Dissertations / Theses on the topic 'Tetrahydrocannabinol'

The effect of Marinol, which contains the antiemetic tetrahydrocannabinol (THC), was evaluated in five cancer patients undergoing chemotherapy. Subjects rated their nausea and vomiting, food intake, appetite and mood status three times daily. Drug therapy (THC) or no drug was administered for an average of four months during the course of their chemotherapy regimen. Subjects began taking THC the first day of chemotherapy and continued (5mg/three times a day) for an average of two weeks. Subjects reported their nausea and vomiting to be increased while receiving THC which coincided with their period of chemotherapy treatment. Subjective ratings for food intake and appetite varied in each case and did not always correlate with actual caloric intake from food. Food intake in most subjects was approximately the same, or greater with THC even though the period when THC was given coincided with chemotherapy treatment, and the use of emetigenic drugs. This resulted in weight maintenance or minor weight loss in most subjects. The absence of THC during chemotherapy treatment resulted in decreased food intake. Some of the moods reported most frequently by subjects while receiving THC were activity, interaction, and relaxation. Depression, social withdrawal, and anxiety were reported less frequently and usually occurred around the time of chemotherapy. The majority of the moods reported indicated that subjects had positive feelings associated with THC therapy. The results of this study indicated that THC benefitted cancer patients by increasing food intake during chemotherapy regimens without causing adverse behavioral changes.
Master of Science

The pharmacokinetics of ethanol and (-)-trans-isomer of 9-tetrahydrocannabinol (THC), and the pharmacokinetic interaction between them were characterized using statistical models in this thesis. In chapter II, a semi-mechanistic absorption rate dependent hepatic extraction model was developed to characterize ethanol pharmacokinetics. The statistical analysis conducted based on this model indicated no association between ethanol disposition and subject age or sex, and a 23% higher typical Vmax value, a 12.5% lower typical Km value for heavy drinkers compared with moderate drinkers. In chapter III, a parent-metabolite pharmacokinetic model was developed to simultaneously describe the concentration time profile of THC and its active metabolite 11-OH-THC. A parent-metabolite model with 3-compartment pharmacokinetic model for THC and a 2-compartment model for 11-OH-THC was found to best describe the pharmacokinetics of THC and 11-OH-THC simultaneously. In chapter IV, the pharmacokinetic interactions of ethanol on THC, 11-OH-THC and 11-nor-COOH-THC were evaluated using linear mixed effects models. The results suggested that co-administration of ethanol caused an increase in THC and 11-OH-THC systemic exposure, failed to influence the terminal elimination processes of THC and 11-OH-THC, and did not affect the pharmacokinetics of 11-nor-9-COOH-THC.

It has been suggested that the CB1 G-protein-coupled receptor is internalized following agonist binding and activation of the second messenger pathways. The process of desensitization and resensitization is intimately involved with receptor internalization. Phosphorylation alters tolerance to cannabinoids thus contributing to tolerance. It is proposed that phosphorylation enhances the down-regulation of the CB1 receptor. These findings led us to look at which kinase(s) may be involved in cannabinoid tolerance. We therefore hypothesize that by preventing phosphorylation of the CB1 receptor, we may reverse tolerance. We evaluated our hypothesis by testing the role of several kinases in tolerance: protein kinase A (PKA), protein kinase C (PKC), protein kinase C (PKG). Beta Adrenergic Receptor Kinase (β-ARK), Phosphatidylinositol 3-kinase (PI3K) and the src family tyrosine kinase. We also looked at cAMP and cGMP analogs. We evaluated PKA using KT5720, a PKA inhibitor; PKC using bisindolylmaleimide I, HCI] (bis), a PKC inhibitor; PKG using KT5823, a PKG inhibitor; β-ARK using Low molecular weight heparin (LMWH), a β-ARK inhibitor; PI3K using LY294002, a PI3K inhibitor and PP1 a src family tyrosine kinase inhibitor. The cAMP analog was dibutyryl-cAMP and the cGMP analog was dibutyryl-cGMP. ICR mice were rendered tolerant to △9- tetrahydrocannabinol (△9-THC) by administering injections of 20mg/kg △9-THC s.c. every 12 hours for 6.5 days. The mice were subsequently challenged 24 hours later with an ED8O of △9-THC at 20μg/mouse (i.t.). Antinociception was measured by the tail-flick test, %MPE’s and ED5O’s were calculated. The PKG inhibitor, KT5823, showed no significant change in %MPE. The β-ARK inhibitor, LMWH, showed no significant change in the %MPE. The PI3K inhibitor, LY294002, showed no significant change in the %MPE. Inhibition of PKC, by bis had no effect on tolerance, but at a higher dose attenuated the antinociceptive effect of △9-THC in non-tolerant mice. PPl, the src family tyrosine kinase inhibitor, reversed tolerance. KT5720, the PKA inhibitor reversed △9- THC tolerance. These data support a role for PKA and tyrosine kinase in phosphorylation events in THC tolerant mice. (Supported by NIDA grants K02DA00186 and P5ODA05274).

Memory is a physiological brain function crucial for adaptive behaviour of individuals. Memory alterations are described as impairments in the processes by which memory is perceived, encoded, consolidated, retrieved, or used. there are countless situations that can lead to memory alterations. In this thesis we used specific murine mouse models to study the cellular and molecular mechanisms involved in learning and memory performance in specific situations where memory is compromised. Specifically, we described that repeated non-amnesic low doses of Δ9-tetrahydrocannabinol (THC) affect memory performance through serotonergic signalling in mice. Moreover, we reported specific memory alterations associated to the genetic inhibition of protein kinase C (PKC) gamma gene. Additionally, we revealed the involvement of the PKC-gamma isoform in the amnesic-like effects produced by THC in mice. Overall, combining behavioural, biochemical, and pharmacological approaches we have advanced in the understanding of relevant mechanisms for memory function and dysfunction associated to cannabis exposure.
La memoria es una función fisiológica del cerebro cuyas alteraciones se definen como déficits en la percepción, codificación, consolidación, recuperación o utilización de esta. En esta tesis hemos utilizado modelos específicos de ratón para estudiar los mecanismos celulares y moleculares involucrados en el desempeño del aprendizaje y de la memoria cuando esta se encuentra afectada. Específicamente, describimos que dosis bajas repetidas y no amnésicas de Δ9-tetrahidrocannabinol (THC) afectan al funcionamiento de la memoria a través de la señalización serotoninérgica. Además, reportamos alteraciones de la memoria asociadas a la inhibición genética de la isoforma gamma de la proteína quinasa C (PKC). Por otra parte, mostramos la participación de la isoforma PKC-gamma en los efectos de tipo amnésico producidos por el THC. Combinando enfoques conductuales, bioquímicos y farmacológicos, hemos avanzado en la comprensión de los mecanismos implicados en la función y la disfunción de la memoria asociada a la exposición al cannabis.

Recent evidence suggests that sustained consumption of a high-fat diet is associated with reduced CB1 receptor expression in some brain areas. Many of the neuromodulatory functions of endogenous cannabinoids are mediated by the CB1 receptor. The CB1 receptor also mediates the behavioral and physiological effects of delta-9-tetrahydrocannabinol (delta-9-THC), the primary psychoactive constituent of marijuana. While high-fat diets are associated with region-specific changes in CB1 receptor expression, it is not clear whether such changes are behaviorally relevant. To that end, separate groups of male and female rats were placed on either a high-fat diet or a standard diet. Cannabinoid function was determined in a triad of measures (e.g., hypothermia, gross locomotion, time on bar apparatus) at postnatal day 30 (PD30), PD44, PD68 and PD114. These age points respectively correspond to rodent models of early adolescence, late adolescence, early adulthood and full maturity in humans. Male rats were also tested at PD37 and PD61. Subsequently, the antinociceptive properties of delta-9-THC and the effect of delta-9-THC on food intake were also measured. After 38 days, female rats maintained on a high-fat diet were significantly less sensitive to the psychomotor effects of delta-9-THC than were the female rats maintained on the control diet. These diet-related differences persisted into full maturity. Female rats maintained on a high-fat diet were also less sensitive to changes in food intake caused by delta-9-THC than were female rats maintained on the control diet. In contrast, the hypothermic effects of delta-9-THC were not differentially affected by the type of diet consumed. Likewise, female rats maintained on a high-fat diet exhibited tail-flick latencies that were indistinguishable from those of female rats maintained on the control diet. With two minor exceptions, and in sharp contrast to female rats, sensitivity to the pharmacological effects of delta-9-THC was not differentially affected by the type of diet in male rats. In short, female rats maintained on a high-fat diet appeared to be cross-tolerant to the psychomotor and hyperphagic effects of delta-9-THC while male rats maintained on a high-fat diet exhibited responses to delta-9-THC that were virtually indistinguishable from control animals.

