Poly-ε-caprolactone microspheres as a drug delivery system for cannabinoid administration: development, characterization and in vitro evaluation of their antitumoral efficacy.

Cannabinoids show promise for the treatment of various medical conditions such as emesis, anorexia, pain, cancer, multiple sclerosis, Parkinson's disease and glaucoma. However, their high lipohilicity and instability complicate their handling and dosing, and restrict their use as pharmaceuticals. The objective of the present work was to assess the feasibility of developing cannabinoid loaded poly-ε-caprolactone (PCL) microparticles prepared by the oil-in-water emulsion-solvent evaporation technique as a suitable dosage form for their administration. Spherical microparticles with a size range of 20-50 µm, and high entrapment efficiency (around 100%) were obtained. Cannabidiol (CBD) dissolved in the polymeric matrix of the microspheres was slowly released in vitro within 10 days. In vitro cell viability studies demonstrated the antitumoral activity of CBD released from microparticles. After 4 and 7 days of incubation, CBD in microspheres significantly inhibited the growth of MDA-MB-231 cells by 60% as compared to the 50% attained with free drug. The results suggest that PCL microparticles could be an alternative delivery system for long-term cannabinoid administration, showing potential therapeutic advantages over free drug.

Altered endocannabinoid signalling after a high-fat diet in Apoe(-/-) mice: relevance to adipose tissue inflammation, hepatic steatosis and insulin resistance.

AIMS/HYPOTHESIS: Apolipoprotein E (ApoE) deficiency is associated with reduced fat accumulation in white adipose tissue (WAT) and high liver triacylglycerol content. Elevated levels of endocannabinoids and cannabinoid receptor type 1 (CB(1)) receptors in the liver and in epididymal vs subcutaneous WAT are associated with fatty liver, visceral adipose tissue, inflammatory markers and insulin resistance. METHODS: We investigated, in Apoe (-/-) and wild-type (WT) mice, the effect of a high-fat diet (HFD) on: (1) subcutaneous and epididymal WAT accumulation, liver triacylglycerols, phospholipid-esterified fatty acids, inflammatory markers in WAT and liver, and insulin resistance; and (2) endocannabinoid levels, and the gene expression levels of the Cb ( 1 ) receptor and endocannabinoid metabolic enzymes in liver and WAT. RESULTS: After a 16 week HFD, Apoe (-/-) mice exhibited lower body weight, WAT accumulation and fasting leptin, glucose and insulin levels, and higher hepatic steatosis, than WT mice. Glucose clearance and insulin-mediated glucose disposal following the HFD were slower in WT than Apoe (-/-) mice, which exhibited higher levels of mRNA encoding inflammatory markers (tumour necrosis factor-α [TNF-α], monocyte chemoattractant protein-1 [MCP-1], cluster of differentiation 68 [CD68] and EGF-like module-containing mucin-like hormone receptor-like 1 [EMR1]) in the liver, but lower levels in epididymal WAT. HFD-induced elevation of endocannabinoid levels in the liver or epididymal WAT was higher or lower, respectively, in Apoe (-/-) mice, whereas HFD-induced decrease of subcutaneous WAT endocannabinoid and CB(1) receptor levels was significantly less marked. Alterations in endocannabinoid levels reflected changes in endocannabinoid catabolic enzymes in WAT, or the availability of phospholipid precursors in the liver. CONCLUSIONS/INTERPRETATION: Liver and adipose tissue endocannabinoid tone following an HFD is altered on Apoe deletion and strongly associated with inflammation, insulin resistance and hepatic steatosis, or lack thereof.

Anandamide-loaded nanoparticles: preparation and characterization.

OBJECTIVE: Preparation and characterization of anandamide (N-arachidonoyl-ethanolamine, AEA) loaded polycaprolactone nanoparticles (PCL NP) as a research tool to clarify the presence of an AEA transporter in cell membranes and to avoid AEA plastic adsorption and instability. MATERIALS AND METHODS: High performance liquid chromatography and light scattering were used to determine encapsulation efficiency, particle size, drug release, permeability and stability. RESULTS: A high encapsulation efficiency 96.05 ± 1.77% and a particle size of 83.52 ± 21.38 nm were obtained. Nearly 40% of AEA remained in the NP after a 99.9% dilution and only 50% was released after 24 h at 37 °C with a 99% dilution. PCL NP prevented the adsorption of the drug to polypropylene or polystyrene, but not to acrylic multiwell plates. Drug permeability through artificial membranes was low (10⁻7 to 10⁻8 cm/s) and was affected by the presence of NP. NP increased AEA stability in suspension (drug half-life 431 h vs. 12 h) and freeze-dried with 5% sucrose. CONCLUSION: This article presents the first study where stable AEA-loaded NP with high encapsulation efficiencies have been obtained.

The endocannabinoid system as a link between homoeostatic and hedonic pathways involved in energy balance regulation.

