Anti-Bacterial and Anti-Biofilm Activities of Anandamide against the Cariogenic Streptococcus mutans.

Streptococcus mutans is a cariogenic bacterium in the oral cavity involved in plaque formation and dental caries. The endocannabinoid anandamide (AEA), a naturally occurring bioactive lipid, has been shown to have anti-bacterial and anti-biofilm activities against Staphylococcus aureus. We aimed here to study its effects on S. mutans viability, biofilm formation and extracellular polysaccharide substance (EPS) production. S. mutans were cultivated in the absence or presence of various concentrations of AEA, and the planktonic growth was followed by changes in optical density (OD) and colony-forming units (CFU). The resulting biofilms were examined by MTT metabolic assay, Crystal Violet (CV) staining, spinning disk confocal microscopy (SDCM) and high-resolution scanning electron microscopy (HR-SEM). The EPS production was determined by Congo Red and fluorescent dextran staining. Membrane potential and membrane permeability were determined by diethyloxacarbocyanine iodide (DiOC2(3)) and SYTO 9/propidium iodide (PI) staining, respectively, using flow cytometry. We observed that AEA was bactericidal to S. mutans at 12.5 µg/mL and prevented biofilm formation at the same concentration. AEA reduced the biofilm thickness and biomass with concomitant reduction in total EPS production, although there was a net increase in EPS per bacterium. Preformed biofilms were significantly affected at 50 µg/mL AEA. We further show that AEA increased the membrane permeability and induced membrane hyperpolarization of these bacteria. AEA caused S. mutans to become elongated at the minimum inhibitory concentration (MIC). Gene expression studies showed a significant increase in the cell division gene ftsZ. The concentrations of AEA needed for the anti-bacterial effects were below the cytotoxic concentration for normal Vero epithelial cells. Altogether, our data show that AEA has anti-bacterial and anti-biofilm activities against S. mutans and may have a potential role in preventing biofilms as a therapeutic measure.

Targeting the Achilles' Heel of Multidrug-Resistant Staphylococcus aureus by the Endocannabinoid Anandamide.

Antibiotic-resistant Staphylococcus aureus is a major health issue that requires new therapeutic approaches. Accumulating data suggest that it is possible to sensitize these bacteria to antibiotics by combining them with inhibitors targeting efflux pumps, the low-affinity penicillin-binding protein PBP2a, cell wall teichoic acid, or the cell division protein FtsZ. We have previously shown that the endocannabinoid Anandamide (N-arachidonoylethanolamine; AEA) could sensitize drug-resistant S. aureus to a variety of antibiotics, among others, through growth arrest and inhibition of drug efflux. Here, we looked at biochemical alterations caused by AEA. We observed that AEA increased the intracellular drug concentration of a fluorescent penicillin and augmented its binding to membrane proteins with concomitant altered membrane distribution of these proteins. AEA also prevented the secretion of exopolysaccharides (EPS) and reduced the cell wall teichoic acid content, both processes known to require transporter proteins. Notably, AEA was found to inhibit membrane ATPase activity that is necessary for transmembrane transport. AEA did not affect the membrane GTPase activity, and the GTPase cell division protein FtsZ formed the Z-ring of the divisome normally in the presence of AEA. Rather, AEA caused a reduction in murein hydrolase activities involved in daughter cell separation. Altogether, this study shows that AEA affects several biochemical processes that culminate in the sensitization of the drug-resistant bacteria to antibiotics.

Fenchone Derivatives as a Novel Class of CB2 Selective Ligands: Design, Synthesis, X-ray Structure and Therapeutic Potential.

