Cannabichromene and Δ9-Tetrahydrocannabinolic Acid Identified as Lactate Dehydrogenase-A Inhibitors by in Silico and in Vitro Screening.

Cannabis sativa contains >120 phytocannabinoids, but our understanding of these compounds is limited. Determining the molecular modes of action of the phytocannabinoids may assist in their therapeutic development. Ligand-based virtual screening was used to suggest novel protein targets for phytocannabinoids. The similarity ensemble approach, a virtual screening tool, was applied to target identification for the phytocannabinoids as a class and predicted a possible interaction with the lactate dehydrogenase (LDH) family of enzymes. In order to evaluate this in silico prediction, a panel of 18 phytocannabinoids was screened against two LDH isozymes (LDHA and LDHB) in vitro. Cannabichromene (CBC) and Δ9-tetrahydrocannabinolic acid (Δ9-THCA) inhibited LDHA via a noncompetitive mode of inhibition with respect to pyruvate, with Ki values of 8.5 and 6.5 µM, respectively. In silico modeling was then used to predict the binding site for CBC and Δ9-THCA. Both were proposed to bind within the nicotinamide pocket, overlapping the binding site of the cofactor NADH, which is consistent with the noncompetitive modes of inhibition. Stemming from our in silico screen, CBC and Δ9-THCA were identified as inhibitors of LDHA, a novel molecular target that may contribute to their therapeutic effects.

Allosteric Cannabinoid Receptor 1 (CB1) Ligands Reduce Ocular Pain and Inflammation.

Cannabinoid receptor 1 (CB1) activation has been reported to reduce transient receptor potential cation channel subfamily V member 1 (TRPV1)-induced inflammatory responses and is anti-nociceptive and anti-inflammatory in corneal injury. We examined whether allosteric ligands, can modulate CB1 signaling to reduce pain and inflammation in corneal hyperalgesia. Corneal hyperalgesia was generated by chemical cauterization of cornea in wildtype and CB2 knockout (CB2-/-) mice. The novel racemic CB1 allosteric ligand GAT211 and its enantiomers GAT228 and GAT229 were examined alone or in combination with the orthosteric CB1 agonist Δ8-tetrahydrocannabinol (Δ8-THC). Pain responses were assessed following capsaicin (1 µM) stimulation of injured corneas at 6 h post-cauterization. Corneal neutrophil infiltration was also analyzed. GAT228, but not GAT229 or GAT211, reduced pain scores in response to capsaicin stimulation. Combination treatments of 0.5% GAT229 or 1% GAT211 with subthreshold Δ8-THC (0.4%) significantly reduced pain scores following capsaicin stimulation. The anti-nociceptive effects of both GAT229 and GAT228 were blocked with CB1 antagonist AM251, but remained unaffected in CB2-/- mice. Two percent GAT228, or the combination of 0.2% Δ8-THC with 0.5% GAT229 also significantly reduced corneal inflammation. CB1 allosteric ligands could offer a novel approach for treating corneal pain and inflammation.

The Endocannabinoid System as a Therapeutic Target in Glaucoma.

Glaucoma is an irreversible blinding eye disease which produces progressive retinal ganglion cell (RGC) loss. Intraocular pressure (IOP) is currently the only modifiable risk factor, and lowering IOP results in reduced risk of progression of the disorder. The endocannabinoid system (ECS) has attracted considerable attention as a potential target for the treatment of glaucoma, largely due to the observed IOP lowering effects seen after administration of exogenous cannabinoids. However, recent evidence has suggested that modulation of the ECS may also be neuroprotective. This paper will review the use of cannabinoids in glaucoma, presenting pertinent information regarding the pathophysiology of glaucoma and how alterations in cannabinoid signalling may contribute to glaucoma pathology. Additionally, the mechanisms and potential for the use of cannabinoids and other novel agents that target the endocannabinoid system in the treatment of glaucoma will be discussed.

