Human Cannabinoid CB2 Receptor Research Articles

A Highly Potent, Orally Bioavailable Pyrazole-Derived Cannabinoid CB2 Receptor- Selective Full Agonist for In Vivo Studies.

The cannabinoid CB2 receptor (CB2R) is a potential therapeutic target for distinct forms of tissue injury and inflammatory diseases. To thoroughly investigate the role of CB2R in pathophysiological conditions and for target validation in vivo, optimal pharmacological tool compounds are essential. Despite the sizable progress in the generation of potent and selective CB2R ligands, pharmacokinetic parameters are often neglected for in vivo studies. Here, we report the generation and characterization of a tetra-substituted pyrazole CB2R full agonist named RNB-61 with high potency (K i 0.13-1.81 nM, depending on species) and a peripherally restricted action due to P-glycoprotein-mediated efflux from the brain. 3H and 14C labeled RNB-61 showed apparent K d values of <4 nM toward human CB2R in both cell and tissue experiments. The 6,800-fold selectivity over CB1 receptors and negligible off-targets in vitro, combined with high oral bioavailability and suitable systemic pharmacokinetic (PK) properties, prompted the assessment of RNB-61 in a mouse ischemia-reperfusion model of acute kidney injury (AKI) and in a rat model of chronic kidney injury/inflammation and fibrosis (CKI) induced by unilateral ureteral obstruction. RNB-61 exerted dose-dependent nephroprotective and/or antifibrotic effects in the AKI/CKI models. Thus, RNB-61 is an optimal CB2R tool compound for preclinical in vivo studies with superior biophysical and PK properties over generally used CB2R ligands.

A highly potent, orally bioavailable pyrazole-derived cannabinoid CB2 receptor-selective full agonist for in vivo studies.

The cannabinoid CB2 receptor (CB2R) is a potential therapeutic target for distinct forms of tissue injury and inflammatory diseases. To thoroughly investigate the role of CB2R in pathophysiological conditions and for target validation in vivo, optimal pharmacological tool compounds are essential. Despite the sizable progress in the generation of potent and selective CB2R ligands, pharmacokinetic parameters are often neglected for in vivo studies. Here, we report the generation and characterization of a tetra-substituted pyrazole CB2R full agonist named RNB-61 with high potency (K i 0.13-1.81 nM, depending on species) and a peripherally restricted action due to P-glycoprotein mediated efflux from the brain. 3H and 14C labelled RNB-61 showed apparent K d values < 4 nM towards human CB2R in both cell and tissue experiments. The >6000-fold selectivity over CB1 receptors and negligible off-targets in vitro, combined with high oral bioavailability and suitable systemic pharmacokinetic (PK) properties, prompted the assessment of RNB-61 in a mouse ischemia-reperfusion model of acute kidney injury (AKI) and in a rat model of chronic kidney injury/inflammation and fibrosis (CKI) induced by unilateral ureteral obstruction. RNB-61 exerted dose-dependent nephroprotective and/or antifibrotic effects in the AKI/CKI models. Thus, RNB-61 is an optimal CB2R tool compound for preclinical in vivo studies with superior biophysical and PK properties over generally used CB2R ligands.

Displacement Binding Assay Using Human Cannabinoid CB2 Receptor-Transfected Cells.

Displacement binding assays are nonfunctional assays mostly used with the aim of determining whether a certain compound (plant-derived or synthetic) can bind to a specific receptor with high affinity. Here, we describe the displacement binding assay that is carried out with a radioligand and CHO (Chinese Hamster Ovarian) cells stably transfected with the human cannabinoid CB2 receptor.

Behavioral and binding studies on the quinolinyl ester indoles 5F-PB22 (5F-QUPIC) and BB-22 (QUCHIC) in the mouse model

