Marijuana has been used for medicinal and recreational purposes for thousands of years. Many people think of marijuana in the context of an illegal drug. Because of the antimarijuana attitude, research with cannabinoids was neglected for a long time. Although this substance is related to social problems, scientists are interested in its action and possible medicinal properties. Since the identification of the structure of Î 9--tetrahydrocannabinol, the main psychoactive ingredient of marijuana, there has been increased interest in this compound. Following the discovery of two cannabinoid receptors, CB1 and CB2 receptors, it was determined that CB1 receptors are in high density in the central nervous system while CB2 receptors are found primarily in the immune system. The endogenous cannabinoid ligands, anandamide and 2-arachidonoylglycerol, were observed in the central nervous system and peripheral tissues. Endocannabinoids differ from other "classical" neurotransmitters because they do not appear to be stored in synaptic vesicles, and they act as retrograde messengers within the brain. The endogenous cannabinoid signaling system includes cannabinoid receptors, their endogenous ligands called endocannabinoids, and the proteins for their synthesis and inactivation. The cannabinoid system appears to act as a neuromodulatory system. During the past ten years, the endogenous cannabinoid system has been implicated in a variety of physiological functions including pain reduction, motor regulation, learning, memory, and reward. Because obesity and eating disorders are prevalent, scientists are working at the molecular level to study the mechanisms controlling body weight and regulation of food intake. Several of the neuropeptides present in hypothalamic nuclei contribute to energy balance and food intake regulation. Endogenous cannabinoid and cannobinoid receptors are found in the hypothalamus and are associated with the regulation of food intake. Although the mechanisms whereby cannabinoids influence food intake remain unclear, results suggest that the cannabinoid system will be an important target in future studies in obesity. Most research on cannabinoids has focused on their role in food intake regulation in mammalian species. It is important to determine the role of endocannabinoids in other species. The effect of intracerebroventricular injection of agonists and antagonists of both CB1 and CB2 receptors in 8 to 11 week-old male Single Comb White Leghorn and 3 to 6 weeks old male broilers was investigated. It was found that agonists of both the CB1 and CB2 receptor increased food intake significantly; however, the CB2 receptor agonist had a stronger and longer lasting effect. Antagonists of both receptors decreased food intake significantly. The CB1 receptor antagonist appeared to block both cannabinoid receptors in birds, whereas the CB2 receptor antagonist did not block both receptors. Previous studies have indicated that the CB2 receptor is found only outside the brain and spinal cord, and is involved with the immune system. From the present results, it appears that both cannabinoid receptors are present in the chicken brain. Furthermore, the CB2 receptor may also be localize in the chicken brain. There are also differences in cannabinoid system between Leghorn and broilers. / Master of Science

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The suitability of the electrically stimulated guinea-pig MP-LM preparation, mouse isolated vas deferens and urinary bladder for the study of cannabinoid receptor ligands was investigated. Cannabinoid receptor agonists produced concentration-related inhibition of the contractile responses in all three tissue preparations, demonstrating high potency, chemical- and stereo-selectivity. The rank order of the inhibitory potency of the cannabinoid agonists in all three tissue preparations correlated with their binding affinity for specific cannabinoid CB1 binding sites in rat brain tissue. These results suggested a receptor-mediated mechanism of action for cannabinoid receptor agonists via prejunctional functional cannabinoid CB1 receptors in these three models, in the absence of an antagonist. The endogenous cannabinoid receptor ligand anandamide, also produced concentration-related inhibitory effects in all three tissue preparations. However, anandamide was found to be metabolically less stable in the guinea-pig MP-LM preparation. SR141716A, a potent, CB1 selective cannabinoid receptor antagonist was found to attenuate the inhibitory effects of cannabinoid receptor agonists investigated in all three tissue preparations. This provided further evidence for a receptor-mediated mechanism of action for cannabinoid receptor ligands in these three tissue preparations. However, further studies with SR141716A suggests that, it may be acting as an inverse agonist rather than a pure antagonist in these three preparations. Finally, this study was further extended to characterise some novel cannabinoid receptor ligands in the guinea-pig ML-LM preparation and mouse isolated vas deferens.

Thesis (Ph.D.)--Aberdeen University, 2008. / Title from web page (viewed on July 20, 2009). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 86-98).

