Is It Time to Test the Antiseizure Potential of Palmitoylethanolamide in Human Studies? A Systematic Review of Preclinical Evidence

Antiseizure medications are the cornerstone pharmacotherapy for epilepsy. They are not devoid of side effects. In search for better-tolerated antiseizure agents, cannabinoid compounds and other N-acylethanolamines not directly binding cannabinoid receptors have drawn significant attention. Among these, palmitoylethanolamide (PEA) has shown neuroprotective, anti-inflammatory, and analgesic properties. All studies examining PEA’s role in epilepsy and acute seizures were systematically reviewed. Preclinical studies indicated a systematically reduced PEA tone accompanied by alterations of endocannabinoid levels. PEA supplementation reduced seizure frequency and severity in animal models of epilepsy and acute seizures, in some cases, similarly to available antiseizure medications but with a better safety profile. The peripheral-brain immune system seemed to be more effectively modulated by subchronic pretreatment with PEA, with positive consequences in terms of better responding to subsequent epileptogenic insults. PEA treatment restored the endocannabinoid level changes that occur in a seizure episode, with potential preventive implications in terms of neural damage. Neurobiological mechanisms for PEA antiseizure effect seemed to include the activation of the endocannabinoid system and the modulation of neuroinflammation and excitotoxicity. Although no human study was identified, there is ground for testing the antiseizure potential of PEA and its safety profile in human studies of epilepsy.

Regulation of adult neurogenesis by the endocannabinoid-producing enzyme diacylglycerol lipase alpha (DAGLa)

The endocannabinoid system modulates adult hippocampal neurogenesis by promoting the proliferation and survival of neural stem and progenitor cells (NSPCs). This is demonstrated by the disruption of adult neurogenesis under two experimental conditions: (1) NSPC-specific deletion of cannabinoid receptors and (2) constitutive deletion of the enzyme diacylglycerol lipase alpha (DAGLa) which produces the endocannabinoid 2-arachidonoylglycerol (2-AG). However, the specific cell types producing 2-AG relevant to neurogenesis remain unknown. Here we sought to identify the cellular source of endocannabinoids in the subgranular zone of the dentate gyrus (DG) in hippocampus, an important neurogenic niche. For this purpose, we used two complementary Cre-deleter mouse strains to delete Dagla either in neurons, or in astroglia and NSPCs. Surprisingly, neurogenesis was not altered in mice bearing a deletion of Dagla in neurons (Syn-Dagla KO), although neurons are the main source for the endocannabinoids in the brain. In contrast, a specific inducible deletion of Dagla in NPSCs and astrocytes (GLAST-CreERT2-Dagla KO) resulted in a strongly impaired neurogenesis with a 50% decrease in proliferation of newborn cells. These results identify Dagla in NSPCs in the DG or in astrocytes as a prominent regulator of adult hippocampal neurogenesis. We also show a reduction of Daglb expression in GLAST-CreERT2-Dagla KO mice, which may have contributed to the neurogenesis phenotype.

In Silico Prediction and Validation of CB2 Allosteric Binding Sites to Aid the Design of Allosteric Modulators

Although the 3D structures of active and inactive cannabinoid receptors type 2 (CB2) are available, neither the X-ray crystal nor the cryo-EM structure of CB2-orthosteric ligand-modulator has been resolved, prohibiting the drug discovery and development of CB2 allosteric modulators (AMs). In the present work, we mainly focused on investigating the potential allosteric binding site(s) of CB2. We applied different algorithms or tools to predict the potential allosteric binding sites of CB2 with the existing agonists. Seven potential allosteric sites can be observed for either CB2-CP55940 or CB2-WIN 55,212-2 complex, among which sites B, C, G and K are supported by the reported 3D structures of Class A GPCRs coupled with AMs. Applying our novel algorithm toolset-MCCS, we docked three known AMs of CB2 including Ec2la (C-2), trans-β-caryophyllene (TBC) and cannabidiol (CBD) to each site for further comparisons and quantified the potential binding residues in each allosteric binding site. Sequentially, we selected the most promising binding pose of C-2 in five allosteric sites to conduct the molecular dynamics (MD) simulations. Based on the results of docking studies and MD simulations, we suggest that site H is the most promising allosteric binding site. We plan to conduct bio-assay validations in the future.

