Fatty acid amide hydrolase (FAAH) belongs to a diverse class of enzymes in amidase signature family. In mammals, FAAH is targeted to affect neurological functions because it terminates endocannabinoid signaling by degrading anandamide, a 20C N-acylethanolamine (NAE 20:4). In higher plants, FAAH is known to modulate growth, development and stress tolerance by degrading 12-18C NAEs. Since anandamide was reported to exclusively occur in early land plants, we investigated its metabolic pathway in the moss Physcomitrella patens. Based on the highest identity with ratFAAH, we identified nine orthologs in moss, PpFAAH1 to PpFAAH9. Several bioinformatic tools were used to understand the structural basis of how catalytic residues fold for amidohydrolase activity. Based on these in silico analyses of PpFAAH homologs and their gene expression in response to saturated (NAE16:0) and unsaturated NAE (NAE 20:4) treatment, PpFAAH1 was selected for biochemical characterization. Heterologously expressed PpFAAH1 showed highest amidohydrolase activity at 37°C and pH 8.0. Both in vivo and in vitro studies showed that unsaturated NAE substrate is hydrolyzed faster than the saturated NAE (> 10-fold in vivo and 50-fold in vitro). Additionally, transgenic moss lines over expressing FAAH1 showed slower growth and disrupted gametophyte formation when compared to wild type. These data suggest that PpFAAH1-mediated NAE metabolism is likely involved in developmental transition in moss.

Delta9-tetrahydrocannbinol (THC), the primary active constituent of Cannabis sativa, has long been known to reduce opioid withdrawal symptoms. Although THC produces most of its pharmacological actions through the activation of CB1 and CB2 cannabinoid receptors, the role these receptors play in reducing opioid withdrawal symptoms remains unknown. The endogenous cannabinoids, N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonylglycerol (2-AG), activate both cannabinoid receptors, but are rapidly metabolized by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. The objective of this dissertation was to test whether increasing AEA or 2-AG, via inhibition of their respective hydrolytic enzymes, reduces morphine withdrawal symptoms in in vivo and in vitro models of opiate dependence. Morphine-dependent ICR mice subjected to acute naloxone challenge or abrupt withdrawal (via pellet removal) reliably displayed a profound withdrawal syndrome, consisting of jumping, paw tremors, head shakes, diarrhea, and weight loss. THC and the MAGL inhibitor, JZL184 dose-dependently reduced the intensity of precipitated withdrawal measures through the activation of CB1 receptors. The FAAH inhibitor, PF-3845, reduced the intensity of a subset of precipitated signs through the activation of CB1 receptors, but did not ameliorate the incidence of diarrhea or weight loss. In the next set of experiments, MAGL inhibition dose-dependently reduced the intensity of all spontaneous withdrawal signs (i.e jumps, paw flutters, head shakes, weight loss and diarrhea) in a CB1 receptor dependent manner. However, FAAH inhibition reduced the intensity of head shakes and paw flutters, but did not affect other signs. Strikingly, a combination of low-dose JZL184 and high-dose PF-3845 reduced abrupt withdrawal signs in a manner similar to complete MAGL inhibition, which suggests potential therapeutic advantages of dual enzyme inhibition. This combination elevated appropriate eCB levels and caused moderate CB1 receptor desensitization, but did not affect receptor number in whole brain. Since MAGL, but not FAAH inhibition, blocked diarrhea during opioid withdrawal in vivo, we investigated whether inhibitors of each enzyme would differentially attenuate naloxone-precipitated contractions and secretion in morphine-dependent ilea in vitro. Both enzyme inhibitors attenuated the intensity of naloxone-induced contractions, and blocked naloxone-precipitated hypersecretion. Thus, these models offer useful tools for investigating in vitro end-ponts of withdrawal, but not for elucidating anti-diarrheal mechanism of these inhibitors.If targeting endocannabinoid catabolic enzymes is indeed a viable approach to treat other abuse disorders, it is important to know whether these inhibitors would themselves have abuse or dependence liability. In the final series of experiments we tested whether prolonged elevation of endocannabinoid leads to the development of cannabinoid dependence, based on the occurrence of somatic withdrawal signs upon challenge with rimonabant, a CB1 receptor antagonist. Repeated treatment with high doses, but not low doses, of JZL184 led to cannabinoid dependnece. These results indicate that the strategy of increasing endogenous cannabinoids through the inhibition of their catabolic enzymes represents a promising approach to ameliorate opioid withdrawal symptoms.