Synthesis and functional evaluation of proteinogenic amino acid-derived synthetic cannabinoid receptor agonists related to MPP-5F-PICA, MMB-5F-PICA, and MDMB-5F-PICA.

Synthetic cannabinoid receptor agonists (SCRAs) comprise the second largest class of new psychoactive substances (NPS), and typically α-amino acid moieties are incorporated as part of their design. Limited investigation has been performed into elucidating structure-activity relationships around commonly used α-amino acid-derived head groups, mainly with valine and tert-leucine-derived compounds previously described. As such, proactive synthesis, characterisation and pharmacological evaluation were performed to explore structure-activity relationships of 15 α-amino acid derivatives, with both the natural isomers and their enantiomers at CB1 and CB2 investigated using a fluorescence-based membrane potential assay. This library was based around the detected SCRAs MPP-5F-PICA, MMB-5F-PICA, and MDMB-5F-PICA, with the latter showing significant receptor activation at CB1 (pEC50 = 8.34 ± 0.05 M; E max = 108 ± 3%) and CB2 (pEC50 = 8.13 ± 0.07 M; E max = 99 ± 2%). Most valine and leucine derivatives were potent and efficacious SCRAs, while smaller derivatives generally showed reduced activity at CB1 and CB2, and larger derivatives also showed reduced activity. SAR trends observed were rationalised via in silico induced fit docking. Overall, while natural enantiomers showed equipotent or greater activity than the unnatural isomers in most cases, this was not universal. As such, a number of these compounds should be monitored as emerging NPS, and various substituents described herein.

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.

Structure-Activity Relationships, Deuteration, and Fluorination of Synthetic Cannabinoid Receptor Agonists Related to AKB48, 5F-AKB-48, and AFUBIATA.

Synthetic cannabinoid receptor agonists (SCRAs) are a growing class of new psychoactive substances (NPS) commonly derived from an N-alkylated indole, indazole, or 7-azaindole scaffold. Diversification of this core (at the 3-position) with amide-linked pendant amino acid groups and modular N-alkylation (of the indole/indazole/7-azaindole core) ensures that novel SCRAs continue to enter the illicit drug market rapidly. In response to the large number of SCRAs that have been detected, pharmacological evaluation of this NPS class has become increasingly common. Adamantane-derived SCRAs have consistently appeared throughout the market since 2011, and as such, a systematic set of these derivatives was synthesized and pharmacologically evaluated. Deuterated and fluorinated adamantane derivatives were prepared to evaluate typical hydrogen bioisosteres, as well as evaluation of the newly detected AFUBIATA.

Medicinal cannabis for psychiatry-related conditions: an overview of current Australian prescribing.

Objective: Evidence is accumulating that components of the Cannabis sativa plant may have therapeutic potential in treating psychiatric disorders. Medicinal cannabis (MC) products are legally available for prescription in Australia, primarily through the Therapeutic Goods Administration (TGA) Special Access Scheme B (SAS-B). Here we investigated recent prescribing practices for psychiatric indications under SAS-B by Australian doctors. Methods: The dataset, obtained from the TGA, included information on MC applications made by doctors through the SAS-B process between 1st November 2016 and 30th September 2022 inclusive. Details included the primary conditions treated, patient demographics, prescriber location, product type (e.g., oil, flower or capsule) and the general cannabinoid content of products. The conditions treated were categorized according to the Diagnostic and Statistical Manual of Mental Disorders, 5th edition, text revision (DSM-5-TR). Trends in prescribing for conditions over time were analyzed via polynomial regression, and relationships between categorical variables determined via correspondence analyses. Results: Approximately 300,000 SAS-B approvals to prescribe MC had been issued in the time period under investigation. This included approvals for 38 different DSM-5-TR defined psychiatric conditions (33.9% of total approvals). The majority of approvals were for anxiety disorders (66.7% of psychiatry-related prescribing), sleep-wake disorders (18.2%), trauma- and stressor-related disorders (5.8%), and neurodevelopmental disorders (4.4%). Oil products were most prescribed (53.0%), followed by flower (31.2%) and other inhaled products (12.4%). CBD-dominant products comprised around 20% of total prescribing and were particularly prevalent in the treatment of autism spectrum disorder. The largest proportion of approvals was for patients aged 25-39 years (46.2% of approvals). Recent dramatic increases in prescribing for attention deficit hyperactivity disorder were identified. Conclusion: A significant proportion of MC prescribing in Australia is for psychiatry-related indications. This prescribing often appears somewhat "experimental", given it involves conditions (e.g., ADHD, depression) for which definitive clinical evidence of MC efficacy is lacking. The high prevalence of THC-containing products being prescribed is of possible concern given the psychiatric problems associated with this drug. Evidence-based clinical guidance around the use of MC products in psychiatry is lacking and would clearly be of benefit to prescribers.

