RG7774 (Vicasinabin), an orally bioavailable cannabinoid receptor 2 (CB2R) agonist, decreases retinal vascular permeability, leukocyte adhesion, and ocular inflammation in animal models.

Introduction: Preclinical studies suggest that cannabinoid receptor type 2 (CB2R) activation has a therapeutic effect in animal models on chronic inflammation and vascular permeability, which are key pathological features of diabetic retinopathy (DR). A novel CB2R agonist, triazolopyrimidine RG7774, was generated through lead optimization of a high-throughput screening hit. The aim of this study was to characterize the pharmacology, absorption, distribution, metabolism, elimination, and toxicity (ADMET) profile of RG7774, and to explore its potential for managing the key pathological features associated with retinal disease in rodents. Methods: The in vitro pharmacology of RG7774 was investigated for CB2R binding and receptor activation using recombinant human and mouse CB2R expression in Chinese hamster ovary cells, and endogenous CB2R expression in human Jurkat cells, and rat and mouse spleen cells. The ADMET profile was evaluated and the effects of RG7774 on retinal permeability, leukocyte adhesion, and choroidal neovascularization (CNV) were investigated in rodent models of retinal disease. Pharmacokinetic (PK) parameters and the exposure-response relationship were characterized in healthy animals and in animals with laser-induced CNV. Results: RG7774 was found to be a potent (EC50: 2.8 nM and Ki: 51.3 nM), selective, and full CB2R agonist with no signs of cannabinoid receptor type 1 (CB1R) binding or activation. The ligand showed a favorable ADMET profile and exhibited systemic and ocular exposure after oral delivery. Functional potency in vitro translated from recombinant to endogenous expression systems. In vivo, orally administered RG7774 reduced retinal permeability and leukocyte adhesion in rodents with lipopolysaccharide (LPS)-induced uveitis and streptozotocin (STZ)-induced DR, and reduced lesion areas in rats with laser-induced CNV with an ED50 of 0.32 mg/kg. Anatomically, RG7774 reduced the migration of retinal microglia to retinal lesions. Discussion: RG7774 is a novel, highly selective, and orally bioavailable CB2R agonist, with an acceptable systemic and ocular PK profile, and beneficial effects on retinal vascular permeability, leukocyte adhesion, and ocular inflammation in rodent animal models. Results support the development of RG7774 as a potential treatment for retinal diseases with similar pathophysiologies as addressed by the animal models.

Computational and Experimental Drug Repurposing of FDA-Approved Compounds Targeting the Cannabinoid Receptor CB1.

The cannabinoid receptor 1 (CB1R) plays a pivotal role in regulating various physiopathological processes, thus positioning itself as a promising and sought-after therapeutic target. However, the search for specific and effective CB1R ligands has been challenging, prompting the exploration of drug repurposing (DR) strategies. In this study, we present an innovative DR approach that combines computational screening and experimental validation to identify potential Food and Drug Administration (FDA)-approved compounds that can interact with the CB1R. Initially, a large-scale virtual screening was conducted using molecular docking simulations, where a library of FDA-approved drugs was screened against the CB1R's three-dimensional structures. This in silico analysis allowed us to prioritize compounds based on their binding affinity through two different filters. Subsequently, the shortlisted compounds were subjected to in vitro assays using cellular and biochemical models to validate their interaction with the CB1R and determine their functional impact. Our results reveal FDA-approved compounds that exhibit promising interactions with the CB1R. These findings open up exciting opportunities for DR in various disorders where CB1R signaling is implicated. In conclusion, our integrated computational and experimental approach demonstrates the feasibility of DR for discovering CB1R modulators from existing FDA-approved compounds. By leveraging the wealth of existing pharmacological data, this strategy accelerates the identification of potential therapeutics while reducing development costs and timelines. The findings from this study hold the potential to advance novel treatments for a range of CB1R -associated diseases, presenting a significant step forward in drug discovery research.

