Cannabinoid agonists (e.g., THC) dose-dependently decrease locomotor activity and body temperature and produce antinociception and catalepsy. Drugs that produce this tetrad of effects within a limited dose range are likely to function as CB1 receptor agonists. A structure activity relationship study from our laboratory investigating analogs of the CB1 antagonist rimonabant revealed that certain alterations in the 3-substituent of rimonabant’s pyrazole core conferred agonist-like properties in the tetrad. Interestingly, these effects were present in CB1 -/- mice, and were not reversed by rimonabant in wild-type mice. The present study evaluated two novel 3-substituent rimonabant analogs, O-6629 and O-6658 in the tetrad and drug discrimination, a preclinical model of drug subjective effects that possesses a high degree of pharmacological specificity. Drugs that elicit cannabinergic psychoactive effects in humans are likely to produce THC-like operant responding in animals trained to discriminate between the interoceptive stimuli produced by THC relative to vehicle. O-6629 and O-6658 decreased locomotor activity and body temperature and produced catalepsy. O-6629, but not O-6658 produced significant antinociception. However, these drugs differed from THC in regard to the magnitude of tetrad effects observed. These analogs also failed to elicit THC-like discriminative stimulus effects, nor did they antagonize THC’s discriminative stimulus in mice discriminating 5.6 mg/kg THC from vehicle. Finally, mice were trained to discriminate 5.6 mg/kg O-6629 from vehicle. O-6658 produced full substitution for O-6629, whereas the cannabinoid agonists THC and anandamide did not. O-6629’s discriminative stimulus failed to generalize to rimonabant, cocaine or morphine, whereas WIN 55,212-2 and nicotine evoked partial substitution. These results suggest that these analogs might exert their pharmacological properties through a novel cannabinoid receptor, as has been proposed for WIN 55,212-2 and anandamide. Additionally, O-6629’s discriminative stimulus may involve nicotinic acetylcholine or dopaminergic components. Future directions include determining whether the partial substitution observed with nicotine was mediated through a nicotinic mechanism. Tests with chlorpromazine, an antipsychotic that is a false positive in the tetrad, and diazepam, which produces partial substitution for THC’s discriminative stimulus through a GABAergic mechanism are also planned.

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This thesis reviews the literature on, and describes experimental work carried out with, the drug rimonabant, an antagonist at the cannabinoid CBI receptor. -, Rimonabant was licensed in Europe for the treatment of obesity in 2006, however it <- was withdrawn from the market in 2008, because of unacceptable psychiatric side effects, notably depression and anxiety. The mechanism( s) by which CB 1 antagonists produce such symptoms is unclear. The Introduction to this thesis reviews the literature on clinical depression, with special reference to endocannabinoids, and concludes that there are several plausible neurobiological mechanisms linking CB 1 antagonism to the symptoms of this disease. These include inhibition ofphasic dopamine firing elicited by reward, leading to anhedonia; disinhibition ofthe hypothalamo-pituitary stress axis; interference with the extinction of negative emotional memories; and interference with hippocampal neurogenesis. The remainder of the thesis describes the methods and results of four studies in which healthy volunteers were given rimonabant in double-blind, placebo- , controlled experiments. This work was intended to elucidate possible mechanisms by which rimonabant produces depression, with the twin goals of advancing the understanding ofthe endocannabinoid system, and contributing to our knowledge of the neurobiology of clinical depression. Two studies made use of cognitive psychological tasks in order to investigate the impact of rim on ab ant on responses to positive and negative emotional stimuli. The other two studies used functional magnetic resonance imaging (fMRI) to probe the neural effects of CBI antagonism on various functions ofthe brain. Taken together, the results of these four investigations suggest that rimonabant causes depression via interference with the brain's response to positive stimuli and enhancement ofthe response to negative ones. I conclude by discussing the key role of endocannabinoids in mood. The important implications for our understanding of the pathophysiology of clinical depression are explained.