Activity-based detection of synthetic cannabinoid receptor agonists in plant materials.

BACKGROUND: Since late 2019, fortification of 'regular' cannabis plant material with synthetic cannabinoid receptor agonists (SCRAs) has become a notable phenomenon on the drug market. As many SCRAs pose a higher health risk than genuine cannabis, recognizing SCRA-adulterated cannabis is important from a harm reduction perspective. However, this is not always an easy task as adulterated cannabis may only be distinguished from genuine cannabis by dedicated, often expensive and time-consuming analytical techniques. In addition, the dynamic nature of the SCRA market renders identification of fortified samples a challenging task. Therefore, we established and applied an in vitro cannabinoid receptor 1 (CB1) activity-based procedure to screen plant material for the presence of SCRAs. METHODS: The assay principle relies on the functional complementation of a split-nanoluciferase following recruitment of ß-arrestin 2 to activated CB1. A straightforward sample preparation, encompassing methanolic extraction and dilution, was optimized for plant matrices, including cannabis, spiked with 5 µg/mg of the SCRA CP55,940. RESULTS: The bioassay successfully detected all samples of a set (n = 24) of analytically confirmed authentic Spice products, additionally providing relevant information on the 'strength' of a preparation and whether different samples may have originated from separate batches or possibly the same production batch. Finally, the methodology was applied to assess the occurrence of SCRA adulteration in a large set (n = 252) of herbal materials collected at an international dance festival. This did not reveal any positives, i.e. there were no samples that yielded a relevant CB1 activation. CONCLUSION: In summary, we established SCRA screening of herbal materials as a new application for the activity-based CB1 bioassay. The simplicity of the sample preparation, the rapid results and the universal character of the bioassay render it an effective and future-proof tool for evaluating herbal materials for the presence of SCRAs, which is relevant in the context of harm reduction.

Characterization of the effects of cannabinoid receptor deletion on energy metabolism in female C57BL mice.

Background: Despite the evidence that energy balance is regulated differently in females and that the endocannabinoid system is sexually dimorphic, previous studies on the endocannabinoid system and energy balance predominantly used male models. Here, we characterize the effects of cannabinoid receptor deletion on body weight gain and glucose metabolism in female C57BL mice. Methods: Female mice lacking the cannabinoid-1 receptor (CB1R-/-), cannabinoid-2 receptor (CB2R-/-), or both receptors (CB1R-/-/CB2R-/-) and wild-type (WT) mice were fed with a low (LFD; 10% of calories from fat) or high-fat diet (HFD; 45% of calories from fat) for six weeks. Results: Female WT mice fed with HFD gained significantly more weight than WT mice fed with LFD (p < 0.001). Similar pattern was observed for CB2/- mice fed with HFD compared to CB2R-/- mice fed with LFD (p < 0.001), but not for CB1R-/- fed with HFD vs. LFD (p = 0.22) or CB1R-/-/CB2R-/- fed with HFD vs. LFD (p = 0.96). Comparing the 4 groups on LFD, weight gain of CB1R-/- mice was greater than all other genotypes (p < 0.05). When fed with HFD, the deletion of CB1R alone in females did not attenuate weight gain compared to WT mice (p = 0.72). Female CB1R-/-/CB2R-/- mice gained less weight than WT mice when fed with HFD (p = 0.007) despite similar food intake and locomotor activity, potentially owing to enhanced thermogenesis in the white adipose tissue. No significant difference in weight gain was observed for female CB2R-/- and WT mice on LFD or HFD. Fasting glucose, however, was higher in CB2R-/- mice fed with LFD than all other groups (p < 0.05). Conclusion: The effects of cannabinoid receptor deletion on glucose metabolism in female mice were similar to previously published findings on male mice, yet the effects on body weight gain and thermogenesis were attenuated in CB1R-/- mice.

SGIP1 binding to the α-helical H9 domain of cannabinoid receptor 1 promotes axonal surface expression.