The endocannabinoid system (ECS) and, in particular, cannabinoid CB(1) receptors, their endogenous agonists (the endocannabinoids anandamide and 2-arachidonoylglycerol) and enzymes for the biosynthesis and degradation of the latter mediators are emerging as key players in the control of all aspects of food intake and energy balance. The ECS is involved in stimulating both the homoeostatic (that is, the sensing of deficient energy balance and gastrointestinal load) and the hedonic (that is, the sensing of the salience and the incentive/motivational value of nutrients) aspects of food intake. The orexigenic effects of endocannabinoids are exerted in the brain by CB(1)-mediated stimulatory and inhibitory effects on hypothalamic orexigenic and anorectic neuropeptides, respectively; by facilitatory actions on dopamine release in the nucleus accumbens shell; and by regulating the activity of sensory and vagal fibres in brainstem-duodenum neural connections. In turn, the levels of anandamide and 2-arachidonoylglycerol and/or CB(1) receptors in the brain are under the control of leptin, ghrelin and glucocorticoids in the hypothalamus, under that of dopamine in the limbic forebrain and under that of cholecystokinin and ghrelin in the brainstem. These bi-directional communications between the ECS and other key players in energy balance ensure local mediators such as the endocannabinoids to act in a way coordinated in both 'space' and 'time' to enhance food intake, particularly after a few hours of food deprivation. Alterations of such communications are, however, also among the underlying causes of overactivity of the ECS in hyperphagia and obesity, a phenomenon that provided the rationale for the development of anti-obesity drugs from CB(1) receptor antagonists.

Chemical synthesis, pharmacological characterization, and possible formation in unicellular fungi of 3-hydroxy-anandamide.

The fungal pathogen Candida albicans transforms arachidonic acid (AA) into 3-hydroxyarachidonic acid [3R-HETE], and we investigated if its nonpathogenic and 3R-HETE-producing close relative, Dipodascopsis uninucleata, could similarly transform the endocannabinoid/endovanilloid anandamide into 3-hydroxyanandamide (3-HAEA). We found that D. uninucleata converts anandamide into 3-HAEA, and we therefore developed an enantiodivergent synthesis for this compound to study its pharmacological activity. Both enantiomers of 3-HAEA were as active as anandamide at elevating intracellular Ca2+ via TRPV1 receptors overexpressed in HEK-293 cells, while a approximately 70-90-fold and approximately 45-60-fold lower affinity at cannabinoid CB1 and CB2 receptors was instead observed. Patch clamp recordings showed that 3R-HAEA activates a TRPV1-like current in TRPV1-expressing HEK-293 cells. Thus, 3R-HETE-producing yeasts might convert anandamide released by host cells at the site of infection into 3R-HAEA, and this event might contribute to the inflammatory and algogenous responses associated to fungal diseases.

Antinociceptive effects of tetrazole inhibitors of endocannabinoid inactivation: cannabinoid and non-cannabinoid receptor-mediated mechanisms.

BACKGROUND AND PURPOSE: Tetrazoles were recently developed as inhibitors of the cellular uptake of the endocannabinoid anandamide or of its hydrolysis by fatty acid amide hydrolase (FAAH), but were proposed to act also on non-endocannabinoid-related serine hydrolases. EXPERIMENTAL APPROACH: We tested, in a model of inflammatory pain induced in mice by formalin, five chemically similar inhibitors: (i) OMDM119 and OMDM122, two potent carbamoyl tetrazole FAAH inhibitors with no effect on anandamide uptake; (ii) LY2183240, a carbamoyl tetrazole with activity as both FAAH and uptake inhibitor; (iii) OMDM132, a non-carbamoyl tetrazole with activity only as uptake inhibitor and iv) OMDM133, a non-carbamoyl tetrazole with no activity at either FAAH or uptake. RESULTS: All compounds (2.5-10 mg kg(-1), i.p.) inhibited the second phase of the nocifensive response induced by intraplantar injection of formalin. The effects of OMDM119, OMDM122 and OMDM133 were not antagonized by pretreatment with cannabinoid CB(1) receptor antagonists, such as rimonabant or AM251 (1-3 mg kg(-1), i.p.). The effects of LY2183240 and OMDM132 were fully or partially antagonized by rimonabant, respectively, and the latter compound was also partly antagonized by the CB(2) receptor antagonist, AM630. CONCLUSIONS AND IMPLICATIONS: (i) non-FAAH hydrolases might be entirely responsible for the antinociceptive activity of some, but not all, tetrazole FAAH inhibitors, (ii) the presence of a carbamoylating group is neither necessary nor sufficient for such compounds to act through targets other than FAAH and (iii) inhibition of anandamide uptake is responsible for part of this antinociceptive activity, independently of effects on FAAH.

Prostaglandin ethanolamides (prostamides): in vitro pharmacology and metabolism.

