The endocannabinoid receptors CB1R and CB2R are involved in a plethora of processes, and consequently are involved in many pathological conditions. Their wide distribution makes the CBRs both an... Show moreThe endocannabinoid receptors CB1R and CB2R are involved in a plethora of processes, and consequently are involved in many pathological conditions. Their wide distribution makes the CBRs both an interesting therapeutic target and hard to study. Additional chemical tools are required to study and understand the function and mechanism of CB1R and CB2R. This thesis describes the development of several such tools to improve our insight in the (pathological) roles of the receptors in order to develop novel and improved therapeutics. First evaluation of three dimensional ligand-CB2R complexes made and analysed with Cryo-EM are described. Hotspots that potentially generate selectivity between CB1R and CB2R are evaluated with point-mutations in vitro. Consequently describes the development of the first tools, two-step bifunctional probes based on LEI-121 and LEI 102, is described. As two-step probes are not compatible with every assay, the toolbox is expanded with a one-step fluorescent probe. Briefly touching upon CB1R, ligands were designed with negatively charged phosphonium groups that are potentially selective for mtCB1R. Show less
Posttraumatic stress disorder (PTSD) is an often chronic condition for which currently available medications have limited efficacy. Medical cannabis is increasingly used to treat patients with PTSD... Show morePosttraumatic stress disorder (PTSD) is an often chronic condition for which currently available medications have limited efficacy. Medical cannabis is increasingly used to treat patients with PTSD; however, evidence for the efficacy and safety of cannabinoids is scarce. To learn more about patients' opinions on and experiences with medical cannabis, we organized a focus group discussion among military veterans (N = 7) with chronic PTSD who were treated with medical cannabis. Afterwards, some of their partners (N = 4) joined the group for an evaluation, during which they shared their perspective on their partner's use of medical cannabis. Both sessions were audio-recorded, transcribed verbatim, and analyzed by means of qualitative content analysis. Five overarching themes were identified. The first four themes related to the different phases of medical cannabis use - namely, 1) Consideration; 2) Initiation; 3) Usage; and 4) Discontinuation. The fifth theme related to several general aspects of medical cannabis use. Patients used medical cannabis to manage their symptoms and did not experience an urge to "get high." They used a variety of different cannabis strains and dosages and reported several therapeutic effects, including an increased quality of sleep. Furthermore, discussions about the experienced stigma surrounding cannabis generated insights with implications for the initiation of medical cannabis use. These results underscore the value of qualitative research in this field and are relevant for the design of future clinical trials on the use of medical cannabis for the treatment of PTSD. (c) 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/) Show less
Endocannabinoids, a class of lipid messengers, have emerged as crucial regulators of synaptic communication in the central nervous system (CNS). Dysregulation of these compounds has been... Show moreEndocannabinoids, a class of lipid messengers, have emerged as crucial regulators of synaptic communication in the central nervous system (CNS). Dysregulation of these compounds has been implicated in many brain disorders. Although some studies have identified and quantified a limited number of target compounds, a method that provides comprehensive quantitative information on endocannabinoids and related N-acylethanolamines (NAEs) in CSF is currently lacking as measurements are challenging due to low concentrations under normal physiological conditions. Here we developed and validated a high-throughput nano liquid chromatography-electrospray ionization mass spectrometry (nano LC-ESI-MS/MS) platform for the simultaneous quantification of endocannabinoids (anandamide (AEA), 2-arachidonoyl glycerol (2-AG)), ten related NAEs and eight additional putatively annotated NAEs in human CSF. Requiring only 200 μL of CSF our method has limits of detection from 0.28 to 61.2 pM with precisions of RSD <15% for most compounds. We applied our method to CSF from 45 healthy humans and demonstrated potential age and gender effects on concentrations of endocannabinoids and NAEs. Notably, our results show that docosahexaenoyl ethanolamide (DHEA) concentrations increase with age in males. Our method may offer new opportunities to gain insight into regulatory functions of endocannabinoids in the context of (ab)normal brain function. Show less