A series of novel cannabinoid-type derivatives were synthesized by the coupling of (1S,4R)-(+) and (1R,4S)-(-)-fenchones with various resorcinols/phenols. The fenchone-resorcinol derivatives were fluorinated using Selectfluor and demethylated using sodium ethanethiolate in dimethylformamide (DMF). The absolute configurations of four compounds were determined by X-ray single crystal diffraction. The fenchone-resorcinol analogs possessed high affinity and selectivity for the CB2 cannabinoid receptor. One of the analogues synthesized, 2-(2',6'-dimethoxy-4'-(2″-methyloctan-2″-yl)phenyl)-1,3,3-trimethylbicyclo[2.2.1]heptan-2-ol (1d), had a high affinity (Ki = 3.51 nM) and selectivity for the human CB2 receptor (hCB2). In the [35S]GTPγS binding assay, our lead compound was found to be a highly potent and efficacious hCB2 receptor agonist (EC50 = 2.59 nM, E(max) = 89.6%). Two of the fenchone derivatives were found to possess anti-inflammatory and analgesic properties. Molecular-modeling studies elucidated the binding interactions of 1d within the CB2 binding site.

Bioactive lipids ALIAmides differentially modulate inflammatory responses of distinct subsets of primary human T lymphocytes.

Autacoid local injury antagonist amides (ALIAmides) are a family of endogenous bioactive acyl ethanolamides that include the renowned palmitoyl ethanolamide (PEA), oleoyl ethanolamide (OEA), and stearoyl ethanolamide (SEA), and that are involved in several biologic processes such as nociception, lipid metabolism, and inflammation. The role of ALIAmides in the control of inflammatory processes has recently gained much attention and prompted the use of these molecules or their analogs, and the pharmacologic manipulation of their endogenous levels, as plausible therapeutic strategies in the treatment of several chronic inflammatory conditions. Since chronic inflammation is mainly driven by cells of adaptive immunity, particularly T lymphocytes, we aimed at investigating whether such bioactive lipids could directly modulate T-cell responses. We found that OEA, PEA, and eicosatrienoyl ethanolamide (ETEA) could directly inhibit both T-cell responses by reducing their production of TNF-α and IFN-γ from CD8 T cells and TNF-α, IFN-γ and IL-17 from CD4 T cells. Furthermore, neither SEA nor docosatrienoyl ethanolamide (DTEA) could affect cytokine production from both T cell subsets. Interestingly, unlike OEA and ETEA, PEA was also able to enhance de novo generation of forkhead box P3 (FoxP3)-expressing regulatory T cells from CD4-naive T cells. Our findings show for the first time that specific ALIAmides can directly affect different T-cell subsets, and provide proof of their anti-inflammatory role in chronic inflammation, ultimately suggesting that these bioactive lipids could offer novel tools for the management of T-cell dependent chronic inflammatory diseases.-Chiurchiù, V., Leuti, A., Smoum, R., Mechoulam, R., Maccarrone, M. Bioactive lipids ALIAmides differentially modulate inflammatory responses of distinct subsets of primary human T lymphocytes.

HU-671, a Novel Oleoyl Serine Derivative, Exhibits Enhanced Efficacy in Reversing Ovariectomy-Induced Osteoporosis and Bone Marrow Adiposity.

Oleoyl serine (OS), an endogenous fatty acyl amide (FAA) found in bone, has been shown to have an anti-osteoporotic effect. OS, being an amide, can be hydrolyzed in the body by amidases. Hindering its amide bond by introducing adjacent substituents has been demonstrated as a successful method for prolonging its skeletal activity. Here, we tested the therapeutic efficacy of two methylated OS derivatives, oleoyl α-methyl serine (HU-671) and 2-methyl-oleoyl serine (HU-681), in an ovariectomized mouse model for osteoporosis by utilizing combined micro-computed tomography, histomorphometry, and cell culture analyses. Our findings indicate that daily treatment for 6 weeks with OS or HU-671 completely rescues bone loss, whereas HU-681 has only a partial effect. The increased bone density was primarily due to enhanced trabecular thickness and number. Moreover, the most effective dose of HU-671 was 0.5 mg/kg/day, an order of magnitude lower than with OS. The reversal of bone loss resulted from increased bone formation and decreased bone resorption, as well as reversal of bone marrow adiposity. These results were further confirmed by determining the serum levels of osteocalcin and type 1 collagen C-terminal crosslinks, as well as demonstrating the enhanced antiadipogenic effect of HU-671. Taken together, these data suggest that methylation interferes with OS's metabolism, thus enhancing its effects by extending its availability to its target cells.

CB2 cannabinoid receptor agonist enantiomers HU-433 and HU-308: An inverse relationship between binding affinity and biological potency.

