Classical Cannabinoids Research Articles

Divergent synthesis of fractionated Cannabis sativa extract led to multiple cannabinoids C-&O-glycosides with anti-proliferative/anti-metastatic properties

Here, we present an interesting, previously unreported method for fractionating a particular class of cannabinoids from the crude leaf extract of Cannabis sativa using HP-20 resins. In this study, we report a novel method of divergent synthesis of fractionated Cannabis sativa extract, which allows the generation of multiple cannabinoids C- and O-glycosides which react with the glycosyl donor 2,3,4,6-tetra-O-acetyl-d-mannosyl trichloroacetimidate (TAMTA) to create eight C- and O-β-d-cannabinoids glycosides (COCG), which are separated by HPLC and whose structures are characterized by 1D, 2D NMR, and mass spectrometry. These glycosides exhibit improved anti-proliferative and anti-metastatic effects against numerous cancer cell lines in vitro and are more water-soluble and stable than their parent cannabinoids. The in vitro testing of the pure cannabinoids (1–4) and their C- & O-glycosides (1a-4a) and 1b-4b exhibited anti-proliferative and anti-metastatic activities against a panel of eight human cancer cell lines in contrast to their respective parent molecules. Different cancer cell lines' IC50 values varied significantly when their cell viability was compared. In addition to the others, compounds 2a, 3a, 4a, and 2b, 3b were highly potent, with IC50values ranging from 0.74 µM (3a) to 51.40 µM (4a).Although2a(1.42 µM) and3a(0.74 µM) exhibited lower IC50values in the MiaPaca-2 cell line than4a(2.58 µM). But, in addition to the comparable anti-clonogenic activity of4ain MiaPaca-2 and Panc-1 cells, it manifested remarkable anti-invasive activity than either 2a or 3a.In contrast to 2a, 2b, 3a, and 3b and their respective parent compounds,4ahad substantial anti-invasive/anti-metastatic capabilities and possessed anti-proliferative activity.The effects of 4a treatment on MiaPaca-2 and Panc-1 cells include a dose-dependent increase in the expression of E-cadherin and a significant decrease in the expression of Zeb-1, Vimentin, and Snail1. Our results demonstrate that divergent synthesis of fractionated Cannabis sativa extract is a feasible and efficient strategy to produce a library of novel cannabinoid glycosides with improved pharmacological properties and potential anticancer benefits.

Cannabinoids and the endocannabinoid system in immunotherapy: helpful or harmful?

Numerous studies in various cancer models have demonstrated that ingredients of cannabis can influence tumor growth through the endocannabinoid system (ECS), a network of molecules (mediators, receptors, transporters, enzymes) that maintains homeostasis and protection in many tissues. The main constituents of the ECS are the classical cannabinoid (CB) receptors, such as CB1 and CB2, their endogenous ligands (endocannabinoids), and the endocannabinoids’ synthesizing and degrading enzymes. The role of the ECS in cancer is still unclear and its effects often depend on the tumor entity and the expression levels of CB receptors. Many studies have highlighted the tumor cell-killing potential of CB1 agonists. However, cannabis is also known as an immunosuppressant and some data suggest that the use of cannabis during immunotherapy worsens treatment outcomes in cancer patients. CB receptors are widely present in immune cells, and together with monoacylglycerol lipase, the 2-arachidonoylglycerol degrading enzyme, they could be critically involved in the regulation of the immune cell profile of the tumor microenvironment (TME), and hence in tumor progression. So far, data on the impact of the ECS in the immune-TME are still vague. In this review, we discuss the current understanding of the ECS on immunoregulation during tumor growth, and how it might affect the outcome of cancer immunotherapy.