Theoretical Study of the Structural Stability, Chemical Reactivity, and Protein Interaction for NMP Compounds as Modulators of the Endocannabinoid System

The cannabinoid receptors (CB1/CB2) and the T-type calcium channels are involved in disorders associated with both physiological pain and depressive behaviors. Valuable pharmacological species carbazole derivatives such as the NMP-4, NMP-7, and NMP-181 (Neuro Molecular Production) regulate both biological entities. In this work, DFT calculations were performed to characterize theoretically their structural and chemical reactivity properties using the BP86/cc-pVTZ level of theory. The molecular orbital contributions and the chemical reactivity analysis reveal that a major participation of the carbazole group is in the donor-acceptor interactions of the NMP compounds. The DFT analysis on the NMP compounds provides insights into the relevant functional groups involved during the ligand-receptor interactions. Molecular docking analysis is used to reveal possible sites of interaction of the NMP compounds with the Cav3.2 calcium channel. The interaction energy values and reported experimental evidence indicate that the site denominated as “Pore-blocking”, which is formed mainly by hydrophobic residues and the T586 residue, is a probable binding site for the NMP compounds.

Cannabinoids for Symptom Management in Patients with Kidney Failure

People with kidney failure can experience a range of symptoms that lead to suffering and poor quality of life. Available therapies are limited, and evidence for new treatment options is sparse, often resulting in incomplete relief of symptoms. There is growing interest in the potential for cannabinoids, including cannabidiol and tetrahydrocannabinol, to treat symptoms across a wide range of chronic diseases. As legal prohibitions are withdrawn or minimized in many jurisdictions, patients are increasingly able to access these agents. Cannabinoid receptors, CB1 and CB2, are widely expressed in the body, including within the nervous and immune systems, and exogenous cannabinoids can have anxiolytic, anti-emetic, analgesic and anti-inflammatory effects. Considering their known physiological actions and successful studies in other patient populations, cannabinoids may be viewed as potential therapies for a variety of common symptoms affecting those with kidney failure, including pruritus, nausea, insomnia, chronic neuropathic pain, anorexia, and restless legs syndrome. In this review, we summarize the pharmacology and pharmacokinetics of cannabinoids, along with what is known about the use of cannabinoids for symptom relief in those with kidney disease, and the evidence available concerning their role in management of common symptoms. Presently, while these agents show varying efficacy with a reasonable safety profile in other patient populations, evidence-based prescribing of cannabinoids for people with symptomatic kidney failure is not possible. Given the symptom burden experienced by individuals with kidney failure, there is an urgent need to understand the tolerability and safety of these agents in this population, which must ultimately be followed by robust, randomized controlled trials to determine if they are effective for symptom relief.

Cannabinoid Receptor Modulation in the 3-Acetylpyridine Cerebellar Ataxia Mouse Model

Cerebellar ataxia is a neurodegenerative disorder leading to severe motor incoordination. Recently it has been suggested that cannabinoids play a role in modulation of ataxic symptoms. In order to understand the possible therapeutic effect of cannabinoids for management of cerebellar ataxia, we used cannabinoid agonist/antagonists to target the cannabinoid type 1 receptor (CB1R) in the 3 acetyl pyridine (3AP) mouse model of ataxia. The role of the CB1R was examined by using three different doses of the CB1R agonist, WIN55,212-2 (WIN; 0.1, 0.5, 1 mg/kg) administrated 30 min prior to 3AP (55 mg/kg, i.p.) which leads to motor impairment through destruction of the inferior olive. In some recordings, the CB1R antagonist AM251(1 mg/kg) was given in combination with WIN. Locomotor activity and motor coordination were impaired by 3AP, and the application of WIN did not ameliorate this effect. However, the abnormal gait, rearing and grooming caused by 3AP were prevented by co-administration of AM251 with WIN. While the addition of the CB1R antagonist inhibition improved some ataxic symptoms, there was no effect of AM251 on balance or locomotor activity when co-administrated with WIN. Behavioral testing indicated that not only did WIN fail to exert any protective effect on ataxic symptoms, it exacerbated ataxic symptoms, suggesting that CB1R agonists may not be the ideal therapeutic drug in this disorder. When taken together, the findings from the present study indicate that cannabinoid modulation of ataxia symptoms may not act solely through CB1Rs and other cannabinoid receptors should be consider in future studies.