Δ9-tetrahydrocannabinol (THC) is the major lipophilic cannabinoid in the cannabis plant and is responsible for many of its pharmacological effects. Cannabis and cannabinoids are often administered orally in foods or lipidic formulations. Marinol® is an oral lipidic formulation containing THC dissolved in sesame oil, and is approved to treat nausea associated with cancer chemotherapy. THC exerts its pharmacological effects by binding to cannabinoid receptors 1 and 2, both of which are involved in a large variety of downstream signalling pathways. Some of these pathways regulate cell death. In agreement with this involvement, THC has been shown to be immunosuppressive in vitro and in vivo. Previous work on synthetic cannabinoids has shown that they can be transported in the lymphatic system in significant amounts when administered orally. The lymphatic system is a passive circulatory network of vessels and lymphocyte-rich nodes that has three major functions. The first function is to maintain body fluid balance. The lymphatic system is open-ended, unidirectional, and collects excess fluid at blood capillaries and tissues. Large molecules that cannot re-enter the blood also enter the lymphatic system. The second function of the lymphatic system is to mount immune responses: after lymphocytes are produced in bone marrow and thymus, they migrate to the lymphatic system and are organised into lymph nodes, which are immunological checkpoints that screen passing fluid for antigens, where innate or adaptive immune responses can be mounted onto them. The third function of the lymphatic system is the absorption of dietary lipids. Lipids are a heterogeneous group of molecules that include lipids, triglycerides and vitamins which are found in food. Lipophilic lipids are emulsified in the gastrointestinal tract by bile and phospholipids, forming mixed micelles. The micelles provide an interface for pancreatic lipase to digest triglycerides into fatty acids and glycerol. These micelles dissociate when they approach the unstirred water layer of the intestinal wall, and the contents are absorbed into specialised enterocytes. Absorption of long-chain fatty acids triggers the production of chylomicrons (CM), a large triglyceride-rich lipoprotein which transports digested fats through the lymphatic system. Triglycerides can then be released from CM by the action of lipoprotein lipase, which is expressed in a variety of cells and tissues. Lipophilic molecules and drugs can associate with CM, and share their fate in the body. Transport through the lymphatic system associated with CM bypasses the liver, which could reduce the first-pass metabolism of a drug. Lipophilic drugs with a high association with CM are prime candidates for lymphatic transport if administered orally. The use of Marinol® by cancer patients receiving chemotherapy could target THC to the lymphatic system, further suppress the immune system of those taking it and possibly making them more vulnerable to secondary infection. Therefore, it is important to confirm whether THC is transported by the intestinal lymphatic system, and whether it is selectively targeted there at immunosuppressive concentrations. To address this research question, analytical assays needed to be developed and optimised to detect THC in a variety of different biological matrices. HPLC-UV methods were successfully developed and validated to determine concentrations of THC in plasma, CM emulsions, and simulated intestinal fluid. The first step to determine the lymphatic transport of THC is to assess the fraction available for absorption in the small intestine. As mentioned previously, Marinol® is a lipidic formulation of THC, and the emulsification and digestion of lipids are not taken into account with traditional drug dissolution techniques. However, an in vitro lipolysis model is a technique that mimics lipid digestion and can reliably predict the absorption of a drug dissolved in a lipidic formulation. After establishment and optimisation of the model, up to 45% of THC dissolved in a lipidic formulation (sesame oil) was solubilised and made readily available for absorption in simulated intestinal fluid. As the concentration of THC in formulation increased, the fraction of drug solubilised decreased. The lymphatic transport potential of THC was then assessed using three techniques – association with CM, pharmacokinetic and biodistribution studies. THC had a high association with CM (72.5%), and its absolute oral bioavailability was improved three-fold when administered in a lipidic formulation (18.5%) compared to a lipid-free formulation (5.73%). At times of peak plasma concentration, the level of THC in mesenteric lymph node homogenate was 30 times higher than plasma, and was at immunosuppressive levels shown in previous in vitro studies (8.6 µg/mL). The mesenteric lymph nodes drain the small intestine, and would be the first nodes that dietary fats pass after being absorbed from the small intestine. Finally, the antiproliferative effect of THC was assessed by exposure to peripheral blood mononuclear cells isolated from healthy individuals and cancer patients recovering from chemotherapy. THC produced a dose-dependent reduction in cell proliferation, with significant differences from vehicle above 10 µg/mL. THC had a reduced effect on cells isolated from cancer patients compared to cells isolated from healthy individuals. THC associated with CM emulsion also had a similarly reduced effect to cells isolated from healthy individuals compared to the effect of THC dissolved in organic solvent. Collectively, this work confirms that THC is transported via the intestinal lymphatic system, and can be concentrated there at extremely high levels, shown to be immunosuppressive in previous in vitro studies.

The frontostriatal dopamine system modulates brain function and is affected by both genetic and environmental factors. Dysfunction of this system is associated with many pathological states, including schizophrenia. The enzyme catechol-O- methyltransferase (COMT) metabolises dopamine and its gene contains a polymorphism (Val¹⁵⁸Met) that affects enzyme activity. Delta-9- tetrahydrocannabinol (THC), the main psychoactive component of cannabis, has been suggested to interact with this polymorphism to increase the risk for psychosis and cognitive impairments. Dopaminergic mechanisms are a plausible candidate for mediating this interaction. I used microdialysis coupled with high performance liquid chromatography (HPLC) to examine the effects of THC on extracellular dopamine and its metabolites in the nucleus accumbens, dorsal striatum and medial prefrontal cortex (mPFC) in freely moving mice. Following acute COMT inhibition with tolcapone, THC increased extracellular dopamine levels in the nucleus accumbens in tolcapone-, but not in vehicle-, treated mice. The introduction of the low activity Met allele into the COMT gene produced a highly specific, novel mouse model of the Val158Met polymorphism. In contrast to the effects of acute COMT inhibition, the Met allele protected against THC-induced changes in accumbal dopamine. No interactive neurochemical effects were observed in the dorsal striatum (pharmacological and genetic study) or in a preliminary study of the mPFC (genetic study only). On a progressive ratio task measuring motivational salience, the direction of the interactive effect between COMT genotype and THC differed between 2 independent cohorts and provided tentative leads that stress/arousal-dependent effects on COMT may have a confounding effect. My data provide evidence that COMT activity modulates the effect of THC on accumbal dopamine function, and suggest the mechanism through which this interaction is mediated differs between acute and lifelong reduction in COMT activity. Through the interactive effect on the dopaminergic system, the data provide a potential mechanism for the reported interaction between COMT and cannabis/THC in determining psychosis risk and cognitive impairments.

