The Endocannabinoid Peptide RVD-Hemopressin Is a TRPV1 Channel Blocker.

Neurotransmission is critical for brain function, allowing neurons to communicate through neurotransmitters and neuropeptides. RVD-hemopressin (RVD-Hp), a novel peptide identified in noradrenergic neurons, modulates cannabinoid receptors CB1 and CB2. Unlike hemopressin (Hp), which induces anxiogenic behaviors via transient receptor potential vanilloid 1 (TRPV1) activation, RVD-Hp counteracts these effects, suggesting that it may block TRPV1. This study investigates RVD-Hp's role as a TRPV1 channel blocker using HEK293 cells expressing TRPV1-GFP. Calcium imaging and patch-clamp recordings demonstrated that RVD-Hp reduces TRPV1-mediated calcium influx and TRPV1 ion currents. Molecular docking and dynamics simulations indicated that RVD-Hp interacts with TRPV1's selectivity filter, forming stable hydrogen bonds and van der Waals contacts, thus preventing ion permeation. These findings highlight RVD-Hp's potential as a therapeutic agent for conditions involving TRPV1 activation, such as pain and anxiety.

New Pyridone-Based Derivatives as Cannabinoid Receptor Type 2 Agonists.

The activation of the human cannabinoid receptor type II (CB2R) is known to mediate analgesic and anti-inflammatory processes without the central adverse effects related to cannabinoid receptor type I (CB1R). In this work we describe the synthesis and evaluation of a novel series of N-aryl-2-pyridone-3-carboxamide derivatives tested as human cannabinoid receptor type II (CB2R) agonists. Different cycloalkanes linked to the N-aryl pyridone by an amide group displayed CB2R agonist activity as determined by intracellular [cAMP] levels. The most promising compound 8d exhibited a non-toxic profile and similar potency (EC50 = 112 nM) to endogenous agonists Anandamide (AEA) and 2-Arachidonoylglycerol (2-AG) providing new information for the development of small molecules activating CB2R. Molecular docking studies showed a binding pose consistent with two structurally different agonists WIN-55212-2 and AM12033 and suggested structural requirements on the pyridone substituents that can satisfy the orthosteric pocket and induce an agonist response. Our results provide additional evidence to support the 2-pyridone ring as a suitable scaffold for the design of CB2R agonists and represent a starting point for further optimization and development of novel compounds for the treatment of pain and inflammation.

Central nervous system, peripheral and hemodynamic effects of nanoformulated anandamide in hypertension.

PURPOSE: Hypertensive lesions induce alterations at hemodynamic, peripheral, and central levels. Anandamide (N-arachidonoylethanolamine; AEA) protects neurons from inflammatory damage, but its free administration may cause central adverse effects. AEA controlled release by nanoformulations could reduce/eliminate its side effects. The present study aimed to evaluate the effects of nanoformulated AEA (nf-AEA) on systolic blood pressure (SBP), behavior, and central/peripheral inflammatory, oxidative, and apoptotic state in spontaneously hypertensive rats (SHR). MATERIALS/METHODS: Male rats were used, both Wistar Kyoto (WKY) and SHR (n â€‹= â€‹10 per group), with/without treatment with nf-AEA (obtained by electrospraying) at a weekly dose of 5 â€‹mg/kg IP for 4 weeks. SBP was measured and behavioral tests were performed. Inflammatory/oxidative markers were quantified at the central (brain cortex) and peripheral (serum) level. RESULTS: SHR showed hyperactivity, low anxiety, and high concentrations of central/peripheral inflammatory/oxidative markers, also higher apoptosis of brain cortical cells compared to WKY. As opposed to this group, treatment with nf-AEA in SHR significantly reduced SBP, peripheral/central inflammatory/oxidative makers, and central apoptosis. Nf-AEA also increased neuroprotective mechanisms mediated by intracellular heat shock protein 70 (Hsp70), which were attenuated in untreated SHR. Additionally, nf-AEA reversed the abnormal behaviors observed in SHR without producing central adverse effects. CONCLUSIONS: Our results suggest protective properties of nf-AEA, both peripherally and centrally, through a signaling pathway that would involve the type I angiotensin II receptor, Wilms tumor transcription factor 1, Hsp70, and iNOS. Considering non-nf-AEA limitations, this nanoformulation could contribute to the development of new antihypertensive and behavioral disorder treatments associated with neuroinflammation.