Novel Psychoactive Substances newly introduced on the drug market are constantly changing patterns of drug consumption. Among these, Synthetic Cannabinoids (SCs) mainly dominated European seizures in the last years. Quinolin-8-yl 1-(5-fluoropentyl)-1H-indole-3-carboxylate (5F-PB22) and quinolin-8-yl 1-(cyclohexylmethyl)-1H-indole-3-carboxylate (BB-22) are SCs whose abuse has been linked to a range of fatal intoxications and hospitalizations. Therefore, this study aims to investigate and compare in vitro and in vivo pharmacodynamics activity of these quinolinyl ester indoles. In vitro competition binding experiments performed on CD-1 murine and human cannabinoid CB1 and CB2 receptors revealed a sub-nanomolar affinity and potency of 5F-PB22 and BB-22. In vivo studies demonstrated that the acute systemic administration of 5F-PB22 and BB-22 (0.001-6 mg/kg) deeply impaired sensorimotor and motor responses, core temperature, breath rate and nociceptive threshold of CD-1 male mice. Pre-treatment with the selective cannabinoid CB1 receptor antagonist/inverse agonist AM-251 (6 mg/kg) fully prevented the effects of both cannabinoids (at 1 mg/kg) suggesting a CB1 receptor mediated action. Relying on these findings, this study showed for the first time the pharmaco-toxicological effects of these substances confirming their potential burden on human health. Furthermore, it revealed that the difference of the chemical structures of the two SCs results in their potency related disparities.

Identification and Quantification of 5-Fluoro ADB and the 5-Fluoro ADB Ester Hydrolysis Metabolite in Postmortem Blood Samples by LC-MS/MS.

5-Fluoro ADB, also known as 5-fluoro MDMB-PINACA, is a potent synthetic cannabinoid that is an agonist to the human cannabinoid CB1 and CB2 receptors. Adverse physiological and psychological effects that have resulted in hospitalization and/or death have been associated with 5-Fluoro ADB use. In addition, analytical confirmation of 5-Fluoro ADB use has been reported in both forensic human performance toxicology and postmortem cases. An analytical method for the identification and quantification of 5-fluoro ADB and the 5-fluoro ADB ester hydrolysis metabolite in human blood samples by liquid chromatography-tandem mass spectrometry was created and validated. The linear range of this assay was determined to be 0.01-10ng/mL for 5-fluoro ADB and 10-500ng/mL for the 5-fluoro ADB ester hydrolysis metabolite. The method met both precision and accuracy requirements. Endogenous and exogenous interferences were not observed. Ion suppression exceeding 25% was observed for 5-fluoro ADB. However, additional experiments were performed to ensure that the observed suppression did not affect other method validation parameters such as limit of detection and accuracy. Blood samples from 36 postmortem cases were analyzed utilizing this methodology. The average blood concentration of 5-fluoro ADB was 0.29ng/mL in central blood specimens and 0.05ng/mL in peripheral blood specimens. The average blood concentration of the 5-fluoro ADB ester hydrolysis metabolite was 49ng/mL in central blood specimens and 21ng/mL in peripheral blood specimens. A serum sample was also analyzed and had a serum concentration of 0.12ng/mL for 5-fluoro ADB and 42ng/mL for the 5-fluoro ADB ester hydrolysis metabolite. As the concentration of the 5-fluoro ADB ester hydrolysis metabolite was found at a greater concentration than that of 5-fluoro ADB, this metabolite may be a useful marker to monitor in an attempt to confirm 5-fluoro ADB use in toxicological investigations.

Testing for AB-PINACA in human hair: Distribution in head hair versus pubic hair.

Synthetic cannabinoid receptor agonists were first identified in herbal products in 2008 advertised as a legal replacement for cannabis. These herbal incense are usually called "spice" and among these, one product in particular has gained popularity: AB-PINACA (N-[(2S)-1-Amino-3-methyl-1-oxobutan-2-yl]-1-pentyl-1H-indazole-3-carboxamide). This drug has been discovered to have a stronger binding to human cannabinoid CB1 and CB2 receptors than ∆9 -THC.While some articles have been published regarding the presence of AB-PINACA in biological fluids such as blood and urine, none reports the presence of AB-PINACA in hair. We have developed and validated a method for detection of AB-PINACA in hair using a liquid chromatography-tandem mass spectrometry system and applied it to head and pubic hair obtained in a case of intoxication. The validation procedure demonstrated a limit of detection and a limit of quantification of 0.5 and 1pg/mg, respectively and acceptable linearity, repeatability, and reproducibility. AB-PINACA tested positive in the blood (5.7ng/mL) and less than 1ng/mL was found in urine. The analysis of the hair specimens resulted in an unusual distribution of the drug between head and pubic hair. AB-PINACA was identified at a higher concentration in head hair (195pg/mg) versus in pubic hair (5pg/mg). The very low concentration of AB-PINACA in the urine after consumption, due to rapid metabolism, could explain this infrequent distribution, as pubic hair can be contaminated by urine. In any case, it cannot be excluded that the high concentration in head hair may be due to environmental contamination.