Comprehensive Characterization of a Systematic Library of Alkyl and Alicyclic Synthetic Cannabinoids Related to CUMYL-PICA, CUMYL-BUTICA, CUMYL-CBMICA, and CUMYL-PINACA.

Over 200 synthetic cannabinoid receptor agonists (SCRAs) have been identified as new psychoactive substances. Effective monitoring and characterization of SCRAs are hindered by the rapid pace of structural evolution. Ahead of possible appearance on the illicit drug market, new SCRAs were synthesized to complete a systematic library of cumyl-indole- (e.g., CUMYL-CPrMICA, CUMYL-CPMICA) and cumyl-indazole-carboxamides (e.g., CUMYL-CPrMINACA, CUMYL-CPMINACA), encompassing butyl, pentyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, and cyclohexylmethyl tails. Comprehensive pharmacological characterization was performed with three assay formats, monitoring the recruitment of either wild-type or C-terminally truncated (ßarr2d366) ß-arrestin2 to the activated cannabinoid 1 receptor (CB1) or monitoring Gßγ-mediated membrane hyperpolarization. Altered compound characterization was observed when comparing derived potency (EC50) and efficacy (Emax) values from both assays monitoring the same or a different signaling event, whereas ranges and ranking orders were similar. Structure-activity relationships (SAR) were assessed in threefold, resulting in the identification of the pendant tail as a critical pharmacophore, with the optimal chain length for CB1 activation approximating an n-pentyl (e.g., cyclopentylmethyl or cyclohexylmethyl tail). The activity of the SCRAs encompassing cyclic tails decreased with decreasing number of carbons forming the cyclic moiety, with CUMYL-CPrMICA showing the least CB1 activity in all assay formats. The SARs were rationalized via molecular docking, demonstrating the importance of the optimal steric contribution of the hydrophobic tail. While SAR conclusions remained largely unchanged, the differential compound characterization by both similar and different assay designs emphasizes the importance of detailing specific assay characteristics to allow adequate interpretation of potencies and efficacies.

Off-target pharmacological profiling of synthetic cannabinoid receptor agonists including AMB-FUBINACA, CUMYL-PINACA, PB-22, and XLR-11.