Endocannabinoid signalling mediated by cannabinoid receptor 1 (CB1R, also known as CNR1) is critical for homeostatic neuromodulation of both excitatory and inhibitory synapses. This requires highly polarised axonal surface expression of CB1R, but how this is achieved remains unclear. We previously reported that the α-helical H9 domain in the intracellular C terminus of CB1R contributes to axonal surface expression by an unknown mechanism. Here, we show in rat primary neuronal cultures that the H9 domain binds to the endocytic adaptor protein SGIP1 to promote CB1R expression in the axonal membrane. Overexpression of SGIP1 increases CB1R axonal surface localisation but has no effect on CB1R lacking the H9 domain (CB1RΔH9). Conversely, SGIP1 knockdown reduces axonal surface expression of CB1R but does not affect CB1RΔH9. Furthermore, SGIP1 knockdown diminishes CB1R-mediated inhibition of presynaptic Ca2+ influx in response to neuronal activity. Taken together, these data advance mechanistic understanding of endocannabinoid signalling by demonstrating that SGIP1 interaction with the H9 domain underpins axonal CB1R surface expression to regulate presynaptic responsiveness.

A Humanized CB1R Yeast Biosensor Enables Facile Screening of Cannabinoid Compounds.

Yeast expression of human G-protein-coupled receptors (GPCRs) can be used as a biosensor platform for the detection of pharmaceuticals. Cannabinoid receptor type 1 (CB1R) is of particular interest, given the cornucopia of natural and synthetic cannabinoids being explored as therapeutics. We show for the first time that engineering the N-terminus of CB1R allows for efficient signal transduction in yeast, and that engineering the sterol composition of the yeast membrane modulates its performance. Using an engineered cannabinoid biosensor, we demonstrate that large libraries of synthetic cannabinoids and terpenes can be quickly screened to elucidate known and novel structure-activity relationships. The biosensor strains offer a ready platform for evaluating the activity of new synthetic cannabinoids, monitoring drugs of abuse, and developing therapeutic molecules.

Conditional Knockout of Neurexins Alters the Contribution of Calcium Channel Subtypes to Presynaptic Ca2+ Influx.

Presynaptic Ca2+ influx through voltage-gated Ca2+ channels (VGCCs) is a key signal for synaptic vesicle release. Synaptic neurexins can partially determine the strength of transmission by regulating VGCCs. However, it is unknown whether neurexins modulate Ca2+ influx via all VGCC subtypes similarly. Here, we performed live cell imaging of synaptic boutons from primary hippocampal neurons with a Ca2+ indicator. We used the expression of inactive and active Cre recombinase to compare control to conditional knockout neurons lacking either all or selected neurexin variants. We found that reduced total presynaptic Ca2+ transients caused by the deletion of all neurexins were primarily due to the reduced contribution of P/Q-type VGCCs. The deletion of neurexin1α alone also reduced the total presynaptic Ca2+ influx but increased Ca2+ influx via N-type VGCCs. Moreover, we tested whether the decrease in Ca2+ influx induced by activation of cannabinoid receptor 1 (CB1-receptor) is modulated by neurexins. Unlike earlier observations emphasizing a role for ß-neurexins, we found that the decrease in presynaptic Ca2+ transients induced by CB1-receptor activation depended more strongly on the presence of α-neurexins in hippocampal neurons. Together, our results suggest that neurexins have unique roles in the modulation of presynaptic Ca2+ influx through VGCC subtypes and that different neurexin variants may affect specific VGCCs.

Inhibiting the CB1 receptor in CIH-induced animal model alleviates colon injury.