Road fatalities related to marijuana intoxication have steadily increased over the last 10 years (Drummer, 1994; Drummer, 1998; Drummer & Gerostamoulos, 1999). This has led to the introduction of sobriety testing in Victoria, Australia to test for driving impairment caused by marijuana and other psychotropic drugs. Surveys have reported an increase in community concern in Australia over the use of marijuana and an increase in the prevalence and use of marijuana (National Campaign Against Drug Abuse Survey; 1985, 1988, 1991, 1993; National Drug Household Survey; 1995, 1998). Commensurate with the increase in the use of marijuana in society, road statistics indicated that the number of road accidents and deaths involving the presence of THC (the active ingredient in marijuana) in driver specimens has also increased (Drummer & Gerostamoulos, 1999). Consistent with these mortality statistics, past research examining the effects of THC on driving ability indicate that THC impairs both car control (Moskowitz, 1985), and the maintenance of the lateral position of a vehicle (Ramaekers et al., 2000). Intoxication by THC is more likely to result in the crashing into obstacles on a driving course than when not intoxicated (Hansteen et al., 1976). These findings indicate that marijuana impairs driving ability and since the prevalence of marijuana use is increasing this poses a significant risk on our roads. It is essential therefore, that a tool that detects levels of THC in drivers, similar to breath analysis instruments used for the detection of alcohol in drivers, is introduced. To date, there is no such reliable instrument, that could be used on the roadside, and that accurately measures the level of THC in humans. For this reason, some government departments have considered the use of sobriety tests to detect impaired driving. In particular, the Standardised Field Sobriety test (SFSTs) that comprises the Horizontal Gaze Nystagmus test (HGN), Walk and Turn test (WAT) and the One Leg Stand test (OLS) were implemented in Victoria, Australia from December 1st 2000. The validity of these tests have been previously examined by other researchers and their conclusions suggest that sobriety tests have a varied accuracy in detecting impairment caused by drugs, ranging from 44% to 94% (Heishman et al., 1996; Compton, 1986). The present study examines the efficiency of sobriety tests to detect impairment in driving caused by marijuana. The SFSTs were examined, as well as the Romberg Balance test (RB) and the Finger to Nose test (FTN) taken from the Drug Evaluation and Classification Program (DECP) (Los Angeles Police Department, USA). The present study was conducted by Swinburne University, Victoria, Australia. The National Institute on Drug Abuse in the USA (NIDA) provided the marijuana cigarettes. The major objectives of the study were to examine the influence of cannabis on driving performance and on performance on the sobriety tests. The relationship between simulated driving performance and sobriety test performance was then examined to establish the accuracy of sobriety tests to predict driving ability. The present study also examined whether any differences in performance either on the driving tests or on the sobriety tests exist between regular cannabis users and non-regular cannabis users. Driving stress was an additional variable assessed to establish whether individuals with low, normal or high driver stress perform differently on the driving task after the consumption of a low and high dose of cannabis. We tested 40 participants comprising 14 females and 26 males. All participants completed a medical examination questionnaire, demographics questionnaire, Frequency of Cannabis Use Questionnaire and Intoxication Rating Questionnaire. All participants completed 3 marijuana sessions involving the administration of a placebo cigarette (0% THC, weight 702mg, .000gm ∆-9-THC; 0.0mg/kg THC), the administration of a low THC cigarette (1.74% THC, weight 779mg, .813gm ∆-9-THC; 0.2mg/kg THC) and the administration of a high THC marijuana cigarette (2.93% THC, weight 790mg, 1.776gm ∆-9-THC; 0.73mg/kg THC). All sessions were randomised (using Latin-square design), counter-balanced and double-blind. In each session, participants completed 3 sobriety tests and 2 driving simulator tests. Sobriety tests were scored by allocating a score of 1 for each sign (error, e.g., hopping during test performance to maintain balance) observed by the administrator. Generally, a score of 2 or more constituted impairment to a degree equivalent to a blood alcohol concentration (BAC) above 0.10%. The driving simulator test comprised 36 variables. Each time the participant performed an error, a loading factor was added to the corresponding variable (e.g., collision (variable) loading factor is 10, if a collision occurred twice a score of 20 was allocated to this variable). The sum of all 36 variables constituted the level of overall driving impairment. Blood samples were taken throughout each session approximately 20 minutes apart. Intoxication Rating Questionnaires revealed that participants reported that the subjective effect of placebo cigarettes was much weaker than the cigarettes that they usually smoke and that no psychological (such as time distortion) and physiological (such as increased heart rate) changes were experienced. For the low THC cigarettes most participants described the strength, and the effects, as similar to cannabis that they usually smoke. The high THC cigarette was described by most participants as being much stronger, and having some different symptoms, when compared to cannabis that they usually smoked. There were however, some differences in the description of the low THC and the high THC cannabis cigarettes between regular and non-regular cannabis users. Regular users reported that the high THC cigarette was more similar to the cannabis that they usually smoke, whereas non-regular users stated that this was more likely to be the case for the low THC cigarette. Results from the driving simulator task revealed that THC impaired the driving variables: �straddling the solid line� and �straddling the barrier line�. The results indicated that increasing levels of THC increasingly impaired the ability to maintain the steady position of a vehicle within the correct traffic lane. The consumption of low and high doses of THC resulted in two or more wheels of the vehicle moving over a solid line marked out for traffic moving in the opposite direction. Low and high doses of THC also resulted in two or more wheels of the vehicle moving over a broken/barrier line marked out for traffic moving in the same direction. Increasing levels of THC appear to impair both balance and attention required to control the position of a vehicle in traffic. These results are consistent with past research that indicates that THC impairs car control (Moskowitz, 1985) and increases the standard deviation of the lateral position of a vehicle (Smiley et al., 1981; Ramaekers et al., 2000). Research into the effects of THC on brain cannabinoid receptors indicate that THC interferes with normal functioning of the cerebellum, the brain region responsible for balance, posture, and the coordination of movement (Childers & Breivogel, 1998). When driving ability was impaired the level of THC in the blood was between 3 and 5 ng/ml. These findings are consistent with previous research that has reported that driving is maximally impaired by THC plasma levels of 13 ng/ml (approximately 8ng/ml in blood, using a multiplication factor of 1.6 (Giroud, et al., 2001) (Berghaus et al., 1995). The results of the present study also indicated that THC impairs performance on sobriety tests with more individuals impaired with increasing levels of THC (e.g., at Time 1; placebo: 2.5%, low THC: 23.1%, and high THC: 46.2%). Performances on the sobriety tests RB and FTN were unrelated to the level of THC. The test most related to the level of THC was the OLS test, where almost all signs of this test were observed, after the consumption of both low and high THC cigarettes. The accuracy of a �new� sign in the scoring procedure of the HGN test: head moves/jerks (HMJ) was also identified. Including HMJ increased the percentage of individuals scored as impaired after the consumption of low and high THC cigarettes (e.g., at Time 1; placebo: 2.5%, low THC: 38.5% and high THC: 56.4%). Including HMJ as a sign significantly improved the accuracy of the SFSTs to detect impairment associated with the level of THC. The mean level of THC in the blood, when the highest number of participants were classified as impaired, was 70 ng/ml. Differences in performance were observed between regular cannabis users and nonregular cannabis users. Non-regular cannabis users were more impaired on the driving simulator task after the consumption of low and high levels of THC when compared to regular users. Non-regular users recorded significantly longer RTs to emergency situations, more collisions, and shorter distances between the vehicle and an object (after an emergency stop) when compared to regular cannabis users. Signs exhibited during sobriety test performance were related to the level of THC more often for nonregular users compared to regular users. The level of THC in the blood was higher in regular users, compared to non-regular users, at all times in both THC conditions. When driving ability was impaired and significantly related to the level of THC, the SFSTs were also related to level of THC. Sobriety test performance was related to driving impairment, because, as driving impairment increased with the level of THC, so did the number of signs present during the performance of the sobriety tests. Since nonregular users performed more poorly on the driving task compared to regular users, it is no surprise that they exhibited a larger number of signs during the sobriety testing. Although there was a positive linear relationship between driving ability and sobriety tests, such as the relationship between straddling barrier lines and the OLS test, the validity of sobriety tests to predict driving impairment in part depends upon the size of this relationship. Using performance on the SFSTs to assess �impairment�, 46.7% of individuals in the high THC condition were impaired. A discriminant analysis was performed to determine whether the remaining 53.3% of participants were also impaired but not classified as impaired, or whether the SFSTs correctly classified them as not impaired. The results indicated that the sobriety tests (SFSTs; HGN, WAT and OLS) correctly assessed 76.3% of participants in the high THC condition as either impaired on driving or not impaired on driving. Specifically, this percentage included the correct identification of 84% of impaired drivers as impaired, but only 61.5% of unimpaired drivers as unimpaired. The best predictor of driving impairment was the OLS test. In the low THC condition the sobriety tests correctly classified 100% of impaired drivers as impaired, but this occurred at the expense of falsely classifying most unimpaired drivers as also impaired. This finding suggests that sobriety tests detect the presence of THC even when driving is not impaired. Examining the utility of including the �new� sign HMJ in the SFSTs indicated that when identifying impairment on the driving task performed at Time 2, in both the low and high THC condition, the SFSTs were a better predictor of driving impairment when HMJ was included than when the sign was not included. This finding suggests that the inclusion of HMJ in SFSTs scoring procedure increases the likelihood of detecting drivers who are impaired by THC. In conclusion, the results suggest that THC impairs driving ability by reducing one�s ability to maintain a safe position in traffic. At this time THC blood levels are between 3 and 5 ng/ml. THC also impairs driving ability differently for non-regular and regular users of cannabis, where non-regular users are more impaired by THC than regular users. When this occurs, THC blood levels in non-regular users are between 2 and 12 ng/ml, and in regular users between 5 and 16 ng/ml. Performance on the sobriety tests is also impaired by increasing levels of THC. The OLS test is the most sensitive test in detecting the presence of THC. In the present study the SFST battery and each individual test that it comprises are moderate predictors of driving impairment but do misclassify 16% of impaired individuals and 38.5% of not impaired individuals. In addition, the results suggest that sobriety tests are more sensitive to the presence of THC than actual driving impairment. This was revealed by the large number of individuals judged as impaired on driving in the low and high THC conditions even when driving was unaffected. It is important to note that when this occurred, the sobriety tests were accurate in detecting 100% of impaired individuals. Finally, the introduction of the �new� sign HMJ is likely to increase the accuracy of the SFSTs to detect individuals impaired by THC and this sign should be considered for inclusion by policing agencies.