Pharmacological evaluation of new constituents of \u201cSpice\u201d: synthetic cannabinoids based on indole, indazole, benzimidazole and carbazole scaffolds

PurposeIn the present study we characterized a series of synthetic cannabinoids containing various heterocyclic scaffolds that had been identified as constituents of “Spice”, a preparation sold on the illicit drug market. All compounds were further investigated as potential ligands of the orphan receptors GPR18 and GPR55 that interact with some cannabinoids.MethodsThe compounds were studied in radioligand binding assays to determine their affinity for human cannabinoid CB1 and CB2 receptors expressed in CHO cells, and in cAMP accumulation assays to study their functionality.ResultsStructure-activity relationships were analyzed. The most potent CB1 receptor agonist of the present series MDMB-FUBINACA (12) (Ki = 98.5 pM) was docked into the human CB1 receptor structure, and a plausible binding mode was identified showing high similarity with that of the co-crystallized THC derivatives. MDMB-CHMCZCA (41) displayed a unique profile acting as a full agonist at the CB1 receptor subtype, but blocking the CB2 receptor completely. Only a few weakly potent antagonists of GPR18 and GPR55 were identified, and thus all compounds showed high CB receptor selectivity, mostly interacting with both subtypes, CB1 and CB2.ConclusionsThese results will be useful to assess the compounds’ toxicological risks and to guide legislation. Further studies on 41 are warranted.

Binding and Signaling Studies Disclose a Potential Allosteric Site for Cannabidiol in Cannabinoid CB2 Receptors.

The mechanism of action of cannabidiol (CBD), the main non-psychotropic component of Cannabis sativa L., is not completely understood. First assumed that the compound was acting via cannabinoid CB2 receptors (CB2Rs) it is now suggested that it interacts with non-cannabinoid G-protein-coupled receptors (GPCRs); however, CBD does not bind with high affinity to the orthosteric site of any GPCR. To search for alternative explanations, we tested CBD as a potential allosteric ligand of CB2R. Radioligand and non-radioactive homogeneous binding, intracellular cAMP determination and ERK1/2 phosphorylation assays were undertaken in heterologous systems expressing the human version of CB2R. Using membrane preparations from CB2R-expressing HEK-293T (human embryonic kidney 293T) cells, we confirmed that CBD does not bind with high affinity to the orthosteric site of the human CB2R where the synthetic cannabinoid, [3H]-WIN 55,212-2, binds. CBD was, however, able to produce minor but consistent reduction in the homogeneous binding assays in living cells using the fluorophore-conjugated CB2R-selective compound, CM-157. The effect on binding to CB2R-expressing living cells was different to that exerted by the orthosteric antagonist, SR144528, which decreased the maximum binding without changing the KD. CBD at nanomolar concentrations was also able to significantly reduce the effect of the selective CB2R agonist, JWH133, on forskolin-induced intracellular cAMP levels and on activation of the MAP kinase pathway. These results may help to understand CBD mode of action and may serve to revisit its therapeutic possibilities.

The Cyclic AMP Assay Using Human Cannabinoid CB2 Receptor-Transfected Cells.

The cyclic AMP assay is a functional assay that is commonly used to determine the pharmacological behavior (agonists, antagonists, inverse agonists) of G-protein-coupled receptor (GPCR) ligands. Here, we describe the cyclic AMP assay that is carried out with commercially available non-radioligand ready-to-use kits and Chinese hamster ovarian (CHO) cells stably transfected with the human cannabinoid CB2 receptor.

The Displacement Binding Assay Using Human Cannabinoid CB2 Receptor-Transfected Cells.

Displacement binding assays are nonfunctional assays mostly used with the aim of determining whether a certain compound (plant-derived or synthetic) is able to bind to a specific receptor with high affinity. Here, we describe the displacement binding assay that is carried out with a radioligand and CHO (Chinese Hamster Ovarian) cells stably transfected with the human cannabinoid CB2 receptor.

