Autoantibodies to BACE1 promote Aß accumulation and neurodegeneration in Alzheimer's disease.

The profile of autoantibodies is dysregulated in patients with Alzheimer's disease (AD). Autoantibodies to beta-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) are present in human blood. This study aims to investigate the clinical relevance and pathophysiological roles of autoantibodies to BACE1 in AD. Clinical investigations were conducted in two independent cohorts, the Chongqing cohort, and the Australian Imaging, Biomarkers, and Lifestyle (AIBL) cohort. The Chongqing cohort included 55 AD patients, 28 patients with non-AD dementia, and 70 cognitively normal subjects (CN). The AIBL cohort included 162 Aß-PET- CN, 169 Aß-PET+ cognitively normal subjects (preclinical AD), and 31 Aß-PET+ cognitively impaired subjects (Clinical AD). Plasma autoantibodies to BACE1 were determined by one-site Elisa. The associations of plasma autoantibodies to BACE1 with brain Aß load and cognitive trajectory were investigated. The effects of autoantibodies to BACE1 on AD-type pathologies and underlying mechanisms were investigated in APP/PS1 mice and SH/APPswe/PS1wt cell lines. In the Chongqing cohort, plasma autoantibodies to BACE1 were higher in AD patients, in comparison with CN and non-AD dementia patients. In the AIBL cohort, plasma autoantibodies to BACE1 were highest in clinical AD patients, followed by preclinical AD and CN subjects. Higher autoantibodies to BACE1 were associated with an increased incidence of brain amyloid positivity conversion during follow-up. Autoantibodies to BACE1 exacerbated brain amyloid deposition and subsequent AD-type pathologies, including Tau hyperphosphorylation, neuroinflammation, and neurodegeneration in APP/PS1 mice. Autoantibodies to BACE1 increased Aß production by promoting BACE1 expression through inhibiting PPARγ signaling. These findings suggest that autoantibodies to BACE1 are pathogenic in AD and the upregulation of these autoantibodies may promote the development of the disease. This study offers new insights into the mechanism of AD from an autoimmune perspective.

Systemic perturbations of the kynurenine pathway precede progression to dementia independently of amyloid-ß.

Increasing evidence suggests that kynurenine pathway (KP) dyshomeostasis may promote disease progression in dementia. Studies in Alzheimer's disease (AD) patients confirm KP dyshomeostasis in plasma and cerebrospinal fluid (CSF) which correlates with amyloid-ß and tau pathology. Herein, we performed the first comprehensive study assessing baseline levels of KP metabolites in participants enrolling in the Australian Imaging Biomarkers Flagship Study of Aging. Our purpose was to test the hypothesis that changes in KP metabolites may be biomarkers of dementia processes that are largely silent. We used a cross-sectional analytical approach to assess non-progressors (N = 73); cognitively normal (CN) or mild cognitive impairment (MCI) participants at baseline and throughout the study, and progressors (N = 166); CN or MCI at baseline but progressing to either MCI or AD during the study. Significant KP changes in progressors included increased 3-hydroxyanthranilic acid (3-HAA) and 3-hydroxyanthranilic acid/anthranilic acid (3-HAA/AA) ratio, the latter having the largest effect on the odds of an individual being a progressor (OR 35.3; 95% CI between 14 and 104). 3-HAA levels were hence surprisingly bi-phasic, high in progressors but low in non-progressors or participants who had already transitioned to MCI or dementia. This is a new, unexpected and interesting result, as most studies of the KP in neurodegenerative disease show reduced 3-HAA/AA ratio after diagnosis. The neuroprotective metabolite picolinic acid was also significantly decreased while the neurotoxic metabolite 3-hydroxykynurenine increased in progressors. These results were significant even after adjustment for confounders. Considering the magnitude of the OR to predict change in cognition, it is important that these findings are replicated in other populations. Independent validation of our findings may confirm the utility of 3-HAA/AA ratio to predict change in cognition leading to dementia in clinical settings.

Design, synthesis and in vitro and in vivo biological evaluation of flurbiprofen amides as new fatty acid amide hydrolase/cyclooxygenase-2 dual inhibitory potential analgesic agents.

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.

