Jenny L. Wilkerson

Intrathecal cannabilactone CB2R agonist, AM1710, controls pathological pain and restores basal cytokine levels

Summary Spinal AM1710 suppresses allodynia with corresponding anti‐inflammatory and anti‐MAGL (monoacylglycerol lipase) effects in the spinal cord and dorsal root ganglia. ABSTRACT Spinal glial and proinflammatory cytokine actions are strongly implicated in pathological pain. Spinal administration of the anti‐inflammatory cytokine interleukin (IL)‐10 abolishes pathological pain and suppresses proinflammatory IL‐1β and tumor necrosis factor alpha (TNF‐α). Drugs that bind the cannabinoid type‐2 receptor (CB2R) expressed on spinal glia reduce mechanical hypersensitivity. To better understand the CB2R‐related anti‐inflammatory profile of key anatomical nociceptive regions, we assessed mechanical hypersensitivity and protein profiles following intrathecal application of the cannabilactone CB2R agonist, AM1710, in 2 animal models; unilateral sciatic nerve chronic constriction injury (CCI), and spinal application of human immunodeficiency virus‐1 glycoprotein 120 (gp120), a model of peri‐spinal immune activation. In CCI animals, lumbar dorsal spinal cord and corresponding dorsal root ganglia (DRG) were evaluated by immunohistochemistry for expression of IL‐10, IL‐1β, phosphorylated p38‐mitogen‐activated‐kinase (p‐p38MAPK), a pathway associated with proinflammatory cytokine production, glial cell markers, and degradative endocannabinoid enzymes, including monoacylglycerol lipase (MAGL). AM1710 reversed bilateral mechanical hypersensitivity. CCI revealed decreased IL‐10 expression in dorsal spinal cord and DRG, while AM1710 resulted in increased IL‐10, comparable to controls. Adjacent DRG and spinal sections revealed increased IL‐1β, p‐p38MAPK, glial markers, and/or MAGL expression, while AM1710 suppressed all but spinal p‐p38MAPK and microglial activation. In spinal gp120 animals, AM1710 prevented bilateral mechanical hypersensitivity. For comparison to immunohistochemistry, IL‐1β and TNF‐α protein quantification from lumbar spinal and DRG homogenates was determined, and revealed increased DRG IL‐1β protein levels from gp120, that was robustly prevented by AM1710 pretreatment. Cannabilactone CB2R agonists are emerging as anti‐inflammatory agents with pain therapeutic implications.

Immunofluorescent spectral analysis reveals the intrathecal cannabinoid agonist, AM1241, produces spinal anti-inflammatory cytokine responses in neuropathic rats exhibiting relief from allodynia.

During pathological pain, the actions of the endocannabinoid system, including the cannabinoid 2 receptor (CB2R), leads to effective anti‐allodynia and modifies a variety of spinal microglial and astrocyte responses. Here, following spinal administration of the CB2R compound, AM1241, we examined immunoreactive alterations in markers for activated p38 mitogen‐activated protein kinase, interleukin‐1β (IL‐1β), the anti‐inflammatory cytokine, interleukin‐10 (IL‐10) as well as degradative endocannabinoid enzymes, and markers for altered glial responses in neuropathic rats. In these studies, the dorsal horn of the spinal cord and dorsal root ganglia were examined. AM1241 produced profound anti‐allodynia with corresponding immunoreactive levels of p38 mitogen‐activated kinase, IL‐1β, IL‐10, the endocannabinoid enzyme monoacylglycerol lipase, and astrocyte activation markers that were similar to nonneuropathic controls. In contrast, spinal AM1241 did not suppress the increased microglial responses observed in neuropathic rats. The differences in fluorescent markers were determined within discrete anatomical regions by applying spectral analysis methods, which virtually eliminated nonspecific signal during the quantification of specific immunofluorescent intensity. These data reveal expression profiles that support the actions of intrathecal AM1241 control pathological pain through anti‐inflammatory mechanisms by modulating critical glial factors, and additionally decrease expression levels of endocannabinoid degradative enzymes.