Activation of the CB2 receptor is apparently an endogenous protective mechanism. Thus, it restrains inflammation and protects the skeleton against age-related bone loss. However, the endogenous cannabinoids, as well as Δ(9)-tetrahydrocannabinol, the main plant psychoactive constituent, activate both cannabinoid receptors, CB1 and CB2. HU-308 was among the first synthetic, selective CB2 agonists. HU-308 is antiosteoporotic and antiinflammatory. Here we show that the HU-308 enantiomer, designated HU-433, is 3-4 orders of magnitude more potent in osteoblast proliferation and osteoclast differentiation culture systems, as well as in mouse models, for the rescue of ovariectomy-induced bone loss and ear inflammation. HU-433 retains the HU-308 specificity for CB2, as shown by its failure to bind to the CB1 cannabinoid receptor, and has no activity in CB2-deficient cells and animals. Surprisingly, the CB2 binding affinity of HU-433 in terms of [(3)H]CP55,940 displacement and its effect on [(35)S]GTPγS accumulation is substantially lower compared with HU-308. A molecular-modeling analysis suggests that HU-433 and -308 have two different binding conformations within CB2, with one of them possibly responsible for the affinity difference, involving [(35)S]GTPγS and cAMP synthesis. Hence, different ligands may have different orientations relative to the same binding site. This situation questions the usefulness of universal radioligands for comparative binding studies. Moreover, orientation-targeted ligands have promising potential for the pharmacological activation of distinct processes.

Effect of the synthetic cannabinoid HU-210 on quorum sensing and on the production of quorum sensing-mediated virulence factors by Vibrio harveyi.

BACKGROUND: Bacterial populations communicate through the cell density-dependent mechanism of quorum sensing (QS). Vibrio harveyi, one of the best studied model organisms for QS, was used to explore effects of the synthetic cannabinoid HU-210 on QS and different QS-regulated physiological processes in bacteria. RESULTS: Analysis of QS-regulated bioluminescence in wild-type and mutant strains of V. harveyi revealed that HU-210 affects the autoinducer-2 (AI-2) pathway, one of three known QS cascades of V. harveyi. Furthermore, QS-mediated biofilm formation and swimming motility in the mutant strain BB152 (AI-1(-), AI-2(+)) were significantly reduced in the presence of HU-210. HU-210 inhibited QS-mediated virulence factor production without any inhibitory effect on bacterial growth. It also alters the expression of several genes, which are regulated by QS, specifically downregulating the genes of the AI-2 QS cascade. CONCLUSION: First evidence is being provided for interference of bacterial signal-transduction systems by a synthetic cannabinoid. The effect of HU-210 was specific to the AI-2 cascade in V. harveyi. AI-2 is known as a "universal autoinducer" and interference with its activity opens a broad spectrum of applications for synthetic cannabinoids in future research as a potential anti-QS agent.

Oleoyl serine, an endogenous N-acyl amide, modulates bone remodeling and mass.

Bone mass is determined by a continuous remodeling process, whereby the mineralized matrix is being removed by osteoclasts and subsequently replaced with newly formed bone tissue produced by osteoblasts. Here we report the presence of endogenous amides of long-chain fatty acids with amino acids or with ethanolamine (N-acyl amides) in mouse bone. Of these compounds, N-oleoyl-l-serine (OS) had the highest activity in an osteoblast proliferation assay. In these cells, OS triggers a Gi-protein-coupled receptor and Erk1/2. It also mitigates osteoclast number by promoting osteoclast apoptosis through the inhibition of Erk1/2 phosphorylation and receptor activator of nuclear-κB ligand (RANKL) expression in bone marrow stromal cells and osteoblasts. In intact mice, OS moderately increases bone volume density mainly by inhibiting bone resorption. However, in a mouse ovariectomy (OVX) model for osteoporosis, OS effectively rescues bone loss by increasing bone formation and markedly restraining bone resorption. The differential effect of exogenous OS in the OVX vs. intact animals is apparently a result of an OVX-induced decrease in skeletal OS levels. These data show that OS is a previously unexplored lipid regulator of bone remodeling. It represents a lead to antiosteoporotic drug discovery, advantageous to currently available therapies, which are essentially either proformative or antiresorptive.

