Control of analgesic and anti-inflammatory pathways by fatty acid amide hydrolase

Abstract

The endogenous cannabinoids (endocannabinoids) arachidonoyl ethanolamide (AEA) and 2-arachidonoyl glycerol (2-AG) regulate neurotransmission and inflammation by activating the CB1 cannabinoid receptors in the central nervous system and the CB2 cannabinoid receptors on immune cells. Endocannabinoids are inactivated via a 2-step process: firstly undergoing reuptake into the cell and then hydrolysis by fatty acid amide hydrolase (FAAH) for AEA or by monoacylglycerol lipase (MAGL) for 2-AG. Inhibition of FAAH is an effective treatment for inflammatory pain and is a target for the development of novel analgesics. FAAH and MAGL produce arachidonic acid by hydrolysis of endocannabinoids. In inflammation arachidonic acid is oxidised by cyclooxygenase 2 (COX-2) to synthesise the prostaglandins. Both FAAH and MAGL inhibition were showed to indirectly suppress prostaglandin E2 synthesis in activated macrophages. This effect was found to be dependent on the amount of lipopolysaccharide used to activate the macrophages. The FAAH substrates, AEA, oleoyl ethanolamide (OEA) and palmitoyl ethanolamide (PEA), were measured in samples from an in vivo model of inflammatory pain to screen various FAAH inhibitors to prove their mechanism of action. Liquid chromatography-tandem mass spectrometry methods were developed and validated for the measurement of these three substrates in brain and whole blood. Blood OEA and PEA concentrations were found to be indicative of brain FAAH activity and brain AEA concentrations. FAAH is capable of conjugating arachidonic acid to paracetamol to form the active metabolite AM404, an inhibitor of AEA reuptake. It was shown that FAAH can also conjugate oleic acid and palmitic acid to paracetamol, producing novel metabolites. A clinical trial was conducted to comprehensively investigate the pharmacokinetics, metabolism and mechanism of action of intravenous paracetamol in cerebrospinal fluid and plasma. Paracetamol, its major metabolites, AM404, prostaglandin E2, leukotriene B4, serotonin and the FAAH substrates were measured. For the first time AM404 was demonstrated to be formed by humans and be present in the cerebrospinal fluid.