Involvement of β-arrestin-2 and clathrin in agonist-mediated internalization of the human cannabinoid CB2 receptor.

The CB2 cannabinoid receptor is a promising therapeutic target for the treatment of inflammatory diseases, neuropathic pain, liver diseases, cancer and cardiovascular diseases. Obtaining detailed information on the internalization and trafficking of the human CB2 receptor in response to agonist will have a significant impact on drug discovery. Visualization and quantitative detection of EGFP-tagged CB2 receptor showed that, upon WIN55,212-2 stimulation, the CB2 receptor was rapidly internalized in a dose- and time-dependent manner from the cell membrane into the cytoplasm. Pretreatment with hypertonic sucrose, MDC clathrin inhibitor, or siRNA-mediated knock-down of clathrin heavy chain led to significant inhibition of agonist-induced CB2 internalization. Using the RNA interference method, we showed that knockdown of β-arrestin2 expression significantly impaired receptor internalisation. Further investigation demonstrated that the internalized CB2 receptors were co-localized with the early endosome probe and were recycled to the cell surface after the removal of agonist, but treatment with specific cell-permeable proteasome inhibitor MG132 a inhibited the recycling of internalized CB2 receptor, suggesting that the proteasome-mediated degradation pathway may be involved in CB2 internalization. Moreover, the single residue Ser(352) and residue cluster S(335)S(336)T(338)T(340) at the C-terminal tail are shown to be essential for receptor phosphorylation and β-arrestin2 association. These data provide new insights into the mechanisms regulating agonist-mediated internalization and trafficking of the human CB2 receptor.

Inhibition of colon carcinogenesis by a standardized Cannabis sativa extract with high content of cannabidiol

PurposeColon cancer is a major public health problem. Cannabis-based medicines are useful adjunctive treatments in cancer patients. Here, we have investigated the effect of a standardized Cannabis sativa extract with high content of cannabidiol (CBD), here named CBD BDS, i.e. CBD botanical drug substance, on colorectal cancer cell proliferation and in experimental models of colon cancer in vivo. MethodsProliferation was evaluated in colorectal carcinoma (DLD-1 and HCT116) as well as in healthy colonic cells using the MTT assay. CBD BDS binding was evaluated by its ability to displace [3H]CP55940 from human cannabinoid CB1 and CB2 receptors. In vivo, the effect of CBD BDS was examined on the preneoplastic lesions (aberrant crypt foci), polyps and tumours induced by the carcinogenic agent azoxymethane (AOM) as well as in a xenograft model of colon cancer in mice. ResultsCBD BDS and CBD reduced cell proliferation in tumoral, but not in healthy, cells. The effect of CBD BDS was counteracted by selective CB1 and CB2 receptor antagonists. Pure CBD reduced cell proliferation in a CB1-sensitive antagonist manner only. In binding assays, CBD BDS showed greater affinity than pure CBD for both CB1 and CB2 receptors, with pure CBD having very little affinity. In vivo, CBD BDS reduced AOM-induced preneoplastic lesions and polyps as well as tumour growth in the xenograft model of colon cancer. ConclusionsCBD BDS attenuates colon carcinogenesis and inhibits colorectal cancer cell proliferation via CB1 and CB2 receptor activation. The results may have some clinical relevance for the use of Cannabis-based medicines in cancer patients.

Correction: The Second Intracellular Loop of the Human Cannabinoid CB2 Receptor Governs G Protein Coupling in Coordination with the Carboxyl Terminal Domain

Correction: The Second Intracellular Loop of the Human Cannabinoid CB2 Receptor Governs G Protein Coupling in Coordination with the Carboxyl Terminal Domain

The Second Intracellular Loop of the Human Cannabinoid CB2 Receptor Governs G Protein Coupling in Coordination with the Carboxyl Terminal Domain