Evaluation of Sex Differences in the Potential of Δ9-Tetrahydrocannabinol, Cannabidiol, Cannabidiolic Acid, and Oleoyl Alanine to Reduce Nausea-Induced Conditioned Gaping Reactions in Sprague-Dawley Rats.

Introduction: Cancer patients report nausea as a side effect of their chemotherapy treatment. Using the pre-clinical rodent model of acute nausea-lithium chloride (LiCl)-induced conditioned gaping-our group has demonstrated that exogenous cannabinoids may have antinausea potential. Materials and Methods: With the goal of evaluating the role of sex as a factor in pre-clinical research, we first compared the conditioned gaping reactions produced by varying doses of LiCl in male and female rats using the taste reactivity test (Experiment 1). Results: LiCl produced dose-dependent conditioned gaping similarly in male and female rats with the highest dose (127.2 mg/kg) producing robust conditioned gaping, with this dose used in subsequent experiments. Next, we examined the antinausea potential of THC (Experiment 2), CBD (Experiment 3), cannabidiolic acid (CBDA; Experiment 4) and oleoyl alanine (OlAla; Experiment 5) in both male and female rats. THC, CBD, CBDA, and OlAla dose dependently reduced conditioned gaping in both male and female rats in a similar manner. Conclusions: These results suggest that cannabinoids may be equally effective in treating nausea in both males and females.

N-Oleoyl Glycine and Its Derivatives Attenuate the Acquisition and Expression of Cocaine-Induced Behaviors.

Introduction: The endocannabinoid system (ECS) plays a key modulatory role during synaptic plasticity and homeostatic processes in the brain and plays an important role in the neurobiological processes underlying drug addiction. Impaired endocannabinoid (eCB) signaling contributes to dysregulated synaptic plasticity, increased stress responsivity, and craving that propel addiction. Therefore, we hypothesized that boosting the ECS by exogenous administration of selective eCBs will attenuate cocaine-induced behaviors. Materials and Methods: The behavioral paradigms included psychomotor sensitization (PS) and conditioned place preference (CPP). Liquid chromatography-mass spectrometry analysis was used for quantitative profiling of eCBs in mouse brain. Results: We first measured the levels of eCBs in different brain areas of the reward system following chronic cocaine treatment. We found that following daily administration of cocaine, the levels of N-oleoyl glycine (OlGly) were significantly elevated in the nucleus accumbens (NAc) in a region-specific manner. We next tested whether administration of OlGly will attenuate cocaine-induced behaviors. We found that administration of OlGly during withdrawal, but not during acquisition of PS, attenuated the expression of cocaine sensitization. In addition, the administration of OlGly during the acquisition of cocaine CPP, but not during withdrawal, attenuated the expression of cocaine-conditioned reward. To enhance the stability of OlGly and its duration of action, two methylated derivatives of OlGly were synthesized, the monomethylated OlGly (HU-595) and dimethylated OlGly (HU-596). We found that the effect of administration of HU-595 or HU-596 during cocaine conditioning did not differ from the OlGly-induced decrease in the expression of CPP. Conclusion: Our findings suggest that the ECS is involved in the common neurobiological mechanisms underlying the development and expression of cocaine reward and drug-seeking. Boosting the ECS exogenously has beneficial effects against cocaine-induced behaviors.

N-oleoyl glycine and N-oleoyl alanine attenuate alcohol self-administration and preference in mice.