Radiometric Assay of NAPE-PLD Activity.

N-Acyl-phosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D (NAPE-PLD) is a prominent enzyme involved in the biosynthesis of fatty acid amides, a family of bioactive lipids including anandamide as the prototypical member. Here, we describe a NAPE-PLD assay based on radioactive substrates and product separation by thin layer chromatography (TLC).

Chemical Proteomics Reveals Off-Targets of the Anandamide Reuptake Inhibitor WOBE437.

Anandamide or N-arachidonoylethanolamine (AEA) is a signaling lipid that modulates neurotransmitter release via activation of the type 1 cannabinoid receptor (CB1R) in the brain. Termination of anandamide signaling is thought to be mediated via a facilitated cellular reuptake process that utilizes a purported transporter protein. Recently, WOBE437 has been reported as a novel, natural product-based inhibitor of AEA reuptake that is active in cellular and in vivo models. To profile its target interaction landscape, we synthesized pac-WOBE, a photoactivatable probe derivative of WOBE437, and performed chemical proteomics in mouse neuroblastoma Neuro-2a cells. Surprisingly WOBE437, unlike the widely used selective inhibitor of AEA uptake OMDM-1, was found to increase AEA uptake in Neuro-2a cells. In line with this, WOBE437 reduced the cellular levels of AEA and related N-acylethanolamines (NAEs). Using pac-WOBE, we identified saccharopine dehydrogenase-like oxidoreductase (SCCPDH), vesicle amine transport 1 (VAT1), and ferrochelatase (FECH) as WOBE437-interacting proteins in Neuro-2a cells. Further genetic studies indicated that SCCPDH and VAT1 were not responsible for the WOBE437-induced reduction in NAE levels. Regardless of the precise mechanism of action of WOB437 in AEA transport, we have identified SSCPHD, VAT1, and FECH as unprecedented off-targets of this molecule which should be taken into account when interpreting its cellular and in vivo effects.

Selective Fatty Acid Amide Hydrolase Inhibitors as Potential Novel Antiepileptic Agents.

Temporal lobe epilepsy is the most common form of epilepsy, and current antiepileptic drugs are ineffective in many patients. The endocannabinoid system has been associated with an on-demand protective response to seizures. Blocking endocannabinoid catabolism would elicit antiepileptic effects, devoid of psychotropic effects. We herein report the discovery of selective anandamide catabolic enzyme fatty acid amide hydrolase (FAAH) inhibitors with promising antiepileptic efficacy, starting from a further investigation of our prototypical inhibitor 2a. When tested in two rodent models of epilepsy, 2a reduced the severity of the pilocarpine-induced status epilepticus and the elongation of the hippocampal maximal dentate activation. Notably, 2a did not affect hippocampal dentate gyrus long-term synaptic plasticity. These data prompted our further endeavor aiming at discovering new antiepileptic agents, developing a new set of FAAH inhibitors (3a-m). Biological studies highlighted 3h and 3m as the best performing analogues to be further investigated. In cell-based studies, using a neuroblastoma cell line, 3h and 3m could reduce the oxinflammation state by decreasing DNA-binding activity of NF-kB p65, devoid of cytotoxic effect. Unwanted cardiac effects were excluded for 3h (Langendorff perfused rat heart). Finally, the new analogue 3h reduced the severity of the pilocarpine-induced status epilepticus as observed for 2a.

In Silico and In Vitro Analysis of Major Cannabis-Derived Compounds as Fatty Acid Amide Hydrolase Inhibitors.