The Implications of Cannabinoid-Induced Metabolic Dysregulation for Cellular Differentiation and Growth

The endocannabinoid system (ECS) governs and coordinates several physiological processes through an integrated signaling network, which is responsible for inducing appropriate intracellular metabolic signaling cascades in response to (endo)cannabinoid stimulation. This intricate cellular system ensures the proper functioning of the immune, reproductive, and nervous systems and is involved in the regulation of appetite, memory, metabolism, and development. Cannabinoid receptors have been observed on both cellular and mitochondrial membranes in several tissues and are stimulated by various classes of cannabinoids, rendering the ECS highly versatile. In the context of growth and development, emerging evidence suggests a crucial role for the ECS in cellular growth and differentiation. Indeed, cannabinoids have the potential to disrupt key energy-sensing metabolic signaling pathways requiring mitochondrial-ER crosstalk, whose functioning is essential for successful cellular growth and differentiation. This review aims to explore the extent of cannabinoid-induced cellular dysregulation and its implications for cellular differentiation.

Immunomodulatory Actions of Cannabinoids: Clinical Correlates and Therapeutic Opportunities for Allergic Inflammation

Endogenously produced cannabinoids as well as phytocannabinoids broadly exhibit anti-inflammatory actions. Recent emergence of cannabis for multiple medical issues combined with reports on potent immunomodulatory actions of distinct components has underscored the therapeutic potential of cannabis. Although synthetic cannabinoids that are based on structural similarities to the existing class of cannabinoids have been on the rise, their application in therapeutics have been limited owing to toxicity concerns. Herein, we review the current literature that details the immunomodulatory actions of cannabinoids. Further, we highlight the complexities of cannabinoid biology and examine the potential inflammatory risks associated with the use of cannabis including potential for toxic interactions between distinct constituents of cannabis.

Cannabinoids: an exploratory review

Since 2000, the synthetic cannabinoids drug market is in a continuous expansion and development. Unfortunately, addictive trends include not only the adult population but also adolescents. The new synthetic cannabinoids compounds have similar effects as the Cannabis plant, but are more potent and present a higher risk for consumers. Globally, the fight against drug production, distribution and use involves several directions, including the development of new analytical tools able to identify these new illicit substances. The aim of this paper is to present a systematic review regarding the class of cannabinoids in terms of their physico-chemical properties and biological effects.

The Enteric Glia and Its Modulation by the Endocannabinoid System, a New Target for Cannabinoid-Based Nutraceuticals?

The enteric nervous system (ENS) is a part of the autonomic nervous system that intrinsically innervates the gastrointestinal (GI) tract. Whereas enteric neurons have been deeply studied, the enteric glial cells (EGCs) have received less attention. However, these are immune-competent cells that contribute to the maintenance of the GI tract homeostasis through supporting epithelial integrity, providing neuroprotection, and influencing the GI motor function and sensation. The endogenous cannabinoid system (ECS) includes endogenous classical cannabinoids (anandamide, 2-arachidonoylglycerol), cannabinoid-like ligands (oleoylethanolamide (OEA) and palmitoylethanolamide (PEA)), enzymes involved in their metabolism (FAAH, MAGL, COX-2) and classical (CB1 and CB2) and non-classical (TRPV1, GPR55, PPAR) receptors. The ECS participates in many processes crucial for the proper functioning of the GI tract, in which the EGCs are involved. Thus, the modulation of the EGCs through the ECS might be beneficial to treat some dysfunctions of the GI tract. This review explores the role of EGCs and ECS on the GI tract functions and dysfunctions, and the current knowledge about how EGCs may be modulated by the ECS components, as possible new targets for cannabinoids and cannabinoid-like molecules, particularly those with potential nutraceutical use.