mestrado em Bioquímica
Com a descoberta do sistema endocanabinóide (ECS), a sinalização canabinóide tem sido identificada em vários processos fisiológicos e patológicos. Este sistema é formado pelos recetores canabinóides do tipo 1 e 2 (CB1 e CB2), pelos endocanabinóides anandamida (AEA) e 2-araquidonoilglicerol (2-AG), e pelas enzimas de síntese e degradação destes endocanabinóides. A sinalização endocanabinóide é essencial para a fertilidade, implantação e decidualização, e poderá também estar envolvida no desenvolvimento da placenta, sendo necessários mais estudos nesta área. O desenvolvimento da placenta é um processo altamente regulado e dinâmico, que envolve a proliferação, diferenciação e apoptose dos trofoblastos, as células especializadas da placenta. Este orgão, além de ser responsável pela proteção imune do feto, e pela troca de gases/nutrientes, desempenha um papel endócrino, produzindo hormonas essenciais para o estabelecimento e manutenção de uma gravidez saudável. Os endocanabinóides poderão ser um dos muitos fatores intervenientes na complexa regulação da produção de hormonas pelos trofoblastos. A cannabis é a droga mais utilizada pelas grávidas e o seu consumo durante a gravidez está associado a várias complicações nomeadamente atraso do crescimento intra-uterino e parto prematuro. Contudo, os mecanismos bioquímicos responsáveis por estas complicações estão, ainda, por desvendar. O principal fitocanabinóide, o Δ9- tetrahidrocanabinol (THC) poderá levar a um desequilíbrio da homeostasia do sistema endocanabinóide e da função endócrina da placenta. Com este trabalho pretendemos analisar qual o efeito do THC, nos trofoblastos, em especial na sua função endócrina. Para isso foram utilizados explantes de placenta e uma linha celular de trofoblastos, as células BeWo. Os resultados obtidos por qRT-PCR demonstram um aumento da transcrição de genes que codificam para a leptina, proteína placentária 13, progesterona e estradiol, hormonas essenciais para o desenvolvimento placentário e crescimento fetal. Foi também observado um aumento da secreção de gonadotrofina coriónica e estradiol. Esta desregulação hormonal poderá originar alterações nas vias de sinalização que regulam a proliferação, diferenciação e apoptose dos trofoblastos dando origem a uma deficiente placentação associada a complicações na gravidez.
With the discovery of the endocannabinoid system (ECS), the relevance of cannabinoid signaling has been recognized in several physiological and pathological processes including reproduction. This system consists of cannabinoid receptors type 1 and 2 (CB1 and CB2), the endocannabinoids anandamide (AEA) and 2- arachidonoylglycerol (2-AG) and the enzymes for degradation and synthesis of these endocannabinoids. Cannabinoid signaling is essential for fertility, implantation and decidualization and possibly also for placental development, which is a research area that needs future studies. The formation and development of the placenta is a highly regulated and dynamic process that involves proliferation, differentiation and apoptosis of trophoblasts, the specialized cells of placenta. Besides being responsible for the immune protection of the fetus, and for the exchange of gases and nutrients, the placenta plays an endocrine role, producing hormones essential for proper establishment and maintenance of a healthy pregnancy. The endocannabinoids may be involved in the complex network that regulates hormone production by trophoblast cells. Cannabis is the drug most commonly used by pregnant women and its consumption during pregnancy is associated with complications, such as, intrauterine growth restriction and preterm delivery. Nevertheless, the biochemical mechanisms that lead to these complications are yet to be unveiled. The major phytocannabinoid, Δ9-tetrahydrocannabinol (THC) may have an impact in the endocannabinoid system homeostasis and in placenta endocrine function. With this work it was intended to analyze the effect of THC on trophoblasts, especially in its endocrine function. For this it was used placental explants and a trophoblast cell line, BeWo cells. Quantitative PCR results demonstrated an increase in the transcription of genes that encode hormones such as leptin, placental protein 13, progesterone and estradiol fundamental for placental development and fetal growth. It was also observed an increase in the secretion of beta-chorionic gonadotropin and estradiol This hormonal deregulation can have an impact on the signaling pathways for trophoblasts proliferation, differentiation and apoptosis, resulting in an incorrect placentation, which is associated with poor pregnancy outcomes.

In the past ten years, synthetic cannabinoids (SC) have emerged as drugs of abuse. Unlike D9-tetrahydrocannabinol (THC), many SCs are associated with serious health complications and death. One way in which THC and SCs differ lies with their enhanced potency and efficacy at the CB1 receptor. No current methods exist to measure efficacy at the CB1 receptor in vivo, and the abuse-related properties of SC cannabinoids are not well explored. Here, we utilized CB1 wild type (WT), heterozygous (HET), and knockout (KO) mice. By employing CB1 ligands which differ in efficacy we have developed a method to explore the relationship between efficacy and the ability to produce cannabimimetic (catalepsy, hypothermia, and antinociception) effects when CB1 expression was reduced by half. Additionally, the intracranial self-stimulation procedure (ICSS) was utilized to investigate the effects of enhanced efficacy at CB1 upon reward processes using representative SC CP55,940. As predicted, the potency shift between WT and HET mice inversely correlated with the efficacy of the test drug for both hypothermia and antinociception, but not catalepsy. This efficacy stratification was correlated with the agonist-stimulated [35S]GTPgS binding assay, demonstrating this model as an effective tool to ascertain in vivo efficacy differences at CB1. In ICSS, CP55,940 elicited only rate-decreasing effects acutely, although tolerance developed following repeated dosing, with no evidence for spontaneous or rimonabant-precipitated withdrawal. Together, these data indicate that highly efficacious cannabinoid ligands require few receptors to produce cannabimimetic effects, and that the model provides an effective means to quickly ascertain differences in efficacy.