Improvement of spinal non-viral IL-10 gene delivery by D-mannose as a transgene adjuvant to control chronic neuropathic pain

BackgroundPeri-spinal subarachnoid (intrathecal; i.t.) injection of non-viral naked plasmid DNA encoding the anti-inflammatory cytokine, IL-10 (pDNA-IL-10) suppresses chronic neuropathic pain in animal models. However, two sequential i.t. pDNA injections are required within a discrete 5 to 72-hour period for prolonged efficacy. Previous reports identified phagocytic immune cells present in the peri-spinal milieu surrounding the i.t injection site that may play a role in transgene uptake resulting in subsequent IL-10 transgene expression.MethodsIn the present study, we aimed to examine whether factors known to induce pro-phagocytic anti-inflammatory properties of immune cells improve i.t. IL-10 transgene uptake using reduced naked pDNA-IL-10 doses previously determined ineffective. Both the synthetic glucocorticoid, dexamethasone, and the hexose sugar, D-mannose, were factors examined that could optimize i.t. pDNA-IL-10 uptake leading to enduring suppression of neuropathic pain as assessed by light touch sensitivity of the rat hindpaw (allodynia).ResultsCompared to dexamethasone, i.t. mannose pretreatment significantly and dose-dependently prolonged pDNA-IL-10 pain suppressive effects, reduced spinal IL-1β and enhanced spinal and dorsal root ganglia IL-10 immunoreactivity. Macrophages exposed to D-mannose revealed reduced proinflammatory TNF-α, IL-1β, and nitric oxide, and increased IL-10 protein release, while IL-4 revealed no improvement in transgene uptake. Separately, D-mannose dramatically increased pDNA-derived IL-10 protein release in culture supernatants. Lastly, a single i.t. co-injection of mannose with a 25-fold lower pDNA-IL-10 dose produced prolonged pain suppression in neuropathic rats.ConclusionsPeri-spinal treatment with D-mannose may optimize naked pDNA-IL-10 transgene uptake for suppression of allodynia, and is a novel approach to tune spinal immune cells toward pro-phagocytic phenotype for improved non-viral gene therapy.

Selective Monoacylglycerol Lipase Inhibitors: Antinociceptive versus Cannabimimetic Effects in Mice

The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) plays an important role in a variety of physiologic processes, but its rapid breakdown by monoacylglycerol lipase (MAGL) results in short-lived actions. Initial MAGL inhibitors were limited by poor selectivity and low potency. In this study, we tested JZL184 [4-nitrophenyl 4-[bis(2H-1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate] and MJN110 [2,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate], MAGL inhibitors that possess increased selectivity and potency, in mouse behavioral assays of neuropathic pain [chronic constriction injury (CCI) of the sciatic nerve], interoceptive cannabimimetic effects (drug-discrimination paradigm), and locomotor activity in an open field test. MJN110 (1.25 and 2.5 mg/kg) and JZL184 (16 and 40 mg/kg) significantly elevated 2-AG and decreased arachidonic acid but did not affect anandamide in whole brains. Both MAGL inhibitors significantly reduced CCI-induced mechanical allodynia with the following potencies [ED50 (95% confidence limit [CL]) values in mg/kg: MJN110 (0.43 [0.30–0.63]) > JZL184 (17.8 [11.6–27.4])] and also substituted for the potent cannabinoid receptor agonist CP55,940 [2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol] in the drug-discrimination paradigm [ED50 (95% CL) values in mg/kg: MJN110 (0.84 [0.69–1.02]) > JZL184 (24.9 [14.6–42.5])]; however, these compounds elicited differential effects on locomotor behavior. Similar to cannabinoid 1 (CB1) receptor agonists, JZL184 produced hypomotility, whereas MJN110 increased locomotor behavior and did not produce catalepsy or hypothermia. Although both drugs substituted for CP55,940 in the drug discrimination assay, MJN110 was more potent in reversing allodynia in the CCI model than in producing CP55,940-like effects. Overall, these results suggest that MAGL inhibition may alleviate neuropathic pain, while displaying limited cannabimimetic effects compared with direct CB1 receptor agonists.