Development of a new green microextraction technique, switchable hydrophilicity solvent-based homogenous liquid–liquid microextraction, to analyze synthetic cannabinoids in plasma

Synthetic cannabinoid receptor agonists (SCRA) are currently the most popular class of new psychoactive substances (NPS) in several countries, including Brazil. Notwithstanding that, they pose a threat to public health, as little of their toxicological properties is known. Moreover, these SCRAs have different presentations compared to classic cannabinoids and are not usually detected by conventional analytical methods due to their chemical variety. Thus, the development of new methodologies fit to analyze SCRAs is extremely warranted. In that regard, the use of environmentally benign or green approaches has been a trend in the last decades and has gained attention in Forensic Toxicology – so much so that guidelines have been established for that purpose, e.g. green analytical toxicology (GAT). With that in mind, the aim of this work was to develop and validate a quantitative method to analyze SCRAs in plasma by a novel green technique that agrees with the principles of GAT. A switchable hydrophilicity solvent-based homogenous liquid–liquid microextraction (SHS-HLLME) was developed and statistical tools were used to optimize it. Analysis of Variance (ANOVA) was chosen to find the best switchable hydrophilicity solvent (SHS) and Full Factorial and Central Composite design to study the following variables: volume of SHS:HCl 6 M mixture (200–500 μL) and NaOH 10 M (200–500 μL), salting-out effect, and time of extraction (1–5 min). The ANSI/ASB Standard 036, 1st edition 2019 guidelines were used for method validation. All analyses were performed using a UPLC-MS/MS with the multiple reaction monitoring approach. Ethical Committee approval No. 46404121.8.3001.0067. Funded by CNPq 142056/2020-0, CAPES/INSPEQT 16/2020, and FAPESP 21/09857-8. ANOVA showed that between two SHS tested, N,N-dimethylcyclohexylamine and dipropylamine, the latter yielded higher analyte recovery ( P < 0.05). Moreover, the Design of Experiment tools herein used showed that the salting-out effect and time of extraction had no major impact on analyte recovery; in contrast, 500 μL of both SHS:HCl mixture and NaOH yielded higher analyte recovery ( P < 0.05). Once optimized, the proposed technique was successfully validated according to international guidelines with the following parameters: limits of detection: 0.01–0.08 ng/mL; limit of quantitation: 0.1 ng/mL; linearity: 0.1–10 ng/mL; bias and precision < 15%; no carryover was observed and the method proved to be selective for the aimed SCRAs. The matrix effect, recovery efficiency, and process efficiency studies are being currently carried out. The microextraction technique herein developed is based on an acid-base neutralization between HCl and NaOH, making it an approach significantly more sustainable when compared to conventional choices that rely entirely on organic solvents. In that regard, only 250 μL of dipropylamine was required–an organic solvent considerably less toxic than those used in common extractions, organochlorides for example. In addition, a small volume was used compared to more conventional techniques in forensic analyses, such as solid-phase extraction (> 500 μL). Hence, we were able to successfully detect and quantitate 31 SCRAs at extremely low concentrations (0.01–0.1 ng/mL, respectively) using only 300 μL of plasma, showing that the SHS-HLLME can be used for the analysis of multiple SCRAs in a single run, thus could be a valuable tool in forensic cases. We demonstrated for the first time the application of the novel SHS-HLLME for synthetic cannabinoids. The microextraction technique herein developed has promising applications in Forensic Toxicology, as it consists of a simple and fast procedure that yields high sensitivity with only a few microliters of a biological sample. Also, it avoids the use of highly toxic chemicals and generates less residue, thus it fits the growing trend of using sustainable analytical methodologies, established by GAT and other similar guidelines.

