Prostamide F2α receptor antagonism combined with inhibition of FAAH may block the pro-inflammatory mediators formed following selective FAAH inhibition

Background and PurposeProstamides are lipid mediators formed by COX-2-catalysed oxidation of the endocannabinoid anandamide and eliciting effects often opposed to those caused by anandamide. Prostamides may be formed when hydrolysis of anandamide by fatty acid amide hydrolase (FAAH) is physiologically, pathologically or pharmacologically decreased. Thus, therapeutic benefits of FAAH inhibitors might be attenuated by concomitant production of prostamide F_2α. This loss of benefit might be minimized by compounds designed to selectively antagonize prostamide receptors and also inhibiting FAAH.Experimental ApproachInhibition of FAAH by a series of selective antagonists of prostamide receptors, including AGN 204396, AGN 211335 and AGN 211336, was assessed using rat, mouse and human FAAH in vitro, together with affinity for human recombinant CB₁ and CB₂ receptors. Effects in vivo were measured in a model of formalin-induced inflammatory pain in mice.Key ResultsThe prostamide F_2α receptor antagonists were active against mouse and rat FAAH in the low μM range and behaved as non-competitive and plasma membrane-permeant inhibitors. AGN 211335, the most potent inhibitor of rat FAAH (IC₅₀ = 1.2 μM), raised exogenous anandamide levels in intact cells and also bound to cannabinoid CB₁ receptors. Both AGN 211335 and AGN 211336 (0.25–1 mg·kg⁻¹, i.p.) inhibited the formalin-induced nociceptive response in mice.Conclusions and ImplicationsSynthetic compounds with indirect agonist activity at cannabinoid receptors and antagonist activity at prostamide receptors can be developed. Such compounds could be used as alternatives to selective FAAH inhibitors to prevent the possibility of prostamide F_2α-induced inflammation and pain.Linked ArticlesThis article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-6     

Inhibition of COX-2-mediated eicosanoid production plays a major role in the anti-inflammatory effects of the endocannabinoid N-docosahexaenoylethanolamine (DHEA) in macrophages

BACKGROUND AND PURPOSE

N-docosahexaenoylethanolamine (DHEA) is the ethanolamine conjugate of the long-chain polyunsaturated n-3 fatty acid docosahexaenoic (DHA; 22: 6n-3). Its concentration in animal tissues and human plasma increases when diets rich in fish or krill oil are consumed. DHEA displays anti-inflammatory properties in vitro and was found to be released during an inflammatory response in mice. Here, we further examine possible targets involved in the immune-modulating effects of DHEA.

EXPERIMENTAL APPROACH

Antagonists for cannabinoid (CB)₁ and CB₂ receptors and PPAR_γ were used to explore effects of DHEA on NO release by LPS-stimulated RAW264.7 cells. The possible involvement of CB₂ receptors was studied by comparing effects in LPS-stimulated peritoneal macrophages obtained from CB₂^−/− and CB₂^+/+ mice. Effects on NF-κB activation were determined using a reporter cell line. To study DHEA effects on COX-2 and lipoxygenase activity, 21 different eicosanoids produced by LPS-stimulated RAW264.7 cells were quantified by LC-MS/MS. Finally, effects on mRNA expression profiles were analysed using gene arrays followed by Ingenuity® Pathways Analysis.

KEY RESULTS

CB₁ and CB₂ receptors or PPARs were not involved in the effects of DHEA on NO release. NF-κB and IFN-β, key elements of the myeloid differentiation primary response protein D88 (MyD88)-dependent and MyD88-independent pathways were not decreased. By contrast, DHEA significantly reduced levels of several COX-2-derived eicosanoids. Gene expression analysis provided support for an effect on COX–2-mediated pathways.

CONCLUSIONS AND IMPLICATIONS

Our findings suggest that the anti-inflammatory effects of DHEA in macrophages predominantly take place via inhibition of eicosanoids produced through COX-2.