The research presented in this thesis focused on the effects of cannabis on mental functions and the brain. Specifically, the investigation aimed at exploring how cannabis affects creative... Show moreThe research presented in this thesis focused on the effects of cannabis on mental functions and the brain. Specifically, the investigation aimed at exploring how cannabis affects creative thinking, awareness of errors, and the neurotransmitter dopamine among regular cannabis users. It was discovered that cannabis does not improve creativity and that it can decrease error awareness and impair dopaminergic functioning. Consequently, these findings allow for a better understanding of the nature of subtle mental impairments among chronic cannabis users. Show less
Abboussi, O.; Said, N.; Fifel, K.; Lakehayli, S.; Tazi, A.; Ganouni, S. el 2016
The endocannabinoid system has only been discovered during the last few decades, and scientific progress in understanding the relevance of this system in health and disease has been limited and... Show moreThe endocannabinoid system has only been discovered during the last few decades, and scientific progress in understanding the relevance of this system in health and disease has been limited and slow. CB1 antagonists were considered a __miracle drug__ for the treatment of obesity and smoking with __blockbuster__ potential. But due to central side effects (such as depression and suicidal behaviour) and a lack of systematic clinical pharmacologic research, market access of a CB1 antagonists failed. In this thesis, we explored some improvements in the early development of cannabinoids, and by systematically investigating, we found that the new cannabinoid antagonist TN38837 seems effective with a reduced propensity for central side effects, and that a new oral THC formulation enhances the pharmacological activities by its seemingly superior pharmacokinetics. Also, we experiment with new methodology to optimise effect measurement, including resting state-FMRI which we found suitable for early phase cannabinoid research, and including new concentration-effect models to improve the simulation and prediction of future studies. The research in this thesis shows that a revival of research on the cannabinoid system requires novel approaches to the administration of cannabinoids, to the measurements and the study designs, and to the analyses of the effects. This reflects the complexity of the highly integrated endocannabinoid system, but also sets the stage for other innovative drug development programs Show less
Cannabinoids are a group of terpenophenolic compounds and are naturally found in the cannabis plant (Cannabis sativa L). _9-Tetrahydrocannabinol (_9-THC) is the psychoactive cannabinoid. The high... Show moreCannabinoids are a group of terpenophenolic compounds and are naturally found in the cannabis plant (Cannabis sativa L). _9-Tetrahydrocannabinol (_9-THC) is the psychoactive cannabinoid. The high lipophilicity of _9-THC is a hindering factor in the further development of this compound into a large scale pharmaceutical product. In an effort to generate highly polar derivatives of _9-THC, we screened 206 alkane degrading bacterial strains. In total, eight derivatives were produced on milligram. All of these biotransformation products harbored modified alkyl chains, with hydroxy, carboxy and ester functionalities. We also investigated the potential of cell suspension culture of Catharanthus Roseus to transform _9-THC. Unlike bacterial strains, Catharanthus Roseus cells transformed _9-THC to its glycosylated and additionally hydroxylated derivatives. The behavioral effects of __9-THC are mediated by the centra l CB1 receptor in rats. CB1 receptors show high degree of sequence conservation in vertebrates. We used zebrafish embryos as a model to study the behavioral effects of cannabinoids. We conclude that cannabinoids have behavioural effects in zebrafish that are comparable to some of those reported in the literature for mammals. In particular, the acute exposure response resembles behavioural effects reported for adult. This suggests that some similarity in cannabinoid response pathways between zebrafish and mammals exists. We also performed the metabolomic profiling of zebrafish embryos treated with the concentrations of CB1 agonist and antagonist used in behavioural assays. The results of behavioural assays were successfully correlated with the metabolomic data of zebrafish embryos. Show less