Prospects of Cannabis and its Cultivation in Sri Lanka

Cannabis (Cannabis sativa L) is cultivated commercially in various parts of the world, predominantly for its medicinal properties (medicinal-type) and as a source of fibre and oilseed (fibre-type). The plant is known to contain over 567 chemical compounds out of which about one hundred chemicals belong to the highly valued unique class of Cannabinoids. At present, more than 25,000 Cannabis-based products worth 344 billion US$ are available at the world market. In a situation where the environmental agriculture is principally promoted, the present paper reviewed the applicability and potential worth of cultivating Cannabis as a medicinal plant in Sri Lanka. At present, Cannabis is restricted to be used in Sri Lanka, though the country spends about US$ 27,000 annually to import Cannabis for medicinal preparations. Cannabis is well- known for its wide range of environmental adaptation, higher nutrient and water use efficiency, low dependency on external inputs such as fertilizers and pesticides, high economic return for a unit area of land, and ability to cultivate both in indoors and outdoors. Considering the genetic resources and inherent knowledge, along with favourable climatic conditions available in the dry zone, suggest that Cannabis is an ideal crop, which could assure higher economic benefits under low inputs. While serving as a venture of foreign earnings, local cultivation would halt the country’s dependency on importation of Cannabis-based raw materials. However, legalization and cultivation should be rigorously monitored by an authorized body to ensure that the production meets the health regulations and utilized accordingly.

Epidemiology of Δ8THC-Related Carcinogenesis in USA: A Panel Regression and Causal Inferential Study.

The use of Δ8THC is increasing at present across the USA in association with widespread cannabis legalization and the common notion that it is “legal weed”. As genotoxic actions have been described for many cannabinoids, we studied the cancer epidemiology of Δ8THC. Data on 34 cancer types was from the Centers for Disease Control Atlanta Georgia, substance abuse data from the Substance Abuse and Mental Health Services Administration, ethnicity and income data from the U.S. Census Bureau, and cannabinoid concentration data from the Drug Enforcement Agency, were combined and processed in R. Eight cancers (corpus uteri, liver, gastric cardia, breast and post-menopausal breast, anorectum, pancreas, and thyroid) were related to Δ8THC exposure on bivariate testing, and 18 (additionally, stomach, Hodgkins, and Non-Hodgkins lymphomas, ovary, cervix uteri, gall bladder, oropharynx, bladder, lung, esophagus, colorectal cancer, and all cancers (excluding non-melanoma skin cancer)) demonstrated positive average marginal effects on fully adjusted inverse probability weighted interactive panel regression. Many minimum E-Values (mEVs) were infinite. p-values rose from 8.04 × 10−78. Marginal effect calculations revealed that 18 Δ8THC-related cancers are predicted to lead to a further 8.58 cases/100,000 compared to 7.93 for alcoholism and −8.48 for tobacco. Results indicate that between 8 and 20/34 cancer types were associated with Δ8THC exposure, with very high effect sizes (mEVs) and marginal effects after adjustment exceeding tobacco and alcohol, fulfilling the epidemiological criteria of causality and suggesting a cannabinoid class effect. The inclusion of pediatric leukemias and testicular cancer herein demonstrates heritable malignant teratogenesis.

Congenital anomaly epidemiological correlates of Δ8THC across USA 2003-16: panel regression and causal inferential study.

Δ8-Tetrahydrocannabinol (Δ8THC) is marketed in many US states as ‘legal weed’. Concerns exist relating to class-wide genotoxic cannabinoid effects. We conducted an epidemiological investigation of Δ8THC-related genotoxicity expressed as 57 congenital anomaly (CA) rates (CARs) in the USA. CARs were taken from the Centers for Disease Control, Atlanta, Georgia. Drug exposure data were taken from the National Survey of Drug Use and Health, with a response rate of 74.1%. Ethnicity and income data were taken from the US Census Bureau. National cannabinoid exposure was taken from Drug Enforcement Agency publications and multiplied by state cannabis use data to derive state-based estimates of Δ8THC exposure. At bivariate continuous analysis, Δ8THC was associated with 23 CAs on raw CA rates, 33 CARs after correction for early termination for anomaly estimates and 41 on a categorical analysis comparing the highest and lowest exposure quintiles. At inverse probability weighted multivariable additive and interactive models lagged to 0, 2 and 4 years, Δ8THC was linked with 39, 8, 4 and 9 CAs. Chromosomal, cardiovascular, gastrointestinal, genitourinary, limb, central nervous system (CNS) and face systems were particularly affected. The minimum E-values ranged to infinity. Both the number of anomalies implicated and the effect sizes demonstrated were much greater for Δ8THC than for tobacco and alcohol combined. Δ8THC appears epidemiologically to be more strongly associated with many CAs than for tobacco and alcohol and is consistent with a cannabinoid class genotoxic/epigenotoxic effect. Quantitative causality criteria were fulfilled, and causal relationships either for Δ8THC or for cannabinoid/s, for which it is a surrogate marker, may be in operation.