Introduction: Synthetic cannabinoid receptor agonists (SCRAs) are a diverse class of new psychoactive substances that have been associated with multiple instances and types of toxicity. Some SCRAs appear to carry a greater toxicological burden than others, or compared to the prototypical cannabis-derived agonist Δ9-tetrahydrocannabinol (Δ9-THC), despite a common primary mechanism of action via cannabinoid 1 (CB1) receptors. "Off-target" (i.e., non-CB1 receptor) effects could underpin this differential toxicity, although there are limited data around the activity of SCRAs at such targets. Methods: A selection of 7 SCRAs (AMB-FUBINACA, XLR11, PB-22, AKB-48, AB-CHMINICA, CUMYL-PINACA, and 4F-MDMB-BUTINACA), representing several distinct chemotypes and toxicological profiles, underwent a 30 µM single-point screen against 241 G protein-coupled receptor (GPCR) targets in antagonist and agonist mode using a cellular ß-arrestin recruitment assay. Strong screening "hits" at specific GPCRs were followed up in detail using concentration-response assays with AMB-FUBINACA, a SCRA with a particularly notable history of toxicological liability. Results: The single-point screen yielded few hits in agonist mode for any compound aside from CB1 and CB2 receptors, but many hits in antagonist mode, including a range of chemokine receptors, the oxytocin receptor, and histamine receptors. Concentration-response experiments showed that AMB-FUBINACA inhibited most off-targets only at the highest 30 µM concentration, with inhibition of only a small subset of targets, including H1 histamine and α2B adrenergic receptors, at lower concentrations (≥1 µM). AMB-FUBINACA also produced concentration-dependent CB1 receptor signaling disruption at concentrations higher than 1 µM, but did not produce overt cytotoxicity beyond CP55,940 or Δ9-THC in CB1 expressing cells. Discussion: These results suggest that while some "off-targets" could possibly contribute to the SCRA toxidrome, particularly at high concentrations, CB1-mediated cellular dysfunction provides support for hypotheses concerning on-target, rather than off-target, toxicity. Further investigation of non-GPCR off-targets is warranted.

Medicinal Cannabis Prescribing in Australia: An Analysis of Trends Over the First Five Years.

A regulatory framework allowing legal access to medicinal cannabis (MC) products has operated in Australia since November 2016. MC prescribing by healthcare practitioners (HCPs) is primarily conducted through the Special Access Scheme - Category B (SAS-B) pathway, through which prescribers apply to the Therapeutic Goods Administration (TGA-the federal regulator) for approval to prescribe a category of product to an individual patient suffering from a specific indication. The dataset collected by the TGA provides a unique opportunity to examine MC prescribing trends over time in the Australian population. Here we analysed this TGA SAS-B dataset since inception with respect to age, gender, product type (e.g., oil, flower, etc.), CBD content, indication treated, and prescriber location. Results are presented descriptively as well as being analysed using non-linear regression models. Relationship between variables were explored via correspondence analyses. Indications were classified with reference to the International Statistical Classification of Diseases and Related Health Problems (10th Revision). As of 31 August 2021, a total of 159,665 SAS-B approvals had been issued for MC products, 82.4% of were since January 2020. Leading indications for approvals were for pain, anxiety, and sleep disorders. Oil products were the most popular product type, while CBD-dominant products (≥98% CBD) accounted for 25.1% of total approvals. Approvals for flower products increased markedly during 2020-2021, as did approvals involving younger age groups (18-31 years old), male patients, and non-CBD dominant products. A disproportionate number of SAS-B MC applications (around 50%) came from HCPs in the state of Queensland. Associations between patient gender and age and/or indication with product type were found. For example, approvals for oil products were commonly associated with approvals for pain. While, overall prescribing increased dramatically over the last 2 years of analysis, stabilization of approval numbers is evident for some indications, such as pain. Current prescribing practices do not always reflect provided TGA guidance documents for MC prescribing. While acknowledging some limitations around the SAS-B dataset, it provides a unique and valuable resource with which to better understand current prescribing practices and utilisation of MC products within Australia.

Synthesis and pharmacological evaluation of newly detected synthetic cannabinoid receptor agonists AB-4CN-BUTICA, MMB-4CN-BUTINACA, MDMB-4F-BUTICA, MDMB-4F-BUTINACA and their analogs.