Accumulated evidence suggests that enhancing the endocannabinoid (eCB) tone, in particular of anandamide (N-arachidonoylethanolamine, AEA), has therapeutic potential in many human diseases. Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme principally responsible for the degradation of AEA, and thus it represents a relevant target to increase signaling thereof. In recent years, different synthetic and natural compounds have been developed and tested on rat FAAH, but little is known of their effect on the human enzyme. Here, we sought to investigate six major cannabis-derived compounds to compare their action on rat and human FAAHs. To this aim, we combined an in silico analysis of their binding mode and affinity, with in vitro assays of their effect on enzyme activity. This integrated approach allowed to disclose differences in efficacy towards rat and human FAAHs, and to highlight the role of key residues involved in the inhibition of both enzymes. This study suggests that the therapeutic efficacy of compounds targeted towards FAAH should be always tested in vitro on both rat and human enzymes.

Bisphenol A Deranges the Endocannabinoid System of Primary Sertoli Cells with an Impact on Inhibin B Production.

Bisphenol A (BPA) is an endocrine disruptor that negatively affects spermatogenesis, a process where Sertoli cells play a central role. Thus, in the present study we sought to ascertain whether BPA could modulate the endocannabinoid (eCB) system in exposed mouse primary Sertoli cells. Under our experimental conditions, BPA turned out to be cytotoxic to Sertoli cells with an half-maximal inhibitory concentration (IC50) of ~6.0 µM. Exposure to a non-cytotoxic dose of BPA (i.e., 0.5 µM for 48 h) increased the expression levels of specific components of the eCB system, namely: type-1 cannabinoid (CB1) receptor and diacylglycerol lipase-α (DAGL-α), at mRNA level, type-2 cannabinoid (CB2) receptor, transient receptor potential vanilloid 1 (TRPV1) receptors, and DAGL-ß, at protein level. Interestingly, BPA also increased the production of inhibin B, but not that of transferrin, and blockade of either CB2 receptor or TRPV1 receptor further enhanced the BPA effect. Altogether, our study provides unprecedented evidence that BPA deranges the eCB system of Sertoli cells towards CB2- and TRPV1-dependent signal transduction, both receptors being engaged in modulating BPA effects on inhibin B production. These findings add CB2 and TRPV1 receptors, and hence the eCB signaling, to the other molecular targets of BPA already known in mammalian cells.

Author Correction: The endocannabinoid hydrolase FAAH is an allosteric enzyme.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Advances in the discovery of fatty acid amide hydrolase inhibitors: what does the future hold?

INTRODUCTION: Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme, that inactivates endogenous signaling lipids of the fatty acid amide family, including the endocannabinoid anandamide (N-arachidonoylethanolamine, AEA). The latter compound has been shown to regulate a number of important pathophysiological conditions in humans, like feeding, obesity, immune response, reproductive events, motor coordination, and neurological disorders. Hence, direct manipulation of the endocannabinoid tone is thought to have therapeutic potential. A new opportunity to develop effective drugs may arise from multi-target directed ligand (MTDL) strategies, which brings the concept that a single compound can recognize different targets involved in the cascade of pathophysiological events. AREAS COVERED: This review reports the latest advances in the development of new single targeted and dual-targeted FAAH inhibitors over the past 5 years. EXPERT OPINION: In recent years, several FAAH inhibitors have been synthesized and investigated, yet to date none of them has reached the market as a systemic drug. Due to the diligence of inherent redundancy and robustness in many biological networks and pathways, multitarget inhibitors present a new prospect in the pharmaceutical industry for treatment of complex diseases.

Characterization of Endocannabinoid System and Interleukin Profiles in Ovine AEC: Cannabinoid Receptors Type-1 and Type-2 as Key Effectors of Pro-Inflammatory Response.