Here we demonstrate that both exogenous and endogenous cannabinoids affect different aspects of learning and memory in the rat.  For example, the potent CB₁ receptor agonist, WIN-2 was able to delay-dependently impair short-term memory (STM) sparing reference memory (RM).  This demonstrates that it is the STM but  not RM processes that are more sensitive to the effects of cannabinoids. In addition, given that cannabinoids were able to hinder the recruitment of hippocampal firing correlates that are crucial for correct performance of a STM task, suppress hippocampal principal cell firing during the encoding phase of a STM task, reduce spontaneous bursting and disrupt synchronous firing of hippocampal principal cells respectively, confirm that they do alter the neurophysiology of the hippocampus.  These cannabinoid induced alterations in hippocampal neuronal activity may well explain the observed deficits across numerous other working memory (WM) and STM tasks. The results also revealed that cannabinoid-induced deficits in learning and memory are brought about due to an interaction between cannabinoid and cholinergic systems.  Although endocannabinoids failed to produce impairments in STM under normal physiological conditions, STM deficits were observed when anadamide levels were pharmacologically elevated beyond normal physiological levels.  Moreover, results demonstrate that the endocannabinoid system is involved in behavioural flexibility (i.e. reversal learning) and modulation of acquisition and/or consolidation of certain spatial elements that are necessary to perform an operant conditioning risk. Overall, the results in this thesis show that cannabinoid induced deficits in learning and memory are produced as a result of their direct effects on hippocampal processing.  The exact mechanisms that mediate these cannabinoid-induced deficits in memory are yet unclear and remain to be determined.

Type 1 cannabinoid receptor (CB1) levels decline in the striatum of animal models of Huntington’s disease (HD) and in the brains of human patients suffering from HD prior to other pathogenic changes. CB1 levels can be elevated by treatment with cannabinoids in non-neuronal cells. We wanted to determine: 1) whether cannabinoid treatment could induce CB1 expression in a striatal cell line, and 2) determine the molecular mechanisms by which cannabinoids and mutant huntingtin regulate CB1 expression. Treatment of striatal cell lines with CB1-specific agonists produced a CB1 receptor-, Akt-, and NF-?B-dependent increase in CB1 promoter activity and mRNA expression that was attenuated in the presence of mutant huntingtin. Cannabinoid treatment was associated with increased expression of the trophic factor BDNF-2 and the mitochondrial regulator PGC1? in the cell types tested. In vivo, cannabinoids may initiate a positive feedback loop increasing receptor expression and restoring cannabinoid-dependent inhibition of neurotransmitter release.

Synthetic cannabinoids such as Δ8-Tetrahydrocannabinol (Δ8) are becoming increasingly popular. Δ8 was once studied for medicinal value and showed promise as an antiemetic. The typical synthesis of Δ8 includes a Cannabidiol (CBD) dissolved in a nonpolar solvent then reacted with a strong acid and washed in a basic solution to remove contaminants. We investigated the effects of varying the acid and solvent along with the washing method used in synthesizing Δ8 to produce novel synthetic cannabinoids and increase purity of Δ8. CBD was reacted with p-Toluenesulfonic acid monohydrate or Phosphotungstic acid dissolved in Toluene, Cyclohexene, or Cyclohexane. Samples were either washed with sodium bicarbonate or extracted from the non-polar layer when added to a biphasic solution of cyclohexane and either acetonitrile or ethanol. Samples were taken at 1-, 24-, 72-, 96-, 168-, and 288-hour increments. Samples were diluted with DCM and analyzed via GCMS. Notable cannabinoids synthesized include CBC, CBN, isomers of CBD, and unknown hydrated cannabinoids. Through comparative GCMS analysis cyclohexane was discovered to be the commercial solvent of choice. Residual cyclohexane (<4%) was identified in all similar samples and poses unknown toxicity. The biphasic extraction and wash produced samples with greater purity while also reducing byproduct contamination when compared to commercial samples. This work will be useful in identifying cannabinoid byproducts in the production of Δ8 from which future drugs may be developed. This work will also be key in highlighting hidden toxic compounds related to of unregulated synthetic cannabinoid production.