LINKED ARTICLES

This article is part of a themed section on Cannabinoids 2013 published in volume 171 issue 6. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.2014.171.issue-6/issuetoc     

Peripheral interactions between cannabinoid and opioid systems contribute to the antinociceptive effect of crotalphine

Background and Purpose

Crotalphine is an antinociceptive peptide that, despite its opioid-like activity, does not induce some of the characteristic side effects of opioids, and its amino acid sequence has no homology to any known opioid peptide. Here, we evaluated the involvement of the peripheral cannabinoid system in the crotalphine effect and its interaction with the opioid system.

Experimental Approach

Hyperalgesia was evaluated using the rat paw pressure test. Involvement of the cannabinoid system was determined using a selective cannabinoid receptor antagonist. Cannabinoid and opioid receptor activation were evaluated in paw slices by immunofluorescence assays using conformation state-sensitive antibodies. The release of endogenous opioid peptides from skin tissue was measured using a commercial enzyme immunoassay (EIA).

Key Results

Both p.o. (0.008–1.0 μg·kg⁻¹) and intraplantar (0.0006 μg per paw) administration of crotalphine induced antinociception in PGE₂-induced hyperalgesia. Antinociception by p.o. crotalphine (1 μg·kg⁻¹) was blocked by AM630 (50 μg per paw), a CB₂ receptor antagonist, and by antiserum anti-dynorphin A (1 μg per paw). Immunoassay studies confirmed that crotalphine increased the activation of both κ-opioid (51.7%) and CB₂ (28.5%) receptors in paw tissue. The local release of dynorphin A from paw skin was confirmed by in vitro EIA and blocked by AM630.

Conclusions and Implications

Crotalphine-induced antinociception involves peripheral CB₂ cannabinoid receptors and local release of dynorphin A, which is dependent on CB₂ receptor activation. These results enhance our understanding of the mechanisms involved in the peripheral effect of crotalphine, as well as the interaction between the opioid and cannabinoid systems.     

Methyl-β-cyclodextrin suppresses the monocyte-endothelial adhesion triggered by lipopolysaccharide (LPS) or oxidized low-density lipoprotein (oxLDL)

ContextRecent studies demonstrated the anti-atherosclerotic efficacy of cyclodextrin. However, it remains unclear whether cyclodextrin exerts the anti-atherosclerotic effect via regulating monocyte-endothelial adhesion.ObjectiveTo answer that question by recruiting methyl-β-cyclodextrin (MβCD) as a cyclodextrin representative.Materials and methodsHuman umbilical vein endothelial cells (HUVECs) were not treated, or treated with 1 µg/mL liposaccharide (LPS) or 50 µg/mL oxidized low-density lipoprotein (oxLDL) for 12 h, 5 mM MβCD for 1 h, and LPS/oxLDL (1 and 50 µg/mL, respectively for 12 h) plus MβCD (5 mM for 1 h), respectively. The effects of MβCD on LPS/oxLDL-triggered monocyte-endothelial adhesion and related molecules in signalling pathways were evaluated via confocal microscopy, flow cytometry, RT-PCR, western blotting, and cell adhesion assay.ResultsMβCD with an IC₅₀ of 27.66 mM (1 h treatment) exerted no significant cytotoxicity at ≤5 mM for ≤2 h. Compared with the control, both LPS and oxLDL induced an ∼2–3-fold increase in adhesion molecule expression (ICAM-1 and VCAM-1 at protein and mRNA levels) and NF-κB phosphorylation (p-NF-κB/pP65), an increase in IκB kinase (IKK), and a decrease in phosphorylated protein kinase B (p-Akt), respectively. Moreover, more monocytes (2-fold higher for LPS and 15% higher for oxLDL) were attached on LPS/oxLDL-stimulated HUVECs. 5 mM MβCD reversed the LPS/oxLDL-induced changes back to the control levels.ConclusionsMβCD significantly suppresses the LPS/oxLDL-triggered monocyte-endothelial adhesion by downregulating adhesion molecule expression probably via LPS-IKK-NF-κB or oxLDL-Akt-NF-κB pathway. This study demonstrates a potential mechanism of the anti-atherosclerotic efficacy of cyclodextrin from the angle of monocyte-endothelial adhesion.     

Inhibition by cannabinoid receptor agonists of acetylcholine release from the guinea-pig myenteric plexus

1. The dose-related inhibition of the twitch responses of the myenteric plexus-longitudinal muscle preparation of the guinea-pig small intestine by cannabinoid (CB) agonists, (+)-WIN 55212 and CP 55940 during stimulation at 0.1 Hz with supramaximal voltage was confirmed. These agonists inhibited acetylcholine (ACh) release in the presence of physostigmine (7.7 μM) thus indicating a prejunctional site of action.
2. Inhibition of twitch responses and ACh release by CB agonists was reversed by the CB₁-selective cannabinoid receptor antagonist, SR141716A. Dose-response curves to (+)-WIN 55212 and CP 55940 were shifted to the right, with no reduction of maximal response, by pretreatment with SR141716A (31.6–1000 nM), but not its vehicle, Tween 80 (1 μM). However, at very high concentrations (25–400 μM), Tween 80 itself caused a dose-related inhibition of the twitch response which was significantly reduced in the presence of SR141716A (1 μM). The opioid receptor antagonist, naloxone (1 μM) had no significant effect on the inhibition by CP 55940 of the twitch response.
3. (+)-WIN 55212, CP 55940 and Tween 80 (50 μM) had no effect on responses to exogenous ACh, confirming that their actions were prejunctional. SR141716A (1 μM) did not increase the sensitivity of the longitudinal muscle to either ACh or histamine, but inhibited the responses to high doses of ACh.
4. The (−)-enantiomer of WIN 55212, was approximately 300 times less active than the (+) enantiomer in inhibiting the twitch response, had no CB₁ antagonist activity against the active isomer and did not inhibit the release of ACh in the presence of physostigmine.
5. The dissociation constant (K_D) values for SR 141716A against the inhibitory effect of (+)-WIN 55212 and CP 55940 on the twitch response were 12.07 nM (95% confidence intervals 8.55 and 20.83) and 6.44 nM (95% confidence intervals 4.70 and 10.24), respectively. In experiments in which the release of ACh was inhibited by (+)-WIN 55212, the K_D values were 9.21 nM and 10.53 nM at SR141716A concentrations of 31.6 nM and 100 nM, respectively. The K_D values for the antagonism by naloxone of the inhibition of the twitch responses and the inhibition of ACh release by normorphine in this preparation were found to be 2.38±0.69 nM and 2.00±0.9 nM, respectively.
6. During maximal inhibition of ACh release by (+)-WIN 55212, the addition of normorphine (400 nM) caused a further significant decrease in ACh output.
7. SR141716A alone produced a significant increase in ACh release in both the absence and presence of exogenous cannabinoid drugs, hence we conclude that it has a presynaptic site of action. We also conclude that SR141716A acts either by antagonizing the effect of an endogenous CB₁ receptor agonist or by having an inverse agonist effect at these receptors.

     

Long-term consequences of perinatal fatty acid amino hydrolase inhibition

Background and PurposeFatty acid amide hydrolase inhibitors show promise as a treatment for anxiety, depression and pain. Here we investigated whether perinatal exposure to URB597, a fatty acid amide hydrolase inhibitor, alters brain development and affects behaviour in adult mice.Experimental ApproachMouse dams were treated daily from gestational day 10.5 to 16.5 with 1, 3 or 10 mg kg⁻¹ URB597. MS was used to measure a panel of endocannabinoids and related lipid compounds and brain development was assessed at embryonic day 16.5. Separate cohorts of mouse dams were treated with 10 mg kg⁻¹ URB597, from gestational day 10.5 to postnatal day 7, and the adult offspring were assessed with a battery of behavioural tests.Key ResultsPerinatal URB597 exposure elevated anandamide and related N-acyl amides. URB597 did not induce signs of toxicity or affect dam weight gain, neurogenesis or axonal development at embryonic day 16.5. It did lead to subtle behavioural deficits in adult offspring, manifested by reduced cocaine-conditioned preference, increased depressive behaviours and impaired working memory. Anxiety levels, motor function and sensory-motor gating were not significantly altered.Conclusions and ImplicationsTaken together, the present results highlight how exposure to elevated levels of anandamide and related N-acyl amides during brain development can lead to subtle alterations in behaviour in adulthood.Linked ArticlesThis article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-6     

A novel fluorophosphonate inhibitor of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol with potential anti-obesity effects

Background and Purpose

The development of potent and selective inhibitors of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) via DAG lipases (DAGL) α and β is just starting to be considered as a novel and promising source of pharmaceuticals for the treatment of disorders that might benefit from a reduction in endocannabinoid tone, such as hyperphagia in obese subjects.

Experimental Approach

Three new fluorophosphonate compounds O-7458, O-7459 and O-7460 were synthesized and characterized in various enzymatic assays. The effects of O-7460 on high-fat diet intake were tested in mice.

Key Results

Of the new compounds, O-7460 exhibited the highest potency (IC₅₀ = 690 nM) against the human recombinant DAGLα, and selectivity (IC₅₀ > 10 μM) towards COS-7 cell and human monoacylglycerol lipase (MAGL), and rat brain fatty acid amide hydrolase. Competitive activity-based protein profiling confirmed that O-7460 inhibits mouse brain MAGL only at concentrations ≥10 μM, and showed that this compound has only one major ‘off-target’, that is, the serine hydrolase KIAA1363. O-7460 did not exhibit measurable affinity for human recombinant CB₁ or CB₂ cannabinoid receptors (K_i > 10 μM). In mouse neuroblastoma N18TG2 cells stimulated with ionomycin, O-7460 (10 μM) reduced 2-AG levels. When administered to mice, O-7460 dose-dependently (0–12 mg·kg⁻¹, i.p.) inhibited the intake of a high-fat diet over a 14 h observation period, and, subsequently, slightly but significantly reduced body weight.

Conclusions and Implications

O-7460 might be considered a useful pharmacological tool to investigate further the role played by 2-AG both in vitro and in vivo under physiological as well as pathological conditions.

Linked Articles

This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-4 & http://dx.doi.org/10.1111/bph.2012.167.issue-8     

Shikonin Isolated from Lithospermum erythrorhizon Downregulates Proinflammatory Mediators in Lipopolysaccharide-Stimulated BV2 Microglial Cells by Suppressing Crosstalk between Reactive Oxygen Species and NF-κB

According to the expansion of lifespan, neuronal disorder based on inflammation has been social problem. Therefore, we isolated shikonin from Lithospermum erythrorhizon and evaluated anti-inflammatory effects of shikonin in lipopolysaccharide (LSP)-stimulated BV2 microglial cells. Shikonin dose-dependently inhibits the expression of the proinflammatory mediators, nitric oxide (NO), prostaglandin E₂ (PGE₂), and tumor necrosis factor-α (TNF-α) as well as their main regulatory genes and products such as inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α in LPS-stimulated BV2 microglial cells. Additionally, shikonin suppressed the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) to regulate the key regulatory genes of the proinflammatory mediators, such as iNOS, COX-2, and TNF-α, accompanied with downregulation of reactive oxygen species (ROS) generation. The results indicate that shikonin may downregulate the expression of proinflammatory genes involved in the synthesis of NO, PGE₂, and TNF-α in LPS-treated BV2 microglial cells by suppressing ROS and NF-κB. Taken together, our results revealed that shikonin exerts downregulation of proinflammatory mediators by interference the ROS and NF-κB signaling pathway.     

Decreased Cocaine Motor Sensitization and Self-Administration in Mice Overexpressing Cannabinoid CB2 Receptors

The potential involvement of the cannabinoid CB₂ receptors (CB₂r) in the adaptive responses induced by cocaine was studied in transgenic mice overexpressing the CB₂r (CB₂xP) and in wild-type (WT) littermates. For this purpose, the acute and sensitized locomotor responses to cocaine, conditioned place preference, and cocaine intravenous self-administration were evaluated. In addition, we assessed whether CB₂r were localized in neurons and/or astrocytes, and whether they colocalized with dopamine D1 and D2 receptors (D1Dr and D2Dr). Dopamine (DA) extracellular levels in the nucleus accumbens (NAcc), and gene expression of tyrosine hydroxylase (TH) and DA transporter (DAT) in the ventral tegmental area (VTA), and μ-opioid and cannabinoid CB₁ receptors in the NAcc were also studied in both genotypes. CB₂xP mice showed decreased motor response to acute administration of cocaine (10–20 mg/kg) and cocaine-induced motor sensitization compared with WT mice. CB₂xP mice presented cocaine-induced conditioned place aversion and self-administered less cocaine than WT mice. CB₂r were found in neurons and astrocytes and colocalized with D2Dr in the VTA and NAcc. No significant differences in extracellular DA levels in the NAcc were observed between genotypes after cocaine administration. Under baseline conditions, TH and DAT gene expression was higher and μ-opioid receptor gene expression was lower in CB₂xP than in WT mice. However, both genotypes showed similar changes in TH and μ-opioid receptor gene expression after cocaine challenge independently of the pretreatment received. Importantly, the cocaine challenge decreased DAT gene expression to a lesser extent in cocaine-pretreated CB₂xP than in cocaine-pretreated WT mice. These results revealed that CB₂r are involved in cocaine motor responses and cocaine self-administration, suggesting that this receptor could represent a promising target to develop novel treatments for cocaine addiction.     

The monoacylglycerol lipase inhibitor JZL184 attenuates LPS-induced increases in cytokine expression in the rat frontal cortex and plasma: differential mechanisms of action

Background and purpose

JZL184 is a selective inhibitor of monoacylglycerol lipase (MAGL), the enzyme that preferentially catabolizes the endocannabinoid 2-arachidonoyl glycerol (2-AG). Here, we have studied the effects of JZL184 on inflammatory cytokines in the brain and plasma following an acute immune challenge and the underlying receptor and molecular mechanisms involved.

Experimental approach

JZL184 and/or the CB₁ receptor antagonist, AM251 or the CB₂receptor antagonist, AM630 were administered to rats 30 min before lipopolysaccharide (LPS). 2 h later cytokine expression and levels, MAGL activity, 2-AG, arachidonic acid and prostaglandin levels were measured in the frontal cortex, plasma and spleen.

Key results

JZL184 attenuated LPS-induced increases in IL-1β, IL-6, TNF-α and IL-10 but not the expression of the inhibitor of NFkB (IκBα) in rat frontal cortex. AM251 attenuated JZL184-induced decreases in frontal cortical IL-1β expression. Although arachidonic acid levels in the frontal cortex were reduced in JZL184-treated rats, MAGL activity, 2-AG, PGE₂ and PGD₂ were unchanged. In comparison, MAGL activity was inhibited and 2-AG levels enhanced in the spleen following JZL184. In plasma, LPS-induced increases in TNF-α and IL-10 levels were attenuated by JZL184, an effect partially blocked by AM251. In addition, AM630 blocked LPS-induced increases in plasma IL-1β in the presence, but not absence, of JZL184.

Conclusion and implications

Inhibition of peripheral MAGL in rats by JZL184 suppressed LPS-induced circulating cytokines that in turn may modulate central cytokine expression. The data provide further evidence for the endocannabinoid system as a therapeutic target in treatment of central and peripheral inflammatory disorders.

Linked Articles

This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-4 & http://dx.doi.org/10.1111/bph.2012.167.issue-8     

Compound Danshen Dripping Pill inhibits high altitude-induced hypoxic damage by suppressing oxidative stress and inflammatory responses

ContextPrevious studies indicate that compound Danshen Dripping Pill (CDDP) improves the adaptation to high-altitude exposure. However, its mechanism of action is not clear.ObjectiveTo explore the protective effect of CDDP on hypobaric hypoxia (HH) and its possible mechanism.Materials and methodsA meta-analysis of 1051 human volunteers was performed to evaluate the effectiveness of CDDP at high altitudes. Male Sprague-Dawley rats were randomized into 5 groups (n = 6): control at normal pressure, model, CDDP-170 mg/kg, CDDP-340 mg/kg and acetazolamide groups. HH was simulated at an altitude of 5500 m for 24 h. Animal blood was collected for arterial blood-gas analysis and cytokines detection and their organs were harvested for pathological examination. Expression levels of AQP1, NF-κB and Nrf2 were determined by immunohistochemical staining.ResultsThe meta-analysis data indicated that the ratio between the combined RR of the total effective rate and the 95% CI was 0.23 (0.06, 0.91), the SMD and 95% CI of SO₂ was 0.37 (0.12, 0.62). Pre-treatment of CDDP protected rats from HH-induced pulmonary edoema and heart injury, left-shifted oxygen-dissociation curve and decreased P50 (30.25 ± 3.72 vs. 37.23 ± 4.30). Mechanistically, CDDP alleviated HH-reinforced ROS by improving SOD and GPX1 while inhibiting pro-inflammatory cytokines and NF-κB expression. CDDP also decreased HH-evoked D-dimer, erythrocyte aggregation and blood hemorheology, promoting AQP1 and Nrf2 expression.Discussion and conclusionsPre-treatment with CDDP could prevent HH-induced tissue damage, oxidative stress and inflammatory response. Suppressed NF-κB and up-regulated Nrf2 might play significant roles in the mechanism of CDDP.     

Central antinociception induced by μ-opioid receptor agonist morphine,but not δ- or κ-, is mediated by cannabinoid CB1 receptor

Background and purpose:

It has been demonstrated that cannabinoids evoke the release of endogenous opioids to produce antinociception; however, no information exists regarding the participation of cannabinoids in the antinociceptive mechanisms of opioids. The aim of the present study was to determine whether endocannabinoids are involved in central antinociception induced by activation of µ-, δ- and κ-opioid receptors.

Experimental approach:

Nociceptive threshold to thermal stimulation was measured according to the tail-flick test in Swiss mice. Morphine (5 µg), SNC80 (4 µg), bremazocine (4 µg), AM251 (2 and 4 µg), AM630 (2 and 4 µg) and MAFP (0.1 and 0.4 µg) were administered by the intracerebroventricular route.

Key results:

The CB₁-selective cannabinoid receptor antagonist AM251 completely reversed the central antinociception induced by morphine in a dose-dependent manner. In contrast, the CB₂-selective cannabinoid receptor antagonist AM630 did not antagonize this effect. Additionally, the administration of the anandamide amidase inhibitor, MAFP, significantly enhanced the antinociception induced by morphine. In contrast, the antinociceptive effects of δ- and κ-opioid receptor agonists were not affected by the cannabinoid antagonists. The antagonists alone caused no hyperalgesic or antinociceptive effects.

Conclusions and implications:

The results provide evidence for the involvement of cannabinoid CB₁ receptors in the central antinociception induced by activation of µ-opioid receptors by the agonist morphine. The release of endocannabinoids appears not to be involved in central antinociception induced by activation of κ- and δ-opioid receptors.     

Compound FLZ inhibits lipopolysaccharide-induced inflammatory effects via down-regulation of the TAK-IKK and TAK-JNK/p38MAPK pathways in RAW264.7 macrophages

Aim:

The aim of this study was to investigate the effect of the squamosamide derivative FLZ (N-2-(4-hydroxy-phenyl)-ethyl-2-(2,5-dimethoxy-phenyl)-3-(3-methoxy-4-hydroxy-phenyl)-acrylamide) on lipopolysaccharide (LPS)-induced inflammatory mediator production and the underlying mechanism in RAW264.7 macrophages.

Methods:

RAW264.7 cells were preincubated with non-toxic concentrations of compound FLZ (1, 5, and 10 μmol/L) for 30 min and then stimulated with 10 μg/L LPS. The production of nitric oxide (NO), the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), and the activation of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways were examined.

Results:

FLZ significantly inhibited the LPS-induced production of NO, as well as the expression of iNOS and COX-2 at both the RNA and the protein levels in RAW264.7 cells. The LPS-induced increase in the DNA binding activity of NF-κB and activator protein 1 (AP-1), the nuclear translocation of NF-κB p65, the degradation of the inhibitory κBα protein (IκBα) and the phosphorylation of IκBα, IκB kinase (IKK) α/β, c-Jun NH₂-terminal kinase (JNK) and p38 MAPKs were all suppressed by FLZ. However, the phosphorylation of extracellular signal-regulated kinase (ERK) was not affected. Further study revealed that FLZ inhibited the phosphorylation of transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1), which is an upstream signaling molecule required for IKKα/β, JNK and p38 activation.

Conclusion:

FLZ inhibited the LPS-induced production of inflammatory mediators at least partly through the downregulation of the TAK-IKK and TAK-JNK/p38MAPK pathways.     

In vivo characterization of the highly selective monoacylglycerol lipase inhibitor KML29: antinociceptive activity without cannabimimetic side effects

Background and PurposeSince monoacylglycerol lipase (MAGL) has been firmly established as the predominant catabolic enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG), a great need has emerged for the development of highly selective MAGL inhibitors. Here, we tested the in vivo effects of one such compound, KML29 (1,1,1,3,3,3-hexafluoropropan-2-yl 4-(bis(benzo[d][1,3]dioxol-5-yl)(hydroxy)methyl)piperidine-1-carboxylate).Experimental ApproachIn the present study, we tested KML29 in murine inflammatory (i.e. carrageenan) and sciatic nerve injury pain models, as well as the diclofenac-induced gastric haemorrhage model. KML29 was also evaluated for cannabimimetic effects, including measurements of locomotor activity, body temperature, catalepsy, and cannabinoid interoceptive effects in the drug discrimination paradigm.Key ResultsKML29 attenuated carrageenan-induced paw oedema and completely reversed carrageenan-induced mechanical allodynia. These effects underwent tolerance after repeated administration of high-dose KML29, which were accompanied by cannabinoid receptor 1 (CB₁) receptor desensitization. Acute or repeated KML29 administration increased 2-AG levels and concomitantly reduced arachidonic acid levels, but without elevating anandamide (AEA) levels in the whole brain. Furthermore, KML29 partially reversed allodynia in the sciatic nerve injury model and completely prevented diclofenac-induced gastric haemorrhages. CB₁ and CB₂ receptors played differential roles in these pharmacological effects of KML29. In contrast, KML29 did not elicit cannabimimetic effects, including catalepsy, hypothermia and hypomotility. Although KML29 did not substitute for Δ⁹-tetrahydrocannabinol (THC) in C57BL/6J mice, it fully and dose-dependantly substituted for AEA in fatty acid amide hydrolase (FAAH) (−/−) mice, consistent with previous work showing that dual FAAH and MAGL inhibition produces THC-like subjective effects.Conclusions and ImplicationsThese results indicate that KML29, a highly selective MAGL inhibitor, reduces inflammatory and neuropathic nociceptive behaviour without occurrence of cannabimimetic side effects.Linked ArticlesThis article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-6     

The antinociceptive triterpene β-amyrin inhibits 2-arachidonoylglycerol (2-AG) hydrolysis without directly targeting cannabinoid receptors

BACKGROUND AND PURPOSE

Pharmacological activation of cannabinoid CB₁ and CB₂ receptors is a therapeutic strategy to treat chronic and inflammatory pain. It was recently reported that a mixture of natural triterpenes α- and β-amyrin bound selectively to CB₁ receptors with a subnanomolar K_i value (133 pM). Orally administered α/β-amyrin inhibited inflammatory and persistent neuropathic pain in mice through both CB₁ and CB₂ receptors. Here, we investigated effects of amyrins on the major components of the endocannabinoid system.

EXPERIMENTAL APPROACH

We measured CB receptor binding interactions of α- and β-amyrin in validated binding assays using hCB₁ and hCB₂ transfected CHO-K1 cells. Effects on endocannabinoid transport in U937 cells and breakdown using homogenates of BV2 cells and pig brain, as well as purified enzymes, were also studied.

KEY RESULTS

There was no binding of either α- or β-amyrin to hCB receptors in our assays (K_i > 10 µM). The triterpene β-amyrin potently inhibited 2-arachidonoyl glycerol (2-AG) hydrolysis in pig brain homogenates, but not that of anandamide. Although β-amyrin only weakly inhibited purified human monoacylglycerol lipase (MAGL), it also inhibited α,β-hydrolases and more potently inhibited 2-AG breakdown than α-amyrin and the MAGL inhibitor pristimerin in BV2 cell and pig brain homogenates.

CONCLUSIONS AND IMPLICATIONS

We propose that β-amyrin exerts its analgesic and anti-inflammatory pharmacological effects via indirect cannabimimetic mechanisms by inhibiting the degradation of the endocannabinoid 2-AG without interacting directly with CB receptors. Triterpenoids appear to offer a very broad and largely unexplored scaffold for inhibitors of the enzymic degradation of 2-AG.

LINKED ARTICLES

This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.167.issue-8