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / Como as doenças cardiovasculares (DCV) constituem a principal causa de morte na maioria dos países e as tendências de mortalidade não se apresentam totalmente elucidadas nos países em desenvolvimento, torna-se adequado explorar a evolução da mortalidade das DCV, dando ênfase ao acidente vascular cerebral (AVC) no Brasil. Devido à prevalência de AVC e também devido à associação causal entre sobrepeso ou obesidade e AVC não ser clara, é importante avaliar o efeito da perda de peso na prevenção primária de AVC. Baseado no fato do rimonabant ser a primeira droga de uma nova classe de medicamentos promissora não apenas na redução de peso, mas por sua influência sobre os fatores de risco cardiovascular, torna-se pertinente estabelecer sua eficácia e segurança. Inicialmente, para traçar um panorama sobre a epidemiologia das DCV no Brasil, com ênfase em AVC, foram realizados dois estudos com as tendências temporais de mortalidade por DCV ao longo das três últimas décadas, investigando as diferenças entre as regiões do país e entre indivíduos de diversas faixas etárias e de ambos os sexos, (artigo I e II). Além disso, duas revisões sistemáticas foram realizadas: uma para avaliar o efeito da perda de peso na prevenção primária de AVC; a segunda para investigar o uso do medicamento rimonabant no tratamento da obesidade (artigo III e IV). As taxas de mortalidade de AVC diminuíram substancialmente nas últimas três décadas, de 68,2 a 40,9 por 100 000 habitantes. Essa redução foi detectada em ambos os sexos de todas as faixas etárias, e nas diferentes regiões do país, sendo mais acentuadas nas regiões mais ricas (artigo I). A mesma tendência foi observada nas demais DCV, que em geral apresentaram uma redução anual média de 3,9%. As maiores reduções foram encontradas para AVC (média de 4,0% ao ano) seguido por doença coronariana (média de 3,6% ao ano) (artigo II). Não existem estudos avaliando o efeito da redução de peso na prevenção primária de AVC (artigo III). Houve um efeito doseresposta com o uso do rimonabant: comparado com placebo, 20 mg da droga produziu uma redução de peso maior (4,9 kg) em 4 ensaios clínicos com duração de 1 ano. Foram observadas melhoras nos marcadores de risco cardiovascular. Porém 5 mg comparado com placebo mostrou apenas uma redução de 1,3 kg a mais do peso. A maior dose também provocou maiores efeitos adversos. Perdas no seguimento foram de aproximadamente 40% (artigo IV). Durante as últimas décadas, a mortalidade por DCV em geral e AVC diminiu consistentemente no Brasil, porém a magnitude do declínio variou de acordo com as diferenças socioeconômicas. Amplas intervenções poderiam ter mais êxito se planejadas de acordo com as desigualdades sociais e diferenças culturais. Os achados apontam para a necessidade da realização de ensaios clínicos randomizados controlados avaliando a perda de peso na prevenção primária do AVC, devido à alta relevância dessa condição. Como intervenções não são totamente eficazes no tratamento da obesidade, a prevenção, englobando um conjunto articulado de ações, permanece a forma mais eficiente de controlá-la. O medicamento rimonabant apresentou modesta perda de peso, porém os resultados obtidos devem ser interpretados com cautela de acordo com as deficiências na qualidade metodológica apresentadas por todos os estudos. São necessárias pesquisas de alta qualidade para avaliar a eficácia e a segurança do rimonabant em períodos mais longos. / Cardiovascular diseases (DCV) are the leading cause of death in the world. Although mortality rates declined gradually in developed countries, the scenario is less clear in developing countries. We describe the trends in cardiovascular mortality in Brazil foccusing on stroke. Given the prevalence of stroke and the enormous health and economic cost of the disease, and the causal association between overweight or obesity and stroke is unclear, it is important to evaluate the effect of the weight loss in the primary prevention of stroke. Due to the fact that rimonabant is the first drug of a new class promising not only for weight reduction but also for the reduction of cardiovascular risk factors, it is important to establish its possible efficacy and safety. Firstly, to better understand current epidemiological aspects of CVD in Brazil, focusing on stroke, two studies were carried out evaluating the secular trends of CVD mortality along the last three decades and possible differences according to