The major effects of cannabinoids and endocannabinoids are mediated via two G protein-coupled receptors, CB1 and CB2, elucidation of the mechanism and structural determinants of the CB2 receptor coupling with G proteins will have a significant impact on drug discovery. In the present study, we systematically investigated the role of the intracellular loops in the interaction of the CB2 receptor with G proteins using chimeric receptors alongside the characterization of cAMP accumulation and ERK1/2 phosphorylation. We provided evidence that ICL2 was significantly involved in G protein coupling in coordination with the C-terminal end. Moreover, a single alanine substitution of the Pro-139 in the CB2 receptor that corresponds to Leu-222 in the CB1 receptor resulted in a moderate impairment in the inhibition of cAMP accumulation, whereas mutants P139F, P139M and P139L were able to couple to the Gs protein in a CRE-driven luciferase assay. With the ERK activation experiments, we further found that P139L has the ability to activate ERK through both Gi- and Gs-mediated pathways. Our findings defined an essential role of the second intracellular loop of the CB2 receptor in coordination with the C-terminal tail in G protein coupling and receptor activation.

Structural basis of human cannabinoid CB1 G protein‐coupled receptor activation

Transmembrane signal transduction is achieved by activation of G protein‐coupled receptors (GPCRs) like the human cannabinoid CB1 receptor. These receptors exist in an ensemble of conformations, each of which might bind to different signaling molecules. Mutating a single residue, Thr 210, to Ile in the third transmembrane (TM3) domain, makes it more active then WT, whereas mutating it to Ala makes it fully inactive. We used the Gensemble method to predict 3D structures of these receptors. We find conformational differences that explain the CB1 receptor's activation mechanism. These predictions were validated by designing double mutants that were expected to switch the inactive T210A back to WT levels of activation. The 2nd mutation for T210A is predicted to cause an important salt‐bridge between TMs 2 and 6 to break. GTPδS binding assays show a large increase in G protein‐coupling for the double mutants indicating increased activation. We docked known agonists to these receptors and then performed 50 ns of molecular dynamics. The inactive T210A receptor with the docked agonist WIN55212‐2 maintains two stable interhelical salt‐bridges. Similarly, the WT receptor maintains a salt‐bridge between TMs 3 and 6, which suggests that a G protein is necessary to stabilize the active conformation.This work was funded by gifts from PharmSelex/Accelerator and NIH grants (R01NS071112, R01NS073115, R01AI040567, and DA020763).

Effect of Anionic Lipids on Structure and Function of Cannabinoid CB2 Receptor in Micelles and in Reconstituted Liposomes

Human cannabinoid CB2 receptor functions in plasma membranes of cells in immune and hematopoietic systems upon binding of cannabinoid ligands. Previously we reported on functional reconstitution of the recombinant CB2 receptor into liposomes. During solubilization and purification, structure of the receptor in micelles is stabilized by adding anionic cholesteryl hemisuccinate (CHS), that is used widely to maintain the functionality of recombinant GPCR. Here we show that the functional structure of CB2 receptor in micelles can be significantly preserved by replacing CHS with phospholipids prior to reconstitution. By ligand binding studies on the reconstituted receptor performed by 2H MAS NMR, that distinguishes specific binding to the receptor and nonspecific binding to the lipid matrix, and by G protein activation tests, we demonstrated that the anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) can be almost as efficient as CHS in stabilizing the receptor in micelles while the zwitterionic PC counterpart, POPC, was much less effective. Our results obtained with various phospholipid headgroup and acyl-chain species suggest that the net negative charge as well as the specific molecular structure of the PS headgroup plays a key role in maintaining the structural integrity of the receptor in micelles. On the reconstituted CB2 receptor in liposomes, effect of negative electric potential of membrane surface on G protein activation was investigated by systematically changing the content of anionic CHS or PS. The activation was highest at an anionic lipid content of about 50 mol%. There was no correlation between the efficiency of G protein activation and an increase of hydrocarbon chain order induced by CHS or cholesterol. The results highlight importance of anionic lipids in regulating signal transduction by CB2 receptor and possibly other class-A GPCR.

Identification of a Potent and Selective Cannabinoid CB1 Receptor Antagonist from Auxarthron reticulatum.

The fungus Auxarthron reticulatum derived from the marine sponge Ircinia variabilis produced the diketopiperazine alkaloid amauromine (1) and the quinolinone methyl-penicinoline (2). Compound 2 is identical to the reported methyl-marinamide, whose structure is herewith revised. In radioligand binding studies at human cannabinoid CB1 and CB2 receptors recombinantly expressed in Chinese hamster ovary (CHO) cells, amauromine (1) was found to exhibit high affinity and selectivity for the CB1 receptor (K i = 178 nM). The compound was shown to be a neutral CB1 antagonist with a K b value of 66.6 nM determined in cAMP assays. Compound 2 exhibited only weak or no effects at CB receptors. To the best of our knowledge, compound 1 is the first fungal natural product that shows affinity for cannabinoid CB1 receptors. Because of its high antagonistic potency and selectivity, it may have potential for use as a drug and/or serve as a lead structure for drug development.