Obstructive sleep apnea (OSA) can lead to intestinal injury, endotoxemia, and disturbance of intestinal flora. Additionally, as a crucial component of the endocannabinoid system, some studies have demonstrated that cannabinoid 1 (CB1) receptors are closely linked to the multiple organ dysfunction triggered by OSA. However, the role of the CB1 receptor in alleviating OSA-induced colon injury remains unclear. Here, through the construction of the OSA classic model, we found that the colon tissue of chronic intermittent hypoxia (CIH)-induced mice exhibited an overexpression of the CB1 receptor. The results of hematoxylin-eosin staining and transmission electron microscopy revealed that inhibition of the CB1 receptor could decrease the gap between the mucosa and muscularis mucosae, alleviate mitochondrial swelling, reduce microvilli shedding, and promote the recovery of tight junctions of CIH-induced mice. Furthermore, CB1 receptor inhibition reduced the levels of metabolic endotoxemia and inflammatory responses, exhibiting significant protective effects on the colon injury caused by CIH. At the molecular level, through western blotting and real-time polymerase chain reaction techniques, we found that inhibiting the CB1 receptor can significantly increase the expression of ZO-1 and Occludin proteins, which are closely related to the maintenance of intestinal mucosal barrier function. Through 16S rRNA high-throughput sequencing and short-chain fatty acid (SCFA) determination, we found that inhibition of the CB1 receptor increased the diversity of the microbial flora and controlled the makeup of intestinal flora. Moreover, butyric acid concentration and the amount of SCFA-producing bacteria, such as Ruminococcaceae and Lachnospiraceae, were both markedly elevated by CB1 receptor inhibition. The results of the spearman correlation study indicated that Lachnospiraceae showed a positive association with both ZO-1 and Occludin but was negatively correlated with the colon CB1 receptor, IL-1ß, and TNF-α. According to this study, we found that inhibiting CB1 receptor can improve CIH-induced colon injury by regulating gut microbiota, reducing mucosal damage and promoting tight junction recovery. KEY POINTS: •CIH leads to overexpression of CB1 receptor in colon tissue. •CIH causes intestinal flora disorder, intestinal mucosal damage, and disruption of tight junctions. •Inhibition of CB1 receptor can alleviate the colon injury caused by CIH through regulating the gut microbiota, reducing mucosal injury, and promoting tight junction recovery.

Structure-based identification of a G protein-biased allosteric modulator of cannabinoid receptor CB1.

Cannabis sativa is known for its therapeutic benefit in various diseases including pain relief by targeting cannabinoid receptors. The primary component of cannabis, Δ9-tetrahydrocannabinol (THC), and other agonists engage the orthosteric site of CB1, activating both Gi and ß-arrestin signaling pathways. The activation of diverse pathways could result in on-target side effects and cannabis addiction, which may hinder therapeutic potential. A significant challenge in pharmacology is the design of a ligand that can modulate specific signaling of CB1. By leveraging insights from the structure-function selectivity relationship (SFSR), we have identified Gi signaling-biased agonist-allosteric modulators (ago-BAMs). Further, two cryoelectron microscopy (cryo-EM) structures reveal the binding mode of ago-BAM at the extrahelical allosteric site of CB1. Combining mutagenesis and pharmacological studies, we elucidated the detailed mechanism of ago-BAM-mediated biased signaling. Notably, ago-BAM CB-05 demonstrated analgesic efficacy with fewer side effects, minimal drug toxicity and no cannabis addiction in mouse pain models. In summary, our finding not only suggests that ago-BAMs of CB1 provide a potential nonopioid strategy for pain management but also sheds light on BAM identification for GPCRs.

Cannabinoid receptor 1 positive allosteric modulator ZCZ011 shows differential effects on behavior and the endocannabinoid system in HIV-1 Tat transgenic female and male mice.