Pain is a debilitating condition that presents a problem of clinical relevance. Common analgesics include opioids and non-steroidal anti-inflammatory drugs (NSIADs). Despite different degrees of effectiveness, a major drawback of these analgesic classes is their side effects. For example, side effects associated with opioids include pruritus, respiratory depression, hyperalgesia, constipation, dependence. In addition, chronic use of NSAIDs can cause gastric ulcers. Delta-9 tetrahydrocannabinol (THC), the primary psychoactive constituent of marijuana, produces antinociception in various preclinical models of pain. Similarly, many synthetic cannabinoid receptor agonists produce antinociception in preclinical models of pain. However, their psychomimetic effects dampen interest for their therapeutic development. THC and these cannabinoids act upon the endocannabinoid system, which is comprised of the cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptors, endogenous ligands arachidonoylethanolamide (anandamide; AEA), 2-arachidonoyolglycerol (2-AG), and endocannabinoid biosynthetic and catabolic enzymes. In the present study, we evaluated the impact of inhibiting one of the major biosynthetic enzymes of 2-AG, diacylglycerol lipase-b (DAGL-b), and two primary endocannabinoid catabolic enzymes, monoacylglycerol lipase (MAGL), and fatty acid amide hydrolase (FAAH). MAGL is responsible for degrading 2-AG and FAAH is the principal degradative enzyme for anandamide. We hypothesized that inhibiting these enzymes will produce anti-edematous and anti-allodynic effects in preclinical models of inflammatory pain. In Chapter 2, we tested whether the selective MAGL inhibitor JZL184 and FAAH inhibitor PF-3845 would reduce nociceptive behavior in the carrageenan test. JZL184 and PF-3845 significantly attenuated carrageenan-induced paw edema and mechanical allodynia (a nociceptive response to normally non-noxious stimuli), whether administered before or after carrageenan. Complementary genetic and pharmacological approaches revealed that JZL184’s anti-allodynic effects required both CB1 and CB2 receptors, but only CB2 receptors mediated its anti-edematous actions. Anti-edematous effects of PF-3845 were mediated through CB2 receptors. Importantly, the anti-edematous and anti-allodynic effects of JZL184 underwent tolerance following repeated injections of high dose JZL184 (16 or 40 mg/kg), but repeated administration of low dose JZL184 (4 mg/kg) retained efficacy. Although the data in the first set of studies demonstrate that inhibition of MAGL or FAAH reduces inflammatory pain, JZL184 and PF-3845 only produced partial effects. In an attempt to increase efficacy, Chapter 3 tested whether combined blockade of FAAH and MAGL would produce enhanced anti-edematous and anti-allodynic effects in the carrageenan model of inflammatory pain. Partial blockade of MAGL, with a low dose of JZL184 (4 mg/kg), and full blockade of FAAH, with a high dose of PF-3845 (10 mg/kg), enhanced the anti-allodynic effects, but no further increases in the anti-edematous effects were found. Importantly, repeated administration of this combination did not result in tolerance. A novel FAAH-MAGL dual inhibitor SA-57, which is far more potent in inhibiting FAAH than MAGL, reversed carrageenan-induced allodynia. Taken together, these findings suggest that dual MAGL and FAAH inhibition represents a promising avenue for the treatment of inflammatory pain. Chapter 4 of this dissertation tested whether inhibition of DAGL-b, a major biosynthetic enzyme of 2-AG, would reverse inflammatory pain. Two DAGL-b inhibitors, KT109, and KT172, which have been previously shown to reduce arachidonic acid, prostaglandins, and TNF-a levels in lipopolysaccharide (LPS)-stimulated murine macrophages, were used to test whether these compounds would elicit antinociceptive effects in the LPS model of inflammatory pain model. Because these drugs also inhibit ABHD6, we assessed KT195, a selective ABHD6 inhibitor that is inactive against DAGL-b. KT109, but not KT172 or KT195, significantly