Characterization of a novel and selective cannabinoid CB1 receptor inverse agonist, Imidazole 24b, in rodents.

We document in vitro and in vivo effects of a novel, selective cannabinoid CB(1) receptor inverse agonist, Imidazole 24b (5-(4-chlorophenyl)-N-cyclohexyl-4-(2,4-dichlorophenyl)-1-methyl-imidazole-2-carboxamide). The in vitro binding affinity of Imidazole 24b for recombinant human and rat CB(1) receptor is 4 and 10 nM, respectively. Imidazole 24b binds to human cannabinoid CB(2) receptor with an affinity of 297 nM; in vitro, it is a receptor inverse agonist at both cannabinoid CB(1) and CB(2) receptors as it causes a further increase of forskolin-induced cAMP increase. Oral administration of Imidazole 24b blocked CP-55940-induced hypothermia, demonstrating cannabinoid CB(1) receptor antagonist efficacy in vivo. Using ex vivo autoradiography, Imidazole 24b resulted in dose-dependent increases in brain cannabinoid CB(1) receptor occupancy (RO) at 2h post-dosing in rats, indicating that approximately 50% receptor occupancy is sufficient for attenuation of receptor agonist-induced hypothermia. Imidazole 24b administered to C57Bl/6 mice and to dietary-induced obese (DIO) Sprague-Dawley rats attenuated overnight food intake with a minimal effective dose of 10 mg/kg, p.o. Administration had no effect in cannabinoid CB(1) receptor-deficient mice. DIO rats were dosed orally with vehicle, Imidazole 24b (1, 3 or 10 mg/kg), or dexfenfluramine (3 mg/kg) for 2 weeks. At 3 mg/kg, Imidazole 24b reduced cumulative food intake, leading to a non-significant decrease in weight gain. Imidazole 24b at 10 mg/kg and dexfenfluramine treatment inhibited food intake and attenuated weight gain. These findings suggest that selective cannabinoid CB(1) receptor inverse agonists such as Imidazole 24b have potential for the treatment of obesity.

Characterization of the NOD/scid-[Tg]DR1 mouse expressing HLA-DRB1*01 transgene: a model of SCID-hu mouse for vaccine development.

OBJECTIVE: We previously showed enhanced engraftment of human T cells in the transgenic NonObese Diabetic/severe combined immunodeficient (NOD/scid)-DR1 mice, compared to NOD/scid mice. We now characterize their immunobiology, innate immunity, and intrahepatic neonatal engraftment of cord blood mononuclear cells (CBMNC), and test immune responses of these chimeric mice to an experimental cancer vaccine. METHODS: Fluorescence in situ hybridization analysis, blood biochemistry, hematology, and fluorescein-activated cell sorting analyses of cellular subsets were performed on NOD/scid-DR1 mice, in comparison to parental NOD/scid mice. Innate immunity and lifespan were examined. Histology of engrafted tissues and short-term intrahepatic engraftment of CBMNC were performed. Intracellular interferon-gamma (IFN-gamma) production was assessed in mice immunized with cancer vaccine. RESULTS: The DR1 transgene was located on chromosome 5 and no significant changes were observed in blood chemistry, peripheral blood counts, lymphoid subsets, natural killer cell and lipopolysaccharide response, and antigen presentation in the NOD/scid-DR1 mice, compared to NOD/scid mice. Interestingly, NOD/scid-DR1 mice had a significantly longer lifespan (approximately 14 months) than NOD/scid mice (approximately 8.5 months). Engraftment with human cord blood cells resulted in slight changes in the architecture/structure of spleens. No correlation was found between DR1 genotype of the donor CBMNC and extent of engraftment of human T cells. Enhanced engraftment of human cells was observed with intrahepatic injections of CBMNC in neonatal NOD/scid DR1 mice. Intracellular IFN-gamma was detected in human cells, when chimeric mice were immunized with a cancer vaccine. CONCLUSION: NOD/scid-DR1 mice were similar in most of the physiological parameters as the NOD/scid mice, with the exception of longer lifespan. Intrahepatic engraftment of neonatal mice is the preferred protocol of xenotransplantation in this model and the engrafted human cells can respond to a cancer vaccine.

Intra-thymic/splenic engraftment of human T cells in HLA-DR1 transgenic NOD/scid mice.

A HLA-DR1 transgenic mouse (NOD/scid-DR1) was derived by breeding the existing B10.M/J-[Tg]DR1 mouse with the NOD/scid mouse. The intention was to enhance engraftment of human T cells by providing human class II elements in the tissues. Thymus and spleen fragments from adult NOD/scid-DR1 mice were transplanted under the syngeneic kidney capsules, followed by injection of human cord blood mononuclear cells (CBMNC) into transplanted tissues. FACS analyses showed that human T and B cells were consistently detected in the peripheral blood and spleen, of the chimeric mice. An average of 20% of human cells was found in the spleen and the engrafted thymus/spleen tissues. Furthermore, human cells from these tissues could proliferate with anti-human CD3 antibody and these mice could generate humoral and cellular responses to allogeneic human cells. Cytokines, such as IL-10, GMCSF, IFN-gamma, and TNF-alpha were also detected in the supernatants of the cultured human cells from the chimeric mice, when they were stimulated with allogeneic cells. Therefore, a novel mouse model with functional circulating human T and B cells was established that would facilitate the exploration of vaccine, the disease processes of autoimmunity, HIV infection, and human cancer.