The cannabinoid receptor type 1 (CB1R) is a promising therapeutic target for various neurodegenerative diseases, including HIV-1-associated neurocognitive disorder (HAND). However, the therapeutic potential of CB1R by direct activation is limited due to its psychoactive side effects. Therefore, research has focused on indirectly activating the CB1R by utilizing positive allosteric modulators (PAMs). Studies have shown that CB1R PAMs (ZCZ011 and GAT211) are effective in mouse models of Huntington's disease and neuropathic pain, and hence, we assess the therapeutic potential of ZCZ011 in a well-established mouse model of neuroHIV. The current study investigates the effect of chronic ZCZ011 treatment (14 days) on various behavioral paradigms and the endocannabinoid system in HIV-1 Tat transgenic female and male mice. Chronic ZCZ011 treatment (10 mg/kg) did not alter body mass, locomotor activity, or anxiety-like behavior regardless of sex or genotype. However, differential effects were noted in hot plate latency, motor coordination, and recognition memory in female mice only, with ZCZ011 treatment increasing hot plate latency and improving motor coordination and recognition memory. Only minor effects or no alterations were seen in the endocannabinoid system and related lipids except in the cerebellum, where the effect of ZCZ011 was more pronounced in female mice. Moreover, AEA and PEA levels in the cerebellum were positively correlated with improved motor coordination in female mice. In summary, these findings indicate that chronic ZCZ011 treatment has differential effects on antinociception, motor coordination, and memory, based on sex and HIV-1 Tat expression, making CB1R PAMs potential treatment options for HAND without the psychoactive side effects.

Cannabidiol Protects against the Reinstatement of Oxycodone-Induced Conditioned Place Preference in Adolescent Male but Not Female Rats: The Role of MOR and CB1R.

Cannabidiol (CBD), a phytocannabinoid, appeared to satisfy several criteria for a safe approach to preventing drug-taking behavior, including opioids. However, most successful preclinical and clinical results come from studies in adult males. We examined whether systemic injections of CBD (10 mg/kg, i.p.) during extinction of oxycodone (OXY, 3 mg/kg, i.p.) induced conditioned place preference (CPP) could attenuate the reinstatement of CPP brought about by OXY (1.5 mg/kg, i.p.) priming in adolescent rats of both sexes, and whether this effect is sex dependent. Accordingly, a priming dose of OXY produced reinstatement of the previously extinguished CPP in males and females. In both sexes, this effect was linked to locomotor sensitization that was blunted by CBD pretreatments. However, CBD was able to prevent the reinstatement of OXY-induced CPP only in adolescent males and this outcome was associated with an increased cannabinoid 1 receptor (CB1R) and a decreased mu opioid receptor (MOR) expression in the prefrontal cortex (PFC). The reinstatement of CCP in females was associated with a decreased MOR expression, but no changes were detected in CB1R in the hippocampus (HIP). Moreover, CBD administration during extinction significantly potentialized the reduced MOR expression in the PFC of males and showed a tendency to potentiate the reduced MOR in the HIP of females. Additionally, CBD reversed OXY-induced deficits of recognition memory only in males. These results suggest that CBD could reduce reinstatement to OXY seeking after a period of abstinence in adolescent male but not female rats. However, more investigation is required.

Potential of CBD Acting on Cannabinoid Receptors CB1 and CB2 in Ischemic Stroke.

Stroke is one of the leading causes of death. It not only affects adult people but also many children. It is estimated that, every year, 15 million people suffer a stroke worldwide. Among them, 5 million people die, while 5 million people are left permanently disabled. In this sense, the research to find new treatments should be accompanied with new therapies to combat neuronal death and to avoid developing cognitive impairment and dementia. Phytocannabinoids are among the compounds that have been used by mankind for the longest period of history. Their beneficial effects such as pain regulation or neuroprotection are widely known and make them possible therapeutic agents with high potential. These compounds bind cannabinoid receptors CB1 and CB2. Unfortunately, the psychoactive side effect has displaced them in the vast majority of areas. Thus, progress in the research and development of new compounds that show efficiency as neuroprotectors without this psychoactive effect is essential. On the one hand, these compounds could selectively bind the CB2 receptor that does not show psychoactive effects and, in glia, has opened new avenues in this field of research, shedding new light on the use of cannabinoid receptors as therapeutic targets to combat neurodegenerative diseases such as Alzheimer's, Parkinson's disease, or stroke. On the other hand, a new possibility lies in the formation of heteromers containing cannabinoid receptors. Heteromers are new functional units that show new properties compared to the individual protomers. Thus, they represent a new possibility that may offer the beneficial effects of cannabinoids devoid of the unwanted psychoactive effect. Nowadays, the approval of a mixture of CBD (cannabidiol) and Δ9-THC (tetrahydrocannabinol) to treat the neuropathic pain and spasticity in multiple sclerosis or purified cannabidiol to combat pediatric epilepsy have opened new therapeutic possibilities in the field of cannabinoids and returned these compounds to the front line of research to treat pathologies as relevant as stroke.