Cannabinoid CB1 receptor ligand binding and function examined through mutagenesis studies of F200 and S383

The cannabinoid CB1 G protein-coupled receptor has been shown to be a regulator of food consumption and has been studied extensively as a drug target for the treatment of obesity. To advance understanding of the receptor's three-dimensional structure, we performed mutagenesis studies at human cannabinoid CB1 receptor residues F200 and S383 and measured changes in activity and binding affinity of compounds from two recently discovered active chemotypes, arylsulfonamide agonists and tetrahydroquinoline-based inverse agonists, as well as literature compounds. Our results add support to previous findings that both agonists and inverse agonists show varied patterns of binding at the two mutated residue sites, suggesting multiple subsites for binding to the cannabinoid CB1 receptor for both functional types of ligands. We additionally find that an F200L mutation in the receptor largely restores binding affinity to ligands and significantly decreases constitutive activity when compared to F200A, resulting in a receptor phenotype that is closer to the wild-type receptor. The results downplay the importance of aromatic stacking interactions at F200 and suggest that a bulky hydrophobic contact is largely sufficient to provide significant receptor function and binding affinity to cannabinoid CB1 receptor ligands.

Cannabinoid receptor dependent and independent anti-proliferative effects of omega-3 ethanolamides in androgen receptor positive and negative prostate cancer cell lines

There is good evidence that omega-3 fatty acids have anti-tumour effects. In the present investigation we have shown that the omega-3 fatty acid ethanolamides, docosahexaenoyl ethanolamide (DHEA) and eicosapentaenoyl ethanolamide (EPEA), display significantly greater anti-proliferative potency than their parent omega-3 fatty acids, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), in LNCaP and PC3 prostate cancer cells. From experiments with membranes from mouse brain and from Chinese hamster ovary cells transfected with human cannabinoid CB2 receptors, we have also discovered that DHEA and EPEA activate cannabinoid CB1 and CB2 receptors with significant potency, suggesting they are endocannabinoids.

Novel selective antagonist of the cannabinoid CB1 receptor, MJ15, with prominent anti-obesity effect in rodent models

MJ15, a novel cannabinoid CB(1) receptor selective antagonist was discovered. In receptor binding assays, MJ15 displayed a high affinity for rat cannabinoid CB(1) receptor (K(i)=27.2 pM, and IC(50)=118.9 pM), but a much lower affinity for rat cannabinoid CB(2) receptor (only 46% inhibition at 10 microM). At the cellular level, the IC(50) values against activation of cannabinoid CB(1) and CB(2) receptors induced by Win55212-2 in specially designed EGFP-CB(1)_U2OS and EGFP-CB(2)_U2OS cells were 0.11 microM and >10 microM, respectively. In addition, MJ15 dose-dependently blocked Win55212-2 mediated increase of intracellular Ca(2+) levels in hippocampal cells and reversed the inhibitory effects of cannabinoid CB(1) receptor agonist on forskolin-stimulated adenylyl cyclase activity in CHO cells expressing the human cannabinoid CB(1) receptor. In animal experiments, MJ15 demonstrated remarkable effects from 20 to 40 mg/kg, including promoted the small intestine peristalsis in ICR mice and inhibited food intake and body weight increase in diet-induced obesity (DIO) rat and mouse. 40 mg/kg MJ15 significantly reduced food intake at initial 2 weeks of treatment, prevented the increase of body weight and adipose by 46% and 28% respectively in DIO rats, and reduced body weight and adipose gain by 70% and 23% respectively in early onset obesity DIO mice after 4 weeks treatment. Meanwhile, dyslipidemia were ameliorated in both models. Taken together the in vitro and in vivo data, MJ15 is demonstrated to be a potent and selective cannabinoid CB(1) receptor antagonist and holds a prominent potency in obesity and dyslipidemia treatment.