Abnormal Cannabidiol Research Articles

Development of a liquid chromatographic method with a different selectivity for the quantification of eighteen phytocannabinoids in hemp

To quantify phytocannabinoids in hemp, liquid chromatography diode array detector (LC-DAD) methods are favored, but their selectivity depends on baseline separation of all phytocannabinoids and unknown compounds in an extract. Therefore, development of a LC-DAD method with a different selectivity has become highly desirable. Currently, most LC-DAD methods use the water/acetonitrile eluting system, while this study aimed to use the water/methanol eluting system. A systematic investigation of various chromatographic parameters on LC separation of eighteen phytocannabinoids, the maximum number that has been quantified in hemp so far, plus two potential internal standards, led to a four-step isocratic mobile phase that was able to baseline separate the twenty compounds with a significantly different eluting order from published methods. Although changes in the mobile phase composition caused baseline drifts, consequent difficulty in quantification was avoided through detection at wavelengths longer than 230 nm. Subsequently, the method was validated according to the ISO 17025 guidelines, calibrated between 0.04 and 50 µg/mL, and used to analyze phytocannabinoids in nine strains of hemp flowers that were extracted using methanol between 0.04 and 50 % (w/w). Extraction recovery was tracked in real-time by spiking one of the two potential internal standards, i.e., abnormal cannabidiol (ACBD), a cannabinoid not naturally present in hemp. Method selectivity was further assessed by electrospray ionization time-of-flight mass spectrometry (ESI/TOFMS), indicating minimum interferences. In addition, five untargeted/unknown phytocannabinoids were identified by ESI/TOFMS, including two structural isomers of Δ9-tetrahydrocannabinol (Δ9-THC), two structural isomers of Δ9-tetrahydrocannabinolic acid (Δ9-THCA), and one structural isomer of Δ9-THC acetate.

A liquid chromatography electrospray ionization tandem mass spectrometry method for quantification of up to eighteen cannabinoids in hemp-derived products

A LC-ESI/MS/MS method was developed for quantification of up to eighteen cannabinoids, the maximum number published so far. A thorough study of published LC-ESI/MS/MS methods using triple quadrupole mass spectrometers revealed a possible misconception that multiple reaction monitoring (MRM) was able to definitively differentiate structural isomers of cannabinoids, especially Δ8-/Δ9-tetrahydrocannabinol (THC), which explained why many of those methods were developed for a limited number of cannabinoids, as small as two, and did not include Δ8-THC. In this study, the use of a quadrupole time-of-flight (QTOF) mass spectrometer for targeted analysis indicated that MRM could not definitively distinguish structural isomers of Δ9-THC, with a possible exception of cannabicyclol (CBL) for less accurate quantification, so their baseline separation was essential for their accurate quantification. After the developed method was successfully validated according to the ISO 17025 guidelines, it was further applied for the analysis of eighteen hemp-derived products, including drinks, water-soluble oils, topical serum, body lotion, face cream, lip balm, gummies, hard candy, coffee, snacks, and pet treats. The LOQ was 0.00008% (w/w) for drinks with the analysis of 12.5 mg/mL extracts, while the LOQ was 0.008% (w/w) for other samples because 125 μg/mL extracts were analyzed due to higher content of cannabinoids in non-drink samples. For the first-time, extraction recovery and matrix effect were tracked in real-time for each sample being analyzed, obtaining 92.9–106.3% and 91.3–120.2% in triplicate measurements, respectively, by spiking abnormal cannabidiol (ACBD), a cannabinoid not naturally present in hemp, into each sample before extraction and ACBD-d3 into each sample after extraction.

The Cross Reactivity of Cannabinoid Analogs (Delta-8-THC, Delta-10-THC and CBD), their metabolites, and Chiral Carboxy HHC metabolites in Urine of Six Commercially Available Homogeneous Immunoassays.