social and regional disparities, gender and age distribution (articles I and II). Two systematic revisions were carried out: one evaluating the effect of weight loss in the primary prevention of stroke and the other investigating the use of rimonabant in the treatment of obesity (articles III and IV). Stroke mortality rates decreased consistently in the last 3 decades, from 68.2 to 40.9 per 100 000 habitants. The reduction was detected in men and women and in all age strata being evident in all geopolitical regions of the Country, with the wealthiest regions exhibiting more marked reductions (article I). The same trend was observed for all CVD, with a mean annual reduction of 3.9%. The largest average decline found was for stroke (mean of 4.0% per year) followed by coronary disease (mean of 3.6% per year) (article II). There are no studies evaluating the effect of the weight reduction in the primary prevention of stroke (article III). The results concerning the drug rimonabant showed a dose-response effect: compared with placebo, 20 mg produced a greater weight loss (4.9 kg) in 4 clinical trials with 1 year of follow-up. Improvements in cardiomatabolic risk factors were also seen. However, 5 mg only led to a slightly increased weight reduction (1.3 kg more than placebo). Rimonabant 20 mg caused significantly more adverse effects. Attrition rates were approximately 40% (article IV). CVD and stroke consistently decreased in Brazil during the last decades. The reduction is in apparent relationship with indices of increasing social development. Broad interventions may be more succesful if planed according to social inequality and cultural differences. The findings point to the need of randomized controlled clinical trials specifically addressing the effects of weight loss in the primary prevention of stroke, due to the great importance of this condition. As interventions are not totally effective in the treatment of the obesity, prevention remains the most valuable tool to control its harmful effects. Rimonabant could produce modest weight loss. Some caution with the observerd results should be take account since the studies presented some deficiencies in the methodological quality. More methodologically rigorous studies that are powered to examine efficacy and safety are required.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / Como as doenças cardiovasculares (DCV) constituem a principal causa de morte na maioria dos países e as tendências de mortalidade não se apresentam totalmente elucidadas nos países em desenvolvimento, torna-se adequado explorar a evolução da mortalidade das DCV, dando ênfase ao acidente vascular cerebral (AVC) no Brasil. Devido à prevalência de AVC e também devido à associação causal entre sobrepeso ou obesidade e AVC não ser clara, é importante avaliar o efeito da perda de peso na prevenção primária de AVC. Baseado no fato do rimonabant ser a primeira droga de uma nova classe de medicamentos promissora não apenas na redução de peso, mas por sua influência sobre os fatores de risco cardiovascular, torna-se pertinente estabelecer sua eficácia e segurança. Inicialmente, para traçar um panorama sobre a epidemiologia das DCV no Brasil, com ênfase em AVC, foram realizados dois estudos com as tendências temporais de mortalidade por DCV ao longo das três últimas décadas, investigando as diferenças entre as regiões do país e entre indivíduos de diversas faixas etárias e de ambos os sexos, (artigo I e II). Além disso, duas revisões sistemáticas foram realizadas: uma para avaliar o efeito da perda de peso na prevenção primária de AVC; a segunda para investigar o uso do medicamento rimonabant no tratamento da obesidade (artigo III e IV). As taxas de mortalidade de AVC diminuíram substancialmente nas últimas três décadas, de 68,2 a 40,9 por 100 000 habitantes. Essa redução foi detectada em ambos os sexos de todas as faixas etárias, e nas diferentes regiões do país, sendo mais acentuadas nas regiões mais ricas (artigo I). A mesma tendência foi observada nas demais DCV, que em geral apresentaram uma redução anual média de 3,9%. As maiores reduções foram encontradas para AVC (média de 4,0% ao ano) seguido por doença coronariana (média de 3,6% ao ano) (artigo II). Não existem estudos avaliando o efeito da redução de peso na prevenção primária de AVC (artigo III). Houve um efeito doseresposta com o uso do rimonabant: comparado com placebo, 20 mg da droga produziu uma redução de peso maior (4,9 kg) em 4 ensaios clínicos com duração de 1 ano. Foram observadas melhoras nos marcadores