The endocannabinoid system (ECS) plays a key modulatory role during synaptic plasticity and homeostatic processes in the brain and has an important role in the neurobiological processes underlying drug addiction. We have previously shown that an elevated ECS response to psychostimulant (cocaine) is involved in regulating the development and expression of cocaine-conditioned reward and sensitization. We therefore hypothesized that drug-induced elevation in endocannabinoids (eCBs) and/or eCB-like molecules (eCB-Ls) may represent a protective mechanism against drug insult, and boosting their levels exogenously may strengthen their neuroprotective effects. Here, we determine the involvement of ECS in alcohol addiction. We first measured the eCBs and eCB-Ls levels in different brain reward system regions following chronic alcohol self-administration using LC-MS. We have found that following chronic intermittent alcohol consumption, N-oleoyl glycine (OlGly) levels were significantly elevated in the prefrontal cortex (PFC), and N-oleoyl alanine (OlAla) was significantly elevated in the PFC, nucleus accumbens (NAc) and ventral tegmental area (VTA) in a region-specific manner. We next tested whether exogenous administration of OlGly or OlAla would attenuate alcohol consumption and preference. We found that systemic administration of OlGly or OlAla (60 mg/kg, intraperitoneal) during intermittent alcohol consumption significantly reduced alcohol intake and preference without affecting the hedonic state. These findings suggest that the ECS negatively regulates alcohol consumption and boosting selective eCBs exogenously has beneficial effects against alcohol consumption and potentially in preventing relapse.

Effect of oleoyl glycine and oleoyl alanine on lithium chloride induced nausea in rats and vomiting in shrews.

RATIONALE: The fatty acid amide oleoyl glycine (OlGly) and its more stable methylated form oleoyl alanine (OlAla) reduce naloxone-precipitated morphine withdrawal (MWD)-induced conditioned gaping (nausea) responses in rats. In addition, OlGly has been shown to reduce lithium chloride (LiCl)-induced conditioned gaping in rats and vomiting in Suncus murinus (house musk shrews). OBJECTIVES: Here, we compared the potential of these fatty acid amides to maintain their anti-nausea/anti-emetic effect over a delay. The following experiments examined the potential of a wider dose range of OlGly and OlAla to interfere with (1) LiCl-induced conditioned gaping in rats and (2) LiCl-induced vomiting in shrews, when administered 20 or 70 min prior to illness. RESULTS: OlAla (1, 5, 20 mg/kg) reduced LiCl-induced conditioned gaping, with OlGly only effective at the high dose (20 mg/kg), with no effect of pretreatment delay time. At the high dose of 20 mg/kg, OlGly increased passive drips during conditioning suggesting a sedative effect. In shrews, both OlGly and OlAla (1, 5 mg/kg) suppressed LiCl-induced vomiting, with no effect of pretreatment delay. OlAla more effectively suppressed vomiting, with OlAla (5 mg/kg) also increasing the latency to the first vomiting reaction. CONCLUSIONS: OlAla was more effective than OlGly in reducing both LiCl-induced gaping in rats and LiCl-induced vomiting in shrews. These findings provide further evidence that these fatty acid amides may be useful treatments for nausea and vomiting, with OlAla demonstrating superior efficacy.

A Fenchone Derivative Effectively Abrogates Joint Damage Following Post-Traumatic Osteoarthritis in Lewis Rats.

BACKGROUND: In a previous report, we have identified the cannabinoid receptor 2 (CB2) agonist HU308 to possess a beneficial effect in preventing age and trauma-induced osteoarthritis (OA) in mice. The effects of HU308 were largely related to the capacity of this compound to induce cartilage anabolism which was dependent on the CREB/SOX9 axis, and exhibited pro-survival and pro-proliferative hallmarks of articular cartilage following treatment. Here, we utilized the novel cannabinoid-fenchone CB2 agonists (1B, 1D), which were previously reported to render anti-inflammatory effects in a zymosan model. METHODS: Initially, we assessed the selectivity of CB2 using a Gs-protein receptor cAMP potency assay, which was also validated for antagonistic effects dependent on the Gi-protein receptor cAMP pathway. Based on EC50 values, 1D was selected for a zymosan inflammatory pain model. Next, 1D was administered in two doses intra-articularly (IA), in a post-traumatic medial meniscal tear (MMT, Lewis rats) model, and compared to sham, vehicle, and a positive control consisting of fibroblast growth factor 18 (FGF18) administration. The histopathological assessment was carried out according to the Osteoarthritis Research Society International (OARSI) guidelines for rat models following 28 days post-MMT. RESULTS: The G protein receptor assays confirmed that both 1B and 1D possess CB2 agonistic effects in cell lines and in chondrocytes. Co-administering a CB2 antagonists to 25 mg/kg 1D in a paw inflammatory pain model abolished 1D-related anti-swelling effect and partially abolishing its analgesic effects. Using an MMT model, the high dose (i.e., 24 µg) of 1D administered via IA route, exhibited reduced cartilage damage. Particularly, this dose of 1D exhibited a 30% improvement in cartilage degeneration (zonal/total tibial scores) and lesion depth ratios (44%), comparable to the FGF18 positive control. Synovitis scores remained unaffected and histopathologic evaluation of subchondral bone damage did not suggest that 1D treatment changed the load-bearing ability of the rats. Contrary to the anabolic effect of FGF18, synovial inflammation was observed and was accompanied by increased osteophyte size. CONCLUSION: The structural histopathological analysis supports a disease-modifying effect of IA-administered 1D compound without any deleterious effects on the joint structure.