There has been an exponential surge in the presence and use of cannabinoids since the federal legalization of hemp (Agricultural Improvement Act of 2018). This growth is attributed to Delta-9-tetrahydrocannabinol (Delta-9-THC) and cannabidiol (CBD), the most abundant phytocannabinoid components of cannabis and hemp, respectively, but with many other emerging THC analogs. Structurally, these analogs are similar to Delta-9-THC, yet very little information is available about their potency and even less information is available regarding their detectability using commercially available cannabinoid screening kits. Due to their structural similarity, current cannabinoid homogeneous immunoassay screening methods may be able to detect these emerging cannabinoid analogs and their metabolites. Six urine immunoassay kits (Abbott Cannabinoids - Abbott Diagnostics, LZI Cannabinoids (cTHC) Enzyme Immunoassay - Lin-Zhi International, DRI® Cannabinoid Assay and CEDIA™ THC - Thermo Fisher Scientific, ONLINE DAT Cannabinoid II - Roche Diagnostics, and Syva EMIT®II Plus - Siemens Healthineers) were evaluated at two different cutoff concentrations: 50ng/mL and 20 or 25ng/mL, assay dependent. The analysis was performed on an Abbott Architect Plus c4000 (Abbott Diagnostics). Delta-8-THC, CBD, olivetol and their major metabolites, and Delta-10-THC and HHC carboxylic acid chiral analogs were evaluated. The cross reactivity was evaluated by preparing each analyte at 20, 50, 100, and 1000ng/mL in urine. Analytes that did not cross react at 1000ng/mL for a cutoff were considered not detectable. If detected, the lowest concentration was used as the decision point to determine the precision at the immunoassay's cutoff. The six commercially available urine cannabinoid homogeneous immunoassay screening kits cross reacted with the following analogs: Delta-8-THC, 11-OH-Delta-8-THC, 11-COOH-Delta-8-THC, 6-OH-CBD, 7-OH-CBD, all Delta-10-THC and HHC carboxylic acid chiral analogs, and olivetol with varying selectivity depending on the screening kit and cutoff concentration. The kits did not cross react with the following analogs: CBD, 7-COOH-CBD, Abnormal CBD, CBDA-A, and olivetolic acid.

Potency testing of up to sixteen cannabinoids in hemp-infused edibles using liquid chromatography diode array detector with optional confirmation of identity by electrospray ionization time-of-flight mass spectrometry.

A LC-DAD method for potency testing of up to sixteen cannabinoids has been developed, validated, and applied for analysis of twenty hemp-infused edibles encompassing a broad range of complex matrices. The method was validated according to ISO 17025 guidelines and met requirements. Samples or their uniform water-dispersions were extracted by methanol under homogenization through pulverization and/or ultrasonication. By spiking abnormal cannabidiol, a cannabinoid not naturally present in hemp, into each sample, extraction recovery was tracked in real time, obtaining 90 to 108% in triplicates with relative standard deviations of 0.5 to 6.5%. The linear calibration range was between 0.008 and 10% (w/w) for each cannabinoid using a 250µg/mL solution of hemp-infused edibles, except for drinks (sparkling water and tea), where it was between 0.0008 and 1% (w/w) using a 2.5mg/mL solution. ESI/TOFMS confirmed a good method specificity, i.e., without any false positive identification of individual cannabinoid.

Antidiabetic actions of GPR55 agonist Abn-CBD and sitagliptin in obese-diabetic high fat fed mice

GPR55 has been recognized as a novel anti-diabetic target exerting positive effects on beta cell function and mass. This study evaluated the metabolic actions and therapeutic efficacy of GPR55 agonist abnormal cannabidiol (Abn-CBD) administered alone and in combination with sitagliptin in diet-induced obese-diabetic mice.Chronic effects of 21-day oral administration of Abn-CBD (0.1 µmol/kg BW) monotherapy and in combination with sitagliptin (50 mg/kg BW) were assessed in obese-diabetic HFF mice (n = 8). Assessments of plasma glucose, circulating insulin, DPP-IV activity, CRP, amylase, lipids, body weight and food intake were undertaken. Glucose tolerance, insulin sensitivity, DEXA scanning and islet morphology analysis were performed at 21-days.Sitagliptin, Abn-CBD alone and in combination with sitagliptin attenuated plasma glucose by 37–53 % (p < 0.01 – p < 0.001) and enhanced circulating insulin concentrations by 23–31 % (p < 0.001). Abn-CBD alone and with sitagliptin reduced bodyweight by 9–10 % (p < 0.05). After 21-days, Abn-CBD in combination with sitagliptin (44 %; p < 0.01) improved glucose tolerance, whilst enhancing insulin sensitivity by 79 % (p < 0.01). Abn-CBD increased islet area (86 %; p < 0.05), beta cell mass (p < 0.05) and beta cell proliferation (164 %; p < 0.001), whilst in combination with sitagliptin islet area was decreased (50 %; p < 0.01). Abn-CBD alone, in combination with sitagliptin or sitagliptin alone decreased triglycerides by 34–65 % (p < 0.001) and total cholesterol concentrations by 15–25 % (p < 0.001). In addition, Abn-CBD in combination with sitagliptin reduced fat mass by 19 % (p < 0.05) and reduced CRP concentrations (39 %; p < 0.05).These findings advocate Abn-CBD monotherapy and in combination with sitagliptin as a novel and effective approach for bodyweight control and the treatment of glucose intolerance and dyslipidaemia in type-2-diabetes.

