine the effect of ischemia and reperfusion on the distribution of the pro-apoptotic cysteine protease caspase-3 (CPP 32, apopain, Yama) in an in vivo rat model. Male Sprague-Dawley rats (250-400 g) were anesthetized with sodium pentobarbital (65 mg/kg, i.p.), the left external carotid artery was isolated to monitor arterial pressure and a left thoracotomy was performed. Regional myocardial ischemia was induced by occluding the left main coronary artery for 45 min. The heart was reperfused for 0, 60, 120 or 180 min. TUNEL staining of formalin-fixed, paraffin-embedded left ventricle, and DNA fragmentation analysis, showed that apoptosis occurred during 45 min of ischemia alone, but further developed during the 3-h reperfusion period. Immunohistochemical analysis of ischemic/reperfused left ventricle showed caspase-3 levels were substantially elevated and localized in the ischemic/reperfused region, and that caspase-3 co-localized to TUNEL positive myocytes. Therefore, regional myocardial ischemia serves as a stimulus for myocyte apoptosis, and this form of cell death progresses time-dependently after the onset of reperfusion. Our studies implicate caspase-3 to be involved in apoptotic cell death in ischemic/reperfused rat heart.