Pharmacological Evaluation of Cannabinoid Receptor Modulators Using GRABeCB2.0 Sensor.

Cannabinoid receptors CB1R and CB2R are G-protein coupled receptors acted upon by endocannabinoids (eCBs), namely 2-arachidonoylglycerol (2-AG) and N-arachidonoyl ethanolamine (AEA), with unique pharmacology and modulate disparate physiological processes. A genetically encoded GPCR activation-based sensor that was developed recently-GRABeCB2.0-has been shown to be capable of monitoring real-time changes in eCB levels in cultured cells and preclinical models. However, its responsiveness to exogenous synthetic cannabinoid agents, particularly antagonists and allosteric modulators, has not been extensively characterized. This current study expands upon the pharmacological characteristics of GRABeCB2.0 to enhance the understanding of fluorescent signal alterations in response to various functionally indiscriminate cannabinoid ligands. The results from this study could enhance the utility of the GRABeCB2.0 sensor for in vitro as well as in vivo studies of cannabinoid action and may aid in the development of novel ligands.

Placental Cannabinoid Receptor Expression in Preterm Birth.

Background: The cannabinoid receptor (CBR) plays a significant role in oogenesis, pregnancy, and childbirth. It might also play a significant role in preterm birth (PTB). The aim of the study was to investigate the association between the expression of the CBR in the placenta and the incidence of PTB. Methods: This prospective, observational, multicentre preliminary study was conducted on placental samples obtained from 109 women. The study included 95 patients hospitalized due to the high risk of PTB. They were divided into two groups: Group 1, where the expression of the CBR1 and CBR1a was analyzed, and Group 2, in which we examined CBR2 expression. The control group, that is, Group 3, consisted of 14 women who delivered at term, and their placentas were tested for the presence of all three receptor types (CBR1, CBR1a, and CBR2). Results: The study used reverse transcription and real-time PCR methods to assess the expression of CBRs in the placental tissues. The expression of the CBR2, CBR1, and CBR1a receptors was significantly lower in the placentas of women after PTB compared to those after term births, p = 0.038, 0.033, and 0.034, respectively. Conclusions: The presence of CBR mRNA in the human placental tissue was confirmed. The decreased expression of CBRs could serve as an indicator in predicting PTB.

Excessive sucrose consumption reduces synaptic density and increases cannabinoid receptors in Göttingen minipigs.

Diets high in sucrose and fat are becoming more prevalent the world over, accompanied by a raised prevalence of cardiovascular diseases, cancers, diabetes, obesity, and metabolic syndrome. Clinical studies link unhealthy diets with the development of mental health disorders, particularly depression. Here, we investigate the effects of 12 days of sucrose consumption administered as 2 L of 25% sucrose solution daily for 12 days in Göttingen minipigs on the function of brain receptors involved in reward and motivation, regulating feeding, and pre- and post-synaptic mechanisms. Through quantitative autoradiography of cryostat sections containing limbic brain regions, we investigated the effects of sucrose restricted to a 1-h period each morning, on the specific binding of [3H]raclopride on dopamine D2/3 receptors, [3H]UCB-J at synaptic vesicle glycoprotein 2A (SV2A), [3H]MPEPγ at metabotropic glutamate receptor subtype 5 (mGluR5) and [3H]SR141716A at the cannabinoid receptor 1 (CB1). Compared to control diet animals, the sucrose group showed significantly lower [3H]UCB-J and [3H]MPEPγ binding in the prefrontal cortex. The sucrose-consuming minipigs showed higher hippocampal CB1 binding, but unaltered dopamine D2/3 binding compared to the control group. We found that the sucrose diet reduced the synaptic density marker while increasing CB1 binding in limbic brain structures, which may subserve maladaptive changes in appetite regulation and feeding. Further studies of the effects of diets and lifestyle habits on brain neuroreceptor and synaptic density markers are warranted.