The endocannabinoid hydrolysis inhibitor SA-57: Intrinsic antinociceptive effects, augmented morphine-induced antinociception, and attenuated heroin seeking behavior in mice

ABSTRACT Although opioids are highly efficacious analgesics, their abuse potential and other untoward side effects diminish their therapeutic utility. The addition of non‐opioid analgesics offers a promising strategy to reduce required antinociceptive opioid doses that concomitantly reduce opioid‐related side effects. Inhibitors of the primary endocannabinoid catabolic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) show opioid‐sparing effects in preclinical models of pain. As simultaneous inhibition of these enzymes elicits enhanced antinociceptive effects compared with single enzyme inhibition, the present study tested whether the dual FAAH‐MAGL inhibitor SA‐57 [4‐[2‐(4‐chlorophenyl)ethyl]‐1‐piperidinecarboxylic acid 2‐(methylamino)‐2‐oxoethyl ester] produces morphine‐sparing antinociceptive effects, without major side effects associated with either drug class. SA‐57 dose‐dependently reversed mechanical allodynia in the constriction injury (CCI) of the sciatic nerve model of neuropathic pain and carrageenan inflammatory pain model. As previously reported, SA‐57 was considerably more potent in elevating anandamide (AEA) than 2‐arachidonyl glycerol (2‐AG) in brain. Its anti‐allodynic effects required cannabinoid (CB)1 and CB2 receptors; however, only CB2 receptors were necessary for the anti‐edematous effects in the carrageenan assay. Although high doses of SA‐57 alone were required to produce antinociception, low doses of this compound, which elevated AEA and did not affect 2‐AG brain levels, augmented the antinociceptive effects of morphine, but lacked cannabimimetic side effects. Because of the high abuse liability of opioids and implication of the endocannabinoid system in the reinforcing effects of opioids, the final experiment tested whether SA‐57 would alter heroin seeking behavior. Strikingly, SA‐57 reduced heroin‐reinforced nose poke behavior and the progressive ratio break point for heroin. In conclusion, the results of the present study suggest that inhibition of endocannabinoid degradative enzymes represents a promising therapeutic approach to decrease effective doses of opioids needed for clinical pain control, and may also possess therapeutic potential to reduce opioid abuse. HighlightsSA‐57 inhibits fatty acid amide hydrolase and monoacylglycerol lipase.SA‐57 reverses allodynia in mouse neuropathic and inflammatory pain models.CB1 and CB2 receptors mediate the antinociceptive effects of SA‐57 in the carrageenan model of inflammatory pain.SA‐57 enhances morphine‐induced antinociception.SA‐57 reduces heroin self‐administration.

The α7 nicotinic receptor dual allosteric agonist and positive allosteric modulator GAT107 reverses nociception in mouse models of inflammatory and neuropathic pain.

Orthosteric agonists and positive allosteric modulators (PAMs) of the α7 nicotinic ACh receptor (nAChR) represent novel therapeutic approaches for pain modulation. Moreover, compounds with dual function as allosteric agonists and PAMs, known as ago‐PAMs, add further regulation of receptor function.

The novel, orally available and peripherally restricted selective cannabinoid CB2 receptor agonist LEI-101 prevents cisplatin-induced nephrotoxicity.

Here, we have characterized 3‐cyclopropyl‐1‐(4‐(6‐((1,1‐dioxidothiomorpholino)methyl)‐5‐fluoropyridin‐2‐yl)benzyl)imidazolidine‐2,4‐dione hydrochloride (LEI‐101) as a novel, peripherally restricted cannabinoid CB2 receptor agonist, using both in vitro and in vivo models.