Synthesis and preliminary evaluation of a novel positron emission tomography (PET) ligand for imaging fatty acid amide hydrolase (FAAH).

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.

The fatty acid amide hydrolase and cyclooxygenase-inhibitory properties of novel amide derivatives of carprofen.

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 reversible inhibitors of the hydrolysis of 0.5 µM [3H]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.

Exploring the fatty acid amide hydrolase and cyclooxygenase inhibitory properties of novel amide derivatives of ibuprofen.

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 Ki 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.

The Basal Pharmacology of Palmitoylethanolamide.

Palmitoylethanolamide (PEA, N-hexadecanoylethanolamide) is an endogenous compound belonging to the family of N-acylethanolamines. PEA has anti-inflammatory and analgesic properties and is very well tolerated in humans. In the present article, the basal pharmacology of PEA is reviewed. In terms of its pharmacokinetic properties, most work has been undertaken upon designing formulations for its absorption and upon characterising the enzymes involved in its metabolism, but little is known about its bioavailability, tissue distribution, and excretion pathways. PEA exerts most of its biological effects in the body secondary to the activation of peroxisome proliferator-activated receptor-α (PPAR-α), but PPAR-α-independent pathways involving other receptors (Transient Receptor Potential Vanilloid 1 (TRPV1), GPR55) have also been identified. Given the potential clinical utility of PEA, not least for the treatment of pain where there is a clear need for new well-tolerated drugs, we conclude that the gaps in our knowledge, in particular those relating to the pharmacokinetic properties of the compound, need to be filled.

Altered mRNA Expression of Genes Involved in Endocannabinoid Signalling in Squamous Cell Carcinoma of the Oral Tongue.

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.

Low mRNA expression and activity of monoacylglycerol lipase in human SH-SY5Y neuroblastoma cells.

Relatively little is known about the endocannabinoid system in human neuroblastoma cell lines. In the present study, we have investigated the expression of the genes coding for the enzymes involved in the synthesis and catabolism of endocannabinoids in the SH-SY5Y cell line. The expression of MGLL, the gene coding for the 2-arachidonoylglycerol hydrolytic enzyme monoacylglycerol lipase (MAGL), was found to be 85 and 340 fold lower than the expression levels for the genes coding for alpha/beta-hydrolase domain containing 6 and 12 (ABHD6, ABHD12), which are alternative hydrolytic enzymes for this endocannabinoid. In comparison, mRNA levels of MGLL were 1.5 fold higher than ABHD6 and 2 fold lower than the levels of ABHD12 in DU-145 human prostate cells. In functional assays, the hydrolysis of the 2-arachidonoylglycerol homologue 2-oleoylglycerol by intact SH-SY5Y cells was partially inhibited by the ABHD6 inhibitor WWL70, but not by the MAGL inhibitor JZL184, whereas the reverse was true in DU-145 cells. The combination of JZL184 + WWL70 did, however produce a significantly greater inhibition of 2-OG hydrolysis than seen with WWL70 alone in the SH-SY5Y cells. The low MGLL expression in the SH-SY5Y cells was not due to epigenetic silencing, since levels were not affected by treatment with the methylation inhibitor 5-aza-2'-deoxycytidine and/or the histone acetylase inhibitor trichostatin A. The low MGLL expression in SH-SY5Y cells should be taken into account when using these cells in experiments investigating the involvement of the endocannabinoid system in models of physiological and pathological processes.

Benzylamides and piperazinoarylamides of ibuprofen as fatty acid amide hydrolase inhibitors.

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.

N-aryl 2-aryloxyacetamides as a new class of fatty acid amide hydrolase (FAAH) inhibitors.

Fatty acid amide hydrolase (FAAH) is a promising target for the development of drugs to treat neurological diseases. In search of new FAAH inhibitors, we identified 2-(4-cyclohexylphenoxy)-N-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)acetamide, 4g, with an IC50 of 2.6 µM as a chemical starting point for the development of potent FAAH inhibitors. Preliminary hit-to-lead optimisation resulted in 2-(4-phenylphenoxy)-N-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)acetamide, 4i, with an IC50 of 0.35 µM.

Palmitoylethanolamide for the treatment of pain: pharmacokinetics, safety and efficacy.