Cannabinoid receptors as new targets for HTLV-1 associated myelopathy (HAM/TSP) treatment.

BACKGROUND/AIM: The roles of endocannabinoids are described in immune modulation and neuroprotection. HTLV-1-associated myelopathy (HAM/TSP) is an inflammatory neurodegenerative disease. Therefore, in this study, the interactions of HTLV-1 regulatory factors and host cannabinoid receptors (CBRs) were evaluated in HAM/TSP. METHODS: Nineteen HAM/TSPs, 22 asymptomatic carriers (ACs), and 18 healthy controls (HCs) were enrolled. RNA was extracted from PBMCs and then reverse-transcribed to cDNA. The gene expression of CB1R and CB2R, as well as HTLV-1 proviral load (PVL), Tax and HTLV-1 basic leucine zipper factor (HBZ) were assessed by RT-qPCR. RESULTS: The mean expression of CB1R in ACs (8.51 ± 2.76) was significantly higher than HAMTSPs (1.593 ± 0.74, p = 0.05) and also HCs (0.10 ± 0.039, p = 0.001). The CB2R gene expression level in ACs (2.62±0.44) was significantly higher than HAM/TSPs (0.59 ± 0.15, p = 0.001) and HCs (1.00 ± 0.2, p = 0.006). Meanwhile there was a strong correlation between CB1R and CB2R gene expression levels in the HCs and HAM/TSPs (p = 0.001). HTLV-1-Tax expression in HAM/TSPs (386 ± 104) was higher than ACs (75 ± 32) and statistically significant (p = 0.003). While HTLV-1-HBZ was only expressed in three AC subjects and five HAM/TSPs, thus it cannot be analyzed. CONCLUSION: The up-regulation of CB2R has immunomodulatory effects in inflammatory reactions. While CB1R as a neuroprotective agent may suppress inflammatory reactions in ACs, preventing HAM/TSP. It seems that, like multiple sclerosis (MS), cannabinoid medications are beneficial in HAM/TSP.

Presynaptic nanoscale components of retrograde synaptic signaling.

While our understanding of the nanoscale architecture of anterograde synaptic transmission is rapidly expanding, the qualitative and quantitative molecular principles underlying distinct mechanisms of retrograde synaptic communication remain elusive. We show that a particular form of tonic cannabinoid signaling is essential for setting target cell-dependent synaptic variability. It does not require the activity of the two major endocannabinoid-producing enzymes. Instead, by developing a workflow for physiological, anatomical, and molecular measurements at the same unitary synapse, we demonstrate that the nanoscale stoichiometric ratio of type 1 cannabinoid receptors (CB1Rs) to the release machinery is sufficient to predict synapse-specific release probability. Accordingly, selective decrease of extrasynaptic CB1Rs does not affect synaptic transmission, whereas in vivo exposure to the phytocannabinoid Δ9-tetrahydrocannabinol disrupts the intrasynaptic nanoscale stoichiometry and reduces synaptic variability. These findings imply that synapses leverage the nanoscale stoichiometry of presynaptic receptor coupling to the release machinery to establish synaptic strength in a target cell-dependent manner.

Comparative assessment of CacyBP/SIP, ß-catenin and cannabinoid receptors in the adrenals of hypertensive rats.

Taking into account homeostatic disorders resulting from arterial hypertension and the key importance of CacyBP/SIP, ß-catenin and endocannabinoids in the functioning of many organs, it was decided to assess the presence and distribution of CacyBP/SIP, ß-catenin, CB1 and CB2 in the adrenal glands of hypertensive rats of various aetiology. The study was conducted on the adrenal glands of rats with spontaneous and renovascular hypertension. The expression of CacyBP/SIP, ß-catenin, CB1 and CB2 was detected by immunohistochemistry and real-time PCR method. The results of the present study revealed both lower gene expression and immunoreactivity of CacyBP/SIP in the adrenal glands of all hypertensive groups compared to the normotensive rats. This study demonstrated a reduction in the immunoreactivity and expression of the ß-catenin, CB1 and CB2 genes in the adrenals of 2K1C rats. While in SHR, the reaction showing ß-catenin and CB1 was very weak or negative, and the expression of CB2 in the adrenal glands of these rats increased. The results of this study show, for the first time, marked differences in the expression of CacyBP/SIP, ß-catenin and CB1 and CB2 cannabinoid receptors in the adrenal glands of rats with primary (SHR) and secondary hypertension (2K1C).