Sex and dose-dependent antinociceptive effects of the JNK (c-Jun N-terminal kinase) inhibitor SU 3327 are mediated by CB2 receptors in female, and CB1/CB2 receptors in male mice in an inflammatory pain model

Activation of c-Jun N-terminal kinases (JNKs) has been implicated in the development and persistence of inflammatory and neuropathic pain in animal models. Moreover, JNKs have been involved in the maintenance of chronic pain, as well as development of tolerance to antinociceptive agents in the opioid and cannabinoid class of compounds. In this study, we evaluated the antinociceptive effects of the JNK inhibitor SU 3327 (0.3–30 mg/kg) in the formalin pain model with an emphasis on the sex-specific actions of this compound. In wild-type C57BL6J mice, SU 3327 produced strong antinociceptive effects in the formalin pain model which were mediated by CB2 receptors in females, and both CB1 and CB2 receptors in males. SU 3327 at a dose of 10 mg/kg produced antinociception, hypothermia, motor impairment, and hypolocomotion to a similar extent in both males and females. The antinociceptive effects of SU 3327 were more potent in males at lower doses (1 and 3 mg/kg), while females were more sensitive to the hypothermic, and motor-suppression effects at lower (3 mg/kg) doses versus males. Analysis of spinal cords, using qPCR following SU 3327 administration in the formalin test, revealed changes in cannabinoid, tolerance and inflammatory markers in females only, and only in the high (10–30 mg/kg) dose conditions. Indeed, females showed an increase in mRNA levels of cannabinoid (CB2), but a decrease in tolerance (β-arrestin 1) and inflammatory (TNF-α, IL-1β, IL-6)-associated markers. The differences between males and females, in this study, support sex as an important factor in nociception and antinociceptive responses mediated by JNK and the endocannabinoid system.

Revealing Unknown Controlled Substances and New Psychoactive Substances Using High-Resolution LC-MS-MS Machine Learning Models and the Hybrid Similarity Search Algorithm.

High-resolution liquid chromatography-tandem mass spectrometry (LC--MS-MS)-based machine learning models are constructed to address the analytical challenge of identifying unknown controlled substances and new psychoactive substances (NPSs). Using a training set composed of 770 LC-MS-MS barcode spectra (with binary entries 0 or 1) obtained generally by high-resolution mass spectrometers, three classification machine learning models were generated and evaluated. The three models are artificial neural network (ANN), support vector machine (SVM) and k-nearest neighbor (k-NN) models. In these models, controlled substances and NPSs were classified into 13 subgroups (benzylpiperazine, opiate, benzodiazepine, amphetamine, cocaine, methcathinone, classical cannabinoid, fentanyl, 2C series, indazole carbonyl compound, indole carbonyl compound, phencyclidine and others). Using 193 LC-MS-MS barcode spectra as an external test set, accuracy of the ANN, SVM and k-NN models were evaluated as 72.5%, 90.0% and 94.3%, respectively. Also, the hybrid similarity search (HSS) algorithm was evaluated to examine whether this algorithm can successfully identify unknown controlled substances and NPSs whose data are unavailable in the database. When only 24 representative LC-MS-MS spectra of controlled substances and NPSs were selectively included in the database, it was found that HSS can successfully identify compounds with high reliability. The machine learning models and HSS algorithms are incorporated into our home-coded artificial intelligence screener for narcotic drugs and psychotropic substances standalone software that is equipped with a graphic user interface. The use of this software allows unknown controlled substances and NPSs to be identified in a convenient manner.

