Targeting the Endocannabinoid Metabolic Enzymes to Reduce Inflammatory Pain

........................................................................................................................................... i Chapter 1: Introduction ................................................................................................................... 1 Cannabinoid Discovery and Background .................................................................................... 1 Endocannabinoid System ............................................................................................................ 1 Cannabinoid Receptors ............................................................................................................... 2 Biosynthesis and degradation of Endocannabinoids ................................................................... 5 Overview of Pain:........................................................................................................................ 9 Inflammation ............................................................................................................................. 12 Preclinical pain models ............................................................................................................. 13 Analgesics ................................................................................................................................. 16 Cannabis in clinical model of pain: ........................................................................................... 18 Role of direct cannabinoid receptor agonists on pain: .............................................................. 19 Role of DAGL inhibition in antinociceptive effects ................................................................. 21 Role of FAAH inhibition in antinociceptive effects ................................................................. 23 Review of studies examining FAAH inhibitors in preclinical pain assays. .............................. 27 Role of MAGL inhibition in antinociceptive effects ................................................................ 29 Rational and Hypothesis: .......................................................................................................... 32 Carrageenan model of inflammatory pain ................................................................................. 32 Selection of DAGL MAGL and FAAH inhibitors .................................................................... 34 Chapter 2. The monoacylglycerol lipase inhibitor JZL184 suppresses inflammatory pain in the mouse carrageenan model ............................................................................................................. 40 Introduction ............................................................................................................................... 40 Methods ..................................................................................................................................... 42 Results: ...................................................................................................................................... 46 Induction of Edema and allodynia by carrageenan ............................................................... 46 Anti-edematous and anti-allodynic effects of diclofenac, JZL184, and PF-3845 in the carrageenan model ................................................................................................................. 47 Cannabinoid receptors mediate the anti-allodynic and anti-edematous of JZL184 .............. 49 Differential tolerance following repeated administration of low dose and high dose JZL184 ............................................................................................................................................... 54 JZL184 reverses carrageenan-induced anti-edema and allodynia ......................................... 55 Discussion ................................................................................................................................. 58 Chapter 3. Combined inhibition of MAGL and FAAH suppresses edema and produces augmented anti-allodynic effects in the carrageenan mouse model ............................................. 63 Introduction: .............................................................................................................................. 63 Methods: .................................................................................................................................... 66 Results: ...................................................................................................................................... 75 Co-administration of JZL184 and PF-3845 elevates endocannabinoids without decreasing arachidonic acid in the brain. ................................................................................................. 75 Partial MAGL and complete FAAH inhibition produce augmented anti-allodynic effects in the carrageenan model of inflammatory pain ........................................................................ 77 Partial MAGL and complete FAAH inhibition produce augmented anti-allodynic effects in the chronic constriction injury model of neuropathic pain .................................................... 79 Combination JZL184 and PF-3845 mediates its anti-allodynic effects through cannabinoid receptors................................................................................................................................. 80 Repeated administration of the combination of JZL184 and PF-3845 elevate endocannabinoids and decrease arachidonic acid in the brain. ............................................. 82 Consequences of combined partial MAGL inhibition and full FAAH inhibition given repeatedly............................................................................................................................... 83 JZL-184 (4mg/kg) with PF-3845 10 (mg/kg) do not elicit cannabimimetic effects, as assessed in the tetrad assay .................................................................................................... 84 Partial MAGL and complete FAAH blockade does not cause cross-tolerance to the pharmacological effects of THC............................................................................................ 85 CB1 receptor activity and expression are maintained following repeated administration of JZL184 + PF-3845 combination ............................................................................................ 86 CB1 receptor function in the cingulated cortex is maintained following chronic partial MAGL and complete FAAH blockade .................................................................................. 87 Discussion: ................................................................................................................................ 89 Anti-allodynic effects of SA-57, dual FAAH, and MAGL inhibitor with differential potencies, in the carrageenan model of inflammatory pain ........................................................................ 96 Results ....................................................................................................................................... 99 Administration of different doses of SA-57 elevates endocannabinoids and decrease arachidonic acid in the brain .................................................................................................. 99 Anti-edematous and anti-allodynic effects of SA-57 in the carrageenan model ................. 100 Cannabinoid receptors mediate the anti-allodynic and anti-edematous of SA-57 .............. 101 SA-57-induced cannabimimetic effects, as assessed in the tetrad assay ............................. 102 Discussion: .............................................................................................................................. 103 Chapter 4. KT-109, a selective diacylglycerol lipase beta inhibitor reverses LPS-induced allodynia in mice ......................................................................................................................... 107 Introduction ............................................................................................................................. 107 Materials and methods ............................................................................................................ 110 Results: .................................................................................................................................... 113 Anti-allodynic effects of KT-109 is maintained over an extended period .......................... 113 DAGL- inhibition with KT-109 but not KT-172 reverse LPS-induced allodynia, negative control probe KT-195 does produce any effect ................................................................... 114 DAGL knockout mice show anti-allodynic phenotype ....................................................... 116 Repeated administration of KT-109 does not lead to tolerance .......................................... 116 Anti-allodynic effects of KT-109 are not mediated by cannabinoid receptors ................... 117 KT-109 did not produce cannabimimetic effects in tetrad .................................................. 118 DAGL- inhibition with high dose KT-109 does not elicit gastric ulcers in mice ............. 119 Discussion: .............................................................................................................................. 121 Chapter 5. General Discussion .................................................................................................... 124 Inhibition of FAAH or MAGL reduces carrageenan-induced inflammatory pain .................. 127 Simultaneous partial MAGL and complete FAAH inhibition reducing carrageenan-induced inflammatory pain ................................................................................................................... 130 Inhibition of DAGL-, a major biosynthetic enzyme of 2-AG ............................................... 134 Integration of endocannabinoid degradative and biosynthetic enzyme inhibitors .................. 137 Reference .................................................................................................................................... 140