[Cannabinoid receptor 1 agonist arachidonyl-2'-chloroethylamide (ACEA) improves sepsis-associated encephalopathy by inhibiting inflammatory factors].

Objective To investigate the impact of the cannabinoid receptor agonist arachidonyl-2'-chloroethylamide (ACEA) on cognitive function in mice with sepsis-associated encephalopathy (SAE). Methods C57BL/6 mice were randomly divided into artificial cerebrospinal fluid (ACSF) and lipopolysaccharide (LPS) groups. The SAE model was established by intraventricular injection of LPS. The severity of sepsis in mice was assessed by sepsis severity score (MSS) and body mass changes. Behavioral paradigms were used to evaluate motor ability (open field test) and cognitive function (contextual fear conditioning test, Y-maze test). To evaluate the effects of ACEA intervention on SAE, mice were randomly assigned to ACSF group, ACEA intervention combined with ACSF group, LPS group, and ACEA intervention combined with LPS group. The dosage of ACEA intervention was 1.5 mg/kg. Real-time quantitative PCR was used to measure the mRNA expression levels of interleukin 1ß (IL-1ß), IL-6, and tumor necrosis factor α (TNF-α) in mouse hippocampal tissues. Western blot analysis was used to assess the protein levels of IL-6 and TNF-α in the hippocampus. Nissl staining was performed to examine neuronal damage in the CA1 region of the mouse hippocampus. Behavioral paradigms were again employed to evaluate motor ability and cognitive function. Results Three days after intraventricular LPS injection, mice exhibited significant cognitive dysfunction, confirming SAE modeling. Compared to the control group, the LPS group showed significant increases in mRNA of inflammatory factors such as IL-6, TNF-α, and IL-1ß, together with significant increases in IL-6 and TNF-α protein levels in the hippocampus, a decrease in Nissl bodies in the CA1 region, and significant cognitive dysfunction. Compared to the LPS group, the ACEA intervention group showed a significant decrease in the mRNA of IL-6, TNF-α, and IL-1ß, a significant reduction in IL-6 and TNF-α protein levels, an increase in Nissl bodies, and improved cognitive function. Conclusion ACEA improves cognitive function in SAE mice by inhibiting the expression levels of inflammatory factors IL-6 and TNF-α.

Synthesis and Functional Evaluation of Synthetic Cannabinoid Receptor Agonists Related to ADB-HEXINACA.

ADB-HEXINACA has been recently reported as a synthetic cannabinoid receptor agonist (SCRA), one of the largest classes of new psychoactive substances (NPSs). This compound marks the entry of the n-hexyl tail group into the SCRA landscape, which has continued in the market with recent, newly detected SCRAs. As such, a proactive characterization campaign was undertaken, including the synthesis, characterization, and pharmacological evaluation of ADB-HEXINACA and a library of 41 closely related analogues. Two in vitro functional assays were employed to assess activity at CB1 and CB2 cannabinoid receptors, measuring Gßγ-coupled agonism through a fluorescence-based membrane potential assay (MPA) and ß-arrestin 2 (ßarr2) recruitment via a live cell-based nanoluciferase complementation reporter assay. ADB-HEXINACA was a potent and efficacious CB1 agonist (CB1 MPA pEC50 = 7.87 ± 0.12 M; Emax = 124 ± 5%; ßarr2 pEC50 = 8.27 ± 0.14 M; Emax = 793 ± 42.5), as were most compounds assessed. Isolation of the heterocyclic core and alkyl tails allowed for the comprehensive characterization of structure-activity relationships in this compound class, which were rationalized in silico via induced fit docking experiments. Overall, most compounds assessed are possibly emerging NPSs.