The Therapeutic Potential of Cannabis in Counteracting Oxidative Stress and Inflammation.

Significant growth of interest in cannabis (Cannabis sativa L.), especially its natural anti-inflammatory and antioxidative properties, has been observed recently. This narrative review aimed to present the state of the art of research concerning the anti-inflammatory activity of all classes of cannabinoids published in the last five years. Multimodal properties of cannabinoids include their involvement in immunological processes, anti-inflammatory, and antioxidative effects. Cannabinoids and non-cannabinoid compounds of cannabis proved their anti-inflammatory effects in numerous animal models. The research in humans is missing, and the results are unconvincing. Although preclinical evidence suggests cannabinoids are of value in treating chronic inflammatory diseases, the clinical evidence is scarce, and further well-designed clinical trials are essential to determine the prospects for using cannabinoids in inflammatory conditions.

A review on the syntheses of Dronabinol and Epidiolex as classical cannabinoids with various biological activities including those against SARS-COV2

The very important psychoactive phytocannabinoid from Cannabis Δ9 tetrahydrocannabinol (Δ9-THC) and its non-psychotropic member is cannabidiol (CBD). These compounds have a variety of pharmacological activities. THC has been approved for the treatment of nausea caused by chemotherapy, multiple sclerosis and chronic and neuropathic pain and research is underway to use it to treat stimulation of dementia, anorexia nervous and Tourette’s syndrome. CBD has therapeutic benefits in Epilepsy, neuroprotective, cancer, inflammatory and anxiety. Recognizing candidate drugs efficiently in the new SARS-CoV2 disease 2019 (Covid-19) is crucial. Cannabidiol and Δ9-tetrahydrocannabinol have immunomodulatory and anti-inflammatory effects. They can reduce the uncontrolled cytokine production of acute lung injury. Although THD and CBD can be extracted from natural sources due to the disadvantages of this method such as difficulty in purification, cultivation, etc. It has been proven that chemical-synthesis methods of these two compounds can solve these problems. This review briefly summarizes the chemical-synthetic strategies of Dronabinol and Epidiolex from THC and CBD.Graphic abstract

Oxa-adamantyl cannabinoids

As a continuation of earlier work on classical cannabinoids bearing bulky side chains we report here the design, synthesis, and biological evaluation of 3′-functionalized oxa-adamantyl cannabinoids as a novel class of cannabinergic ligands. Key synthetic steps involve nucleophilic addition/transannular cyclization of aryllithium to epoxyketone in the presence of cerium chloride and stereoselective construction of the tricyclic cannabinoid nucleus. The synthesis of the oxa-adamantyl cannabinoids is convenient, and amenable to scale up allowing the preparation of these analogs in sufficient quantities for detailed in vitro evaluation. The novel oxa-adamantyl cannabinoids reported here were found to be high affinity ligands for the CB1 and CB2 cannabinoid receptors. In the cyclase assay these compounds were found to behave as potent and efficacious CB1 receptor agonists. Isothiocyanate analog AM10504 is capable of irreversibly labeling both the CB1 and CB2 receptors.

Preliminary data on the potential for unintentional antidoping rule violations by permitted cannabidiol (CBD) use.