Synthetic cannabinoid receptor agonists (SCRAs) continue to make up a significant portion new psychoactive substances (NPS) detected and seized worldwide. Due to their often potent activation of central cannabinoid receptors in vivo, use of SCRAs can result in severe intoxication, in addition to other adverse health effects. Recent detections of AB-4CN-BUTICA, MMB-4CN-BUTINACA, MDMB-4F-BUTICA and MDMB-4F-BUTINACA mark a continuation in the appearance of SCRAs bearing novel tail substituents. The proactive characterization campaign described here has facilitated the detection of several new SCRAs in toxicological case work. Here we detail the synthesis, characterization, and pharmacological evaluation of recently detected SCRAs, as well as a systematic library of 32 compounds bearing head, tail, and core group combinations likely to appear in future. In vitro radioligand binding assays revealed most compounds showed moderate to high affinity at both CB1 (pK i = < 5 to 8.89 ± 0.09 M) and CB2 (pK i = 5.49 ± 0.03 to 9.92 ± 0.09 M) receptors. In vitro functional evaluation using a fluorescence-based membrane potential assay showed that most compounds were sub-micromolar to sub-nanomolar agonists at CB1 (pEC50 = < 5 to 9.48 ± 0.14 M) and CB2 (pEC50 = 5.92 ± 0.16 to 8.64 ± 0.15 M) receptors. An in silico receptor-ligand docking approach was utilized to rationalize binding trends for CB2 with respect to the tail substituent, and indicated that rigidity in this region (i.e., 4-cyanobutyl) was detrimental to affinity.

Structure-activity relationships of valine, tert-leucine, and phenylalanine amino acid-derived synthetic cannabinoid receptor agonists related to ADB-BUTINACA, APP-BUTINACA, and ADB-P7AICA.

Synthetic cannabinoid receptor agonists (SCRAs) remain one the most prevalent classes of new psychoactive substances (NPS) worldwide, and examples are generally poorly characterised at the time of first detection. We have synthesised a systematic library of amino acid-derived indole-, indazole-, and 7-azaindole-3-carboxamides related to recently detected drugs ADB-BUTINACA, APP-BUTINACA and ADB-P7AICA, and characterised these ligands for in vitro binding and agonist activity at cannabinoid receptor subtypes 1 and 2 (CB1 and CB2), and in vivo cannabimimetic activity. All compounds showed high affinity for CB1 (K i 0.299-538 nM) and most at CB2 (K i = 0.912-2190 nM), and most functioned as high efficacy agonists of CB1 and CB2 in a fluorescence-based membrane potential assay and a ßarr2 recruitment assay (NanoBiT®), with some compounds being partial agonists in the NanoBiT® assay. Key structure-activity relationships (SARs) were identified for CB1/CB2 binding and CB1/CB2 functional activities; (1) for a given core, affinities and potencies for tert-leucinamides (ADB-) > valinamides (AB-) ≫ phenylalaninamides (APP-); (2) for a given amino acid side-chain, affinities and potencies for indazoles > indoles ≫ 7-azaindoles. Radiobiotelemetric evaluation of ADB-BUTINACA, APP-BUTINACA and ADB-P7AICA in mice demonstrated that ADB-BUTINACA and ADB-P7AICA were cannabimimetic at 0.1 mg kg-1 and 10 mg kg-1 doses, respectively, as measured by pronounced decreases in core body temperature. APP-BUTINACA failed to elicit any hypothermic response up to the maximally tested 10 mg kg-1 dose, yielding an in vivo potency ranking of ADB-BUTINACA > ADB-P7AICA > APP-BUTINACA.

Putative Synthetic Cannabinoids MEPIRAPIM, 5F-BEPIRAPIM (NNL-2), and Their Analogues Are T-Type Calcium Channel (CaV3) Inhibitors.

Synthetic cannabinoid receptor agonists (SCRAs) are a large and growing class of new psychoactive substances (NPSs). Two recently identified compounds, MEPIRAPIM and 5F-BEPIRAPIM (NNL-2), have not been confirmed as agonists of either cannabinoid receptor subtype but share structural similarities with both SCRAs and a class of T-type calcium channel (CaV3) inhibitors under development as new treatments for epilepsy and pain. In this study, MEPIRAPIM and 5F-BEPIRAPIM and 10 systematic analogues were synthesized, analytically characterized, and pharmacologically evaluated using in vitro cannabinoid receptor and CaV3 assays. Several compounds showed micromolar affinities for CB1 and/or CB2, with several functioning as low potency agonists of CB1 and CB2 in a membrane potential assay. 5F-BEPIRAPIM and four other derivatives were identified as potential CaV3 inhibitors through a functional calcium flux assay (>70% inhibition), which was further confirmed using whole-cell patch-clamp electrophysiology. Additionally, MEPIRAPIM and 5F-BEPIRAPIM were evaluated in vivo using a cannabimimetic mouse model. Despite detections of MEPIRAPIM and 5F-BEPIRAPIM in the NPS market, only the highest MEPIRAPIM dose (30 mg/kg) elicited a mild hypothermic response in mice, with no hypothermia observed for 5F-BEPIRAPIM, suggesting minimal central CB1 receptor activity.