reversed LPS-induced allodynia. Importantly, we found that DAGL-b knockout mice possess an anti-allodynic phenotype, but KT109 did not elicit any further decrease in allodynia in these animals. The anti-allodynic effects of KT-109 did not require cannabinoid receptors. Additionally, the anti-allodynic effects of KT-109 did not undergo tolerance following repeated administration. KT-109 did not produce any gastric hemorrhagic effects when compared to the NSAID diclofenac, which significantly produced gastric hemorrhages. These results suggest that blockade of DAGL-b leads to antinociceptive effects through a cannabinoid receptor independent mechanism of action, with absence of notable side effects. Collectively, the research presented in this dissertation suggests that the endocannabinoid catabolic enzymes MAGL and FAAH, and the endocannabinoid biosynthetic enzyme DAGL-b, represent promising targets to treat inflammatory pain.

Vascular calcification is strongly correlated with the clinical manifestations of atherosclerosis, heart attacks and strokes. The calcification process resembles bone formation and involves the osteogenic trans-differentiation of smooth muscles cells within the arterial wall. Cannabinoid receptors are known to modulate bone formation and are present in atherosclerotic vessels, suggesting they may also play a role in modulating calcification. Therefore, we evaluated the effects of cannabinoids on the expression of osteogenic proteins by vascular smooth muscle cells undergoing calcification.

The research presented in this thesis addressed knowledge gaps for three medicinal plant taxa, Souroubea spp. (Marcgraviaceae) and Platanus (Platanaceae) as well as Echinacea spp. (Asteraceae). The primary pharmacological mechanism of Souroubea sympetala and Platanus occidentalis were well established, with pentacyclic triterpenes identified as major active principles. My results indicate that major triterpenoids, and crude plant extracts, selectively inhibited monoacyglycerol lipase (MAGL) activity but not fatty acid amide hydrolase (FAAH) activity. These data suggest a possible secondary anxiolytic mechanism of action through the endocannabinoid system (ECS). My study of herb-drug interactions of Souroubea and Plantanus products showed some potential risk when combined with a classic benzodiazepine class drug, diazepam, and I proposed a mechanism through in vitro CYP450 enzyme inhibition. The pharmacokinetic study revealed the difficulty of detecting betulinic acid in animal blood. To support the development a commercial botanical composed of these medicinal plants, an extraction method and a highly sensitive and selectivity HPLC-APCI-MS based quantification method was successfully developed and validated. Part II of this thesis focused on the impact of phytochemical variation and hepatic metabolism on the ECS activity of Echinacea spp. and explored the potential for new applications of Echinacea spp. as a natural health product. My research indicated that considerable variability in the content of phenolic and alkylamide (AKA) compounds reflected similar variability in in vitro bioactivity at ECS-related pharmacological targets. Following biochemometric analysis, several phenolic compounds and AKAs in Echinacea spp. were found to be significant independent variables determining FAAH inhibition and CB receptor activation. Hepatic metabolism was also found to affect the FAAH inhibition of AKA, as increased FAAH inhibitory effects were observed after CYP450-mediated metabolism of both individual AKAs and crude extracts of E angustifolia and E. purpurea, suggesting a “pro-drug” mechanism. Dose dependent activities were observed with oral administration of both E angustifolia and E. purpurea root extract in rat paw model of inflammation and pain. Further tests indicated these activities can be partially blocked by co-administration of CB1 and CB2 receptor antagonists AM251 and AM630, respectively. This evidence suggests activity for peripheral pain was at least partially mediated through the ECS.

