Defense Date

2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Pharmacology & Toxicology

First Advisor

Aron Lichtman

Abstract

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.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

February 2014

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