Anti-Tumorigenic Effect of a Novel Derivative of 2-Hydroxyoleic Acid and the Endocannabinoid Anandamide on Neuroblastoma Cells.

Modulation of the endogenous cannabinoid system has been suggested as a potential anticancer strategy. In the search for novel and less toxic therapeutic options, structural modifications of the endocannabinoid anandamide and the synthetic derivative of oleic acid, Minerval (HU-600), were done to obtain 2-hydroxy oleic acid ethanolamide (HU-585), which is an HU-600 derivative with the anandamide side chain. We showed that treatment of SK-N-SH neuroblastoma cells with HU-585 induced a better anti-tumorigenic effect in comparison to HU-600 as evidenced by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide assay, colony-forming assay, and migration assay. Moreover, HU-585 demonstrated pro-apoptotic properties shown by increased levels of activated caspase-3 following treatment and a better senescence induction effect in comparison to HU-600, as demonstrated by increased activity of lysosomal ß-galactosidase. Finally, we observed that combined treatment of HU-585 with the senolytic drugs ABT-263 in vitro, and ABT-737 in vivo resulted in enhanced anti-proliferative effects and reduced neuroblastoma xenograft growth in comparison to treatment with HU-585 alone. Based on these results, we suggest that HU-585 is a pro-apoptotic and senescence-inducing compound, better than HU-600. Hence, it may be a beneficial option for the treatment of resistant neuroblastoma especially when combined with senolytic drugs that enhance its anti-tumorigenic effects.

Anandamide alters the membrane properties, halts the cell division and prevents drug efflux in multidrug resistant Staphylococcus aureus.

Antibiotic resistance is a serious public health problem throughout the world. Overcoming methicillin and multidrug-resistant Staphylococcus aureus (MRSA/MDRSA) infections has become a challenge and there is an urgent need for new therapeutic approaches. We have previously demonstrated that the endocannabinoid Anandamide (AEA) can sensitize MRSA to antibiotics. Here we have studied the mechanism of action using a MDRSA clinical isolate that are sensitized by AEA to methicillin and norfloxacin. We found that AEA treatment halts the growth of both antibiotic-sensitive and antibiotic-resistant S. aureus. The AEA-treated bacteria become elongated and the membranes become ruffled with many protrusions. AEA treatment also leads to an increase in the percentage of bacteria having a complete septum, suggesting that the cell division is halted at this stage. The latter is supported by cell cycle analysis that shows an accumulation of bacteria in the G2/M phase after AEA treatment. We further observed that AEA causes a dose-dependent membrane depolarization that is partly relieved upon time. Nile red staining of the bacterial membranes indicates that AEA alters the membrane structures. Importantly, 4'-6-diamidino-2-phenylindole (DAPI) accumulation assay and ethidium bromide efflux (EtBr) assay unveiled that AEA leads to a dose-dependent drug accumulation by inhibiting drug efflux. In conclusion, our study demonstrates that AEA interferes with cell division, alters the membrane properties of MDRSA, and leads to increased intracellular drug retention, which can contribute to the sensitization of MDRSA to antibiotics.

Spontaneous and Naloxone-Precipitated Withdrawal Behaviors From Chronic Opiates are Accompanied by Changes in N-Oleoylglycine and N-Oleoylalanine Levels in the Brain and Ameliorated by Treatment With These Mediators.