Amniotic epithelial cells (AEC) have been proposed as promising clinical candidates for regenerative medicine therapies due to their immunomodulatory capacity. In this context, the endocannabinoid system (ECS) has been identified as mediating the immune-stem cell dialogue, even if no information on AEC is available to date. Therefore, this study was designed to assess whether ECS is involved in tuning the constitutive and lipopolysaccharide (LPS)-induced ovine AEC anti-inflammatory and pro-inflammatory interleukin (IL-10, IL-4, and IL-12) profiles. Firstly, interleukins and ECS expressions were studied at different stages of gestation. Then, the role of cannabinoid receptors 1 and 2 (CB1 and CB2) on interleukin expression and release was investigated in middle stage AEC using selective agonists and antagonists. AEC displayed a degradative more than a synthetic endocannabinoid metabolism during the early and middle stages of gestation. At the middle stage, cannabinoid receptors mediated the balance between pro-inflammatory (IL-12) and anti-inflammatory (IL-4 and IL-10) interleukins. The activation of both receptors mediated an overall pro-inflammatory shift-CB1 reduced the anti-inflammatory and CB2 increased the pro-inflammatory interleukin release, particularly after LPS stimulation. Altogether, these data pave the way for the comprehension of AEC mechanisms tuning immune-modulation, crucial for the development of new AEC-based therapy protocols.

The endocannabinoid hydrolase FAAH is an allosteric enzyme.

Fatty acid amide hydrolase (FAAH) is a membrane-bound homodimeric enzyme that in vivo controls content and biological activity of N-arachidonoylethanolamine (AEA) and other relevant bioactive lipids termed endocannabinoids. Parallel orientation of FAAH monomers likely allows both subunits to simultaneously recruit and cleave substrates. Here, we show full inhibition of human and rat FAAH by means of enzyme inhibitors used at a homodimer:inhibitor stoichiometric ratio of 1:1, implying that occupation of only one of the two active sites of FAAH is enough to fully block catalysis. Single W445Y substitution in rat FAAH displayed the same activity as the wild-type, but failed to show full inhibition at the homodimer:inhibitor 1:1 ratio. Instead, F432A mutant exhibited reduced specific activity but was fully inhibited at the homodimer:inhibitor 1:1 ratio. Kinetic analysis of AEA hydrolysis by rat FAAH and its F432A mutant demonstrated a Hill coefficient of ~1.6, that instead was ~1.0 in the W445Y mutant. Of note, also human FAAH catalysed an allosteric hydrolysis of AEA, showing a Hill coefficient of ~1.9. Taken together, this study demonstrates an unprecedented allosterism of FAAH, and represents a case of communication between two enzyme subunits seemingly controlled by a single amino acid (W445) at the dimer interface. In the light of extensive attempts and subsequent failures over the last decade to develop effective drugs for human therapy, these findings pave the way to the rationale design of new molecules that, by acting as positive or negative heterotropic effectors of FAAH, may control more efficiently its activity.

Development of novel multipotent compounds modulating endocannabinoid and dopaminergic systems.

Polypharmacology approaches may help the discovery of pharmacological tools for the study or the potential treatment of complex and multifactorial diseases as well as for addictions and also smoke cessation. In this frame, following our interest in the development of molecules able to modulate either the endocannabinoid or the dopaminergic system, and given the multiple and reciprocal interconnections between them, we decided to merge the pharmacophoric elements of some of our early leads for identifying new molecules as tools able to modulate both systems. We herein describe the synthesis and biological characterization of compounds 5a-j inspired by the structure of our potent and selective fatty acid amide hydrolase (FAAH) inhibitors (3a-c) and ligands of dopamine D2 or D3 receptor subtypes (4a,b). Notably, the majority of the new molecules showed a nanomolar potency of interaction with the targets of interest. The drug-likeliness of the developed compounds (5a-j) was investigated in silico while hERG affinity, selectivity profile (for some proteins of the endocannabinoid system), cytotoxicity profiles (on fibroblast and astrocytes), and mutagenicity (Ames test) were experimentally determined. Metabolic studies also served to complement the preliminary drug-likeliness profiling for compounds 3a and 5c. Interestingly, after assessing the lack of toxicity for the neuroblastoma cell line (IMR 32), we demonstrated a potential anti-inflammatory profile for 3a and 5c in the same cell line.

Activity-based protein profiling reveals off-target proteins of the FAAH inhibitor BIA 10-2474.