Medicinal cannabis in the treatment of chronic pain.

BACKGROUND: Chronic pain is a major health issue, adversely affecting millions of Australians and costing billions of dollars annually. Current pharmaceutical treatments may be limiting, and in some cases ineffective, while carrying substantial liabilities. Medicinal cannabis is an increasingly popular, albeit controversial, alternative. OBJECTIVE: The aim of this article is to briefly review the scientific evidence related to medicinal cannabis for the treatment of chronic pain and update physicians on relevant issues and optimal prescribing practices. DISCUSSION: To date, >130,000 medicinal cannabis approvals have been issued in Australia, mostly by general practitioners, with approximately 65% of these to treat chronic non-cancer pain. Available products deliver Δ9-tetrahydrocannabinol (THC) and/or cannabidiol (CBD). Despite robust supportive data from animal models, current clinical trial evidence for THC and CBD efficacy in chronic pain is incomplete. In their prescribing decisions, doctors must balance patient demand and curiosity with caution regarding potential risks and limited efficacy.

Systematic evaluation of a panel of 30 synthetic cannabinoid receptor agonists structurally related to MMB-4en-PICA, MDMB-4en-PINACA, ADB-4en-PINACA, and MMB-4CN-BUTINACA using a combination of binding and different CB1 receptor activation assays: Part I-Synthesis, analytical characterization, and binding affinity for human CB1 receptors.

Synthetic cannabinoid receptor agonists (SCRAs) are one of the largest and most structurally diverse classes of new psychoactive substances (NPS). Despite this, pharmacological data are often lacking following the identification of a new SCRA in drug markets. In this first of a three-part series, we describe the synthesis, analytical characterization, and binding affinity of a proactively generated, systematic library of 30 indole, indazole, and 7-azaindole SCRAs related to MMB-4en-PICA, MDMB-4en-PINACA, ADB-4en-PINACA, and MMB-4CN-BUTINACA featuring a 4-pentenyl (4en-P), butyl (B/BUT), or 4-cyanobutyl (4CN-B/BUT) tail and a methyl l-valinate (MMB), methyl l-tert-leucinate (MDMB), methyl l-phenylalaninate (MPP), l-valinamide (AB), l-tert-leucinamide (ADB), l-phenylalaninamide (APP), adamantyl (A), or cumyl head group. Competitive radioligand binding assays demonstrated that the indazole core conferred the highest CB1 binding affinity (Ki = 0.17-39 nM), followed by indole- (Ki = 0.95-160 nM) and then 7-azaindole-derived SCRAs (Ki = 5.4-271 nM). Variation of the head group had the greatest effect on binding, with tert-leucine amides and methyl esters (Ki = 0.17-14 nM) generally showing the greatest affinities, followed by valine derivatives (Ki = 0.72-180 nM), and then phenylalanine derivatives (Ki = 2.5-271 nM). Adamantyl head groups (Ki = 8.8-59 nM) were suboptimal for binding, whereas the cumyl analogues consistently conferred high affinity (Ki = 0.62-36 nM). Finally, both butyl (Ki = 3.1-163 nM) and 4-cyanobutyl (Ki = 5.5-44 nM) tail groups were less favorable for CB1 binding than their corresponding 4-pentenyl counterparts (Ki = 0.72-25 nM).

NNL-3: A Synthetic Intermediate or a New Class of Hydroxybenzotriazole Esters with Cannabinoid Receptor Activity?