Cannabis sativa L. plants produce a diverse array of secondary metabolites, which have been grouped in cannabinoids, flavonoids, stilbenoids, terpenoids, alkaloids and lignans; the cannabinoids are... Show moreCannabis sativa L. plants produce a diverse array of secondary metabolites, which have been grouped in cannabinoids, flavonoids, stilbenoids, terpenoids, alkaloids and lignans; the cannabinoids are the best known group of natural products from this plant. The pharmacological aspects of this secondary metabolite group have been extensively studied and the cannabinoid biosynthetic pathway has been partially elucidated. Although, it is known that the geranyl diphosphate (GPP) and the olivetolic acid are initial precursors in this route the biosynthesis of the olivetolic acid has not been found yet. It has been suggested that the olivetolic acid biosynthesis could be initiated by a polyketide synthase (PKS). This thesis was focused on the characterization of PKSs in cannabis plants. More than 480 compounds have been identified from C. sativa but only 247 are considered as secondary metabolites. These latter are grouped into cannabinoids, flavonoids, stilbenoids, terpenoids, alkaloids and lignans. However, what do we know about their biosynthesis and role in the plant? Chapter 1 summarizes the natural compounds in cannabis from a biosynthetic view. It seems that enzymes belonging to the polyketide synthase group could be involved in the biosynthesis of the initial precursors from the cannabinoid, flavonoid and stilbenoid biosynthetic pathways. The Polyketide Synthases (PKSs) are condensing enzymes which form a myriad of polyketide compounds. In plants several PKSs have been identified and studied. Aspects such as specificity, reaction mechanisms, structure, as well as evolution are reviewed in Chapter 2. In Chapter 3 polyketide synthase (PKS) enzymatic activities were analyzed in crude protein extracts from cannabis plant tissues. Differences in activities of chalcone synthase (CHS), stilbene synthase (STS) and olivetol-forming PKS were observed during the development and growth of glandular trichomes on the female flowers. Although, cannabinoid biosynthesis and accumulation take place in glandular trichomes no activity for an olivetolic acid-forming PKS was detected in this tissue. Content analyses of cannabinoids and flavonoids from different tissues revealed differences in their distribution, suggesting a diverse regulatory control on the biosynthetic fluxes of their biosynthetic pathways in the plant. Chapter 4 reports in silicio expression analysis of a PKS gene isolated from glandular trichomes. The deduced amino acid sequence showed 51-72% identity to other CHS/STS type sequences of the PKS family. Further phylogenetic analysis revealed that this PKS (PKSG2) grouped with other non-chalcone and stilbene-producing PKSs. Homology modeling analyses of this cannabis PKS predicts a 3D overall fold similar to alfalfa CHS2 with small steric differences on the residues that shape the active site of the cannabis PKSG2. Cannabis sativa cell culture induction has been reported for several purposes. However, cannabinoids have not been detected in cell cultures so far. Although, elicitation has been employed in the cell cultures for inducing and/or improving secondary metabolites there are no reports concerning elicitation effect on secondary metabolite production in C. sativa cell cultures. In Chapter 5 the effect of elicitation on secondary metabolism of the plant cell cultures is reported. Metabolic profiles analyzed by 1H-NMR spectroscopy and principal component analyses (PCA) showed variations in some of the metabolite pools. However, no cannabinoids were found in both control and elicited cannabis cell cultures. THCA synthase gene expression was monitored during a time course. Results suggest that other components in the signaling pathway can be controlling the cannabinoid pathway. Show less
The cannabis plant (Cannabis sativa L.) has a long history as a recreational drug, but also as part of traditional medicine in many cultures. Nowadays, it is used by a large number of patients... Show moreThe cannabis plant (Cannabis sativa L.) has a long history as a recreational drug, but also as part of traditional medicine in many cultures. Nowadays, it is used by a large number of patients worldwide, to ameliorate the symptoms of diseases varying from cancer and AIDS to multiple sclerosis and migraine. The discovery of cannabinoid-receptors and the endocannabinoid system have opened up a new and exciting field of research. But despite the pharmaceutical potential of cannabis, its classification as a narcotic drug has prevented the successful development of cannabis into modern medicine. Although a huge number of scientific papers has been published on cannabis, there is currently no scientific consensus on the usefulness of medicinal cannabis. In 2004, The Netherlands became the first country to make herbal cannabis available as a prescription drug. The phytochemical research presented in this thesis has been possible because of the availability of these high-grade cannabis plants. This thesis has a specific focus on the cannabinoids and on analytical problems that currently obstruct advanced study of the cannabis plant. Furthermore, it deals with much needed methods for quality control and with administration forms of medicinal cannabis. In general, it may be considered a general guidebook, covering all the basic phytochemical aspects of medicinal cannabis. Show less