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 49 days, the current clinical data argue against serious adverse drug reactions (ADRs) at an incidence of 1/200 or greater. For treatment lasting more than 60 days, 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.

Association between plasma concentrations of linoleic acid-derived oxylipins and the perceived pain scores in an exploratory study in women with chronic neck pain.

BACKGROUND: Chronic musculoskeletal pain may be associated with changes in the balance of algogenic and anti-nociceptive compounds, and such changes may be visible in plasma samples. We have undertaken an exploratory study to measure the levels of endocannabinoids, related N-acylethanolamines and oxylipins (primarily those derived from linoleic acid) in plasma samples from women with chronic neck pain (NP) and chronic widespread pain (CWP), and to investigate whether the observed levels are associated with the pain experienced by these women. METHODS: Blood samples from 35 women with NP, 15 with CWP and 27 age-matched controls were analysed for the lipids using ultra performance liquid chromatography coupled to tandem mass spectrometry. Current pain ("NRSday") and the average pain during the last week ("NRSweek") were rated by the participants using a numerical rating scale. RESULTS: There were no significant differences in the plasma concentrations of the fifteen lipids investigated between the women with pain and the controls. However, significant correlations were seen for the NP group between the NRSday scores and the plasma concentrations of the linoleic acid derivatives 9- and 13-hydroxy-octadecadienoic acid (Spearman's rho values 0.51 [P = 0.0016]) and 0.53 [P = 0.0011], respectively). CONCLUSIONS: The data obtained in this exploratory study indicate that although no group differences are seen in plasma lipid concentrations, there is an association between the NRSday scores and the 9- and 13-hydroxy-octadecadienoic acid levels. Whether or not the association reflects a causality (i.e. that the circulating lipids contribute to the perceived pain of the pain participants), requires further investigation.

Zinc affects the proteolytic stability of Apolipoprotein E in an isoform-dependent way.

The pathological role of zinc in Alzheimer's disease (AD) is not yet fully elucidated, but there is strong evidence that zinc homeostasis is impaired in the AD brain and that this contributes to disease pathogenesis. In this study we examined the effects of zinc on the proteolysis of synthetic Apolipoprotein E (ApoE), a protein whose allelic variants differentially contribute to the onset/progression of disease. We have demonstrated that zinc promotes the proteolysis (using plasma kallikrein, thrombin and chymotrypsin) of synthetic ApoE in an isoform-specific way (E4>E2 and E3), resulting in more ApoE fragments, particularly for ApoE4. In the absence of exogenous proteases there was no effect of metal modulation on either lipidated or non-lipidated ApoE isoforms. Thus, increased zinc in the complex milieu of the ageing and AD brain could reduce the level of normal full-length ApoE and increase other forms that are involved in neurodegeneration. We further examined human plasma samples from people with different ApoE genotypes. Consistent with previous studies, plasma ApoE levels varied according to different genotypes, with ApoE2 carriers showing the highest total ApoE levels and ApoE4 carriers the lowest. The levels of plasma ApoE were not affected by either the addition of exogenous metals (copper, zinc or iron) or by chelation. Taken together, our study reveals that zinc may contribute to the pathogenesis of AD by affecting the proteolysis of ApoE, which to some extent explains why APOE4 carriers are more susceptible to AD.

Relative and absolute reliability of measures of linoleic acid-derived oxylipins in human plasma.

Modern analytical techniques allow for the measurement of oxylipins derived from linoleic acid in biological samples. Most validatory work has concerned extraction techniques, repeated analysis of aliquots from the same biological sample, and the influence of external factors such as diet and heparin treatment upon their levels, whereas less is known about the relative and absolute reliability of measurements undertaken on different days. A cohort of nineteen healthy males were used, where samples were taken at the same time of day on two occasions, at least 7 days apart. Relative reliability was assessed using Lin's concordance correlation coefficients (CCC) and intraclass correlation coefficients (ICC). Absolute reliability was assessed by Bland-Altman analyses. Nine linoleic acid oxylipins were investigated. ICC and CCC values ranged from acceptable (0.56 [13-HODE]) to poor (near zero [9(10)- and 12(13)-EpOME]). Bland-Altman limits of agreement were in general quite wide, ranging from ±0.5 (12,13-DiHOME) to ±2 (9(10)-EpOME; log10 scale). It is concluded that relative reliability of linoleic acid-derived oxylipins varies between lipids with compounds such as the HODEs showing better relative reliability than compounds such as the EpOMEs. These differences should be kept in mind when designing and interpreting experiments correlating plasma levels of these lipids with factors such as age, body mass index, rating scales etc.