de risco cardiovascular. Porém 5 mg comparado com placebo mostrou apenas uma redução de 1,3 kg a mais do peso. A maior dose também provocou maiores efeitos adversos. Perdas no seguimento foram de aproximadamente 40% (artigo IV). Durante as últimas décadas, a mortalidade por DCV em geral e AVC diminiu consistentemente no Brasil, porém a magnitude do declínio variou de acordo com as diferenças socioeconômicas. Amplas intervenções poderiam ter mais êxito se planejadas de acordo com as desigualdades sociais e diferenças culturais. Os achados apontam para a necessidade da realização de ensaios clínicos randomizados controlados avaliando a perda de peso na prevenção primária do AVC, devido à alta relevância dessa condição. Como intervenções não são totamente eficazes no tratamento da obesidade, a prevenção, englobando um conjunto articulado de ações, permanece a forma mais eficiente de controlá-la. O medicamento rimonabant apresentou modesta perda de peso, porém os resultados obtidos devem ser interpretados com cautela de acordo com as deficiências na qualidade metodológica apresentadas por todos os estudos. São necessárias pesquisas de alta qualidade para avaliar a eficácia e a segurança do rimonabant em períodos mais longos. / Cardiovascular diseases (DCV) are the leading cause of death in the world. Although mortality rates declined gradually in developed countries, the scenario is less clear in developing countries. We describe the trends in cardiovascular mortality in Brazil foccusing on stroke. Given the prevalence of stroke and the enormous health and economic cost of the disease, and the causal association between overweight or obesity and stroke is unclear, it is important to evaluate the effect of the weight loss in the primary prevention of stroke. Due to the fact that rimonabant is the first drug of a new class promising not only for weight reduction but also for the reduction of cardiovascular risk factors, it is important to establish its possible efficacy and safety. Firstly, to better understand current epidemiological aspects of CVD in Brazil, focusing on stroke, two studies were carried out evaluating the secular trends of CVD mortality along the last three decades and possible differences according to social and regional disparities, gender and age distribution (articles I and II). Two systematic revisions were carried out: one evaluating the effect of weight loss in the primary prevention of stroke and the other investigating the use of rimonabant in the treatment of obesity (articles III and IV). Stroke mortality rates decreased consistently in the last 3 decades, from 68.2 to 40.9 per 100 000 habitants. The reduction was detected in men and women and in all age strata being evident in all geopolitical regions of the Country, with the wealthiest regions exhibiting more marked reductions (article I). The same trend was observed for all CVD, with a mean annual reduction of 3.9%. The largest average decline found was for stroke (mean of 4.0% per year) followed by coronary disease (mean of 3.6% per year) (article II). There are no studies evaluating the effect of the weight reduction in the primary prevention of stroke (article III). The results concerning the drug rimonabant showed a dose-response effect: compared with placebo, 20 mg produced a greater weight loss (4.9 kg) in 4 clinical trials with 1 year of follow-up. Improvements in cardiomatabolic risk factors were also seen. However, 5 mg only led to a slightly increased weight reduction (1.3 kg more than placebo). Rimonabant 20 mg caused significantly more adverse effects. Attrition rates were approximately 40% (article IV). CVD and stroke consistently decreased in Brazil during the last decades. The reduction is in apparent relationship with indices of increasing social development. Broad interventions may be more succesful if planed according to social inequality and cultural differences. The findings point to the need of randomized controlled clinical trials specifically addressing the effects of weight loss in the primary prevention of stroke, due to the great importance of this condition. As interventions are not totally effective in the treatment of the obesity, prevention remains the most valuable tool to control its harmful effects. Rimonabant could produce modest weight loss. Some caution with the observerd results should be take account since the studies presented some deficiencies in the methodological quality. More methodologically rigorous studies that are powered to examine efficacy and safety are required.