According to the World Anti-Doping Agency (WADA) regulations, cannabinoids use is prohibited in competition except for cannabidiol (CBD) use. For an adverse analytical finding (AAF) in doping control, cannabinoid misuse is based on identification of the pharmacologically inactive metabolite 11-nor-delta-9-carboxy-tetrahydrocannabinol-9-carboxylic acid (carboxy-THC) in urine at a concentration greater than 180 ng/ml. All other (minor) cannabinoids are reported as AAF when identified, except for CBD that has been explicitly excluded from the class of cannabinoids on WADA's Prohibited List since 2018. However, due to the fact that CBD isolated from cannabis plants may contain additional minor cannabinoids, the permissible use of CBD can lead to unintentional violations of antidoping regulations. An assay for the detection of 16 cannabinoids in human urine was established. The sample preparation consisted of enzymatic hydrolysis of glucuronide conjugates, liquid-liquid extraction, trimethylsilylation, and analysis by gas chromatography/tandem mass spectrometry (GC-MS/MS). Spot urine samples from CBD users, as well as specimens obtained from CBD administration studies conducted with 15 commercially available CBD products, were analyzed, and assay characteristics such as selectivity, reproducibility of detection at the minimum required performance level, limit of detection, and limit of identification were determined. An ethical committee approved controlled single dose commercially available CBD products administration study was conducted to identify 16 cannabinoids in urine samples collected after ingestion or application of the CBD products as well as their presence in spot urine samples of habitual CBD users. Variable patterns of cannabinoids or their metabolites were observed in the urine samples, especially when full spectrum CBD products were consumed. The presence of minor cannabinoids or their metabolites in an athlete's in-competition urine sample represents a substantial risk of an antidoping rule violation.

(282) Sex Differences in the Development of Tolerance to Arachidonyl-2-Chloroethalamide (ACEA) in the Mouse Formalin Pain Model

Interest in the cannabinoid class of compounds is growing rapidly as an option to treat a variety of conditions, including pain. Arachidonyl-2-chloroethalamide (ACEA), a CB1 specific compound and analog of the endocannabinoid anandamide, has demonstrated efficacy in a variety of animal pain models. Our study evaluated ACEA in the formalin model of pain in male and female C57BL6 mice. Intraplantar injection of 2.5% formalin produces a biphasic pain response modeling both acute and inflammatory pain, measured as time spent attending to the injected paw over the course of one hour. Since development of tolerance to the analgesic effects of cannabinoids is well documented in animal models and an important clinical consideration, we evaluated the analgesic effects of ACEA in both control mice, as well as mice pretreated with ACEA (doses of 0.1, 0.5 and 1 mg/kg) at different time point (ranging from 1 to 8 days) and tested with formalin on the last day of treatment. Our preliminary results reveal sex differences between male and female mice treated with an acute dose of ACEA (0.1, 0.5 and 1 mg/kg I.P.) 30 minutes prior to formalin injection. We also note sex differences in the development of tolerance in male and female mice pretreated with ACEA for different number of days prior to the formalin test. Notably, the development of tolerance to the analgesic effects of ACEA corresponds with an alteration in the estrous cycle in female mice, suggesting that a hormonal component may be involved in this process. Further studies are needed to investigate the mechanisms behind these preclinical interesting findings.

Novel inverse agonists for the orphan G protein-coupled receptor 6

The orphan G protein-coupled receptor 6 (GPR6) displays unique promise as a therapeutic target for the treatment of neuropsychiatric disorders due to its high expression in the striatopallidal neurons of the basal ganglia. GPR6, along with closely related orphan receptors GPR3 and GPR12, are phylogenetically related to CB1 and CB2 cannabinoid receptors. In the current study, we performed concentration-response studies on the effects of three different classes of cannabinoids: endogenous, phyto-, and synthetic, on both GPR6-mediated cAMP accumulation and β-arrestin2 recruitment. In addition, structure-activity relationship studies were conducted on cannabidiol (CBD), a recently discovered inverse agonist for GPR6. We have identified four additional cannabinoids, cannabidavarin (CBDV), WIN55212-2, SR141716A and SR144528, that exert inverse agonism on GPR6. Furthermore, we have discovered that these cannabinoids exhibit functional selectivity toward the β-arrestin2 recruitment pathway. These novel, functionally selective inverse agonists for GPR6 can be used as research tools and potentially developed into therapeutic agents.