The medical use of marijuana is increasing, yet little is known about the exposure-response relationships resulting in its psychoactive effects. Δ9-tetrahydrocannabinol (THC) and its active metabolite (11-hydroxy-THC; THC-OH) are the principal psychoactive components in marijuana. It is well known that the plasma concentrations of the psychoactive components of marijuana do not directly relate to the observed psychoactive effects. The presence of a counter-clockwise hysteresis in the plasma concentrations-effect plot demonstrates a temporal delay between the plasma concentrations and observed effect following the intravenous administration of THC. The overarching objective of this research was to better understand the relationship between the plasma and brain concentrations of the psychoactive components (THC and THC-OH) and the observable psychoactive effects after intravenous administration of THC, utilizing model-based approaches. Specifically, the pharmacokinetics were explored using population pharmacokinetic (Pop PK) and physiologically-based pharmacokinetic (PBPK) modeling whereas the pharmacodynamics (PD) of the psychoactive effect (“highness”) were explored using effect-compartment modeling and linking the PD to the PBPK-derived concentrations predicted in the brain and an assumed effect-site. A “hypothetical” effect compartment model was developed to characterize the observed delay in peak “highness” ratings. A direct relationship was established between the reported psychoactive effects (“highness” or intoxication) and the predicted effect-site concentrations of both components (THC and THC-OH) using this effect-compartment modeling approach. The faster plasma to effect compartment equilibration for THC-OH indicated a more rapid equilibration of the active metabolite between plasma and the effect-site (biophase) than for the parent THC. In addition, a PBPK modeling approach was pursued to predict and relate the brain concentrations of THC and THC-OH to the psychoactive effect. The relationship between the effect and the predicted unbound brain concentration of THC indicated an indirect relationship, suggesting a temporal delay between brain concentrations of THC and observed effect. However, a direct relationship was observed between the observed effect and the unbound brain THC-OH concentrations. In addition, the unbound concentrations of THC-OH in the brain were predicted to be higher than the corresponding THC concentrations. These findings highlight the importance for the inclusion of THC-OH, in addition to THC, when relating the observed effect to the concentrations of the psychoactive components of marijuana. These models contribute to the understanding of the PK-PD relationships associated with marijuana use and are important steps in the prediction of the pharmacodynamic effects related to the psychoactive components in marijuana and establish an approach for investigating other THC-related effects.

Purpose: Marijuana is the most widely used illicit drug in the United States. However, marijuana and cannabinoid derivatives have potential therapeutic uses. Studies in cannabis users have yielded contradictory results with regard to long-term effects on cognitive functions. There is no prospective study assessing this issue, and such studies may raise ethical issues in humans, whereas mice have been shown to exhibit similar cannabinoid-mediated behaviors as humans. The purpose of this study was to assess the consequences of chronic administration of Δ9-THC, the major psychoactive component of marijuana, in a mouse memory model. Methods: In Experiment 1, the dose-response relationship of Δ9-THC was assessed in the object recognition task, a well-documented rodent memory model. In Experiment 2, mice were treated repeatedly with either escalating doses of Δ9-THC or vehicle for one week, and then challenged with the drug to assess whether tolerance had developed. Results: Acute Δ9-THC dose-dependently interfered with memory as assessed in the object recognition task (ED50 95% C.I. = 0.5 (0.1 to1.7) mg/kg). No tolerance to the memory disruptive effects of 1 mg/kg Δ9-THC was evident after chronic treatment. Conclusions: Considerably low doses of Δ9-THC impaired memory. The failure of chronic Δ9-THC to produce tolerance in this model was surprising considering that a similar dosing regimen has been reported to produce tolerance in non-mnemonic behaviors. The results suggest that memory is particularly sensitive to the disruptive effects of Δ9-THC and chronic cannabis use is likely to elicit persistent impairment of cognitive function. Caution should be applied in advocating chronic use of medicinal cannabinoids. Potential solutions lie in reinforcing education on the harm caused by cannabis use and availability of alternative solution to cannabis users, especially among youth that have shown to be more vulnerable to this drug.

The idea that humans and laboratory animals can become physically dependent on marijuana or its primary psychoactive constituent, delta-9-tetrahydrocannabinol (THC), is gaining acceptance. However, there are no currently approved pharmacotherapies to treat cannabinoid withdrawal. The objective of this thesis was to evaluate whether elevating endogenous anandamide levels using mice lacking fatty acid amide hydrolase (FAAH), the enzyme responsible for anandamide metabolism, would ameliorate THC dependence. Mice were treated subchronically with a low or high THC dosing regimen and challenged with the CB1 receptor antagonist, rimonabant, to precipitate withdrawal. Following subchronic THC treatment, rimonabant precipitated a significant increase in paw flutters that was dependent on THC dose. However, FAAH-/- mice displayed a similar magnitude of withdrawal responses as wild type control mice, regardless of subchronic dosing regimen. Finally, rimonabant was equipotent in precipitating withdrawal responses in both genotypes. Collectively, these results demonstrate that FAAH-/- and +/+ mice show identical THC dependence, thus arguing against the notion that elevating anandamide levels through FAAH suppression will reduce cannabinoid withdrawal.

A maconha é a droga ilícita mais consumida no mundo, tem como substância psicoativa o delta-9-tetraidrocanabinol (9-THC). Os testes colorimétricos são normalmente realizados em amostras de maconha (e para qualquer outra droga ilícita) apreendidas. A adição de reagentes específicos em uma solução de extrato de maconha pode indicar a presença de substância ilícita mediante mudança de cor. No entanto, a literatura recentemente tem relatado ocorrências de resultados falso-positivos quando os testes colorimétricos são aplicados em algumas folhagens de diversas espécies de plantas. Este estudo utilizou as técnicas voltamétricas em meio orgânico N-N-dimetilformamida utilizando tetrafluoroborato de tetrabutilamônio como eletrólito de suporte e eletrodos de trabalho de disco de carbono vítreo e platina. Aplicando-se um potencial de pré concentração em - 0,5 V, verifica-se um pico de corrente anódica em ± 0,01 V vs Ag/AgCl ,KCl(sat). Utilizando eletrodo de disco de carbono vítreo como eletrodo de trabalho, na modalidade de onda quadrada, obteve-se uma dependência linear na faixa de concentração entre 1,0 x 10-9 mol L-1 a 2,2 x 10-8 mol L-1, com um coeficiente de correlação linear em 0,999 e um limite de detecção de 6,2 x 10-10 mol L-1. Tais resultados possibilitaram a determinação de 9-THC na ordem de nmol L-1.
Marijuana, the illicit drug that is most consumed worldwide, contains 9-tetrahydrocannabinol (9-THC) as the main psychoactive substance. Presumptive colorimetric tests are usually performed on seized marijuana (or any illicit drug) samples: the addition of specific reagents to the marijuana extract solution prompts a change in. This study was developed using voltammetric techniques in organic medium (N,N-dimethylformamide) using tetrabutylammonium tetrafluoroborate (TBATFB) as supporting electrolyte, and glassy carbon and platinum disc as working electrode. Applying a pre-concentration potential of -0.5 by square-wave voltammetry, we detected a well-defined anodic peak current in ± 0.01 V versus Ag/AgCl, KCl(sat); 9-THC detection presented linear dependence at concentrations ranging from 1.0 × 10-9 mol L-1 to 2.2 × 10-8 mol L-1, with a linear correlation coefficient 0.999 and a detection limit of 6.2 × 10-10 mol L-1, using the glassy carbon disc working electrode.

Cannabinoids and cannabinoid receptors have been shown to play important roles in immune regulation particularly as modulators of anti-inflammatory cytokines and antibody production. The predominant cannabinoid receptor involved in this immune regulation is cannabinoid receptor 2 (CB2), which is robustly expressed in B cells. Utilizing a combination of bioinformatics, 5' RACE, real time RT-qPCR, and reporter assays, we showed that human B cells from peripheral blood mononuclear cells (PBMC) expressed one CB2 transcript while mouse B cells from spleen express three CB2 transcripts. Alignment of the sequenced B cell RACE products to either the mouse or human genome, along with the GenBank mRNA sequences, revealed that the transcripts isolated in this study contained previously unidentified transcriptional start sites (TSSs). In addition, expression construct testing of the genomic region containing the TSSs of the mouse CB2 exon 1 and 2 transcripts showed a significant increase of promoter activity. Bioinformatics analysis for cis-sequences in the promoter regions identified DNA binding sites for NF-kB, STAT6, and Elk1 transcription factors activated by LPS, IL-4 and anti-CD40. Regarding variations in CB2 transcript expression among the immune cell subtypes, RACE analysis showed that the exon 1b transcript is seen in B cells but not in T cells, dendritic cells or macrophages. Furthermore, RT-qPCR showed variations in transcript expression during B cell development as well as in resting versus LPS or IL-4/anti-CD40 stimulated B cells. The exon 1a transcript was predominant in pre-, immature and resting B cells whereas the exon 1b and 2 transcripts were enhanced in mature and activated B cells. These data showed for the first time that human B cells use one TSS for CB2 expression while mouse B cells use multiple TSSs for the expression of three CB2 transcripts, in which the expression of the individual transcript is related to immune cell type and/or cell activation state. Additionally, this is the first report in mouse B cells defining TSSs that are in genomic areas with promoter activity thus suggesting the location of two promoter regions. Defining the CB2 transcript expression during various stages of B cell activation provide clues to therapeutic methods.