Diacylglycerol lipase β inhibition reverses nociceptive behaviour in mouse models of inflammatory and neuropathic pain.

Inhibition of diacylglycerol lipase (DGL)β prevents LPS‐induced pro‐inflammatory responses in mouse peritoneal macrophages. Thus, the present study tested whether DGLβ inhibition reverses allodynic responses of mice in the LPS model of inflammatory pain, as well as in neuropathic pain models.

The selective monoacylglycerol lipase inhibitor MJN110 produces opioid sparing effects in a mouse neuropathic pain model

Serious clinical liabilities associated with the prescription of opiates for pain control include constipation, respiratory depression, pruritus, tolerance, abuse, and addiction. A recognized strategy to circumvent these side effects is to combine opioids with other antinociceptive agents. The combination of opiates with the primary active constituent of cannabis (Δ9-tetrahydrocannabinol) produces enhanced antinociceptive actions, suggesting that cannabinoid receptor agonists can be opioid sparing. Here, we tested whether elevating the endogenous cannabinoid 2-arachidonoylglycerol through the inhibition of its primary hydrolytic enzyme monoacylglycerol lipase (MAGL), will produce opioid-sparing effects in the mouse chronic constriction injury (CCI) of the sciatic nerve model of neuropathic pain. The dose-response relationships of i.p. administration of morphine and the selective MAGL inhibitor 2,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate (MJN110) were tested alone and in combination at equieffective doses for reversal of CCI-induced mechanical allodynia and thermal hyperalgesia. The respective ED50 doses (95% confidence interval) of morphine and MJN110 were 2.4 (1.9–3.0) mg/kg and 0.43 (0.23–0.79) mg/kg. Isobolographic analysis of these drugs in combination revealed synergistic antiallodynic effects. Acute antinociceptive effects of the combination of morphine and MJN110 required μ-opioid, CB1, and CB2 receptors. This combination did not reduce gastric motility or produce subjective cannabimimetic effects in the drug discrimination assay. Importantly, combinations of MJN110 and morphine given repeatedly (i.e., twice a day for 6 days) continued to produce antiallodynic effects with no evidence of tolerance. Taken together, these findings suggest that MAGL inhibition produces opiate-sparing events with diminished tolerance, constipation, and cannabimimetic side effects.

The Endogenous Cannabinoid System: A Budding Source of Targets for Treating Inflammatory and Neuropathic Pain

A great need exists for the development of new medications to treat pain resulting from various disease states and types of injury. Given that the endogenous cannabinoid (that is, endocannabinoid) system modulates neuronal and immune cell function, both of which play key roles in pain, therapeutics targeting this system hold promise as novel analgesics. Potential therapeutic targets include the cannabinoid receptors, type 1 and 2, as well as biosynthetic and catabolic enzymes of the endocannabinoids N-arachidonoylethanolamine and 2-arachidonoylglycerol. Notably, cannabinoid receptor agonists as well as inhibitors of endocannabinoid-regulating enzymes fatty acid amide hydrolase and monoacylglycerol lipase produce reliable antinociceptive effects, and offer opioid-sparing antinociceptive effects in myriad preclinical inflammatory and neuropathic pain models. Emerging clinical studies show that ‘medicinal’ cannabis or cannabinoid-based medications relieve pain in human diseases such as cancer, multiple sclerosis, and fibromyalgia. However, clinical data have yet to demonstrate the analgesic efficacy of inhibitors of endocannabinoid-regulating enzymes. Likewise, the question of whether pharmacotherapies aimed at the endocannabinoid system promote opioid-sparing effects in the treatment of pain reflects an important area of research. Here we examine the preclinical and clinical evidence of various endocannabinoid system targets as potential therapeutic strategies for inflammatory and neuropathic pain conditions.