Rationale: The endocannabinoidome mediators, N-Oleoylglycine (OlGly) and N-Oleoylalanine (OlAla), have been shown to reduce acute naloxone-precipitated morphine withdrawal affective and somatic responses. Objectives: To determine the role and mechanism of action of OlGly and OlAla in withdrawal responses from chronic exposure to opiates in male Sprague-Dawley rats. Methods: Opiate withdrawal was produced: 1) spontaneously 24 h following chronic exposure to escalating doses of morphine over 14 days (Experiments 1 and 2) and steady-state exposure to heroin by minipumps for 12 days (Experiment 3), 2) by naloxone injection during steady-state heroin exposure (Experiment 4), 3) by naloxone injection during operant heroin self-administration (Experiment 5). Results: In Experiment 1, spontaneous morphine withdrawal produced somatic withdrawal reactions. The behavioral withdrawal reactions were accompanied by suppressed endogenous levels of OlGly in the nucleus accumbens, amygdala, and prefrontal cortex, N-Arachidonylglycerol and OlAla in the amygdala, 2-arachidonoylglycerol in the nucleus accumbens, amygdala and interoceptive insular cortex, and by changes in colonic microbiota composition. In Experiment 2, treatment with OlAla, but not OlGly, reduced spontaneous morphine withdrawal responses. In Experiment 3, OlAla attenuated spontaneous steady-state heroin withdrawal responses at both 5 and 20 mg/kg; OlGly only reduced withdrawal responses at the higher dose of 20 mg/kg. Experiment 4 demonstrated that naloxone-precipitated heroin withdrawal from steady-state exposure to heroin (7 mg/kg/day for 12 days) is accompanied by tissue-specific changes in brain or gut endocannabinoidome mediator, including OlGly and OlAla, levels and colonic microbiota composition, and that OlAla (5 mg/kg) attenuated behavioural withdrawal reactions, while also reversing some of the changes in brain and gut endocannabinoidome and gut microbiota induced by naloxone. Experiment 5 demonstrated that although OlAla (5 mg/kg) did not interfere with operant heroin self-administration on its own, it blocked naloxone-precipitated elevation of heroin self-administration behavior. Conclusion: These results suggest that OlAla and OlGly are two endogenous mediators whose brain concentrations respond to chronic opiate treatment and withdrawal concomitantly with changes in colon microbiota composition, and that OlAla may be more effective than OlGly in suppressing chronic opiate withdrawal responses.

Cannabinoid receptor-dependent and -independent anti-proliferative effects of omega-3 ethanolamides in androgen receptor-positive and -negative prostate cancer cell lines.

The omega-3 fatty acid ethanolamides, docosahexaenoyl ethanolamide (DHEA) and eicosapentaenoyl ethanolamide (EPEA), displayed greater anti-proliferative potency than their parent omega-3 fatty acids, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), in LNCaP and PC3 prostate cancer cells. DHEA and EPEA activated cannabinoid CB(1) and CB(2) receptors in vitro with significant potency, suggesting that they are endocannabinoids. Both LNCaP and PC3 cells expressed CB(1) and CB(2) receptors, and the CB(1)- and CB(2)-selective antagonists, AM281 and AM630, administered separately or together, reduced the anti-proliferative potencies of EPEA and EPA but not of DHEA or DHA in PC3 cells and of EPA but not of EPEA, DHEA or DHA in LNCaP cells. Even so, EPEA and EPA may not have inhibited PC3 or LNCaP cell proliferation via cannabinoid receptors since the anti-proliferative potency of EPEA was well below the potency it displayed as a CB(1) or CB(2) receptor agonist. Indeed, these receptors may mediate a protective effect because the anti-proliferative potency of DHEA in LNCaP and PC3 cells was increased by separate or combined administration of AM281 and AM630. The anandamide-metabolizing enzyme, fatty acid amide hydrolase (FAAH), was highly expressed in LNCaP but not PC3 cells. Evidence was obtained that FAAH metabolizes EPEA and DHEA and that the anti-proliferative potencies of these ethanolamides in LNCaP cells can be enhanced by inhibiting this enzyme. Our findings suggest that the expression of cannabinoid receptors and of FAAH in some tumour cells could well influence the effectiveness of DHA and EPA or their ethanolamide derivatives as anticancer agents.