Despite the widespread use of Highly Active Anti-retroviral Therapy (HAART) to combat Human Immunodeficiency Virus (HIV), the causative agent of acquired immunodeficiency syndrome (AIDS), HIV-Associated Neurocognitive Disorders (HANDs) still remain a dire issue. HIV can enter the brain through infected monocytes and infect microglia, the resident macrophages within that compartment. Due to its pro-inflammatory properties, HIV regulatory protein Tat (Transactivating protein) may play a role in the progression of neurocognitive disorders. The aim of this project was to determine whether the select cannabinoids THC and CP55,940 could ablate the inflammatory response on BV-2 mouse microglia cells caused by Tat. Within the constraints of the experiment, no major effects of cannabinoid treatment were observed at the level of the proteome as tested by two-dimensional gel electrophoresis . In contrast, these cannabinoids ablated the inflammatory response caused by the HIV protein Tat at the level of the secretome and at the level of gene expression. These collective observations suggest that select cannabinoids have the potential to down-regulate the elicitation of proinflammatory gene products that are engendered by the HIV protein Tat. Furthermore, the results suggest a potential for cannabinoid agonists at cannabinoid receptors to serve as adjunct ablative agents in the treatment of HIV-associated neuropathological processes.

O Canabidiol (CBD), que representa aproximadamente 40% dos canabinóides encontrados na planta Cannabis sativa, é desprovido dos efeitos psicológicos e cognitivos típicos do 9-Tetraidrocanabinol (9THC). Estudos sugerem que o CBD apresenta propriedades ansiolíticas, porém esta substância nunca foi testada na ansiedade clínica. Do mesmo modo, não se sabe como estes possíveis efeitos seriam mediados centralmente em pacientes com transtorno de ansiedade social (TAS). Diante das evidências da existência de um sistema canabinóide em humanos e do crescente interesse terapêutico no uso do CBD, justifica-se um estudo para desenvolvimento e validação de uma metodologia analítica para a determinação e quantificação de CBD e 9THC empregando a técnica de cromatografia em fase gasosa/espectrometria de massas. O método desenvolvido e validado se mostrou rápido, simples, de baixo custo com boa sensibilidade e apropriado para aplicação na área da toxicologia clínica. O método demonstrou ser linear no intervalo de concentração de 5 a 500 ng/0,5 mL de plasma para o CBD (r2 = 0,99) e de 5 a 300 ng/0,5 mL (r2 = 0,98) para o 9THC. Os limites de detecção e quantificação foram respectivamente 0,1 ng/0,5 mL e 0,5 ng/0,5 mL para o CBD e, 5 ng/0,5 mL e 10 ng/0,5 mL para o 9THC. Valores de precisão inter e intra ensaio estão respectivamente, na faixa de 5,5% a 12,7%, e de 2,1% a 8,1%. Valores de exatidão inter e intra ensaio estão respectivamente, na faixa de 1,2% a 12,0, e de 1,2 a 14,5 para CBD e 9THC. A eficiência da extração foi obtida na faixa de 54,6 a 93,2% de recuperação para os analitos. A metodologia validada foi empregada em um estudo clínico para correlacionar a dose após a administração controlada de CBD em pacientes com transtorno de ansiedade social.
Dissertation (Master). Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo, Ribeirão Preto, 2008. The Cannabidiol (CBD), that represents about 40% of the cannabinoids found in Cannabis sativa plant, is devoided of the typical psychological and cognitive effects of 9-Tetrahydrocannabinol (9THC). Researches suggest that CBD shows ansiolitic properties, but this substance was never tested on clinical anxiety. It is unknown how possible CBD effects could be centrally mediated in social anxiety disorder (SAD) patients. There are evidences about the existence of a cannabinoid system in humans and there is also increasing interest on therapeutic CBD application. The present study was conducted to develop and validate an analytical methodology for determination and quantification of CBD and 9THC, employing gas phase chromatography/mass spectrometry technique. The validated method was fast, easy, low cost, with a good sensibility and appropriate for clinical toxicology applications. The method was linear on the range of 5 to 500 ng/0,5 mL of plasma for CBD (r2=0,99) and 5 to 300 ng/0,5 mL (r2=0,98) for 9THC. The detection and quantification limits were 0,1 ng/0,5 mL and 0,5 ng/0,5 mL for CBD and 5ng/0,5 mL and 10 ng/0,5 mL for 9THC respectively. Inter and intraday reproducibility were between 5,5% to 12,7% and 2,1% to 8,1%, respectively. Inter and intraday accuracy was between 1,2% to 12% and 1,2% to 14,5% for CBD and 9THC respectively. The recovery of extraction was between 54, 6% to 93,2% of recovery for the analytes The validated methodology was applied in a clinical trial to correlate the doses after the controlled cannabidiol administration to patients with social anxiety disorders.

Historically, cannabis has been used as a pharmaceutical drug for a variety of conditions including rheumatism, depression, convulsions, and malaria. Since the 1970s, randomised, controlled clinical trials have shown cannabis to be effective in the treatment of debilitating medical conditions including nausea and vomiting resulting from cancer chemotherapy, wasting syndrome associated with HIV/AIDS, and chronic pain. Despite scientific evidence, as of 2011, when the material for this thesis was collected, only 17 states of the United States (U.S.) and the District of Columbia had enacted medical cannabis laws allowing patients with specific medical conditions to use cannabis without being criminally prosecuted. This thesis examines two components of the medical cannabis policy: the medical cannabis policy process in five representative states of the U.S., and the factors influencing the formation of such a process. The first part of the thesis chronologically documents the passing, attempts to pass, and failure to pass medical cannabis policies in five U.S. states; two with a current medical cannabis law; one where attempts to pass a law have been made, but a law has not yet been passed; and two states where no or few attempts at passing a medical cannabis law have been made. The second part of the thesis used a questionnaire to elicit the factors influencing policies as perceived by three groups. Group one comprised individuals directly involved in the medical cannabis policy process in at least one of the five states referred to above and group two comprised individuals participating in research in the alcohol and other drug field. Group four comprised members of the International Society for the Study of Drug Policy (ISSDP). The study found that, despite the expectation that the same rules would apply to cannabis as other medicine, the medical cannabis process appears to be less medically and more politically driven, with scientific evidence having limited influence. The results suggest that there are a number of interlinking factors which played a role in the passing or failure to pass medical cannabis laws in U.S. states, and the level of influence of these factors can vary according to context or conditions placed on them. Three major themes emerged in relation to the factors influencing policy: the role of scientific evidence, the political process, and the interaction between factors. It is hoped that this thesis will be viewed as an observation of the medical cannabis process, not only from the researcher’s point of view but from the views of those who participated in the process, researched the process, or observed the changes in medical cannabis laws over the years