Synthesis, physicochemical characterisation and biological activity of anandamide/ɛ-polycaprolactone nanoparticles obtained by electrospraying.

Drug encapsulation in nanocarriers such as polymeric nanoparticles (Nps) may help to overcome the limitations associated with cannabinoids. In this study, the authors' work aimed to highlight the use of electrospraying techniques for the development of carrier Nps of anandamide (AEA), an endocannabinoid with attractive pharmacological effects but underestimated due to its unfavourable physicochemical and pharmacokinetic properties added to its undesirable effects at the level of the central nervous system. The authors characterised physicochemically and evaluated in vitro biological activity of anandamide/ɛ-polycaprolactone nanoparticles (Nps-AEA/PCL) obtained by electrospraying in epithelial cells of the human proximal tubule (HK2), to prove the utility of this method and to validate the biological effect of Nps-AEA/PCL. They obtained particles from 100 to 900 nm of diameter with a predominance of 200-400 nm. Their zeta potential was -20 ± 1.86 mV. They demonstrated the stable encapsulation of AEA in Nps-AEA/PCL, as well as its dose-dependent capacity to induce the expression of iNOS and NO levels and to decrease the Na+/K+ ATPase activity in HK2 cells. Obtaining Nps-AEA/PCL by electrospraying would represent a promising methodology for a novel AEA pharmaceutical formulation development with optimal physicochemical properties, physical stability and biological activity on HK2 cells.

Potential metabolic and behavioural roles of the putative endocannabinoid receptors GPR18, GPR55 and GPR119 in feeding.

Endocannabinoids are ancient biomolecules involved in several cellular (e.g., metabolism) and physiological (e.g., eating behaviour) functions. Indeed, eating behaviour alterations in marijuana users have led to investigate the orexigenic/anorexigenic effects of cannabinoids in animal/ human models. This increasing body of research suggests that the endocannabinoid system plays an important role in feeding control. Accordingly, within the endocannabinoid system, cannabinoid receptors, enzymes and genes represent potential therapeutic targets for dealing with multiple metabolic and behavioural dysfunctions (e.g., obesity, anorexia, etc.). Paradoxically, our understanding on the endocannabinoid system as a cellular mediator is yet limited. For example: (i) only two cannabinoid receptors have been classified, but they are not enough to explain the pharmacological profile of several experimental effects induced by cannabinoids; and (ii) several orphan G protein-coupled receptors (GPCRs) interact with cannabinoids and we do not know how to classify them (e.g., GPR18, GPR55 and GPR119; amongst others). On this basis, the present review attempts to summarize the lines of evidence supporting the potential role of GPR18, GPR55 and GPR119 in metabolism and feeding control that may explain some of the divergent effects and puzzling data related to cannabinoid research. Moreover, their therapeutic potential in feeding behaviour alterations will be considered.

Anandamide-nanoformulation obtained by electrospraying for cardiovascular therapy.

Anandamide (AEA), an endogenous cannabinoid, has a relevant antihypertensive effect. However, its cardioprotective role has been barely explored due to unfavorable physico-chemical properties and, sometimes, undesirable psychoactive effects. In this context, drug encapsulation in nanocarriers could overcome the limitations associated with the administration of AEA in free form. The aim of the present study was to encapsulate AEA in poly-ε-caprolactone/Pluronic® F127 nanoparticles (AEA/PCL/PF127 NPs) by means of electrospraying, to characterize their physico-chemical properties and cytocompatibility and to evaluate their effect in an in vivo model of cardiovascular remodeling caused by hypertension. AEA/PCL/PF127 NPs were characterized in terms of morphology, size, polydispersity, Z-potential, hydrophilicity, thermal and spectroscopic properties. Also, the encapsulation and loading efficiencies and in vitro release of AEA were analyzed. AEA/PCL/PF127 NPs (700-1000 nm) showed adequate cytocompatibility. For the cardiovascular remodeling studies, normotensive (WKY) and hypertensive (SHR) male rats were treated or not with AEA/PCL/PF127 NPs (5 mg/Kg, intraperitoneal injection) weekly for 1 month. Inflammatory markers and hemodynamic, structural and cardiac functional parameters were monitored. In SHR, the treatment with AEA/PCL/PF127 NPs reversed all altered cardiovascular markers and parameters (p < 0.05). Overall, nanoformulated AEA obtained by electrospraying proved to be effective for the treatment of hypertension and its comorbidities, especially cardiovascular remodeling.