Potency testing of up to twenty cannabinoids by liquid chromatography diode array detector with optional electrospray ionization time-of-flight mass spectrometry

The growing popularity of Cannabis sativa L. and its widespread use for medical and recreational purposes have created an urgent demand of accurate and reliable analytical methods to identify and quantify a growing number of cannabinoids. To meet this demand, a liquid chromatography diode array detector (LC-DAD) method has been developed, validated, and applied in analysis of cannabinoids in nine samples of plant materials of marijuana, six samples of marijuana cigarettes, five samples of hemp flowers, one sample of hemp cigarette, and two samples of Δ8-tetrahydrocannabinol (Δ8-THC) fortified hemp flowers. The method has achieved significant improvements over published methods, which was characterized of 20 targeted cannabinoids, 18 quantified cannabinoids, baseline resolution among quantified cannabinoids, a low limit of quantification (0.02 μg mL−1), a wide linear range (0.02–25 μg mL−1 or 0.04–50% (w/w)), a unique experiment to track the recovery of sample preparation in real time by spiking abnormal cannabidiol (CBD), and a well-assessed specificity by electrospray ionization time-of-flight mass spectrometry (ESI/TOFMS). Precision and accuracy were assessed using quality control (QC) samples at three concentration levels, i.e., 0.02, 0.5, and 12.5 μg mL−1, in triplicates with inter-day and intra-day precision of less than 15% relative standard deviation (RSD) and accuracy of less than ±15% relative error, therefore meeting the requirements by the ISO 17025 standards. Additionally, ESI/TOFMS has discovered seven unknown cannabinoids, including one structural isomer of cannabigerol (CBG), one structural isomer of cannabinolic acid (CBNA), four structural isomers of Δ9-THC, and one structural isomer of Δ9-THC acetate. Furthermore, it uncovered that one of the two samples of Δ8-THC fortified hemp flowers contained 5.16% (w/w) Δ9-THC, an alarm to the current Δ8-THC craze by the public.

Abnormal cannabidiol ameliorates inflammation preserving pancreatic beta cells in mouse models of experimental type 1 diabetes and beta cell damage.

The atypical cannabinoid Abn-CBD improves the inflammatory status in preclinical models of several pathologies, including autoimmune diseases. However, its potential for modulating inflammation in autoimmune type 1 diabetes (T1D) is unknown. Herein we investigate whether Abn-CBD can modulate the inflammatory response during T1D onset using a mouse model of T1D (non-obese diabetic- (NOD)-mice) and of beta cell damage (streptozotocin (STZ)-injected mice). Six-week-old female NOD mice were treated with Abn-CBD (0.1-1mg/kg) or vehicle during 12 weeks and then euthanized. Eight-to-ten-week-old male C57Bl6/J mice were pre-treated with Abn-CBD (1mg/kg of body weight) or vehicle for 1 week, following STZ challenge, and euthanized 1 week later. Blood, pancreas, pancreatic lymph nodes (PLNs) and T cells were collected and processed for analysis. Glycemia was also monitored. In NOD mice, treatment with Abn-CBD significantly reduced the severity of insulitis and reduced the pro-inflammatory profile of CD4+ T cells compared to vehicle. Concomitantly, Abn-CBD significantly reduced islet cell apoptosis and improved glucose tolerance. In STZ-injected mice, Abn-CBD decreased circulating proinflammatory cytokines and ameliorated islet inflammation reducing intra-islet phospho-NF-κB and TXNIP. Abn-CBD significantly reduced 2 folds intra-islet CD8+ T cells and reduced Th1/non-Th1 ratio in PLNs of STZ-injected mice. Islet cell apoptosis and intra-islet fibrosis were also significantly reduced in Abn-CBD pre-treated mice compared to vehicle. Altogether, Abn-CBD reduces circulating and intra-islet inflammation, preserving islets, thus delaying the progression of insulitis. Hence, Abn-CBD and related compounds emerge as new candidates to develop pharmacological strategies to treat the early stages of T1D.

A molecular explanation of cardiovascular protection through abnormal cannabidiol: Involving the dysfunctional β-adrenergic and ATP-sensitive K+ channel activity in cardiovascular compromised preterm infants.