This study was aimed at the development of novel CB1 cannabinoid receptor antago­nists that may have clinical applications for the treatment of cannabinoid and psychostimulant addiction. The rationale for the design for our target was to incorporate a bioisosteric 1,2,3-triazole ring into the vicinal diaryl group revealed in the prototypical antagonist/inverse agonist SR141716 (Rimonabant) that was pre­sumed to interact with a unique region in the CB1 receptors. Based on our prelimi­nary results we identified a novel series of 1,2,3-triazole ester and keto deriva­tives as lead compounds for biological evaluation. Here in the design rationale, syn­thesis and CB1 receptor affinity for a series of 4,5-diaryl-1-substituted-1,2,3-triazoles of ester and ketones is described. These derivatives were synthesized via a one-pot regiospecific click/acylation reaction sequence from 1-azido-2,4-dichlorobenzene and commercially available arylacetylenes. From the structure-activity studies the 5-(4-chlorophenyl) congeners exhibited the most potent CB1 receptor affinities relative to other 5-(substituted-phenyl) moieties. The 1-(2,4-dichlorophenyl)-5-(4-chlorophenyl)-4-propylcarbonyl-1,2,3-triazole (­31a) was found to be the most potent (Ki = 4.6 nM) CB1 receptor ligand of the series and exhibited high CB1 selectivity (CB2/CB1 = 417). The triazole ester 31a was further characterized as a cannabinoid antagonist in locomotor-activity studies by blocking the locomotor-reducing effects of cannabinoid agonist WIN55,212-2. In addition, unlike the prototypical cannabinoid antagonist SR141716A (Rimonabant), the triazole ester 31a did not exhibit increased activity in locomotor activ­ity studies, thus indicating the potential for a neutral antagonist profile.

La stéatose hépatique est une pathologie associée à l'obésité et à l'insulinorésistance. Les mécanismes à l'origine du développement de la stéatose hépatique sont loin d'être élucidés. Cependant, une altération de la fonction mitochondriale pourrait contribuer à l'accumulation de lipides dans le foie. Par conséquent, l'objectif de ce travail est d'évaluer la fonction mitochondriale dans deux modèles animaux de stéatose hépatique ainsi que les effets de deux molécules, le rimonabant et le resvératrol. Dans un modèle génétique d'obésité, le rat Zucker, aucune altération de la phosphorylation oxydative n'est observée. Dans un modèle plus physiologique, le rat alimenté avec un régime riche en lipides pendant différentes durées, dans les premiers temps d'un apport accru en lipides, le métabolisme hépatique est orienté de manière à utiliser ou à stocker l'excès de lipides. Cette première adaptation est ensuite inversée et une diminution de l'oxydation des acides gras mais également une diminution du stockage de triglycérides sont observées. Le traitement avec du rimonabant de rats alimentés avec un régime en gras, a un effet bénéfique sur la fonction mitochondriale hépatique notamment en augmentant l'entrée des acides gras dans la mitochondrie et donc leur oxydation. Enfin, le traitement par du resvératrol de cellules HepG2, entraîne une augmentation de la consommation d'oxygène mitochondriale et une augmentation de l'activité du complexe I mais sans modification de la biogenèse mitochondriale. Même si la mitochondrie semble peu affectée au cours du développement de la stéatose hépatique, la considérer comme une cible thérapeutique reste pertinent.