Polymeric ionic liquid open tubular capillary column for on-line in-tube SPME coupled with UHPLC-MS/MS to determine endocannabinoids in plasma samples.

This manuscript describes the development of wall-coated open tubular capillary column with polymeric ionic liquids (PILs) for on-line in-tube solid phase microextraction coupled with ultra high-performance liquid chromatography tandem mass spectrometry (in-tube SPME/UHPLC-MS/MS) to determine anandamide (AEA) and 2-arachidonoyl glycerol (2 A G) in plasma samples. Selective PILs were synthetized from the [VC6IM][Cl], [VC16IM][Br], and [(VIM)2C10]2 [Br] - ionic liquids - by in-situ thermal-initiated polymerization in a fused silica capillary column for in-tube SPME. The synthesis procedure was optimized, and the capillary columns were characterized using spectroscopic and chromatography techniques. The chemically bonded and cross-linked PIL-based sorbent phase (thickness coating: 1.7 µm) presented high chemical and mechanical stability. Among the sorbents evaluated, the PIL-based capillary, [VC16IM][Br]/[(VIM)2C10]2 [Br] presented the best performance with a sorption capacity of 37,311 ng cm-3 and 48,307 ng cm-3 for AEA and 2 A G, respectively. This capillary was reused more than ninety times without significant changes in extraction efficiency. The in-tube SPME-UHPLC-MS/MS method presented a linear range from 0.1 ng mL-1 to 100 ng mL-1 for AEA, and from 0.05 ng mL-1 to 100 ng mL-1 for 2 A G, with coefficients of determination higher than 0.99, p-value for Lack-of-fit test higher than 0.05 (α of 0.05), precision with coefficient of variation (CV) values ranging from 1.6 to 14.0% and accuracy with relative standard deviation (RSD) values from -19.6% to 13.2%. This method was successfully applied to determine AEA and 2 A G in plasma patients with Parkinson's disease. The concentrations in these plasma samples ranged from 0.14 to 0.46 ng mL-1 for AEA and from <0.05 ng mL-1 to 0.51 ng mL-1 for 2-AG.

The endocannabinoid system--back to the scene of cardiometabolic risk factors control?

This review examines the impact of the endocannabinoid signaling system on metabolic and cardiovascular health and the new therapeutic strategies that selectively target dysfunctional endocannabinoid action in peripheral tissues, without causing the undesirable central nervous system effects that occurred with the first-generation of CB1 receptor blockers. We first review the components of the endocannabinoid system and the enzymes that synthesize and degrade the endocannabinoids, the critical role of the system in the homeostasis of energy balance, and its hedonic aspects related to the incentive and motivational value of food. Second, we describe the central and peripheral actions of the endocannabinoid system and its interactions with other biological modulators, such as ghrelin and leptin. Third, we summarize data from human clinical trials with the CB1 inverse agonist rimonabant, showing that the drug, although effective in increasing weight loss with accompanying improvements in the metabolic profile of the participants in the RIO (Rimonabant In Obesity) trials, was withdrawn from the market because of the risk of serious adverse events. Finally, we describe: 1) the development of new selective peripheral blockers that interrupt endocannabinoid action selectively in peripheral tissues and that have been suggested as an alternative approach to treat the metabolic consequences of obesity and related diseases, without undesirable central nervous system effects, and 2) the potential for inhibition of enzymes of synthesis, as well as the possible role of endocannabinoid congeners, with opposing effects as compared to CB1 receptor agonists, in the control of metabolic disorders.