We investigated whether prostaglandin ethanolamides (prostamides) E(2), F(2alpha), and D(2) exert some of their effects by 1) activating prostanoid receptors either per se or after conversion into the corresponding prostaglandins; 2) interacting with proteins for the inactivation of the endocannabinoid N-arachidonoylethanolamide (AEA), for example fatty acid amide hydrolase (FAAH), thereby enhancing AEA endogenous levels; or 3) activating the vanilloid receptor type-1 (TRPV1). Prostamides potently stimulated cat iris contraction with potency approaching that of the corresponding prostaglandins. However, prostamides D(2), E(2), and F(2alpha) exhibited no meaningful interaction with the cat recombinant FP receptor, nor with human recombinant DP, EP(1-4), FP, IP, and TP prostanoid receptors. Prostamide F(2alpha) was also very weak or inactive in a panel of bioassays specific for the various prostanoid receptors. None of the prostamides inhibited AEA enzymatic hydrolysis by FAAH in cell homogenates, or AEA cellular uptake in intact cells. Furthermore, less than 3% of the compounds were hydrolyzed to the corresponding prostaglandins when incubated for 4 h with homogenates of rat brain, lung, or liver, and cat iris or ciliary body. Very little temperature-dependent uptake of prostamides was observed after incubation with rat brain synaptosomes or RBL-2H3 cells. We suggest that prostamides' most prominent pharmacological actions are not due to transformation into prostaglandins, activation of prostanoid receptors, enhancement of AEA levels, or gating of TRPV1 receptors, but possibly to interaction with novel receptors that seem to be functional in the cat iris.

The inhibitory effect of propolis and caffeic acid phenethyl ester on cyclooxygenase activity in J774 macrophages.

The effect of an ethanolic extract of propolis, with and without CAPE, and some of its components on cyclooxygenase (COX-1 and COX-2) activity in J774 macrophages has been investigated. COX-1 and COX-2 activity, measaured as prostaglandin E2 (PGE2) production, were concentration-dependently inhibited by propolis (3 x 10(-3) - 3 x 10(2) microgml(-1)) with an IC50 of 2.7 microgml(-1) and 4.8 x 10(-2) microgml(-1), respectively. Among the compounds tested pinocembrin and caffeic, ferulic, cinnamic and chlorogenic acids did not affect the activity of COX isoforms. Conversely, CAPE (2.8 x 10(-4) - 28 microgml(-1); 10(-9) - 10(-4) M) and galangin (2.7 x 10(-4) - 27 microgml(-1); 10(-9) - 10(-4) M) were effective, the last being about ten-twenty times less potent. In fact the IC50 of CAPE for COX-1 and COX-2 were 4.4 x 10(-1) microgml(-1) (1.5 x 10(-6) M) and 2 x 10(-3) microgml(-1) (6.3 x 10(-9) M), respectively. The IC50 of galangin were 3.7 microgml(-1) (15 x 10(-6) M) and 3 x 10(-2) microgml(-1) (120 x 10(-9) M), for COX-1 and COX-2 respectively. To better investigate the role of CAPE, we tested the action of the ethanolic extract of propolis deprived of CAPE, which resulted about ten times less potent than the extract with CAPE in the inhibition of both COX-1 and COX-2, with an IC50 of 30 microgml(-1) and 5.3 x 10(-1) microgml(-1), respectively. Moreover the comparison of the inhibition curves showed a significant difference (p < 0.001). These results suggest that both CAPE and galangin contribute to the overall activity of propolis, CAPE being more effective.

Anandamide receptors.

Anandamide (N -arachidonoyl-ethanolamine, AEA) was the first endogenous ligand of cannabinoid receptors to be discovered. Yet, since early studies, AEA appeared to exhibit also some effects that were not mediated by cannabinoid CB(1) or CB(2) receptors. Indeed, AEA exerts some behavioral actions also in mice with genetically disrupted CB(1) receptors, whereas in vitro it is usually a partial agonist at these receptors and a weak activator of CB(2) receptors. Nevertheless, several pharmacological effects of AEA are mediated by CB(1) receptors, which, by being coupled to G-proteins, can be seen as AEA "metabotropic" receptors. Furthermore, at least two different, and as yet uncharacterized, G-protein-coupled AEA receptors have been suggested to exist in the brain and vascular endothelium, respectively. AEA is also capable of directly inhibiting ion currents mediated by L-type Ca(2+) channels and TASK-1 K(+) channels. However, to date the only reasonably well characterized, non-cannabinoid site of action for AEA is the vanilloid receptor type 1 (VR1), a non-selective cation channel gated also by capsaicin, protons and heat. VR1 might be considered as an AEA "ionotropic" receptor and, under certain conditions, mediates effects ranging from vasodilation, broncho-constriction, smooth muscle tone modulation and nociception to stimulation of hippocampal pair-pulse depression, inhibition of tumor cell growth and induction of apoptosis.