There is good pharmacological evidence that cannabinoids caused cellular changes by interacting with specific cannabinoid receptors (CBR) (Klein et al., 2000). To date, two CBRs have been identified in the human body, designated Cannabinoid Receptor 1 (CB1) and Cannabinoid Receptor 2 (CB2) (Begg et al., 2005). Endogenously occurring compounds with action at the CBRs also exist and they are called endocannabinoids. One of the four known endocannabinoids is anandamide (AEA). The endocannabinoid system, present in the human body, plays a significant role in altering the physiology of the immune system. Enhancement of this system’s anti-inflammatory effect could possibly present a vital therapeutic target for central and peripheral inflammatory disorders. A number of synthetic CB1 or CB2 specific antagonists have been developed including the highly specific CB1 receptor antagonist/reverse agonist named Rimonabant/ SR141716A. SR compounds are considered unique because these compounds not only inhibit the binding and function of cannabimimetic agents, but also act as inverse agonists. Activation of CB1 receptors produces inappropriate CNS side effects including psychoactivity, dependence and sedation (Clayton et al., 2002) whereas CB1 receptor antagonists/inverse agonists avoid or prevent these side effects. Taking the above information into consideration, Rimonabant has the potential to offer an effective long term treatment of chronic inflammatory disorders without the serious side effects of commonly used treatments. The main aim of this study is to investigate the in vitro effects of Rimonabant alone and in combination with anandamide on inflammatory associated cytokine production by human umbilical vein endothelial cells (HUVEC) and macrophage cultures. After careful consideration of the evidence stating that endothelial cells produce several important molecules vital to the inflammatory response of the body and the confirmation that CB1 receptor mRNA is generally present in endothelial cells, the use of HUVEC was deemed to be satisfactory for this study. The first phase of the study was dedicated to establishing the technique to isolate HUVEC from fresh human umbilical cord within the local laboratory and to maintain these in culture for further use during experimental procedures to test the effects of CB1 ligands. The isolation procedure, trypsinising, freezing away and thawing methods used during this experiment produced healthy HUVEC in sufficient numbers for further use. The next step was to determine the maximum in vitro concentrations at which Rimonabant and anandamide had insignificant cytotoxic effect on selected human cells and in doing so, determine suitable concentrations for further experimentation. Both compounds had a dose related anti-proliferative response when tested on HUVEC. The same dose related response was observed during the Rimonabant exposure to human lymphocytes, but no decrease in lymphocyte viability was observed when treated with anandamide at the concentrations tested. It is evident from the results that there was an almost ten times difference in the IC50 value of the two different products (14.3 μM for Rimonabant and 124.2 μM for anandamide) which was statistically significant. Flow cytometry was used to determine the effects of Rimonabant and anandamide on the surface expression of the CR3 complement receptor by human neutrophils. Neither Rimonabant nor anandamide significantly affect CR3 expression on the surface of freshly isolated human neutrophils and would exclude the CR3 expression pathways as a potential mechanism of action for the anti-inflammatory effects of these compounds. The in vitro effect of Rimonabant and anandamide alone and in combination on the production of cytokines by human macrophages and by HUVEC was determined. Anandamide was shown to inhibit the production of all the detectable cytokines (IL-8, IL-1β and IL-6 in both cell types and IL-10 and TNF-α in macrophages). Furthermore this inhibitory effect was attenuated by pre-treatment Rimonabant. These results would suggest that anandamide could induce anti-inflammatory effects observed in macrophages and HUVEC, through cannabinoid receptors. Rimonabant also inhibited the production of all the detectable cytokines following treatment with 0.5 μM and 3 μM respectively. The anti-inflammatory effects of anandamide were attenuated when combined with 1 μM of Rimonabant. Throughout the various cytokine responses, the dose-response relationship appeared to follow a bell-shaped dose-response. This occurrence proposes that Rimonabant displaces anandamide and blocks the anti-inflammatory effects of the agonist. Flow cytometry was used to determine the effects of Rimonabant and anandamide alone and in combination on the extracellular surface expression of ICAM-1 by HUVEC. Neither Rimonabant nor anandamide had any significant inhibitory effect on the expression of ICAM-1 by HUVEC. Considering the low levels for ICAM-1 expressed by the HUVEC during this experiment and the literature supporting more effective methods of activating the ICAM-1 gene and subsequent up-regulation of ICAM-1 proteins, TNF-α stimulation of HUVEC might produce a different result compared to IL-1β stimulation. The final phase of the project was to determine the effects of Rimonabant and anandamide on the adhesion of human neutrophils to HUVEC. There was no significant difference with relation to the neutrophil adhesion to HUVEC following the treatment with various combination concentrations of the compounds, and also no significant effect following treatment with either test compound individually. Although a specific mechanism of action for Rimonabant could not be uncovered during this study, there is evidence that several possible mechanisms can be excluded. The results support observations made by other researchers and the hypothesis that Rimonabant has anti-inflammatory effects. The results provide motivation for further experimentation to better understand these anti-inflammatory actions of Rimonabant. / Dissertation (MSc)--University of Pretoria, 2012. / Pharmacology / unrestricted

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