This thesis investigated the pharmacology and phytochemistry of Achillea millefolium L. (yarrow) flowers, roots, stems, and leaves based on ethnobotanical reports in North America, with a focus on applications in a respiratory model. Seasonal changes in the phytochemical profile of yarrow were also assessed. A comprehensive dataset of medicinal Asteraceae was created after collecting ethnobotanical reports from the Native American Ethnobotany (NAEB) database. Using residual and binomial analyses, 14 tribes of Asteraceae were quantitatively evaluated and ranked within ten therapeutic categories as either over- or under-selected for treatment by North American indigenous peoples. Flora belonging to the Anthemideae tribe were over-utilized as pulmonary aids, particularly species of Achillea. Yarrow was selected for further analysis in the subsequent chapters of this thesis. The respiratory pharmacology of yarrow was examined by testing the immunomodulatory effects of four plant parts in an in vitro assay using BEAS-2B human bronchial epithelial cells. Concentrations of the pro-inflammatory cytokines IL-6 and IL-8 were quantified using ELISA kits. Flowers demonstrated significant anti-inflammatory activity at 40 μg/ml in both assays, and also at 20 μg/ml in the IL-8 assay, suggesting a dose-dependent response. Roots displayed significant pro-inflammatory activity at all concentrations. A second mechanism of action via the endocannabinoid system was tested through inhibitory enzyme assays for fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), in which the flowers and roots were most active. Since extracts of the four plant parts exhibited significantly different bioactivities, active metabolites previously identified in yarrow were quantified in each part through the targeted profiling of phenolics and alkylamides using analytical chromatographic techniques. Phenolic compounds were found at highest concentrations in the flowerheads, while alkylamides were detected predominantly within roots. An accompanying phenological analysis of alkylamide and phenolic levels in all parts was explored. Collectively, this research provides the first integrated comparison of yarrow ethnobotany, bioactivity, and phytochemistry across different parts of the plant, contributing novel insights into the traditional, contemporary, and future uses of one of North America’s most important medicinal plants.

Les astrocytes sont des partenaires clés des neurones. Dans l’hippocampe, et tout particulièrement au niveau des synapses CA3-CA1, en libérant la D-sérine, ces cellules gliales régulent l’activité des récepteurs glutamatergiques de type N-methyl-D-aspartate (NMDA) et de ce fait la mémoire synaptique, aussi connue sous le nom de plasticité synaptique à long terme. Cependant, le signal synaptique à l’origine de la libération de la D-sérine par les astrocytes reste à ce jour méconnu. De par des données rapportées dans la littérature nous nous sommes tout particulièrement intéressés aux récepteurs astrocytaires aux ephrins de type B3 (EphB3) et aux endocannabinoïdes de type 1 (CB1). Pour ce faire nous avons principalement utilisé une approche électrophysiologique sur des tranches aiguës d’hippocampe de souris adulte. Dans une première étude, nos données indiquent que l’activation des récepteurs EphB3 augmente la présence de D-sérine synaptique et en conséquence l’activité des récepteurs NMDA synaptiques. A l’inverse, leur inhibition diminue à la fois l’activité des récepteurs NMDA synaptiques et la potentialisation à long-terme qui en dépend (LTP ; une forme de plasticité synaptique à long terme). L’interaction EphB3-ephrinB3 contrôle donc la LTP en contrôlant la disponibilité en D-sérine synaptique. Dans une seconde étude, nous avons utilisé un modèle transgénique permettant d’inhiber l’expression des récepteurs CB1 astrocytaires (souris GFAP-CB1-KO). Nous avons découvert que la suppression de ces récepteurs diminue la disponibilité en D-sérine synaptique. De plus, nos travaux montrent que les récepteurs CB1 astrocytaires sont nécessaires à l’induction de la LTP via la D-serine. En conclusion, ces travaux de Thèse révèlent que les récepteurs astrocytaires EphB3 et CB1 régulent