Cannabidiol, a novel inverse agonist for GPR12

GPR12 is a constitutively active, Gs protein-coupled receptor that currently has no confirmed endogenous ligands. GPR12 may be involved in physiological processes such as maintenance of oocyte meiotic arrest and brain development, as well as pathological conditions such as metastatic cancer. In this study, the potential effects of various classes of cannabinoids on GPR12 were tested using a cAMP accumulation assay. Our data demonstrate that cannabidiol (CBD), a major non-psychoactive phytocannabinoid, acted as an inverse agonist to inhibit cAMP accumulation stimulated by the constitutively active GPR12. Thus, GPR12 is a novel molecular target for CBD. The structure-activity relationship studies of CBD indicate that both the free hydroxyl and the pentyl side chain are crucial for the effects of CBD on GPR12. Furthermore, studies using cholera toxin, which blocks Gs protein and pertussis toxin, which blocks Gi protein, revealed that Gs, but not Gi is involved in the inverse agonism of CBD on GPR12. CBD is a promising novel therapeutic agent for cancer, and GPR12 has been shown to alter viscoelasticity of metastatic cancer cells. Since we have demonstrated that CBD is an inverse agonist for GPR12, this provides novel mechanism of action for CBD, and an initial chemical scaffold upon which highly potent and efficacious agents acting on GPR12 may be developed with the ultimate goal of blocking cancer metastasis.

Characterization of structurally novel G protein biased CB1 agonists: Implications for drug development

The human cannabinoid subtype 1 receptor (hCB1R) is highly expressed in the CNS and serves as a therapeutic target for endogenous ligands as well as plant-derived and synthetic cannabinoids. Unfortunately, acute use of hCB1R agonists produces unwanted psychotropic effects and chronic administration results in development of tolerance and dependence, limiting the potential clinical use of these ligands. Studies in β-arrestin knockout mice suggest that interaction of certain GPCRs, including μ-, δ-, κ-opioid and hCB1Rs, with β-arrestins might be responsible for several adverse effects produced by agonists acting at these receptors. Indeed, agonists that bias opioid receptor activation toward G-protein, relative to β-arrestin signaling, produce less severe adverse effects. These observations indicate that therapeutic utility of agonists acting at hCB1Rs might be improved by development of G-protein biased hCB1R agonists. Our laboratory recently reported a novel class of indole quinulidinone (IQD) compounds that bind cannabinoid receptors with relatively high affinity and act with varying efficacy. The purpose of this study was to determine whether agonists in this novel cannabinoid class exhibit ligand bias at hCB1 receptors. Our studies found that a novel IQD-derived hCB1 receptor agonist PNR-4-20 elicits robust G protein-dependent signaling, with transduction ratios similar to the non-biased hCB1R agonist CP-55,940. In marked contrast to CP-55,940, PNR-4-20 produces little to no β-arrestin 2 recruitment. Quantitative calculation of bias factors indicates that PNR-4-20 exhibits from 5.4-fold to 29.5-fold bias for G protein, relative to β-arrestin 2 signaling (when compared to G protein activation or inhibition of forskolin-stimulated cAMP accumulation, respectively). Importantly, as expected due to reduced β-arrestin 2 recruitment, chronic exposure of cells to PNR-4-20 results in significantly less desensitization and down-regulation of hCB1Rs compared to similar treatment with CP-55,940. PNR-4-20 (i.p.) is active in the cannabinoid tetrad in mice and chronic treatment results in development of less persistent tolerance and no significant withdrawal signs when compared to animals repeatedly exposed to the non-biased full agoinst JWH-018 or Δ9-THC. Finally, studies of a structurally similar analog PNR- 4-02 show that it is also a G protein biased hCB1R agonist. It is predicted that cannabinoid agonists that bias hCB1R activation toward G protein, relative to β-arrestin 2 signaling, will produce fewer and less severe adverse effects both acutely and chronically.