Cannabidiol attenuates seizure susceptibility and behavioural deficits in adult CDKL5R59X knock-in mice.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is caused by a loss-of-function mutation in CDKL5 gene, encoding a serine-threonine kinase highly expressed in the brain. CDD manifests with early-onset epilepsy, autism, motor impairment and severe intellectual disability. While there are no known treatments for CDD, the use of cannabidiol has recently been introduced into clinical practice for neurodevelopmental disorders. Given the increased clinical utilization of cannabidiol, we examined its efficacy in the CDKL5R59X knock-in (R59X) mice, a CDD model based on a human mutation that exhibits both lifelong seizure susceptibility and behavioural deficits. We found that cannabidiol pre-treatment rescued the increased seizure susceptibility in response to the chemoconvulsant pentylenetetrazol (PTZ), attenuated working memory and long-term memory impairments, and rescued social deficits in adult R59X mice. To elucidate a potential mechanism, we compared the developmental hippocampal and cortical expression of common endocannabinoid (eCB) targets in R59X mice and their wild-type littermates, including cannabinoid type 1 receptor (CB1R), transient receptor potential vanilloid type 1 (TRPV1) and 2 (TRPV2), G-coupled protein receptor 55 (GPR55) and adenosine receptor 1 (A1R). Many of these eCB targets were developmentally regulated in both R59X and wild-type mice. In addition, adult R59X mice demonstrated significantly decreased expression of CB1R and TRPV1 in the hippocampus, and TRPV2 in the cortex, while TRPV1 was increased in the cortex. These findings support the potential for dysregulation of eCB signalling as a plausible mechanism and therapeutic target in CDD, given the efficacy of cannabidiol to attenuate hyperexcitability and behavioural deficits in this disorder.

CB1 Receptor Negative Allosteric Modulators as a Potential Tool to Reverse Cannabinoid Toxicity.

While the opioid crisis has justifiably occupied news headlines, emergency rooms are seeing many thousands of visits for another cause: cannabinoid toxicity. This is partly due to the spread of cheap and extremely potent synthetic cannabinoids that can cause serious neurological and cardiovascular complications-and deaths-every year. While an opioid overdose can be reversed by naloxone, there is no analogous treatment for cannabis toxicity. Without an antidote, doctors rely on sedatives, with their own risks, or 'waiting it out' to treat these patients. We have shown that the canonical synthetic 'designer' cannabinoids are highly potent CB1 receptor agonists and, as a result, competitive antagonists may struggle to rapidly reverse an overdose due to synthetic cannabinoids. Negative allosteric modulators (NAMs) have the potential to attenuate the effects of synthetic cannabinoids without having to directly compete for binding. We tested a group of CB1 NAMs for their ability to reverse the effects of the canonical synthetic designer cannabinoid JWH018 in vitro in a neuronal model of endogenous cannabinoid signaling and also in vivo. We tested ABD1085, RTICBM189, and PSNCBAM1 in autaptic hippocampal neurons that endogenously express a retrograde CB1-dependent circuit that inhibits neurotransmission. We found that all of these compounds blocked/reversed JWH018, though some proved more potent than others. We then tested whether these compounds could block the effects of JWH018 in vivo, using a test of nociception in mice. We found that only two of these compounds-RTICBM189 and PSNCBAM1-blocked JWH018 when applied in advance. The in vitro potency of a compound did not predict its in vivo potency. PSNCBAM1 proved to be the more potent of the compounds and also reversed the effects of JWH018 when applied afterward, a condition that more closely mimics an overdose situation. Lastly, we found that PSNCBAM1 did not elicit withdrawal after chronic JWH018 treatment. In summary, CB1 NAMs can, in principle, reverse the effects of the canonical synthetic designer cannabinoid JWH018 both in vitro and in vivo, without inducing withdrawal. These findings suggest a novel pharmacological approach to at last provide a tool to counter cannabinoid toxicity.