Potential combinations of endocannabinoid/endocannabinoid-like compounds and antibiotics against methicillin-resistant Staphylococcus aureus.

Infections caused by antibiotic-resistant strains of Staphylococcus aureus have reached epidemic proportions globally. Our previous study showed antimicrobial effects of anandamide (AEA) and arachidonoyl serine (AraS) against methicillin (MET)-resistant S. aureus (MRSA) strains, proposing the therapeutic potential of these endocannabinoid/endocannabinoid-like (EC/EC-like) agents for the treatment of MRSA. Here, we investigated the potential synergism of combinations of AEA and AraS with different types of antibiotics against MRSA grown under planktonic growth or biofilm formation. The most effective combinations under planktonic conditions were mixtures of AEA and ampicillin (AMP), and of AraS and gentamicin (GEN). The combination with the highest synergy in the biofilm formation against all tested bacterial strains was AEA and MET. Moreover, the combination of AraS and MET synergistically caused default of biofilm formation. Slime production of MRSA was also dramatically impaired by AEA or AraS combined with MET. Our data suggest the novel potential activity of combinations of EC/EC-like agents and antibiotics in the prevention of MRSA biofilm formation.

Anandamide prevents the adhesion of filamentous Candida albicans to cervical epithelial cells.

Candidiasis is a fungal infection caused by Candida species that have formed a biofilm on epithelial linings of the body. The most frequently affected areas include the vagina, oral cavity and the intestine. In severe cases, the fungi penetrate the epithelium and cause systemic infections. One approach to combat candidiasis is to prevent the adhesion of the fungal hyphae to the epithelium. Here we demonstrate that the endocannabinoid anandamide (AEA) and the endocannabinoid-like N-arachidonoyl serine (AraS) strongly prevent the adherence of C. albicans hyphae to cervical epithelial cells, while the endocannabinoid 2-arachidonoylglycerol (2-AG) has only a minor inhibitory effect. In addition, we observed that both AEA and AraS prevent the yeast-hypha transition and perturb hyphal growth. Real-time PCR analysis showed that AEA represses the expression of the HWP1 and ALS3 adhesins involved in Candida adhesion to epithelial cells and the HGC1, RAS1, EFG1 and ZAP1 regulators of hyphal morphogenesis and cell adherence. On the other hand, AEA increased the expression of NRG1, a transcriptional repressor of filamentous growth. Altogether, our data show that AEA and AraS have potential anti-fungal activities by inhibiting hyphal growth and preventing hyphal adherence to epithelial cells.

Comparative Evaluation of Combinatory Interaction between Endocannabinoid System Compounds and Poly-L-lysine against Streptococcus mutans Growth and Biofilm Formation.

Endocannabinoid/endocannabinoid-like (EC/EC-like) are natural endogenous compounds which have been found to affect MRSA pathogenicity. Our previous studies showed that EC/EC-like was able to impair staphylococcal biofilm formation and maintenance as well as to alter biofilm-associated virulence factors. In the present study, we investigated the combinatory effect of the selected EC/EC-like with a natural antimicrobial agent, poly-L-lysine, on cariogenic bacteria Streptococcus mutans growth and biofilm formation. Among four tested EC/EC-like, only two, anandamide (AEA) and oleoylethanolamide (OEA), exhibited synergistic combinatory effect with poly-L-lysine against S. mutans. We attribute this distinct effect to differences in the fatty acid chain structure of the selected EC/EC-like compounds. Moreover, AEA exerted a specific antibiofilm mode of action against S. mutans by effecting total inhibition of biofilm formation while still allowing bacteria viability. Finally, we postulate that the presence of EC/EC-like and poly-L-lysine could enhance the permeability and efficacy of each other via hydrophobic and electrostatic interactions with the S. mutans membrane. In conclusion, we assume that a combination of endogenous natural compounds such as EC/EC-like and poly-L-lysine may benefit oral hygiene by preventing dental plaque.