Revealing genomic changes responsible for cannabinoid receptor loss in parrots: mechanism and functional effects

In vertebrates, an ancient duplication in the genes for cannabinoid receptors (CNRs) allowed the evolution of specialised endocannabinoid receptors expressed in the brain (CNR1) and the periphery (CNR2). While dominantly conserved throughout vertebrate phylogeny, our comparative genomic analysis suggests that certain taxa may have lost either the CNR1 regulator of neural processes or, more frequently, the CNR2 involved in immune regulation. Focussing on conspicuous CNR2 pseudogenization in parrots (Psittaciformes), a diversified crown lineage of cognitively-advanced birds, we highlight possible functional effects of such a loss. Parrots appear to have lost the CNR2 gene at at least two separate occasions due to chromosomal rearrangement. Using gene expression data from the brain and periphery of birds with experimentally-induced sterile inflammation, we compare CNR and inflammatory marker (interleukin 1 beta, IL1B) expression patterns in CNR2-deficient parrots (represented by the budgerigar, Melopsittacus undulatus and five other parrot species) with CNR2-intact passerines (represented by the zebra finch, Taeniopygia guttata). Though no significant changes in CNR expression were observed in either parrots or passerines during inflammation of the brain or periphery, we detected a significant up-regulation of IL1B expression in the brain after stimulation with lipopolysaccharide (LPS) only in parrots. As our analysis failed to show evidence for selection on altered CNR1 functionality in parrots, compared to other birds, CNR1 is unlikely to be involved in compensation for CNR2 loss in modulation of the neuroimmune interaction. Thus, our results provide evidence for the functional importance of CNR2 pseudogenization for regulation of neuroinflammation.

Footshock-Induced Abstinence from Compulsive Methamphetamine Self-administration in Rat Model Is Accompanied by Increased Hippocampal Expression of Cannabinoid Receptors (CB1 and CB2)

Methamphetamine (METH) use disorder (MUD) is characterized by compulsive and repeated drug taking despite negative life consequences. Large intake of METH in humans and animals is accompanied by dysfunctions in learning and memory processes. The endocannabinoid system (ECS) is known to modulate synaptic plasticity and cognitive functions. In addition, the ECS has been implicated in some of the manifestations of substance use disorders (SUDs). We therefore sought to identify potential changes in the expression of various enzymes and of the receptors (CB1 and CB2) that are members of that system. Herein, we used a model of METH self-administration (SA) that includes a punishment phase (footshocks) that helps to separate rats into a compulsive METH phenotype (compulsive) that continues to take METH and a non-compulsive METH (abstinent) group that suppressed or stopped taking METH. Animals were euthanized 2 h after the last METH SA session and their hippocampi were used to measure mRNA levels of cannabinoid receptors (CB/Cnr), as well as those of synthesizing (DAGL-A, DAGL-B, NAPEPLD) and metabolizing (MGLL, FAAH, PTGS2) enzymes of the endocannabinoid cascade. Non-compulsive rats exhibited significant increased hippocampal expression of CB1/Cnr1 and CB2/Cnr2 mRNAs. mRNA levels of the synthesizing enzyme, DAGL-A, and of the metabolic enzymes, MGLL and FAAH, were also increased. Non-compulsive rats also exhibited a significant decrease in hippocampal Ptgs2 mRNA levels. Taken together, these observations implicate the hippocampal endocannabinoid system in the suppression of METH intake in the presence of adverse consequences.

Transcriptomic Profiling in Mice With CB1 receptor Deletion in Primary Sensory Neurons Suggests New Analgesic Targets for Neuropathic Pain

Type 1 and type 2 cannabinoid receptors (CB1 and CB2, respectively) mediate cannabinoid-induced analgesia. Loss of endogenous CB1 is associated with hyperalgesia. However, the downstream targets affected by ablation of CB1 in primary sensory neurons remain unknown. In the present study, we hypothesized that conditional knockout of CB1 in primary sensory neurons (CB1cKO) alters downstream gene expression in the dorsal root ganglion (DRG) and that targeting these pathways alleviates neuropathic pain. We found that CB1cKO in primary sensory neurons induced by tamoxifen in adult Advillin-Cre:CB1-floxed mice showed persistent hyperalgesia. Transcriptome/RNA sequencing analysis of the DRG indicated that differentially expressed genes were enriched in energy regulation and complement and coagulation cascades at the early phase of CB1cKO, whereas pain regulation and nerve conduction pathways were affected at the late phase of CB1cKO. Chronic constriction injury in mice induced neuropathic pain and changed transcriptome expression in the DRG of CB1cKO mice, and differentially expressed genes were mainly associated with inflammatory and immune-related pathways. Nerve injury caused a much larger increase in CB2 expression in the DRG in CB1cKO than in wildtype mice. Interfering with downstream target genes of CB1, such as antagonizing CB2, inhibited activation of astrocytes, reduced neuroinflammation, and alleviated neuropathic pain. Our results demonstrate that CB1 in primary sensory neurons functions as an endogenous analgesic mediator. CB2 expression is regulated by CB1 and may be targeted for the treatment of neuropathic pain.