O consumo de drogas de abuso é grave problema de saúde pública em todo o mundo. No Brasil observa-se aumento no número de usuárias em idade fértil, levando a crescente preocupação com relação à exposição fetal. Efeitos deletérios como diminuição do peso e crescimento fetal, parto prematuro, déficits neurológicos e comportamentais estão associados ao uso de cocaína e cannabis durante a gestação. Portanto, é importante obter informações acerca do consumo de drogas durante a gravidez, permitindo intervenções médicas e psicológicas adequadas. Os relatos fornecidos pelas gestantes são relevantes, porém muitas vezes resultam em dados subestimados devido à omissão de informações por medo de ações punitivas. Desta forma, a confirmação por meio de análises toxicológicas em amostras biológicas se faz necessária. O mecônio, primeiras fezes do recém-nascido, tem sido proposto como matriz adequada para avaliação da exposição fetal, pois apresenta coleta não invasiva, fácil obtenção e fornece informações de longo prazo (2º e 3º trimestre de gestação). Entretanto, é uma matriz complexa, exigindo diversas etapas de purificação para posterior análise. No presente trabalho, métodos analíticos foram desenvolvidos visando à detecção dos biomarcadores da exposição fetal à cocaína e ao tetraidrocanabinol em amostras de mecônio por cromatografia gasosa acoplada à espectrometria de massas (GC-MS). Para ambos os métodos, a extração acelerada por solvente (ASE) foi utilizada para o isolamento dos analitos de interesse de amostras de mecônio, já que esta apresenta vantagens frente às técnicas convencionais, devido sua maior eficiência e menor manipulação da amostra. Associada à ASE, a extração em fase sólida (SPE) foi empregada para purificação e concentração dos analitos de interesse. Na etapa de desenvolvimento, inicialmente foram estabelecidos os procedimentos de derivatização e as condições cromatográficas a serem empregadas nas análises. Posteriormente, realizou-se a otimização dos procedimentos empregados na ASE, através de análise de superfície de resposta. Os métodos foram validados de acordo com o preconizado por referências internacionais, estabelecendo-se os limites de detecção e quantificação, recuperação, linearidade, precisão intra e interensaio e exatidão. O método para detecção dos biomarcadores da cocaína foi aplicado em 342 amostras de mecônio, provenientes do Hospital Universitário da Universidade de São Paulo (HU-USP). Destas, 19 (5,6%) apresentaram resultado positivo para um ou mais biomarcadores da exposição fetal à cocaína. Além disto, foi observada redução estatisticamente significante do peso ao nascimento, comprimento e perímetro cefálico entre os recém-nascidos expostos à cocaína durante a gestação. O método para a detecção da exposição fetal ao tetraidrocanabinol foi aplicado em 6 amostras positivas, obtidas do HU-USP, mostrando a aplicabilidade da técnica desenvolvida.
The use of illicit drugs is a relevant public health problem in the world. In Brazil, the number of women users in fertile age is increasing, which leads to a growing concern regarding fetal drug exposure. Adverse outcomes such as low birth weight, intra-uterine growth restriction, preterm birth, neurobehavioral and developmental deficits are associated with cocaine and cannabis use during pregnancy. Consequently, it would be important to obtain data related to drug misuse during gestation with the aim to plan medical and psychological interventions. Self-report drug use by pregnant women is often inaccurate due to feelings of guilt or fear of punitive actions. Therefore, confirmation by toxicological analysis in biological matrices must be accomplished. Meconium, the first stool of the newborn, has been proposed as a proper matrix to evaluate fetal exposure because it is collected by an easy and non-invasive way and enables the achievement of long-term information regarding fetal exposure. However, meconium is a complex matrix, which requires extensive sample cleaning previously to the analytes identification. In the present research, analytical methods were developed aiming the determination of cocaine and tetrahydrocannabinol biomarkers in meconium samples through gas chromatography-mass spectrometry (GC-MS). Accelerated solvent extraction (ASE) was used for analytes isolation due to its advantages over conventional techniques, such as greater extraction efficiency and minor sample handling. In order to achieve proper analytes selectivity and detectability, solid phase extraction (SPE) was employed for post-extraction clean-up. Initially, the derivatization procedure and chromatographic parameters were established for the development of the methods. Afterwards, ASE procedure was optimized through response surface methodology. The analytical methods were validated in accordance to international references. Limits of detection and quantification, recovery, linearity, precision and accuracy were obtained. The developed method for cocaine was applied in 342 meconium samples collected from the University Hospital of University of São Paulo (HU-USP). Among them, 19 (5.6%) showed positivity result for cocaine biomarkers. Additionally, newborns from mothers exposed to cocaine exhibited statistical significant lower birth weight, length and head circumference when compared with newborns from non-consumer mothers. For the method of tetrahydrocannabinol, applicability and importance was demonstrated by analyzing 6 positive samples from HU-USP.

This thesis focuses on one of the biggest challenges facing drug discovery field: the efficient targeted delivery of therapeutic compounds, and how cell-penetrating peptides (CPPs) acting as drug delivery vectors can overcome pharmacokinetic issues and poor access to difficult targets, such as the central nervous system (CNS). Specifically, we designed and developed an optimized, oral active, CPP-linked peptide-based disruptor targeting cannabinoid CB1 and serotonin 5HT2A receptor heterodimer, which allows medical use of cannabinoids to fight pain while avoiding memory impairment side effects. Similarly, we successfully disturbed the adenosine A2A receptor homodimerization by coupling an interfering peptide to a modified cyclic CPP, as a promising strategy for treatment of CNS disorders. Following also CPP-conjugation, we managed to considerably increase cellular uptake into Leishmania cells of paromomycin, a low bioavailable anti-parasitic drug. Altogether, this work reinforces CPPs relevance as drug carriers and provides valuable insights into CPPs design, optimization, validation and coupling strategies.
Esta tesis se centra en uno de los mayores desafíos que enfrenta el campo del descubrimiento de fármacos: la administración dirigida y eficiente de compuestos terapéuticos; y cómo los péptidos penetrantes de células (CPP), actuando como vectores de liberación, pueden sobreponerse a los problemas farmacocinéticos y el acceso deficiente a objetivos difíciles como el sistema nervioso central (SNC). En particular, diseñamos y desarrollamos un disruptor optimizado, oralmente activo, basado en péptidos y ligado a un CPP, dirigido al heterodímero del receptor de cannabinoide CB1 y serotonina 5HT2A; que permite el uso médico de los cannabinoides para combatir el dolor y evitar los efectos secundarios de deterioro de la memoria. De manera similar, alteramos con éxito la homodimerización del receptor de adenosina A2A utilizando un péptido interferente acoplado a un CPP cíclico modificado, como estrategia prometedora para la exploración de trastornos del SNC. También tras la conjugación con un CPP, logramos aumentar considerablemente la captación celular en células de Leishmania de la paromomicina, un fármaco antiparasitario de baja biodisponibilidad. En conjunto, este trabajo refuerza la relevancia de los CPPs como portadores de medicamentos y proporciona información valiosa sobre el diseño, optimización, validación y las estrategias de acoplamiento de los CPPs.

Nous avons montré : a) qu’il existe un lien entre la consommation de cannabis et la présence de sténoses artérielles intracrâniennes multifocales chez le jeune adulte victime d’infarctus cérébral, b) que la prévalence des sténoses artérielles intracrâniennes atteint un tiers des cas dans une cohorte de 159 infarctus cérébraux du jeune adulte, c) que 13% des infarctus cérébraux dans cette série répondent aux critères angiographiques du syndrome de vasoconstriction cérébrale réversible déclenché majoritairement par la consommation de cannabis, d) que le tétrahydrocannabinol (THC, le principal produit actif du cannabis) inhibe in vitro la chaîne respiratoire mitochondriale de cerveau de rat, et induit une génération significative de peroxyde d’hydrogène. La génération de radicaux libres pourrait être un des mécanismes possibles de toxicité cérébrale du THC en jeu lors d’un infarctus cérébral
We showed that: a) there was a link between cannabis use and intracranial arterial multifocal stenosis in a series of ischemic stroke in the young, b) the prevalence of intracranial arterial stenosis was up to 31% in a series of 159 ischemic strokes in the young, c) 13% of the patients in this series sustained the angiographic criteria of reversible cerebral vasoconstriction syndrome, and that the precipitating factor was the use of cannabis in 67% of cases, d) tetrahydrocannabinol (THC, the main active component in cannabis) inhibits the respiratory mitochondrial chain of the brain in rats and induces a significant production of hydrogen peroxide. These results suggest that one of the mechanisms of brain toxicity induced by cannabis in ischemic stroke patients, may be the high rate of generation of free radicals induced by THC