A recent phase 1 trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474 led to the death of one volunteer and produced mild-to-severe neurological symptoms in four others. Although the cause of the clinical neurotoxicity is unknown, it has been postulated, given the clinical safety profile of other tested FAAH inhibitors, that off-target activities of BIA 10-2474 may have played a role. Here we use activity-based proteomic methods to determine the protein interaction landscape of BIA 10-2474 in human cells and tissues. This analysis revealed that the drug inhibits several lipases that are not targeted by PF04457845, a highly selective and clinically tested FAAH inhibitor. BIA 10-2474, but not PF04457845, produced substantial alterations in lipid networks in human cortical neurons, suggesting that promiscuous lipase inhibitors have the potential to cause metabolic dysregulation in the nervous system.

Palmitoylation of cysteine 415 of CB1 receptor affects ligand-stimulated internalization and selective interaction with membrane cholesterol and caveolin 1.

We previously demonstrated that CB1 receptor is palmitoylated at cysteine 415, and that such a post-translational modification affects its biological activity. To assess the molecular mechanisms responsible for modulation of CB1 receptor function by S-palmitoylation, in this study biochemical and morphological approaches were paralleled with computational analyses. Molecular dynamics simulations suggested that this acyl chain stabilizes helix 8 as well as the interaction of CB1 receptor with membrane cholesterol. In keeping with these in silico data, experimental results showed that the non-palmitoylated CB1 receptor was unable to interact efficaciously with caveolin 1, independently of its activation state. Moreover, in contrast with the wild-type receptor, the lack of S-palmitoylation in the helix 8 made the mutant CB1 receptor completely irresponsive to agonist-induced effects in terms of both lipid raft partitioning and receptor internalization. Overall, our results support the notion that palmitoylation of cysteine 415 modulates the conformational state of helix 8 and influences the interactions of CB1 receptor with cholesterol and caveolin 1, suggesting that the palmitoyl chain may serve as a functional interface for CB1 receptor localization and function.

Cannabinoid CB2 receptor ligand profiling reveals biased signalling and off-target activity.

The cannabinoid CB2 receptor (CB2R) represents a promising therapeutic target for various forms of tissue injury and inflammatory diseases. Although numerous compounds have been developed and widely used to target CB2R, their selectivity, molecular mode of action and pharmacokinetic properties have been poorly characterized. Here we report the most extensive characterization of the molecular pharmacology of the most widely used CB2R ligands to date. In a collaborative effort between multiple academic and industry laboratories, we identify marked differences in the ability of certain agonists to activate distinct signalling pathways and to cause off-target effects. We reach a consensus that HU910, HU308 and JWH133 are the recommended selective CB2R agonists to study the role of CB2R in biological and disease processes. We believe that our unique approach would be highly suitable for the characterization of other therapeutic targets in drug discovery research.

Assay of NAPE-PLD Activity.

N-acyl-phosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D (NAPE-PLD) is a prominent enzyme involved in the biosynthesis of fatty acid amides (FAAs), a family of bioactive lipids including anandamide (AEA) as the prototypical member. Here, we describe a NAPE-PLD assay based on radioactive substrates and product separation by thin-layer chromatography (TLC).

Lipid Discovery by Combinatorial Screening and Untargeted LC-MS/MS.

We present a method for the systematic identification of picogram quantities of new lipids in total extracts of tissues and fluids. It relies on the modularity of lipid structures and applies all-ions fragmentation LC-MS/MS and Arcadiate software to recognize individual modules originating from the same lipid precursor of known or assumed structure. In this way it alleviates the need to recognize and fragment very low abundant precursors of novel molecules in complex lipid extracts. In a single analysis of rat kidney extract the method identified 58 known and discovered 74 novel endogenous endocannabinoids and endocannabinoid-related molecules, including a novel class of N-acylaspartates that inhibit Hedgehog signaling while having no impact on endocannabinoid receptors.