The Potential of Inhibitors of Endocannabinoid Metabolism for Drug Development: A Critical Review.

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.

Tumour epithelial expression levels of endocannabinoid markers modulate the value of endoglin-positive vascular density as a prognostic marker in prostate cancer.

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 15year 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.

Potential upstream regulators of cannabinoid receptor 1 signaling in prostate cancer: a Bayesian network analysis of data from a tissue microarray.

BACKGROUND: The endocannabinoid system regulates cancer cell proliferation, and in prostate cancer a high cannabinoid CB1 receptor expression is associated with a poor prognosis. Down-stream mediators of CB1 receptor signaling in prostate cancer are known, but information on potential upstream regulators is lacking. RESULTS: Data from a well-characterized tumor tissue microarray were used for a Bayesian network analysis using the max-min hill-climbing method. In non-malignant tissue samples, a directionality of pEGFR (the phosphorylated form of the epidermal growth factor receptor) → CB1 receptors were found regardless as to whether the endocannabinoid metabolizing enzyme fatty acid amide hydrolase (FAAH) was included as a parameter. A similar result was found in the tumor tissue, but only when FAAH was included in the analysis. A second regulatory pathway, from the growth factor receptor ErbB2 → FAAH was also identified in the tumor samples. Transfection of AT1 prostate cancer cells with CB1 receptors induced a sensitivity to the growth-inhibiting effects of the CB receptor agonist CP55,940. The sensitivity was not dependent upon the level of receptor expression. Thus a high CB1 receptor expression alone does not drive the cells towards a survival phenotype in the presence of a CB receptor agonist. CONCLUSIONS: The data identify two potential regulators of the endocannabinoid system in prostate cancer and allow the construction of a model of a dysregulated endocannabinoid signaling network in this tumor. Further studies should be designed to test the veracity of the predictions of the network analysis in prostate cancer and other solid tumors.

Cannabidiol improves vasorelaxation in Zucker diabetic fatty rats through cyclooxygenase activation.

Cannabidiol (CBD) decreases insulitis, inflammation, neuropathic pain, and myocardial dysfunction in preclinical models of diabetes. We recently showed that CBD also improves vasorelaxation in the Zucker diabetic fatty (ZDF) rat, and the objective of the present study was to establish the mechanisms underlying this effect. Femoral arteries from ZDF rats and ZDF lean controls were isolated, mounted on a myograph, and incubated with CBD (10 µM) or vehicle for 2 hours. Subsequent vasorelaxant responses were measured in combination with various interventions. Prostaglandin metabolites were detected using enzyme immunoassay. Direct effects of CBD on cyclooxygenase (COX) enzyme activity were measured by oxygraph assay. CBD enhanced the maximum vasorelaxation to acetylcholine (ACh) in femoral arteries from ZDF lean rats (P < 0.01) and especially ZDF rats (P < 0.0001). In ZDF arteries, this enhancement persisted after cannabinoid receptor (CB) type 1, endothelial CB, or peroxisome proliferator-activated receptor-γ antagonism but was inhibited by CB2 receptor antagonism. CBD also uncovered a vasorelaxant response to a CB2 agonist not previously observed. The CBD-enhanced ACh response was endothelium-, nitric oxide-, and hydrogen peroxide-independent. It was, however, COX-1/2- and superoxide dismutase-dependent, and CBD enhanced the activity of both purified COX-1 and COX-2. The CBD-enhanced ACh response in the arteries was inhibited by a prostanoid EP4 receptor antagonist. Prostaglandin E2 metabolite levels were below the limits of detection, but 6-keto prostaglandin F1 α was decreased after CBD incubation. These data show that CBD exposure enhances the ability of arteries to relax via enhanced production of vasodilator COX-1/2-derived products acting at EP4 receptors.