Chronic MCH-1 receptor modulation alters appetite, body weight and adiposity in rats.

Central administration of the neuropeptide melanin-concentrating hormone (MCH) stimulates feeding in rodents. We studied the effects of intracerebroventricular (i.c.v.) administration of an MCH-1 receptor agonist (Compound A) and an MCH-1 receptor antagonist (Compound B) on feeding in satiated rats. Compound B (10 microg, i.c.v.) blocked the acute orexigenic effect of Compound A (5 microg, i.c.v.). In an experiment designed to either stimulate or inhibit MCH-1 receptor signaling over an extended period, rats received continuous i.c.v. infusions of vehicle (saline), Compound A (30 microg/day), Compound B (30 or 48 microg/day) or neuropeptide Y (24 microg/day, as positive control) via implantable infusion pumps. Continuous MCH-1 receptor activation recapitulated the obese phenotype of MCH-over-expressor mice, manifest as enhanced feeding (+23%, P<0.001), caloric efficiency and body weight gain (+38%, P<0.005) over the 14-day period relative to controls. Chronic MCH-1 receptor activation also elevated plasma insulin and leptin levels significantly. Conversely, continuous MCH-1 receptor antagonism led to sustained reductions in food intake (-16%, P<0.001), body weight gain (-35%, P<0.01), and body fat gain relative to controls, without an effect on lean mass. Antagonism of the MCH-1 receptor may be an effective approach for the treatment of obesity.

Neither agouti-related protein nor neuropeptide Y is critically required for the regulation of energy homeostasis in mice.

Agouti-related protein (AgRP), a neuropeptide abundantly expressed in the arcuate nucleus of the hypothalamus, potently stimulates feeding and body weight gain in rodents. AgRP is believed to exert its effects through the blockade of signaling by alpha-melanocyte-stimulating hormone at central nervous system (CNS) melanocortin-3 receptor (Mc3r) and Mc4r. We generated AgRP-deficient (Agrp(-/-)) mice to examine the physiological role of AgRP. Agrp(-/-) mice are viable and exhibit normal locomotor activity, growth rates, body composition, and food intake. Additionally, Agrp(-/-) mice display normal responses to starvation, diet-induced obesity, and the administration of exogenous leptin or neuropeptide Y (NPY). In situ hybridization failed to detect altered CNS expression levels for proopiomelanocortin, Mc3r, Mc4r, or NPY mRNAs in Agrp(-/-) mice. As AgRP and the orexigenic peptide NPY are coexpressed in neurons of the arcuate nucleus, we generated AgRP and NPY double-knockout (Agrp(-/-);Npy(-/-)) mice to determine whether NPY or AgRP plays a compensatory role in Agrp(-/-) or NPY-deficient (Npy(-/-)) mice, respectively. Similarly to mice deficient in either AgRP or NPY, Agrp(-/-);Npy(-/-) mice suffer no obvious feeding or body weight deficits and maintain a normal response to starvation. Our results demonstrate that neither AgRP nor NPY is a critically required orexigenic factor, suggesting that other pathways capable of regulating energy homeostasis can compensate for the loss of both AgRP and NPY.

Melanin-concentrating hormone 1 receptor-deficient mice are lean, hyperactive, and hyperphagic and have altered metabolism.

Melanin-concentrating hormone (MCH) is a cyclic 19-aa hypothalamic neuropeptide derived from a larger prohormone precursor of MCH (Pmch), which also encodes neuropeptide EI (NEI) and neuropeptide GE (NGE). Pmch-deficient (Pmch-/-) mice are lean, hypophagic, and have an increased metabolic rate. Transgenic mice overexpressing Pmch are hyperphagic and develop mild obesity. Consequently, MCH has been implicated in the regulation of energy homeostasis. The MCH 1 receptor (MCH1R) is one of two recently identified G protein-coupled receptors believed to be responsible for the actions of MCH. We evaluated the physiological role of MCH1R by generating MCH1R-deficient (Mch1r-/-) mice. Mch1r-/- mice have normal body weights, yet are lean and have reduced fat mass. Surprisingly, Mch1r-/- mice are hyperphagic when maintained on regular chow, and their leanness is a consequence of hyperactivity and altered metabolism. Consistent with the hyperactivity, Mch1r-/- mice are less susceptible to diet-induced obesity. Importantly, chronic central infusions of MCH induce hyperphagia and mild obesity in wild-type mice, but not in Mch1r-/- mice. We conclude that MCH1R is a physiologically relevant MCH receptor in mice that plays a role in energy homeostasis through multiple actions on locomotor activity, metabolism, appetite, and neuroendocrine function.