Plant-Derived and Endogenous Cannabinoids in Epilepsy.

Cannabis is one of the oldest psychotropic drugs and its anticonvulsant properties have been known since the last century. The aim of this review was to analyze the efficacy of cannabis in the treatment of epilepsy in adults and children. In addition, a description of the involvement of the endocannabinoid system in epilepsy is given in order to provide a biochemical background to the effects of endogenous cannabinoids in our body. General tolerability and adverse events associated with cannabis treatment are also investigated. Several anecdotal reports and clinical trials suggest that in the human population cannabis has anticonvulsant properties and could be effective in treating partial epilepsies and generalized tonic-clonic seizures, still known as "grand mal." They are based, among other factors, on the observation that in individuals who smoke marijuana to treat epilepsy, cessation of cannabis use precipitates the re-emergence of convulsive seizures, whereas resuming consumption of this psychotropic drug controls epilepsy in a reproducible manner. In conclusion, there is some anecdotal evidence for the potential efficacy of cannabis in treating epilepsy. Though there has been an increased effort by patients with epilepsy, their caregivers, growers, and legislators to legalize various forms of cannabis, there is still concern about its efficacy, relative potency, availability of medication-grade preparations, dosing, and potential short- and long-term side effects, including those on prenatal and childhood development.

The novel, orally available and peripherally restricted selective cannabinoid CB2 receptor agonist LEI-101 prevents cisplatin-induced nephrotoxicity.

BACKGROUND AND PURPOSE: Here, we have characterized 3-cyclopropyl-1-(4-(6-((1,1-dioxidothiomorpholino)methyl)-5-fluoropyridin-2-yl)benzyl)imidazolidine-2,4-dione hydrochloride (LEI-101) as a novel, peripherally restricted cannabinoid CB2 receptor agonist, using both in vitro and in vivo models. EXPERIMENTAL APPROACH: We investigated the effects of LEI-101 on binding and functional activity. We assessed its in vitro and in vivo selectivity. Efficacy of LEI-101 was determined in a mouse model of cisplatin-induced nephrotoxicity. KEY RESULTS: LEI-101 behaved as a partial agonist at CB2 receptors using ß-arrestin and GTPγS assays and was ~100-fold selective in CB2 /CB1 receptor-binding assays. It did not display any activity on endocannabinoid hydrolases and nor did it react with serine hydrolases in an activity-based protein profiling assay. In mice, LEI-101 had excellent oral bioavailability reaching high concentrations in the kidney and liver with minimal penetration into the brain. LEI-101 up to a dose of 60 mg·kg(-1) (p.o.) did not exert any CNS-mediated effects in the tetrad assay, in mice. LEI-101 (p.o. or i.p.) at 3 or 10 mg·kg(-1) dose-dependently prevented kidney dysfunction and/or morphological damage induced by cisplatin in mice. These protective effects were associated with improved renal histopathology, attenuated oxidative stress and inflammation in the kidney. These effects were absent in CB2 receptor knockout mice. CONCLUSION AND IMPLICATIONS: These results indicate that LEI-101 is a selective, largely peripherally restricted, orally available CB2 receptor agonist with therapeutic potential in diseases that are associated with inflammation and/or oxidative stress, including kidney disease.

Endocannabinoids, related compounds and their metabolic routes.

Endocannabinoids are lipid mediators able to bind to and activate cannabinoid receptors, the primary molecular targets responsible for the pharmacological effects of the Δ9-tetrahydrocannabinol. These bioactive lipids belong mainly to two classes of compounds: N-acylethanolamines and acylesters, being N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), respectively, their main representatives. During the last twenty years, an ever growing number of fatty acid derivatives (endocannabinoids and endocannabinoid-like compounds) have been discovered and their activities biological is the subject of intense investigations. Here, the most recent advances, from a therapeutic point of view, on endocannabinoids, related compounds, and their metabolic routes will be reviewed.