les fonctions dépendantes des récepteurs NMDA via le contrôle qu’ils exercent sur la disponibilité en D-sérine. / Astrocytes are key partners of neurons. In the hippocampus, and more particularly at CA3-CA1 synapses, by releasing D-serine, these glial cells regulate the activity of synaptic Nmethyl-D-aspartate (NMDA) receptors and thus synaptic memory, also known as long-term synaptic plasticity. Yet, the synaptic signal inducing D-serine release by astrocytes is still unknown. Based on interesting data from the literature we have investigated the role of the astrocytic receptors for ephrinB3 (EphB3) and endocannabinoids (CB1). To this end we used electrophysiological approaches on acute hippocampal slices of adult mice. In a first study, our data indicate on one hand that the activation of EphB3 receptors increases synaptic D-serine availability and in consequences the activity of synaptic NMDA receptor activity. On the other hand, inhibition of EphB3 receptors induces a decrease of synaptic NMDA receptor activity as well as the induction of the long-term potentiation (LTP; a form of long-term plasticity). Thus, EphB3-ephrinB3 interaction controls LTP induction through the availability of synaptic D-serine. In a second study, we used a transgenic model allowing the inhibition of CB1 receptors expression in astrocytes (GFAP-CB1-KO mice). We discovered that their deletion reduced synaptic D-serine availability. Our work shows that astrocytic CB1 receptors are necessary for LTP induction via this D-serine. All together, this PhD work reveals that astrocytic EphB3 and CB1 receptors regulate synaptic NMDA receptor functions through the control of D-serine availability.

In this study, the presence and possible function of endocannabinoid ligands in the planarian is investigated. The endocannabinoids ananadamide (AEA) and 2-arachidonoylglycerol (2-AG) and entourage NAE compounds palmitoylethanolamide (PEA), stearoylethanolamide (SEA) and oleoylethanolamide (OEA) were found in Dugesia dorotocephala. Changes in SEA, PEA, and AEA levels were observed over the initial twelve hours of active regeneration. Exogenously applied AEA, 2-AG and their catabolic inhibition effected biphasic changes in locomotor velocity, analogous to those observed in murines. The genome of a close relative, Schmidtea mediterranea, courtesy of the University of Utah S. med genome database, was explored for cannabinoid receptors, none were found. A putative fatty acid amide hydrolase (FAAH) homolog was found in Schmidtea mediterranea.

Fatty acid amide hydrolase (FAAH) belongs to a diverse class of enzymes in amidase signature family. In mammals, FAAH is targeted to affect neurological functions because it terminates endocannabinoid signaling by degrading anandamide, a 20C N-acylethanolamine (NAE 20:4). In higher plants, FAAH is known to modulate growth, development and stress tolerance by degrading 12-18C NAEs. Since anandamide was reported to exclusively occur in early land plants, we investigated its metabolic pathway in the moss Physcomitrella patens. Based on the highest identity with ratFAAH, we identified nine orthologs in moss, PpFAAH1 to PpFAAH9. Several bioinformatic tools were used to understand the structural basis of how catalytic residues fold for amidohydrolase activity. Based on these in silicoanalyses of PpFAAHhomologs and their gene expression in response to saturated (NAE16:0) and unsaturated NAE (NAE 20:4) treatment, PpFAAH1was selected for biochemical characterization. Heterologously expressed PpFAAH1 showed highest amidohydrolase activity at 37°C and pH 8.0. Both in vivoand in vitrostudies showed that unsaturated NAE substrate is hydrolyzed faster than the saturated NAE (> 10-fold in vivoand50-fold in vitro). Additionally, transgenic moss lines over expressing FAAH1 showed slower growth and disrupted gametophyte formation when compared to wild type. These data suggest that PpFAAH1-mediated NAE metabolism is likely involved in developmental transition in moss.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22365号 / 医博第4606号 / 新制||医||1043(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩田 想, 教授 川上 浩司, 教授 横出 正之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM