Umeå University's logo

Little is known about the endocannabinoid (eCB) system in squamous cell carcinoma of the oral tongue (SCCOT). Here we have investigated, at the mRNA level, expression of genes coding for the components of the eCB system in tumour and non-malignant samples from SCCOT patients. Expression of NAPEPLD and PLA2G4E, coding for eCB anabolic enzymes, was higher in the tumour tissue than in non-malignant tissue. Among genes coding for eCB catabolic enzymes, expression of MGLL was lower in tumour tissue while PTGS2 was increased. It is concluded that the eCB system may be dysfunctional in SCCOT.

BACKGROUND AND PURPOSE CYP1B1 and CYP1A1 are important extra-hepatic cytochromes, expressed in the colon and involved in the metabolism of dietary constituents and exogenous compounds. CYP1B1 expression is increased by pro-inflammatory cytokines, and it has been recently implicated in regulation of blood brain barrier function. We investigated its involvement in the increased permeability of the intestinal epithelial barrier observed in inflammatory conditions. EXPERIMENTAL APPROACH Epithelial monolayers formed by human T84 colon carcinoma cells cultured on transwells, were disrupted by incubation with IFN gamma (10 ng.mL(-1)). Monolayer integrity was measured using transepithelial electrical resistance. CYP1A1 and CYP1B1 inhibitors or inducers were applied apically. Potential mechanisms of action were investigated using RT-qPCR. KEY RESULTS IFN gamma disrupts the barrier integrity of the T84 monolayers and increases CYP1B1 and HIF1 alpha mRNA expression. CYP1B1 induction is inhibited by the NF-kappa B inhibitor ammonium pyrrolidinedithiocarbamate (100 mu M) but not by the HIF1 alpha inhibitor 3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole (50 mu M). Inhibition of CYP1B1 with the selective inhibitor 2,4,3,5-tetramethoxystilbene (100 nM) partly reverses the effects of IFN gamma on epithelial permeability. CONCLUSIONS AND IMPLICATIONS These data suggest that increased expression of CYP1B1 is involved in the effects of IFN gamma on epithelial permeability. Inhibition of CYP1B1 counteracts the alterations of epithelial barrier integrity induced by IFN gamma and could thus have a therapeutic potential in disorders of intestinal permeability associated with inflammation.

Background and purpose: Endocannabinoids and related N-acylethanolamines (NAEs) are involved in regulation of gut function, but relatively little is known as to whether inflammatory cytokines such as IFN affect their levels. We have investigated this in vitro using cultures of T84 colon cancer cells.

Experimental approach: T84 cells, when cultured in monolayers, differentiate to form adult colonic crypt-like cells with excellent permeability barrier properties. The integrity of the permeability barrier in these monolayers was measured using transepithelial electrical resistance (TEER). NAE levels were determined by ultra-performance liquid chromatography-tandem mass spectrometric analysis. Expression of the enzymes involved in NAE and 2-arachidonoylglycerol (2-AG) turnover were assessed with qPCR.

Key results: IFN treatment for 8 or 24h increased levels of both endocannabinoids (anandamide and 2-AG) and the related NAEs. The treatment did not affect the rate of hydrolysis of either anandamide or palmitoylethanolamide by intact cells, and in both cases, fatty acid amide hydrolase (FAAH) rather than NAE-hydrolysing acid amidase (NAAA) was mainly responsible for the hydrolysis of these NAEs. IFN treatment reduced the TEER of the cells in a manner that was not prevented by inhibition of either FAAH or NAAA but was partially reversed by apical administration of the NAE palmitoylethanolamide.

Conclusion and implications: IFN treatment mobilized endocannabinoid and related NAE levels in T84 cells. However, blockade of anandamide or NAE hydrolysis was insufficient to negate the deleterious effects of this cytokine upon the permeability barrier of the cell monolayers.

The large pore ion channel pannexin-1 (Panx1) has been reported to play a role in the cellular uptake and release of anandamide (AEA) in the hippocampus. It is not known whether this is a general mechanism or limited to the hippocampus. We have investigated this pharmacologically using T84 colon cancer cells. The cells expressed Panx1 at the mRNA level, and released ATP in a manner that could be reduced by treatment with the Panx1 inhibitors carbenoxolone and mefloquine and the Panxl substrate SR101. However, no significant effects of these compounds upon the uptake or hydrolysis of exogenously applied AEA was seen. Uptake by T84 cells of the other main endocannabinoid 2-arachidonoylglycerol and the AEA homologue palmitoylethanolamide was similarly not affected by carbenoxolone or mefloquine. Total release of tritium from [H-3]AEA-prelabelled T84 cells over 10 min was increased, rather than inhibited by carbenoxolone and mefloquine. Finally, AEA uptake by PC3 prostate cancer and SH-SY5Y neuroblastoma cells, which express functional Panx1 channels, was not inhibited by carbenoxolone. Thus, in contrast to the hippocampus, Panx1 does not appear to play a role in AEA uptake and release from the cells studied under the conditions used.

There is good evidence that the N-acylethanolamine (NAE)/monoacylglycerol (MAG) signalling systems are involved in the pathogenesis of cancer. However, it is not known how prostate tumours affect these systems in the surrounding non-malignant tissue and vice versa. In the present study we have investigated at the mRNA level 11 components of these systems (three coding for anabolic enzymes, two for NAE/MAG targets and six coding for catabolic enzymes) in rat prostate tissue following orthotopic injection of low metastatic AT1 cells and high metastatic MLL cells. The MLL tumours expressed higher levels of Napepld, coding for a key enzyme in NAE synthesis, and lower levels of Naaa, coding for the NAE hydrolytic enzyme N-acylethanolamine acid amide hydrolase than the AT1 tumours. mRNA levels of the components of the NAE/MAG signalling systems studied in the tissue surrounding the tumours were not overtly affected by the tumours. AT1 cells in culture expressed Faah, coding for the NAE hydrolytic enzyme fatty acid amide hydrolase, at much lower levels than Naaa. However, the ability of the intact cells to hydrolyse the NAE arachidonoylethanolamide (anandamide) was inhibited by an inhibitor of FAAH, but not of NAAA. Treatment of the AT1 cells with interleukin-6, a cytokine known to be involved in the pathogenesis of prostate cancer, did not affect the expression of the components of the NAE/MAG system studied. It is thus concluded that in the model system studied, the tumours show different expressions of mRNA coding for key the components of the NAE/MAG system compared to the host tissue, but that these changes are not accompanied by alterations in the non-malignant tissue.

The endocannabinoid system consists of the cannabinoid (CB) receptors, CB(1) and CB(2), the endogenous ligands anandamide (AEA, arachidonoylethanolamide) and 2-arachidonoylglycerol (2-AG), and their synthetic and metabolic machinery. The use of cannabis has been described in classical and recent literature for the treatment of pain, but the potential for psychotropic effects as a result of the activation of central CB(1) receptors places a limitation upon its use. There are, however, a number of modern approaches being undertaken to circumvent this problem, and this review represents a concise summary of these approaches, with a particular emphasis upon CB(2) receptor agonists. Selective CB(2) agonists and peripherally restricted CB(1) or CB(1)/CB(2) dual agonists are being developed for the treatment of inflammatory and neuropathic pain, as they demonstrate efficacy in a range of pain models. CB(2) receptors were originally described as being restricted to cells of immune origin, but there is evidence for their expression in human primary sensory neurons, and increased levels of CB(2) receptors reported in human peripheral nerves have been seen after injury, particularly in painful neuromas. CB(2) receptor agonists produce antinociceptive effects in models of inflammatory and nociceptive pain, and in some cases these effects involve activation of the opioid system. In addition, CB receptor agonists enhance the effect of mu-opioid receptor agonists in a variety of models of analgesia, and combinations of cannabinoids and opioids may produce synergistic effects. Antinociceptive effects of compounds blocking the metabolism of anandamide have been reported, particularly in models of inflammatory pain. There is also evidence that such compounds increase the analgesic effect of non-steroidal anti-inflammatory drugs (NSAIDs), raising the possibility that a combination of suitable agents could, by reducing the NSAID dose needed, provide an efficacious treatment strategy, while minimizing the potential for NSAID-induced gastrointestinal and cardiovascular disturbances. Other potential "partners" for endocannabinoid modulatory agents include alpha(2)-adrenoceptor modulators, peroxisome proliferator-activated receptor alpha agonists and TRPV1 antagonists. An extension of the polypharmacological approach is to combine the desired pharmacological properties of the treatment within a single molecule. Hopefully, these approaches will yield novel analgesics that do not produce the psychotropic effects that limit the medicinal use of cannabis.

Background:

The endocannabinoid ligand anandamide (AEA) is removed from the extracellular space by a process ofcellular uptake followed by metabolism. In many cells, such as the RBL-2H3 cell line, inhibition of FAAH activity reduces theobserved uptake, indicating that the enzyme regulates uptake by controlling the intra- : extracellular AEA concentrationgradient. However, in other FAAH-expressing cells, no such effect is seen. It is not clear, however, whether these differencesare methodological in nature or due to properties of the cells themselves. In consequence, we have reinvestigated the roleof FAAH in gating the uptake of AEA.Methodology/Principal Findings: The effects of FAAH inhibition upon AEA uptake were investigated in four cell lines: AT1rat prostate cancer, RBL-2H3 rat basophilic leukaemia, rat C6 glioma and mouse P19 embryonic carcinoma cells. SemiquantitativePCR for the cells and for a rat brain lysate confirmed the expression of FAAH. No obvious expression of atranscript with the expected molecular weight of FLAT was seen. FAAH expression differed between cells, but all four couldaccumulate AEA in a manner inhibitable by the selective FAAH inhibitor URB597. However, there was a difference in thesensitivities seen in the reduction of uptake for a given degree of FAAH inhibition produced by a reversible FAAH inhibitor,with C6 cells being more sensitive than RBL-2H3 cells, despite rather similar expression levels and activities of FAAH. Thefour cell lines all expressed FABP5, and AEA uptake was reduced in the presence of the FABP5 inhibitor SB-FI-26, suggestingthat the different sensitivities to FAAH inhibition for C6 and RBL2H3 cells is not due to differences at the level of FABP-5.Conclusions/Significance: When assayed using the same methodology, different FAAH-expressing cells display differentsensitivities of uptake to FAAH inhibition.

Background: The endogenous cannabinoid system is involved in the control of pain. However, little is known as to the integrity of the cannabinoid system in human pain syndromes. Here we investigate the expression of the cannabinoid receptor 1 (CB(1)) in human Achilles tendons from healthy volunteers and from patients with Achilles tendinosis.

Methodology: Cannabinoid CB(1) receptor immunoreactivity (CB(1)IR) was evaluated in formalin-fixed biopsies from individuals suffering from painful Achilles tendinosis in comparison with healthy human Achilles tendons.

Principal Findings: CB(1)IR was seen as a granular pattern in the tenocytes. CB(1)IR was also observed in the blood vessel wall and in the perineurium of the nerve. Quantification of the immunoreactivity in tenocytes showed an increase of CB(1) receptor expression in tendinosis tissue compared to control tissue.

Conclusion: Expression of cannabinoid receptor 1 is increased in human Achilles tendinosis suggesting that the cannabinoid system may be dysregulated in this disorder.

Background: The antifungal compound ketoconazole has, in addition to its ability to interfere with fungal ergosterol synthesis, effects upon other enzymes including human CYP3A4, CYP17, lipoxygenase and thromboxane synthetase. In the present study, we have investigated whether ketoconazole affects the cellular uptake and hydrolysis of the endogenous cannabinoid receptor ligand anandamide (AEA). Methodology/Principal Findings: The effects of ketoconazole upon endocannabinoid uptake were investigated using HepG2, CaCo2, PC-3 and C6 cell lines. Fatty acid amide hydrolase (FAAH) activity was measured in HepG2 cell lysates and in intact C6 cells. Ketoconazole inhibited the uptake of AEA by HepG2 cells and CaCo2 cells with IC50 values of 17 and 18 mu M, respectively. In contrast, it had modest effects upon AEA uptake in PC-3 cells, which have a low expression of FAAH. In cell-free HepG2 lysates, ketoconazole inhibited FAAH activity with an IC50 value (for the inhibitable component) of 34 mu M. Conclusions/Significance: The present study indicates that ketoconazole can inhibit the cellular uptake of AEA at pharmacologically relevant concentrations, primarily due to its effects upon FAAH. Ketoconazole may be useful as a template for the design of dual-action FAAH/CYP17 inhibitors as a novel strategy for the treatment of prostate cancer.

BACKGROUND AND PURPOSE: Drugs used clinically usually have a primary mechanism of action, but additional effects on other biological targets can contribute to their effects. A potentially useful additional target is the endocannabinoid metabolizing enzyme monoacylglycerol lipase (MGL). We have screened a range of drugs for inhibition of MGL and compared the observed potencies using different MGL enzyme assays.

EXPERIMENTAL APPROACH: MGL activity was screened using recombinant human MGL (cell lysates and purified enzyme) with 4-nitrophenyl acetate (NPA) as substrate. 2-Oleolyglycerol metabolism by rat cerebellar cytosolic MGL and by recombinant MGL was also investigated.

KEY RESULTS: Among the 96 compounds screened in the NPA assay, troglitazone, CP55,940, N-arachidonoyl dopamine and AM404 inhibited NPA hydrolysis by the lysates with IC(50) values of 1.1, 4.9, 0.78 and 3.1µM, respectively. The potency for troglitazone is in the same range as its primary pharmacological activity, activation of peroxisome proliferator-activated receptor (PPAR) γ. Among PPARγ ligands, the potency order towards human MGL was troglitazone > ciglitazone > rosiglitazone > 15-deoxy-Δ(12,14) -prostaglandin J(2) ≈ CAY 10415 > CAY 10514. In contrast to the time-dependent inhibitor JZL184, the potency of troglitazone was dependent upon the enzyme assay system used. Thus, troglitazone inhibited rat cytosolic 2-oleoylglycerol hydrolysis less potently (IC(50) 41µM) than hydrolysis of NPA by the human MGL lysates.

CONCLUSIONS AND IMPLICATIONS: 'Hits' in screening programmes for MGL inhibitors should be assessed in different MGL assays. Troglitazone may be a useful lead for the design of novel, dual action MGL inhibitors/PPARγ activators.

A cyclic Leu-enkephalin mimetic containing a 7-membered ring, and two linear analogues, has been prepared on solid phase. In the cyclic mimetic the intramolecular (1–4) hydrogen bond found in crystalline Leu-enkephalin has been replaced by an ethylene bridge. In addition, the amide bond between Tyr1 and Gly2 has been replaced by a methylene ether isostere and Gly3 has been deleted. The two linear analogues both contain the methylene ether isostere instead of the Tyr1-Gly2 amide bond and the shorter of the two lacks Gly3. The three compounds, and a β-turn mimetic analogous to the 7-membered turn mimetic but with Gly3 included, were evaluated for specific binding to µ- and δ-opioid receptors in rat brain membranes. With the exception of the β-turn mimetic the three other Leu-enkephalin analogues all bound with varying affinity to the µ- and δ-opioid receptors. From the results it could be concluded that Leu-enkephalin binds in a turn conformation to the opiate receptors, but that this conformation is not a (1–4) β-turn.

Regulation of appetite and food intake is partly regulated by N-acylethanolamine lipids oleoylethanolamide (OEA), stearoylethanolamide (SEA), and palmitoylethanolamide (PEA), which induce satiety through endogenous formation in the small intestine upon feeding, but also when orally or systemic administered. OEA, SEA, and PEA are present in human milk, and we hypothesized that the content of OEA, SEA, and PEA in mother's milk differed for infants being heavy (high weight-for-age Z-score (WAZ)) or light (low WAZ) at time of milk sample collection. Ultra-high performance liquid chromatography-mass spectrometry was used to determine the concentration of OEA, SEA, and PEA in milk samples collected four months postpartum from mothers to high (n = 50) or low (n = 50) WAZ infants. Associations between OEA, SEA, and PEA concentration and infant anthropometry at four months of age as well as growth from birth were investigated using linear and logistic regression analyses, adjusted for birth weight, early infant formula supplementation, and maternal pre-pregnancy body mass index. Mean OEA, SEA, and PEA concentrations were lower in the high compared to the low WAZ group (all p < 0.02), and a higher concentration of SEA was associated with lower anthropometric measures, e.g., triceps skinfold thickness (mm) (β = -2.235, 95% CI = -4.04, -0.43, p = 0.016), and weight gain per day since birth (g) (β = -8.169, 95% CI = -15.26, -1.08, p = 0.024). This raises the possibility, that the content of satiety factors OEA, SEA, and PEA in human milk may affect infant growth.

Fatty acid amide hydrolase (FAAH) exerts its main function in the catabolism of the endogenous chemical messenger anandamide (AEA), thus modulating the endocannabinoid (eCB) pathway. Inhibition of FAAH may serve as an effective strategy to relieve anxiety and possibly other central nervous system (CNS)-related disorders. Positron emission tomography (PET) would facilitate us to better understand the relationship between FAAH in certain disease conditions, and accelerate clinical translation of FAAH inhibitors by providing in vivo quantitative information. So far, most PET tracers show irreversible binding patterns with FAAH, which would result in complicated quantitative processes. Herein, we have identified a new FAAH inhibitor (1-((1-methyl-1H-indol-2-yl)methyl)piperidin-4-yl)(oxazol-2-yl)methanone (8) which inhibits the hydrolysis of AEA in the brain with high potency (IC₅₀ value 11 nM at a substrate concentration of 0.5 µM), and without showing time-dependency. The PET tracer [¹¹C]8 (also called [¹¹C]FAAH-1906) was successfully radiolabeled with [¹¹C]MeI in 17 ± 6% decay-corrected radiochemical yield (n = 7) with >74.0 GBq/μmol (2 Ci/μmol) molar activity and >99% radiochemical purity. Ex vivo biodistribution and blocking studies of [¹¹C]8 in normal mice were also conducted, indicating good brain penetration, high brain target selectivity, and modest to excellent target selectivity in peripheral tissues. Thus, [¹¹C]8 is a potentially useful PET ligand with enzyme inhibitory and target binding properties consistent with a reversible mode of action.

Monoacylglycerol lipase (MAGL) is a senile hydrolase that degrades 2-arachidonoylglycerol (2-AG) in the endocannabinoid system (eCB). Selective inhibition of MAGL has emerged as a potential therapeutic approach for the treatment of diverse pathological conditions, including chronic pain, inflammation, cancer, and neurodegeneration. Herein, we disclose a novel array of reversible and irreversible MAGL inhibitors by means of "tail switching" on a piperazinyl azetidine scaffold. We developed a lead irreversible-binding MAGL inhibitor 8 and reversible-binding compounds 17 and 37, which are amenable for radiolabeling with C-11 or F-18. [C-11]8 ([C-11]MAGL-2-11) exhibited high brain uptake and excellent binding specificity in the brain toward MAGL. Reversible radioligands [C-11]17 ([C-11]PAD) and [F-18]37 ([F-18]MAGL-4-11) also demonstrated excellent in vivo binding specificity toward MAGL in peripheral organs. This work may pave the way for the development of MAGL-targeted positron emission tomography tracers with tunability in reversible and irreversible binding mechanisms.

Monoacylglycerol lipase (MAGL) is the principle enzyme for metabolizing endogenous cannabinoid ligand 2-arachidonoyglycerol (2-AG). Blockade of MAGL increases 2-AG levels, resulting in subsequent activation of the endocannabinoid system, and has emerged as a novel therapeutic strategy to treat drug addiction, inflammation, and neurodegenerative diseases. Herein we report a new series of MAGL inhibitors, which were radiolabeled by site-specific labeling technologies, including C-11-carbonylation and spirocyclic iodonium ylide (SCIDY) radio fluorination. The lead compound [C-11]10 (MAGL-0519) demonstrated high specific binding and selectivity in vitro and in vivo. We also observed unexpected washout kinetics with these irreversible radiotracers, in which in vivo evidence for turnover of the covalent residue was unveiled between MAGL and azetidine carboxylates. This work may lead to new directions for drug discovery and PET tracer development based on azetidine carboxylate inhibitor scaffold.

In the light of findings indicating that cannabinoids can affect the proliferation of a number of cancer cell types and that cannabinoid receptor expression is higher in prostate cancer cell lines than in non-malignant cells, we investigated whether the level of cannabinoid 1 receptor immunoreactivity (CB(1)IR) in prostate cancer tissues is associated with disease severity and outcome. Formalin-fixed paraffin-embedded non-malignant and tumour tissue samples from patients who were diagnosed with prostate cancer at a transurethral resection for voiding problems were used. CB(1)IR, which was scored in a total of 399 cases, was associated with the epithelial cell membranes, with little staining in the stroma. Patients with a tumour CB(1)IR score greater or equal to the median (2) had a significantly higher proportion of Gleason scores 8-10, metastases at diagnosis, tumour size and rate of cell proliferation at diagnosis than patients with a score<2. For 269 cases, tumour CB(1)IR was measured for patients who only received palliative therapy at the end stages of the disease, allowing the influence of CB(1)IR upon the disease outcome to be determined. Receiver operating characteristic (ROC) curves showed an area under the curve of 0.67 (95% confidence limits 0.59-0.74) for CB(1)IR in the tumour. CB(1)IR in non-malignant tissue was not associated with disease outcome. A tumour CB(1)IR score >or=2 was associated with a significantly lower disease specific survival. A Cox proportional hazards regression indicated that the tumour CB(1)IR score and the Gleason score were independent prognostic variables. It is concluded that a high tumour CB(1)IR score is associated with prostate cancer severity and outcome.

Inhibitors of the metabolism of the endogenous cannabinoid ligand anandamide by fatty acid amide hydrolase (FAAH) reduce the gastric damage produced by non-steroidal anti-inflammatory agents and synergise with them in experimental pain models. This motivates the design of compounds with joint FAAH/cyclooxygenase (COX) inhibitory activity. Here we present data on the N-(3-methylpyridin-2-yl) amide derivatives of flurbiprofen and naproxen (Flu-AM1 and Nap-AM1, respectively) with respect to their properties towards these two enzymes. Flu-AM1 and Nap-AM1 inhibited FAAH-catalysed hydrolysis of [H-3]anandamide by rat brain homogenates with IC50 values of 044 and 0.74 mu M. The corresponding values for flurbiprofen and naproxen were 29 and > 100 mu M, respectively. The inhibition by Flu-AM1 was reversible, mixed-type, with K-slope(i) and K-intercept(i) values of 0.21 and 1.4 mu M, respectively. Flurbiprofen and Flu-AM1 both inhibited COX in the same manner with the order of potencies COX-2 vs. 2-arachidonoylglycerol > COX-1 vs. arachidonic acid > COX-2 vs. arachidonic acid with flurbiprofen being approximately 2-3 fold more potent than Flu-AM 1 in the assays. Nap-AM1 was a less potent inhibitor of COX. Flu-AM1 at low micromolar concentrations inhibited the FAAH-driven uptake of [H-3]anandamide into RBL2H3 basophilic leukaemia cells in vitro, but did not penetrate the brain in vivo sufficiently to block the binding of [F-18]DOPP to brain FAAH. It is concluded that Flu-AM 1 is a dual-action inhibitor of FAAH and COX that may be useful in exploring the optimal balance of effects on these two enzyme systems in producing peripheral alleviation of pain and inflammation in experimental models.

Background: It has been reported that direct activation of the cannabinoid CB₁ receptor in epidermal growth factor (EGR)-stimulated PC-3 prostate cancer cells results in an anti-proliferative effect accompanied by a down-regulation of EGF receptors (EGFR). In the present study, we investigated whether similar effects are seen following inhibition of the endocannabinoid hydrolytic enzyme monoacylglycerol lipase (MGL).

Results: CB₁ receptor expression levels were found to differ greatly between two experimental series conducted using PC-3 cells. The monoacylglycerol lipase inhibitor JZL184 increased levels of 2-arachidonoylglycerol in the PC-3 cells without producing changes in the levels of anandamide and related N-acylethanolamines. In the first series of experiments, JZL184 produced a small mitogenic effect for cells that had not been treated with EGF, whereas an anti-proliferative effect was seen for EGF-treated cells. An anti-proliferative effect for the EGF-treated cells was also seen with the CB receptor agonist CP55,940. In the second batch of cells, there was an interaction between JZL184 and CB₁ receptor expression densities in linear regression analyses with EGFR expression as the dependent variable.

Conclusions: Inhibition of MGL by JZL184 can affect EGFR expression. However, the use in our hands of PC-3 cells as a model to investigate the therapeutic potential of MGL inhibitors and related compounds is compromised by their variability of CB₁ receptor expression.

Background: In prostate cancer, tumour expression of cannabinoid CB1 receptors is associated with a poor prognosis. One explanation for this association comes from experiments with transfected astrocytoma cells, where a high CB receptor expression recruits the Akt signalling survival pathway. In the present study, we have investigated the association between CB1 receptor expression and the Akt pathway in a well-characterised prostate cancer tissue microarray.

Methodology/Principal Findings: Phosphorylated Akt immunoreactivity (pAkt-IR) scores were available in the database. CB1 receptor immunoreactivity (CB1IR) was rescored from previously published data using the same scale as pAkt-IR. There was a highly significant correlation between CB1IR and pAkt-IR. Further, cases with high expression levels of both biomarkers were much more likely to have a more severe form of the disease at diagnosis than those with low expression levels. The two biomarkers had additive effects, rather than an interaction, upon disease-specific survival.

Conclusions/Significance: The present study provides data that is consistent with the hypothesis that at a high CB1 receptor expression, the Akt signalling pathway becomes operative.

The two endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), play independent and nonredundant roles in the body. This makes the development of both selective and dual inhibitors of their inactivation an important priority. In this work we report a new series of inhibitors of monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH). Among them, (+/-)-oxiran-2-ylmethyl 6-(1,1'-biphenyl-4-yl)hexanoate (8) and (2R)-(-)-oxiran-2-ylmethyl(4-benzylphenyl)acetate (30) stand out as potent inhibitors of human recombinant MAGL (IC(50) (8) = 4.1 mu M; IC(50) (30) = 2.4 mu M), rat brain monoacylglycerol hydrolysis (IC(50) (8) = 1.8 mu M; IC(50) (30) = 0.68 mu M), and rat brain FAAH (IC(50) (8) = 5.1 mu M; IC(50) (30) = 0.29 mu M). Importantly, and in contrast to the other previously described MAGL inhibitors, these compounds behave as reversible inhibitors either of competitive (8) or noncompetitive nature (30). Hence, they could be useful to explore the therapeutic potential of reversible MAGL inhibitors.

A series of 32 heterocyclic analogues based on the structure of 2-arachidonoylglycerol (2-AG) were synthesized and tested for their ability to inhibit monoacylglycerol lipase and fatty acid an-tide hydrolase activities. The designed compounds feature a hydrophobic moiety and different heterocyclic subunits that mimic the glycerol fragment. This series has allowed us to carry out the first systematic structure activity relationship study on inhibition of 2-AG hydrolysis. The most promising compounds were oxiran-2-ylmethyl (5Z,8Z,l 11Z,14Z)-icosa-5,8,11,14-tetraenoate (1) and tetrahydro-2H-pyran-2-ylmethyl (5Z,8Z,11Z,14z)-icosa5,8,11,14-tetraenoate (5). They inhibited cytosolic 2-oleoylglycerol (2-OG) hydrolysis completely (IC50 values of 4.5 and 5.6 mu M, respectively). They also blocked, albeit less potently, 2-OG hydrolysis in membrane fractions (IC50 values of 19 and 26,mu M, respectively) and anandamide hydrolysis (IC50 values of 12 and 51 mu M, respectively). These compounds will be useful in delineating the importance of the cytosolic hydrolytic activity in the regulation of 2-AG levels and, hence, its potential as a target for drug development.

Oxylipins and endocannabinoids play important biological roles, including effects upon inflammation. It is not known whether the circulating levels of these lipids are affected by inhalation of the environmental pollutant acrolein. In the present study, we have investigated the consequences of low-level exposure to acrolein on oxylipin, endocannabinoid and related lipid levels in the plasma of healthy individuals and individuals with chemical intolerance (CI), an affliction with a suggested inflammatory origin. Participants were exposed twice (60 min) to heptane and a mixture of heptane and acrolein. Blood samples were collected before exposure, after and 24 h post-exposure. There were no overt effects of acrolein exposure on the oxylipin lipidome or endocannibinoids detectable in the bloodstream at the time points investigated. No relationship between basal levels or levels after exposure to acrolein and CI could be identified. This implicates a minor role of inflammatory mediators on the systemic level in CI.

Aims: The covalent linking of nonsteroidal anti-inflammatory drugs to a hydrogen sulfide (H2S)-releasing moiety has been shown to dramatically reduce gastrointestinal (GI) damage and bleeding, as well as increase anti-inflammatory and analgesic potency. We have tested the hypothesis that an H2S-releasing derivative of ketoprofen (ATB-352) would exhibit enhanced efficacy without significant GI damage in a mouse model of allodynia/hyperalgesia.

Results: ATB-352 was significantly more potent and effective as an analgesic than ketoprofen and did not elicit GI damage. Pretreatment with an antagonist of the CB1 cannabinoid receptor (AM251) significantly reduced the analgesic effects of ATB-352. The CB1 antagonist exacerbated GI damage when coadministered with ketoprofen, but GI damage was not induced by the combination of ATB-352 and the CB1 antagonist. In vitro, ATB-352 was substantially more potent than ketoprofen as an inhibitor of fatty acid amide hydrolase, consistent with a contribution of endogenous cannabinoids to the analgesic effects of this drug. Blood anandamide levels were significantly depressed by ketoprofen, but remained unchanged after treatment with ATB-352.

Innovation: Ketoprofen is a potent analgesic, but its clinical use, even in the short term, is significantly limited by its propensity to cause significant ulceration and bleeding in the GI tract. Covalently linking an H2S-releasing moiety to ketoprofen profoundly reduces the GI toxicity of the drug, while boosting analgesic effectiveness.

Conclusion: This study demonstrates a marked enhancement of the potency and effectiveness of ATB-352, an H2S-releasing derivative of ketoprofen, in part, through the involvement of the endogenous cannabinoid system. This may have significant advantages for the control and management of pain, such as in a postoperative setting.

Fatty Acid Amide Hydrolase (FAAH) is a serine hydrolase that plays a key role in controlling endogenous levels of endocannabinoids. FAAH inhibition is considered a powerful approach to enhance the endocannabinoid signalling, and therefore it has been largely studied as a potential target for the treatment of neurological disorders such as anxiety or depression, or of inflammatory processes. We present two novel series of amide derivatives of ibuprofen designed as analogues of our reference FAAH inhibitor Ibu-AM5 to further explore its structure-activity relationships. In the new amides, the 2-methylpyridine moiety of Ibu-AM5 was substituted by benzylamino and piperazinoaryl moieties. The obtained benzylamides and piperazinoarylamides showed FAAH inhibition ranging from the low to high micromolar potency. The binding of the new amides in the active site of FAAH, estimated using the induced fit protocol, indicated arylpiperazinoamides binding the ACB channel and the cytosolic port, and benzylamides binding the ACB channel.

In experimental animals, inhibition of fatty acid amide hydrolase (FAAH) reduces the gastrointestinal damage produced by non-steroidal anti-inflammatory agents that act by inhibition of cyclooxygenase (COX). This suggests that compounds able to inhibit both enzymes may be potentially useful therapeutic agents. In the present study, we have investigated eight novel amide analogues of carprofen, ketoprofen and fenoprofen as potential FAAH/COX dual action inhibitors. Carpro-AM1 (2-(6-Chloro-9H-carbazol-2-yl)-N-(3-methylpyridin-2-yl)propenamide) and Carpro-AM6 (2-(6-Chloro-9H-carbazol-2-yl)-N-(3-chloropyridin-2-yl)propenamide) were found to be fully re-versible inhibitors of the hydrolysis of 0.5 mu M [H-3]anandamide in rat brain homogenates with IC50 values of 94 and 23 nM, respectively, i.e. 2-3 orders of magnitude more potent than carprofen in this respect. Both compounds inhibited the cyclooxygenation of arachidonic acid by ovine COX-1, and were more potent inhibitors of human recombinant COX-2 when 2-arachidonoylglycerol was used as substrate than when arachidonic acid was used. It is concluded that Carpro-AM1 and Carpro-AM6 are dual-acting FAAH/substrate-selective COX inhibitors.

Compounds combining dual inhibitory action against FAAH and cyclooxygenase (COX) may be potentially useful analgesics. Here, we describe a novel flurbiprofen analogue, N-(3-bromopyridin-2-yl)-2-(2-fluoro-(1,1'-biphenyl)-4-yl)propanamide (Flu-AM4). The compound is a competitive, reversible inhibitor of FAAH with a Ki value of 13 nM and which inhibits COX activity in a substrate-selective manner. Molecular modelling suggested that Flu-AM4 optimally fits a hydrophobic pocket in the ACB region of FAAH, and binds to COX-2 similarly to flurbiprofen. In vivo studies indicated that at a dose of 10 mg/kg, Flu-AM4 was active in models of prolonged (formalin) and neuropathic (chronic constriction injury) pain and reduced the spinal expression of iNOS, COX-2, and NFκB in the neuropathic model. Thus, the present study identifies Flu-AM4 as a dual-action FAAH/substrate-selective COX inhibitor with anti-inflammatory and analgesic activity in animal pain models. These findings underscore the potential usefulness of such dual-action compounds.

Inhibition of fatty acid amide hydrolase (FAAH) reduces the gastrointestinal damage produced by non-steroidal anti-inflammatory agents such as sulindac and indomethacin in experimental animals, suggesting that a dual-action FAAH-cyclooxygenase (COX) inhibitor could have useful therapeutic properties. Here, we have investigated 12 novel amide analogues of ibuprofen as potential dual-action FAAH/COX inhibitors. N-(3-Bromopyridin-2-yl)−2-(4-isobutylphenyl)propanamide (Ibu-AM68) was found to inhibit the hydrolysis of [³H]anandamide by rat brain homogenates by a reversible, mixed-type mechanism of inhibition with a K_i value of 0.26 µM and an α value of 4.9. At a concentration of 10 µM, the compound did not inhibit the cyclooxygenation of arachidonic acid by either ovine COX-1 or human recombinant COX-2. However, this concentration of Ibu-AM68 greatly reduced the ability of the COX-2 to catalyse the cyclooxygenation of the endocannabinoid 2-arachidonoylglycerol. It is concluded that Ibu-AM68 is a dual-acting FAAH/substrate-selective COX inhibitor.

Fatty acid amide hydrolase (FAAH) has a key role in the control of the cannabinoid signaling, through the hydrolysis of the endocannabinoids anandamide and in some tissues 2-arachidonoylglycerol. FAAH inhibition represents a promising strategy to activate the cannabinoid system, since it does not result in the psychotropic and peripheral side effects characterizing the agonists of the cannabinoid receptors. Here we present the discovery of a novel class of profen derivatives, the N-(heteroary1)-2-(4(2-(trifluoromethyl)pyridin-4-y0amino)phenyl)propanamides, as FAAH inhibitors. Enzymatic assays showed potencies toward FAAH ranging from nanomolar to micromolar range, and the most compounds lack activity toward the two isoforms of cyclooxygenase. Extensive structure-activity studies and the definition of the binding mode for the lead compound of the series are also presented. Kinetic assays in rat and mouse FAAH on selected compounds of the series demonstrated that slight modifications of the chemical structure could influence the binding mode and give rise to competitive (TPA1) or noncompetitive (TPA14) inhibition modes.

Aims: To investigate the local antinociceptive effect as well as the possible mechanisms of action of a novel analogue of palmitoylethanolamide (PEA) N-(4-methoxy-2-nitrophenyl)hexadecanamide (HD) in the rat formalin test.

Main methods: The formalin test was used to assess the antinociceptive activity of HD in vivo. The hydrolysis of anandamide catalyzed by fatty acid amide hydrolase (FAAH) was used to determine the action of HD on FAAH activity in vitro.

Key findings: Local peripheral ipisilateral, but not contralateral, administration of HD (10-100 mu g/paw) produced a dose-dependent antinociceptive effect in rats. The CB1 and CB2 receptor antagonists AM281 (0.3-30 mu g/paw) and SR144528 (0.3-30 mu g/paw), respectively, reduced the antinociceptive effect of HD (100 mu g/paw). In addition, methiothepin (0.03-0.3 mu g/paw) and naloxone (5-50 mu g/paw) significantly reduced HD-induced antinociception (100 mu g/paw). In vitro, HD reduced only to a minor extent the hydrolysis of anandamide catalyzed by FAAH.

Significance: HD local administration produces antinociception that probably results from an indirect activation of peripheral CB1 and CB2 cannabinoid receptors. Data suggest that 5-HT1 and opioid receptors also participate in the antinociceptive effect of this compound. HD may have potential as analgesic drug.

(C) 2012 Elsevier Inc. All rights reserved.

The endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol are removed from the extracellular space by a process of cellular uptake followed by metabolism. Although the enzymes responsible for endocannabinoid metabolism have been well characterised, the processes involved in uptake have been the subject of much controversy. Recent studies, however, have identified intracellular transport proteins (fatty acid binding proteins 5 and 7, heat shock protein 70, albumin, and fatty acid amide hydrolase-like AEA transporter protein) that shuttle AEA from the plasma membrane to its metabolic enzymes. Proteins such as the fatty acid amide hydrolase-like anandamide transporter protein may be useful targets for novel therapeutic strategies aimed at potentiating AEA signalling. In this article I review the current state of the art of endocannabinoid uptake.

An Internet search with search words "cannabis cures cancer" produce a wealth of sites claiming that cannabis has this effect. These sites are freely accessible to the general public and thus contribute to public opinion. But do delta⁹-tetrahydrocannabinol (Δ⁹-THC) and cannabidiol (CBD) cure cancer? In the absence of clinical data other than a safety study and case reports, preclinical data should be evaluated in terms of its predictive value. Using a strict approach where only concentrations and/or models relevant to the clinical situation are considered, the current preclinical data do not yet provide robust evidence that systemically administered Δ⁹-THC will be useful for the curative treatment of cancer. There is more support for an intratumoral route of administration of higher doses of Δ⁹-THC. CBD produces effects in relevant concentrations and models, although more data are needed concerning its use in conjunction with other treatment strategies.

The endocannabinoid (eCB) system is involved in processes as diverse as control of appetite, perception of pain and the limitation of cancer cell growth and invasion. The enzymes responsible for eCB breakdown are attractive pharmacological targets, and fatty acid amide hydrolase inhibitors, which potentiate the levels of the eCB anandamide, are now undergoing pharmaceutical development. Drugable selective inhibitors of monoacylglycerol lipase, a key enzyme regulating the levels of the other main eCB, 2-arachidonoylglycerol, were however not identified until very recently. Their availability has resulted in a large expansion of our knowledge concerning the pharmacological consequences of monoacylglycerol lipase inhibition and hence the role(s) played by the enzyme in the body. In this review, the pharmacology of monoacylglycerol lipase will be discussed, together with an analysis of the therapeutic potential of monoacylglycerol lipase inhibitors as analgesics and anticancer agents.

Read any pharmacology textbook and the message is clear: nonsteroidal anti-inflammatory drugs (NSAIDs) act by inhibiting the activity of cyclooxygenase (COX) and thereby the production of prostaglandins. However, evidence is accumulating that NSAIDs involve the endocannabinoid system in their actions, and that such effects may pave the way towards the design of new analgesics that are not plagued with the gastrointestinal and cardiovascular adverse actions that are associated with this class of drugs. In this Opinion article, our current understanding of the involvement of the endocannabinoid system in the actions of NSAIDs is described, and the ways in which this can lead to novel drug development is discussed.

In this review, the state of the art for compounds affecting the endocannabinoid (eCB) system is described with a focus on the treatment of pain. Amongst directly acting CB receptor ligands, clinical experience with ∆⁹-tetrahydracannabinol and medical cannabis in chronic non-cancer pain indicates that there are differences between the benefits perceived by patients and the at best modest effect seen in meta-analyses of randomized controlled trials. The reason for this difference is not known but may involve differences in the type of patients that are recruited, the study conditions that are chosen and the degree to which biases such as reporting bias are operative. Other directly acting CB receptor ligands such as biased agonists and allosteric receptor modulators have not yet reached the clinic. Amongst indirectly acting compounds targeting the enzymes responsible for the synthesis and catabolism of the eCBs anandamide and 2-arachidonoylglycerol, fatty acid amide hydrolase (FAAH) inhibitors have been investigated clinically but were per se not useful for the treatment of pain, although they may be useful for the treatment of post-traumatic stress disorder and cannabis use disorder. Dual-acting compounds targeting this enzyme and other targets such as cyclooxygenase-2 or transient potential vanilloid receptor 1 may be a way forward for the treatment of pain.

The endocannabinoid system, comprising cannabinoid CB1 and CB2 receptors, their endogenous ligands anandamide and 2-arachidonoylglyerol, and their synthetic and metabolic enzymes, are involved in many biological processes in the body, ranging from appetite to bone turnover. Compounds inhibiting the breakdown of anandamide and 2-arachidonoylglycerol increase brain levels of these lipids and thus modulate endocannabinoid signalling. In the present review, the preclinical evidence that these enzymes are good targets for development of novel therapies for anxiety and depression are discussed from a practical, rather than mechanistic, point of view. It is concluded that the preclinical data are promising, albeit tempered by problems of tolerance as well as effects upon learning and memory for irreversible monoacylglycerol lipase inhibitors, and limited by a focus upon male rodents alone. Clinical data so far has been restricted to safety studies with inhibitors of anandamide hydrolysis and a hitherto unpublished study on such a compound in elderly patients with major depressive disorders, but under the dose regimes used, they are well tolerated and show no signs of "cannabis-like" behaviours.

The endocannabinoids anandamide and 2-arachidonoylglycerol are metabolised by both hydrolytic enzymes (primarily fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL)) and oxygenating enzymes (e.g. cyclooxygenase-2, COX-2). In the present article, the in vivo data for compounds inhibiting endocannabinoid metabolism have been reviewed, focussing on inflammation and pain. Potential reasons for the failure of an FAAH inhibitor in a clinical trial in patients with osteoarthritic pain are discussed. It is concluded that there is a continued potential for compounds inhibiting endocannabinoid metabolism in terms of drug development, but that it is wise not to be unrealistic in terms of expectations of success.

Endocannabinoids are readily accumulated from the extracellular space by cells. Although their uptake properties have the appearance of a process of facilitated diffusion, it is by no means clear as to whether there is a plasma membrane transporter dedicated to this task. Intracellular carrier proteins that shuttle the endocannabinoid anandamide from the plasma membrane to its intracellular targets such as the metabolic enzyme, fatty acid amide hydrolase, have been identified. These include proteins with other primary functions, such as fatty-acid-binding proteins and heat shock protein70, and possibly a fatty acid amide hydrolase-like anandamide transporter protein. Thus, anandamide uptake can be adequately described as a diffusion process across the plasma membrane followed by intracellular carrier-mediated transport to effector molecules, catabolic enzymes and sequestration sites, although it is recognized that different cells are likely to utilize different mechanisms of endocannabinoid transport depending upon the utility of the endocannabinoid for the cell in question.

A dual-action cyclooxygenase (COX)-fatty acid amide hydrolase (FAAH) inhibitor may have therapeutic usefulness as an analgesic, but a key issue is finding the right balance of inhibitory effects. This can be done by the design of compounds exhibiting different FAAH/COX-inhibitory potencies. In the present study, eight ibuprofen analogues were investigated. Ibuprofen (1), 2-(4-Isobutylphenyl)-N-(2-(3-methylpyridin-2-ylamino)-2-oxoethyl)propanamide (9) and N-(3-methylpyridin-2-yl)-2-(4'-isobutylphenyl) propionamide (2) inhibited FAAH with IC50 values of 134, 3.6 and 0.52 mu M respectively. The corresponding values for COX-1 were similar to 29, similar to 50 and similar to 60 mu M, respectively. Using arachidonic acid as substrate, the compounds were weak inhibitors of COX-2. However, when anandamide was used as COX-2 substrate, potency increased, with approximate IC50 values of similar to 6, similar to 10 and similar to 19 mu M, respectively. Compound 2 was confirmed to be active in vivo in a murine model of visceral nociception, but the effects of the compound were not blocked by CB receptor antagonists.

In this review, we consider the biosynthetic, hydrolytic, and oxidative metabolism of the endocannabinoids anandamide and 2-arachidonoylglycerol. We describe the enzymes associated with these events and their characterization. We identify the inhibitor profile for these enzymes and the status of therapeutic exploitation, which to date has been limited to clinical trials for fatty acid amide hydrolase inhibitors. To bring the review to a close, we consider whether point block of a single enzyme is likely to be the most successful approach for therapeutic exploitation of the endocannabinoid system.

In recent years, considerable interest has been generated by findings that cannabinoids not only have useful palliative effects, but also can affect the viability and invasivity of a variety of different cancer cells. In the present review, the potential of targeting the cannabinoid system for the treatment of cancer is considered from a practical, rather than a mechanistic viewpoint, addressing questions such as whether human tumour cells express CB receptors; whether the potencies of action of cannabinoids in vitro match the potencies expected on the base of receptor theory; what is known about the in vivo effects of cannabinoids and cancer, and how relevant the experiments undertaken are to the clinical situation; and finally, what approaches can be taken to minimise unwanted effects of cannabinoid treatment. It is concluded that cannabinoids (or agents modulating the endogenous cannabinoid system) are an attractive target for drug development in the cancer area, but that more in vivo studies, particularly those investigating the potential of cannabinoids as an addition to current treatment strategies, are needed.

These data indicate that a high tumour CB(1) receptor expression at diagnosis augments the deleterious effects of a high pEGFR expression upon disease-specific survival.

Fatty acid amide hydrolase (FAAH) is responsible for the hydrolysis of the endogenous cannabinoid (CB) receptor ligand anandamide. Here we have investigated whether the expression levels of FAAH and CB1 receptors influence the prognostic value of markers of angiogenesis in prostate cancer. Data from a cohort of 419 patients who were diagnosed with prostate cancer at transurethral resection for lower urinary tract symptoms, of whom approximately 2/3 had been followed by expectancy, were used. Scores for the angiogenesis markers endoglin and von Willebrand factor (vWf), the endocannabinoid markers fatty acid amide hydrolase (FAAH) and cannabinoid CB1 receptors and the cell proliferation marker Ki-67 were available in the database. For the cases followed by expectancy, the prognostic value of endoglin was dependent upon the tumour epithelial FAAH immunoreactivity (FAAH-IR) and CB1IR scores, and the non-malignant epithelial FAAH-IR scores, but not the non-malignant CB1IR scores or the tumour blood vessel FAAH-IR scores. This dependency upon the tumour epithelial FAAH-IR or CB1IR scores was less apparent for vWf, and was not seen for Ki-67. Using an endoglin cut-off value of 10 positively stained vessels per core and a median split of tumour FAAH-IR, four groups could be generated, with 15 year of disease-specific survival (%) of 68 +/- 7 (low endoglin, low FAAH), 45 +/- 11 (high endoglin, low FAAH), 77 +/- 6 (low endoglin, high FAAH) and 21 +/- 10 (high endoglin, high FAAH). Thus, the cases with high endoglin and high FAAH scores have the poorest rate of disease-specific survival. At diagnosis, the number of cases with tumour stages 1a-1b relative to stages 2-4 was sensitive to the endoglin score in a manner dependent upon the tumour FAAH-IR. It is concluded that the prognostic value of endoglin as a marker of neovascularisation in prostate cancer can be influenced by the expression level of markers of the endocannabinoid system. This article is part of a Special Issue entitled Lipid Metabolism in Cancer.

Although the dominant approach to drug development is the design of compounds selective for a given target, compounds targeting more than one biological process may have superior efficacy, or alternatively a better safety profile than standard selective compounds. Here, this possibility has been explored with respect to the endocannabinoid system and pain. Compounds inhibiting the enzyme fatty acid amide hydrolase (FAAH), by increasing local endocannabinoid tone, produce potentially useful effects in models of inflammatory and possibly neuropathic pain. Local increases in levels of the endocannabinoid anandamide potentiate the actions of cyclooxygenase inhibitors, raising the possibility that compounds inhibiting both FAAH and cyclooxygenase can be as effective as non-steroidal anti-inflammatory drugs but with a reduced cyclooxygenase inhibitory 'load'. An ibuprofen analogue active in models of visceral pain and with FAAH and cyclooxygenase inhibitory properties has been identified. Another approach, built in to the experimental analgesic compound N-arachidonoylserotonin, is the combination of FAAH inhibitory and transient receptor potential vanilloid type 1 antagonist properties. Although finding the right balance of actions upon the two targets is a key to success, it is hoped that dual-action compounds of the types illustrated in this review will prove to be useful analgesic drugs.

There is now a large volume of data indicating that compounds activating cannabinoid CB(1) receptors, either directly or indirectly by preventing the breakdown of endogenous cannabinoids, can protect against neuronal damage produced by a variety of neuronal "insults". Given that such neurodegenerative stimuli result in increased endocannabinoid levels and that animals with genetic deletions of CB(1) receptors are more susceptible to the deleterious effects of such stimuli, a case can be made for an endogenous neuroprotective role of endocannabinoids. However, this is an oversimplification of the current literature, since (a) compounds released together with the endocannabinoids can contribute to the neuroprotective effect; (b) other proteins, such as TASK-1 and PPARalpha, are involved; (c) the CB(1) receptor antagonist/inverse agonist rimonabant has also been reported to have neuroprotective properties in a number of animal models of neurodegenerative disorders. Furthermore, the CB(2) receptor located on peripheral immune cells and activated microglia are potential targets for novel therapies. In terms of the clinical usefulness of targeting the endocannabinoid system for the treatment of neurodegenerative disorders, data are emerging, but important factors to be considered are windows of opportunity (for acute situations such as trauma and ischemia) and the functionality of the target receptors (for chronic neurodegenerative disorders such as Alzheimer's disease).

The anti-inflammatory agent palmitoylethanolamide (PEA) reduces cyclooxygenase (COX) activity in vivo in a model of inflammatory pain. It is not known whether the compound reduces prostaglandin production in RAW264.7 cells, whether such an action is affected by compounds preventing the breakdown of endogenous PEA, whether other oxylipins are affected, or whether PEA produces direct effects upon the COX-2 enzyme. RAW264.7 cells were treated with lipopolysaccharide and interferon-c to induce COX-2. At the level of mRNA, COX-2 was induced > 1000-fold following 24 h of the treatment. Coincubation with PEA (10 mu mol/L) did not affect the levels of COX-2, but reduced the levels of prostaglandins D-2 and E-2 as well as 11- and 15-hydroxyeicosatetraenoic acid, which can also be synthesised by a COX-2 pathway in macrophages. These effects were retained when hydrolysis of PEA to palmitic acid was blocked. Linoleic acidderived oxylipin levels were not affected by PEA. No direct effects of PEA upon the oxygenation of either arachidonic acid or 2-arachidonoylglycerol by COX-2 were found. It is concluded that in lipopolysaccharide and interferon-c-stimulated RAW264.7 cells, PEA reduces the production of COX-2-derived oxylipins in a manner that is retained when its metabolism to palmitic acid is inhibited.

Palmitoylethanolamide (PEA) has been suggested to have useful analgesic properties and to be devoid of unwanted effects. Here, we have examined critically this contention, and discussed available data concerning the pharmacokinetics of PEA and its formulation. Sixteen clinical trials, six case reports/pilot studies and a meta-analysis of PEA as an analgesic have been published in the literature. For treatment times up to 49days, the current clinical data argue against serious adverse drug reactions (ADRs) at an incidence of 1/200 or greater. For treatment lasting more than 60days, the number of patients is insufficient to rule out a frequency of ADRs of less than 1/100. The six published randomized clinical trials are of variable quality. Presentation of data without information on data spread and nonreporting of data at times other than the final measurement were among issues that were identified. Further, there are no head-to-head clinical comparisons of unmicronized vs. micronized formulations of PEA, and so evidence for superiority of one formulation over the other is currently lacking. Nevertheless, the available clinical data support the contention that PEA has analgesic actions and motivate further study of this compound, particularly with respect to head-to-head comparisons of unmicronized vs. micronized formulations of PEA and comparisons with currently recommended treatments.

Purpose. Chronic neck/shoulder pain (CNSP) is one of the most common pain conditions. The understanding of mechanisms, including the peripheral balance between nociceptive and antinociceptive processes, is incomplete. N-acylethanolamines (NAEs) are a class of endogenous compounds that regulate inflammation and pain. The aim of this study was to investigate the levels of two NAEs: the peroxisome proliferator-activated receptor type-a ligand palmitoylethanolamide (PEA) and stearoylethanolamide (SEA) in the muscle interstitium of the trapezius muscle in women with CNSP randomized to two different neck specific training programs and in a healthy pain-free control group (CON). Materials and Methods. Fifty-seven women with CNSP were randomized to strength + stretch or stretch alone exercise programs. Twenty-nine subjects underwent microdialysis procedure before and after 4-6 months of exercise. Twenty-four CON subjects underwent microdialysis procedure before and after 4-6 months without any intervention in between. Microdialysate samples were collected from the trapezius muscle and analyzed by mass spectrometry for PEA and SEA levels. Results. PEA and SEA levels were significantly higher in CNSP patients compared with CON. PEA was significantly higher in CNSP than in CON after both training programs. SEA was significantly higher in CNSP than in CON after stretch alone but not after strength + stretch training. A significant positive correlation was found between changes in pain intensity and in SEA levels in the strength + stretch group, but not in the stretch alone group. Conclusion. Our results indicate that exercise interventions differentially affect the levels of the bioactive lipids PEA and SEA in the interstitium of the trapezius muscle in women with CNSP.