La 3,4-metilendioximetamfetamina (MDMA, èxtasi) i el cannabis són dues drogues les quals es consumeixen conjuntament de manera habitual. Malgrat que tots dos compostos presenten propietats reforçant i potencial addictiu, també tenen propietats farmacològiques oposades. La MDMA es una droga psicoestimulant, la qual causa hiperlocomoció, hipertèrmia, resposted de tipus asiogènic i neurotoxicitat. Per altra banda el Δ9-tetrahydrocannabinol (THC), principal compost psicoactiu del cannabis, posseeix efectes relaxants, hipolocomotors, hipotèrmics i neuroprotectors. Els efectes de la MDMA i el THC al sistema nerviós central es troben mediats per dos mecanismes notablement diferents. La MDMA augmenta els nivells extracel·lulars de dopamina i serotonina, mentre que el THC produeix l'activació del receptor cannabinoide CB1. Cal destacar a més que les interaccions entre els sistemes monoaminèrgic i endocannabinoide s'observa de manera freqüent en l'organisme.
En el present estudi hem explorat la implicació del sistema endocannabinoide i la MDMA en diversos aspectes. Per una banda el receptor cannabinoide CB1 juga un important paper en els efectes hiperlocomotors i hipertèrmics, i en les respostes de tipus ansiogènic produïdes per la MDMA. Curiosament, encara que el receptor CB1 no participa en els efectes recompensants primaris de la MDMA, és imprescindible per que tinguin lloc els seus efectes reforçants. Així mateix, l'alliberació de serotonina per part de la MDMA redueix de manera dosi-depenent la simptomatologia física causada pel síndrome d'abstinència a cannabinoides precipitada per un antagonista del receptor CB1. Finalment, el tractament amb THC era capaç de prevenir la hipertèrmia, activació glial, estrès oxidatiu i pèrdua de terminals causada per la MDMA. Com a conseqüència el THC exerceix un efecte neuroprotector contra la neurotoxicitat induïda per la MDMA.
3,4-methylenedioximethamphetamine (MDMA, ecstasy) and cannabis are two drugs frequently consumed in combination. Despite both compounds have rewarding properties and abuse liability, they show opposite pharmacological properties. On the one hand, MDMA is a psychostimulant drug with hyperlocomotor, hyperthermic, anxiogenic-like and neurotoxic effects. On the other hand, Δ9-tetrahydrocannabinol (THC), the main psychoactive compound of cannabis, has relaxant, hypolocomotor, hypothermic and neuroprotective properties. The effects of MDMA and THC in the central nervous system are mediated by two different mechanisms. MDMA enhances the extracellular levels of dopamine and serotonin, whereas THC activates the CB1 cannabinoid receptor. Likewise, interactions between the monoaminergic and the endogenous cannabinoid system have been frequently observed.
In the current study, we explored the involvement of CB1 cannabinoid receptor on the hyperlocomotor, hyperthermic, anxiogenic-like, rewarding and reinforcing effects of MDMA. We also studied the effect of acute and chronic administration of MDMA on rimonabant-precipitated THC withdrawal syndrome. Furthermore, we explored the neuroprotective effects of THC on MDMA-induced neurotoxicity.
As a result of this study we may conclude that endocannabinoid system and MDMA interact in a wide variety of aspects. CB1 receptor plays an important role on the hyperlocomotor, hyperthermic, and anxiogenic-like effects of MDMA. Interestingly, CB1 receptor is essential for the reinforcing but not the primary rewarding properties of MDMA. In addition, the release of serotonin by MDMA dose-dependently reduced the severity of THC withdrawal syndrome triggered by a CB1 antagonist. Finally, pretreatment with THC prevented the hyperthermia, glial activation, oxidative stress and terminal loss caused by MDMA. Consequently, THC exerts a neuroprotective effect against MDMA-induced neurotoxicity.

L'objectif de cette thèse est de détailler les effets respiratoires induits par les associations de l'éthanol au THC et de l'éthanol au GHB. Les études ont été menées chez l'animal non anesthésié par pléthysmographie corps entier pendant les quatre heures suivant l'administration intrapéritonéale. Dans une première étape, les effets respiratoires de la prise isolée d'éthanol et de GHB ont été étudiés. Ces deux substances modifient le mode respiratoire : l'éthanol provoquant une tachypnée dès 3 g.kg-1, le GHB une respiration apneustique dès 600 mg.kg-1, sans insuffisance respiratoire (PaO2 normale). Les modifications des gaz du sang observées : acidémie pour l'éthanol et alcalose pour le GHB sont d'origine métabolique. A ces doses, ces deux substances perturbent aussi la conscience des animaux et la thermorégulation : l'éthanol induit une hypothermie et le GHB une évolution triphasique de la température : hypothermie/hyperthermie/ hypothermie. Les dosages sanguins et les études cinétiques menés lors de ces études confirment la vraisemblance de notre modèle et sa pertinence clinique et médicolégale. L'étude des associations à l'éthanol montre que les effets respiratoires du THC et du GHB sont conservés, seule leur association à la dose de 3 g.kg-1 d'éthanol a provoqué une baisse de la ventilation minute avec réduction du débit inspiratoire mais selon des mécanismes différents : baisse du volume courant pour l'association THC-éthanol et augmentation de la durée des apnées expiratoires pour celle du GHB à l'éthanol. Pour cette dernière, l'interaction cinétique observée après administration intrapéritonéale n'explique pas l'intensité du phénomène, une potentialisation est probable.

Tetrahydrocannabinol (THC) is the principal psychoactive constituent of cannabis and its effects are modulated by dopaminergic neurotransmission. Dopamine availability in prefrontal cortex is mostly controlled by catechol-o-methyltransferase (COMT) and it has been suggested that THC induced psychosis and cognitive impairment are dependent on COMT genotype. However the effect of THC in COMT activity is not known.This study aims to evaluate the effect of THC on COMT activity and monoamines tissue levels in mice prefrontal cortex. COMT activity in vitro and in vitro ex-vivo was evaluate in the pre-frontal cortex from C57BL/6J mice. For 10 days, mice were treated either with vehicle or THC (10 mg/Kg bw, total daily dose).In vitro, THC produced a concentration dependent inhibition in brain COMT activity with an IC 50 value of 232 µM. In vivo, THC treatment did not change monoamines or dopamine metabolites in prefrontal cortex. Nevertheless a significant decrease in 5-hydroxyindoleacetic acid (0.35±0.1, 0.54±0.02 pmol/mg tissue for control and THC respectively) was observed. In vitro-ex-vivo, COMT activity in the pre-frontal cortex showed no differences in the enzyme kinetics parameters when comparing THC to control animals. Analysis from the saturation curves found that the Vmax values (in nmol/mg prot/h) were 2.73 (2.4, 3.1) and 2.70 (2.3, 3.1) for CT and THC respectively.In conclusion, THC decreases COMT activity in vitro only in high concentrations (above 100 µM), however when administrated in vivo no significant effect was observed in enzyme activity or dopamine prefrontal cortex metabolism.

Tetrahydrocannabinol (THC) is the principal psychoactive constituent of cannabis and its effects are modulated by dopaminergic neurotransmission. Dopamine availability in prefrontal cortex is mostly controlled by catechol-o-methyltransferase (COMT) and it has been suggested that THC induced psychosis and cognitive impairment are dependent on COMT genotype. However the effect of THC in COMT activity is not known.This study aims to evaluate the effect of THC on COMT activity and monoamines tissue levels in mice prefrontal cortex. COMT activity in vitro and in vitro ex-vivo was evaluate in the pre-frontal cortex from C57BL/6J mice. For 10 days, mice were treated either with vehicle or THC (10 mg/Kg bw, total daily dose).In vitro, THC produced a concentration dependent inhibition in brain COMT activity with an IC 50 value of 232 µM. In vivo, THC treatment did not change monoamines or dopamine metabolites in prefrontal cortex. Nevertheless a significant decrease in 5-hydroxyindoleacetic acid (0.35±0.1, 0.54±0.02 pmol/mg tissue for control and THC respectively) was observed. In vitro-ex-vivo, COMT activity in the pre-frontal cortex showed no differences in the enzyme kinetics parameters when comparing THC to control animals. Analysis from the saturation curves found that the Vmax values (in nmol/mg prot/h) were 2.73 (2.4, 3.1) and 2.70 (2.3, 3.1) for CT and THC respectively.In conclusion, THC decreases COMT activity in vitro only in high concentrations (above 100 µM), however when administrated in vivo no significant effect was observed in enzyme activity or dopamine prefrontal cortex metabolism.