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Control of Analgesic and Anti-Inflammatory Pathways by Fatty Acid Amide Hydrolase Long, James Harry

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Control of Analgesic and Anti-Inflammatory Pathways by Fatty Acid Amide Hydrolase Long, James Harry Control of analgesic and anti-inflammatory pathways by fatty acid amide hydrolase Long, James Harry The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author For additional information about this publication click this link. http://qmro.qmul.ac.uk/jspui/handle/123456789/3124 Information about this research object was correct at the time of download; we occasionally make corrections to records, please therefore check the published record when citing. For more information contact [email protected] Control of analgesic and anti-inflammatory pathways by fatty acid amide hydrolase James Harry Long Thesis submitted for the degree of Doctor of Philosophy to the University of London Translational Medicine and Therapeutics William Harvey Research Institute Charterhouse Square, London, EC1M 6BQ Table of contents Table of Contents Declaration VIII Acknowledgements IX Abstract X Abbreviations XI Chapter 1 – Introduction 1 1.1. Pain and analgesia 2 1.1.1. Nociception 2 1.1.2. Inflammatory pain 5 1.1.3. Neuropathic pain 10 1.1.4. Analgesia 10 1.1.5. COX inhibitors 11 1.1.6. Opioid receptor agonists 12 1.1.7. Glucocorticoids 13 1.1.8. Anaesthetics 13 1.1.9. Antidepressants 14 1.1.10. Anticonvulsants 14 1.1.11. Muscle relaxants 15 1.1.12. An alternative analgesic pathway 15 1.2. Endocannabinoid system 16 1.2.1. Cannabinoid receptors 16 1.2.2. Endocannabinoids 18 1.2.3. Endocannabinoid biosynthesis 21 1.2.4. Endocannabinoid metabolism 21 1.2.5. Endocannabinoid signalling 26 1.3. Cannabinoid receptor-mediated analgesia 28 I Table of contents 1.3.1. Cannabinoid receptor knock-out and activation 28 1.3.2. Endogenous cannabinoid receptor ligands 29 1.3.3. Other cannabinoid receptor ligands 29 1.4. FAAH-mediated analgesia 30 1.4.1. The preference of FAAH over MAGL 30 1.4.2. FAAH knock-out and inhibition 31 1.4.3. The multiple mechanisms of FAAH-mediated analgesia 32 1.4.4. The action of OEA via the PPAR-α and TRPV1 receptors 32 1.4.5. The action of PEA via the PPAR-α receptor and other pathways 33 1.4.6. The action of the FAAH substrates via GPR55 33 1.4.7. FAAH as a potential source of arachidonic acid for prostaglandin 34 synthesis 1.4.8. Further analgesic effects of FAAH inhibition 35 1.5. Development of FAAH inhibitors 38 1.5.1. FAAH inhibitors 38 1.5.2. The FAAH substrates as a pharmacodynamic parameter 40 1.5.3. FAAH substrate assays 41 1.6. Paracetamol 42 1.6.1. Paracetamol inhibits COX activity 43 1.6.2. Paracetamol interacts with the endocannabinoid system 44 1.6.3. Paracetamol interacts with serotonin receptors 46 1.6.4. AM404 interacts with COX, iNOS, TRPV1 and sodium channels 47 1.6.5. Pharmacokinetics of intravenous paracetamol in the cerebrospinal 47 fluid 1.6.6. Paracetamol toxicity 49 1.7. Aims 50 II Table of contents Chapter 2 – Inhibition of FAAH and MAGL suppresses prostaglandin E2 51 synthesis by LPS-stimulated macrophages 2.1. Introduction 52 2.2. Materials and methods 53 2.2.1. Materials 53 2.2.2. Macrophages 53 2.2.3. Prostaglandin E2 assay 54 2.2.4. Nitric oxide assay 54 2.2.5. Cell viability assay 55 2.2.6. Western blotting COX-2 55 2.2.7. Statistical analysis 55 2.3. Results 56 2.3.1. Effect of FAAH and MAGL inhibition on the cell viability of LPS- 56 stimulated RAW264.7 macrophages 2.3.2. Effect of FAAH and MAGL inhibition on LPS-induced PGE2 synthesis in 58 RAW264.7 macrophages 2.3.3. Effect of FAAH and MAGL inhibition on LPS-induced nitric oxide 59 synthesis in RAW264.7 macrophages 2.3.4. Effect of MAGL inhibition on LPS-induced COX-2 expression in 60 RAW264.7 macrophages 2.4. Discussion 61 2.4.1. Limitations of the present study 65 2.4.2. Summary 66 Chapter 3 – FAAH substrates as a pharmacodynamic response in an in 67 vivo model of inflammatory pain 3.1. Introduction 68 III Table of contents 3.2. Materials and methods 70 3.2.1. Materials 70 3.2.2. FCA-induced hypersensitivity 70 3.2.3. Drug administration 71 3.2.4. Sample preparation 71 3.2.5. FAAH substrate assay by LC-MS/MS 72 3.2.6. FAAH activity assay 73 3.2.7. Intrinsic clearance assay 73 3.2.8. Protein binding assay 74 3.2.9. Statistical analysis 76 3.3. Results 77 3.3.1. FAAH substrate assay – optimisation 77 3.3.2. FAAH substrate assay – validation 81 3.3.3. Effect of URB597 on brain and blood FAAH substrate levels in an in 85 vivo inflammatory pain