Synthetic cannabinoid receptor agonists (SCRAs) remain a prolific class of new psychoactive substances (NPS) and continue to expand rapidly. Despite the recent identification of hydroxybenzotriazole (HOBt) containing SCRAs in synthetic cannabis samples, there is currently no information regarding the pharmacological profile of these NPS with respect to human CB1 and CB2 receptors. In the current study, a series consisting of seven HOBt indole-, indazole-, and 7-azaindole-carboxylates bearing a range of N-alkyl substituents were synthesized and pharmacologically evaluated. Competitive binding assays at CB1 and CB2 demonstrated that all analogues except a 2-methyl-substituted derivative had low affinity for CB1 (Ki = 3.80-43.7 µM) and CB2 (Ki = 2.75-18.2 µM). A fluorometric functional assay revealed that 2-methylindole- and indole-derived HOBt carboxylates were potent and efficacious agonists of CB1 (EC50 = 12.0 and 63.7 nM; Emax = 118 and 120%) and CB2 (EC50 = 10.9 and 321 nM; Emax = 91 and 126%). All other analogues incorporating indazole and 7-azaindole cores and bearing a range of N1-substituents showed relatively low potency for CB1 and CB2. Additionally, a reporter assay monitoring ß-arrestin 2 (ßarr2) recruitment to the receptor revealed that the 2-methylindole example was the most potent and efficacious at CB1 (EC50 = 131 nM; Emax = 724%) and the most potent at CB2 (EC50 = 38.2 nM; Emax = 51%). As with the membrane potential assay, the indazole and other indole HOBt carboxylates were considerably less potent at both receptors, and analogues comprising a 7-azaindole core showed little activity. Taken together, these data suggest that NNL-3 demonstrates little CB1 receptor activity and is unlikely to be psychoactive in humans. NNL-3 is likely an unintended SCRA manufacturing byproduct. However, the synthesis of NNL-3 analogues proved simple and general, and some of these showed potent cannabimetic profiles in vitro, indicating that HOBt esters of this type may represent an emerging class of SCRA NPS.

Access to cannabidiol without a prescription: A cross-country comparison and analysis.

BACKGROUND: Recent legislative change has allowed increased access to cannabis products in many jurisdictions. In some locations, this includes over-the-counter (OTC) and/or online access to products containing cannabidiol (CBD), a non-intoxicating cannabinoid with therapeutic properties. Here we compared the availability of CBD products and the associated legislative and regulatory background in nine selected countries. METHODS: Accessibility of CBD products was examined in the USA, Canada, Germany, Ireland, United Kingdom, Switzerland, Japan, Australia, and New Zealand as of May 2020. Regulatory and other relevant documents were obtained from government agency websites and related sources. Relevant commercial websites and some physical retailers were visited to verify access to CBD-containing products and the nature of the products available. RESULTS: A range of CBD products appeared to be accessible without prescription in seven out of nine countries reviewed. Australia and New Zealand were the exceptions where clinician prescription was required to access any CBD-containing product. CBD products commonly available without prescription included oils, gel capsules, purified crystal and topical products. The daily recommended doses with orally administered non-prescription products were typically well below 150 mg and substantially lower than the doses reported to have therapeutic effects in published clinical trials (e.g., 300-1500 mg). The legal foundations enabling access in several countries were often unclear, with marketed products sometimes failing to meet legal requirements for sale. There was an obvious disparity between federal directives and available products in both the USA and European countries examined. CONCLUSIONS: There are a variety of approaches in how countries manage access to CBD products. Many countries appear to permit OTC and online availability of CBD products but often without legislative clarity. As consumer demand for CBD escalates, improved legislation, guidelines and quality control of CBD products would seem prudent together with clinical trials exploring the therapeutic benefits of lower-dose CBD formulations.

Positive allosteric modulation of the type 1 cannabinoid receptor reduces the signs and symptoms of Huntington's disease in the R6/2 mouse model.