EFFECTS OF CANNABINOID RECEPTOR INTERACTING PROTEIN (CRIP1a) ON CANNABINOID (CB1) RECEPTOR FUNCTION. By Tricia Hardt Smith, B.S., M.S. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University Virginia Commonwealth University, 2009. Major Director: Dana E. Selley, Ph.D., Department of Pharmacology and Toxicology This dissertation examines modulation of cannabinoid CB1 receptor function by Cannabinoid Receptor Interacting Protein (CRIP1a), a novel protein that binds the C-terminus of CB1 receptors. In Human embryonic kidney cells expressing human CB1 receptors (hCB1-HEK) and hCB1-HEK cells stably co-expressing CRIP1a (hCB1-HEK-CRIP1a), quantitative immunoblotting revealed a CRIP1a/CB1 molar ratio of 5.4 and 0.37, respectively, with no difference in CB1 receptor expression. To test the hypothesis that CRIP1a modulates CB1 receptor signaling, G-protein and effector activity were examined with and without full, partial and inverse agonists. [35S]GTPgS binding, which measures G-protein-coupled receptor (GPCR)-mediated G-protein activation, showed that CRIP1a inhibited constitutive CB1 receptor activity, as indicated by the decreased effect of the inverse agonist SR141716A. CRIP1a also decreased CB1 receptor-mediated G-protein activation by high efficacy agonists, whereas moderate and low efficacy agonists were unaffected. In experiments varying Na+ concentration, CRIP1a decreased spontaneous G-protein activation at low Na+ concentrations, where spontaneous GPCR activity is highest. This effect was eliminated by pertussis toxin pre-treatment, indicating that CRIP1a only inhibits GPCR-mediated activity. To determine whether CRIP1a modulates receptor adaptation, hCB1-HEK (±CRIP1a) cells were pretreated with WIN or THC. Both ligands desensitized CB1 receptor-mediated G-protein activation, but desensitization was unaffected by CRIP1a. In contrast, CRIP1a attenuated downregulation of CB1 receptor binding sites by WIN, but not THC. Downstream, CRIP1a attenuated constitutive CB1 receptor-mediated inhibition of cAMP, as indicated by elimination of SR141716A-stimulated cAMP, without affecting agonist-induced cAMP inhibition. Constitutive inhibition was not due to endocannabinoids because LC-ESI-MS-MS did not detect endocannabinoids in hCB1-HEK (±CRIP1a) cells. To determine whether effects of CRIP1a were conserved among cell types, Chinese Hamster Ovary cells expressing CB1 receptors were stably co-transfected with CRIP1a, and had a CRIP1a/CB1 receptor molar ratio of 15 and 1900 with and without CRIP1a over-expression, respectively. In this model, CRIP1a inhibited constitutive CB1 receptor-mediated G-protein activity, but activation by agonists was enhanced, suggesting CRIP1a effects were dependent on stoichiometry of CRIP1a/CB1 receptor or cell type. Overall, these results indicate that CRIP1a decreases constitutive CB1 receptor activity, modulates agonist efficacy, and inhibits CB1 receptor downregulation, in a ligand- and cellular environment-dependent manner.

Breast cancer, which in advanced stages often leads to bone metastasis, is the most frequent malignant tumor and the second deadliest form of cancer among women in the U.S. Skeletal metastasis is associated with imbalanced bone remodeling and eventual bone fracture that contributes to incapacitating pain and loss of mobility. Bone cancer pain remains a significant health problem due to the limited repertoire of analgesics available to treat this pain without negatively influencing the quality of life and "bone health" of the patient. Bone cancer results in a marked influx of pro- and anti- inflammatory hematological cells into the medullary cavity resulting in activation of nociceptors that express cytokine and chemokine receptors. Thus, blockade of these factors may result in a significant attenuation in bone cancer pain. The sustained release of cytokines by both primary tumor cells and invading leukocytes into the tumor microenvironment shapes the immune response to tumor invasion and ultimately mediates the shift in immune balance to the predominantly immunosuppressive state seen with late stage disease. Activation of cannabinoid receptor 2 (CB2), found on immune cells but not neuronal cells, has been shown to inhibit the release of cytokines from leukocytes; this inhibition plays an important role in CB2 agonist's ability to inhibit pain without producing the CNS side effects commonly associated with CB1. Cannabinoids have also been demonstrated in a number of cancer models to modulate the tumor microenvironment via effects specific to the tumor cells as well as regulation of invading leukocytes. Here, we show that the CB2 specific agonist JWH-015 mediates inflammatory factors in vitro and in vivo in the femoral intramedullary cavity in a murine model of bone cancer while simultaneously attenuating breast cancer induced bone pain and promoting overall health of the bone microenvironment. Further, we demonstrate JWH-015's ability to positively modify the systemic balance of regulatory to effector lymphocytes as well as modulate the suppressive function of regulatory T lymphocytes. We also show that JWH-015 attenuates breast cancer cell proliferation in vitro in a concentration dependent manner. Finally, utilizing a murine in vivo bioluminescence model, we demonstrate that JWH-015 treatment not only attenuates primary tumor growth, but also rate of metastasis. Taken together, these data establish CB2 as an innovative therapeutic target across multiple stages of breast cancer.