| Arachidonoyl ethanolamide (anandamide), an endogenous derivative of arachidonic acid, activates the same molecular targets as the main psychoactive constituent of the cannabis (marijuana) plant and it is therefore classified as an endogenous cannabinoid. Anandamide belongs to a large and therapeutically important signaling system, the endocannabinoid system, which also includes the two known cannabinoid receptors, CB1 and CB2, the endocannabinoid synthetic and degrading enzymes, and the associated signaling pathways regulated by the CB1 and CB2 receptors. The various components of this system represent novel pharmacological targets for the treatment of many neurological, neuropsychiatric, inflammatory and metabolic disorders. Therefore, it is critical to gain a thorough understanding of all the enzymes that could potentially exert control over the anandamide tone in vivo. The cytochrome P450 monooxygenases (P450s) are one such group of enzymes. The central hypothesis of this thesis was that P450s metabolize anandamide to various oxygenated products which may have physiological and pharmacological significance. By using human liver, kidney and brain tissue preparations, in addition to purified P450 enzymes in the reconstituted system, it was determined that anandamide is metabolized by P450s 2D6, 3A4, and 4F2 to form various hydroxylated and epoxygenated products. Epoxide hydrolase and P450 2D6 further metabolized the primary epoxides of anandamide to form several di-oxygenated derivatives. Pharmacological studies revealed that the epoxide of anandamide, 5,6-epoxyeicosatrienoic acid ethanolamide (5,6-EET-EA), is a potent and selective CB2 receptor agonist which has the potential to modulate immune cell function. Activation of CB2 by 5,6-EET-EA may lead to important anti-inflammatory effects under certain pathological situations affecting many organs, such as the liver during ischemia/reperfusion injury or fibrosis, and the brain during stroke or neurodegeneration. The results from this work may also lead to important insights into the mechanisms of action of two novel classes of drug candidates which have the potential to indirectly increase endogenous 5,6-EET-EA levels. Collectively, these findings uncover an endocannabinoid bioactivation pathway and establish the cytochrome P450 enzymes as important players in endocannabinoid signaling. |