model 3.3.4. Effect of the PF-04457845 and two non-covalent inhibitors, NC1 and 90 NC2, on brain and blood FAAH substrate levels in an in vivo inflammatory pain model 3.3.5. Dose response of the non-covalent FAAH inhibitor, NC3, on brain and 92 blood FAAH substrate levels in an in vivo inflammatory pain model 3.3.6. Effect of the COX-2 inhibitor, celecoxib, on brain and blood FAAH 93 substrate levels in an in vivo inflammatory pain model 3.3.7. Oxidative metabolism of the FAAH substrates in liver microsomes 94 3.3.8. Protein binding of the FAAH substrates in brain and liver microsomes 97 3.4. Discussion 98 3.4.1. FAAH substrate assay 98 3.4.2. Effect of FAAH inhibitors on brain and blood FAAH substrate levels in 99 an in vivo inflammatory pain model 3.4.3. Oxidative metabolism and protein binding of AEA, OEA and PEA 102 3.4.4. Summary 104 IV Table of contents Chapter 4 – Pharmacokinetics and metabolism of intravenous 105 paracetamol in the human cerebrospinal fluid and its conjugation to fatty acids via FAAH 4.1. Introduction 106 4.2. Materials and methods 108 4.2.1. Materials 108 4.2.2. Formation and detection of FAAH formed paracetamol metabolites in 108 rat brain homogenate 4.2.3. Patients 109 4.2.4. Sample collection 110 4.2.5. Protein assay 110 4.2.6. Paracetamol assay 110 4.2.7. AM404 and FAAH substrate assay 111 4.2.8. Prostaglandin E2 assay 112 4.2.9. Leukotriene B4 assay 113 4.2.10. 5HT assay 113 4.2.11. Pharmacokinetic calculations 113 4.3. Results 116 4.3.1. Detection of fatty acid paracetamol conjugates in brain homogenate 116 4.3.2. Formation of fatty acid paracetamol conjugates by FAAH in rat brain 119 homogenate 4.3.3. Collection of human CSF and plasma samples 123 4.3.4. Measurement of total protein in CSF 123 4.3.5. Preliminary investigation of a single method for quantitation of 123 paracetamol and its major and minor metabolites 4.3.6. Paracetamol assay by LC-UV – optimisation 124 4.3.7. Paracetamol assay – validation 125 4.3.8. AM404 and FAAH substrate assay – validation 127 4.3.9. 5HT assay – validation 130 4.3.10. Pharmacokinetics of paracetamol in the cerebrospinal fluid and plasma 132 V Table of contents following intravenous administration 4.3.11. Metabolism of paracetamol to paracetamol glucuronide, paracetamol 134 sulphate and AM404 in the cerebrospinal fluid and plasma following intravenous administration 4.3.12. Formation of oleic and palmitic acid derived metabolites of 140 paracetamol in CSF following intravenous administration 4.3.13. FAAH substrate levels in the CSF and plasma following intravenous 142 administration 4.3.14. Prostaglandin E2 levels in the CSF and plasma following intravenous 143 administration 4.3.15. Leukotriene B4 levels in the CSF and plasma following intravenous 144 administration 4.3.16. 5HT levels in the CSF and plasma following intravenous administration 145 4.4. Discussion 146 4.4.1. Conjugation of fatty acids to paracetamol via FAAH in brain 146 homogenates 4.4.2. Pharmacokinetics of paracetamol in human cerebrospinal fluid and 148 plasma 4.4.3. Metabolism of paracetamol in human cerebrospinal fluid and plasma 150 4.4.4. Metabolism of paracetamol to AM404 and other fatty acid conjugates 152 4.4.5. The transfer of paracetamol and its metabolites into the CSF 154 4.4.6. Paracetamol’s effect on endogenous pathways 155 4.4.7. Summary 158 Chapter 5 – General discussion 160 5.1. Overview 161 5.2. The expanding endocannabinoid system 162 5.2.1. New receptors in the endocannabinoid system 163 5.2.2. New ligands in the endocannabinoid system 163 5.2.3. New proteins in the endocannabinoid system 165 VI Table of contents 5.2.4. New actions of the endocannabinoid system 166 5.2.5. New interactions of the endocannabinoid system 167 5.2.6. Summary 168 5.3. The role of the endocannabinoid system in paracetamol’s 168 action 5.4. The therapeutic potential of a FAAH inhibitor 169 5.4.1. FAAH inhibitors as analgesics 170 5.4.2. Alternative uses of FAAH inhibitors 171 5.4.3. Potential adverse effects of FAAH inhibitors 172 5.4.4. Conclusion 172 References 173 Publications 195 Appendix 196 VII Declaration Declaration I declare that the work presented in this thesis, unless otherwise stated, is my own. James Long VIII Acknowledgements Acknowledgements Thank you to Professor David Perrett for giving me the opportunity to do a PhD. I am grateful for your supervision during my time with you. Especially I thank you for teaching me that most things do not work first time and that what you want to happen is not always what will happen.
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