Huntington's disease (HD) is an inherited progressive neurodegenerative disease characterized by motor, cognitive, and behavioural changes. One of the earliest changes to occur in HD is a reduction in cannabinoid 1 receptor (CB1) levels in the striatum, which is strongly correlated with HD pathogenesis. CB1 positive allosteric modulators (PAM) enhance receptor affinity for, and efficacy of activation by, orthosteric ligands, including the endocannabinoids anandamide and 2-arachidonoylglycerol. The goal of this study was to determine whether the recently characterized CB1 allosteric modulators GAT211 (racemic), GAT228 (R-enantiomer), and GAT229 (S-enantiomer), affected the signs and symptoms of HD. GAT211, GAT228, and GAT229 were evaluated in normal and HD cell models, and in a transgenic mouse model of HD (7-week-old male R6/2 mice, 10 mg/kg/d, 21 d, i.p.). GAT229 was a CB1 PAM that improved cell viability in HD cells and improved motor coordination, delayed symptom onset, and normalized gene expression in R6/2 HD mice. GAT228 was an allosteric agonist that did not enhance endocannabinoid signaling or change symptom progression in R6/2 mice. GAT211 displayed intermediate effects between its enantiomers. The compounds used here are not drugs, but probe compounds used to determine the potential utility of CB1 PAMs in HD. Changes in gene expression, and not protein, were quantified in R6/2 HD mice because HD pathogenesis is associated with dysregulation of mRNA levels. The data presented here provide the first proof of principle for the use of CB1 PAMs to treat the signs and symptoms of HD.

The Cannabinoids Δ8THC, CBD, and HU-308 Act via Distinct Receptors to Reduce Corneal Pain and Inflammation.

Background and Purpose: Corneal injury can result in dysfunction of corneal nociceptive signaling and corneal sensitization. Activation of the endocannabinoid system has been reported to be analgesic and anti-inflammatory. The purpose of this research was to investigate the antinociceptive and anti-inflammatory effects of cannabinoids with reported actions at cannabinoid 1 (CB1R) and cannabinoid 2 (CB2R) receptors and/or noncannabinoid receptors in an experimental model of corneal hyperalgesia. Methods: Corneal hyperalgesia (increased pain response) was generated using chemical cauterization of the corneal epithelium in wild-type (WT) and CB2R knockout (CB2R-/-) mice. Cauterized eyes were treated topically with the phytocannabinoids Δ8-tetrahydrocannabinol (Δ8THC) or cannabidiol (CBD), or the CBD derivative HU-308, in the presence or absence of the CB1R antagonist AM251 (2.0 mg/kg i.p.), or the 5-HT1A receptor antagonist WAY100635 (1 mg/kg i.p.). Behavioral pain responses to a topical capsaicin challenge at 6 h postinjury were quantified from video recordings. Mice were euthanized at 6 and 12 h postcorneal injury for immunohistochemical analysis to quantify corneal neutrophil infiltration. Results: Corneal cauterization resulted in hyperalgesia to capsaicin at 6 h postinjury compared to sham control eyes. Neutrophil infiltration, indicative of inflammation, was apparent at 6 and 12 h postinjury in WT mice. Application of Δ8THC, CBD, and HU-308 reduced the pain score and neutrophil infiltration in WT mice. The antinociceptive and anti-inflammatory actions of Δ8THC, but not CBD, were blocked by the CB1R antagonist AM251, but were still apparent, for both cannabinoids, in CB2R-/- mice. However, the antinociceptive and anti-inflammatory actions of HU-308 were absent in the CB2R-/- mice. The antinociceptive and anti-inflammatory effects of CBD were blocked by the 5-HT1A antagonist WAY100635. Conclusion: Topical cannabinoids reduce corneal hyperalgesia and inflammation. The antinociceptive and anti-inflammatory effects of Δ8THC are mediated primarily via CB1R, whereas that of the cannabinoids CBD and HU-308, involve activation of 5-HT1A receptors and CB2Rs, respectively. Cannabinoids could be a novel clinical therapy for corneal pain and inflammation resulting from ocular surface injury.