Growing cannabis efficacy, usage frequency, legal supply, and declining awareness of danger recently led to expanded United States cannabis exposure. In turn, cannabis use among elderly people over 50 has more than tripled in a decade and has contributed toward a positive association of cannabis use with pathological conditions, which include type II diabetes, metabolic syndrome, neurovascular and cardiovascular disease. Remarkably, all these outcome results are mediated by the involvement of the ATP-sensitive K+ channel. Cardiovascular compromise is a common syndrome in preterm infants that leads to incidence and death and has been distinguished by poor systemic flow or hypotension. Conditions of cardiovascular compromise include vasodysregulation and myocardial malfunction through dysfunctional β-adrenergic activity. To avoid organ hypoperfusion progressing to tissue hypoxia-ischemia, inotropic drugs are used. Many premature children, however, respond insufficiently to inotropic activity with adrenergic agonists. The clinical disturbance including myocardial dysfunction through the activation of the ATP-sensitive K+ channel is often involved and the comparative efficacy of the nonpsychotropic cannabinoid, abnormal cannabidiol(Abn-CBD) is not yet known. Therefore, our primary aim was to investigate the molecular exploration of the cannabinoid system specifically Abn-CBD in cardiovascular protection involving dysregulated KATP.

Abnormal Cannabidiol Affects Production of Pro-Inflammatory Mediators and Astrocyte Wound Closure in Primary Astrocytic-Microglial Cocultures.

Abnormal cannabidiol (abn-CBD) exerts neuroprotective effects in vivo and in vitro. In the present study, we investigated the impact of abn-CBD on the glial production of proinflammatory mediators and scar formation within in vitro models. Primary astrocytic-microglial cocultures and astrocytic cultures from neonatal C57BL/6 mice and CB2 receptor knockout mice were stimulated with lipopolysaccharide (LPS), and the concentrations of tumor necrosis factor α (TNFα), interleukin-6 (IL-6) and nitrite were determined. Furthermore, we performed a live cell microscopy-based scratch-wound assay. After LPS stimulation, TNFα, IL-6 and nitrite production was more strongly increased in cocultures than in isolated astrocytes. Abn-CBD treatment attenuated the LPS-induced production of TNFα and nitrite in cocultures, while IL-6 production remained unaltered. In isolated astrocytes, only LPS-induced TNFα production was reduced by abn-CBD. Similar effects were observed after abn-CBD application in cocultures of CB2 knockout mice. Interestingly, LPS-induced TNFα and nitrite levels were far lower in CB2 knockout cultures compared to wildtypes, while IL-6 levels did not differ. In the scratch-wound assay, treatment with abn-CBD decelerated wound closure when microglial cells were present. Our data shows a differential role of abn-CBD for modulation of glial inflammation and astrocytic scar formation. These findings provide new explanations for mechanisms behind the neuroprotective potential of abn-CBD.

Opposite Effects of Neuroprotective Cannabinoids, Palmitoylethanolamide, and 2-Arachidonoylglycerol on Function and Morphology of Microglia.

Various studies performed in cultured cells and in in vivo models of neuronal damage showed that cannabinoids exert a neuroprotective effect. The increase in cannabinoids and cannabinoid like substances after stroke has been postulated to limit the content of neuronal injury. As well-accepted, inflammation, and neuronal damage are coupled processes and microglial cells as the main intrinsic immunological effector within the brain play a central role in their regulation. Treatment with the endocannabinoid, 2-arachidonoylglycerol (2-AG) or the endocannabinoid-like substance, palmitoylethanolamide (PEA) affected microglial cells and led to a decrease in the number of damaged neurons after excitotoxical lesion in organotypic hippocampal slice cultures (OHSC). 2-AG activated abnormal cannabidiol (abn-CBD) receptor, PEA was shown to mediate neuroprotection via peroxisome proliferator-activated receptor (PPAR)α. Despite the known neuroprotective and anti-inflammatory properties, the potential synergistic effect, namely possible entourage effect after treatment with the combination of these two protective cannabinoids has not been examined yet. After excitotoxical lesion OHSC were treated with PEA, 2-AG or a combination of both and the number of damaged neurons was evaluated. To investigate the role of microglial cells in PEA and 2-AG mediated protection, primary microglial cell cultures were treated with lipopolysaccharide (LPS) and 2-AG, PEA or a combination of those. Thereafter, we measured NO production, ramification index, proliferation and PPARα distribution in microglial cells. While PEA or 2-AG alone were neuroprotective, their co-application vanished the protective effect. This behavior was independent of microglial cells. Furthermore, PEA and 2-AG had contrary effects on ramification index and on NO production. No significant changes were observed in the proliferation rate of microglial cells after treatment. The expression of PPARα was not changed upon stimulation with PEA or 2-AG, but the distribution was significantly altered. 2-AG and PEA mediated neuroprotection was abolished when co-applied. Both cannabinoids exert contrary effects on morphology and function of microglial cells. Co-application of both cannabinoids with different targets did not lead to a positive additive effect as expected, presumably due to the contrary polarization of microglial cells.

Antitumor Activity of Abnormal Cannabidiol and Its Analog O-1602 in Taxol-Resistant Preclinical Models of Breast Cancer.

Cannabinoids exhibit anti-inflammatory and antitumorigenic properties. Contrary to most cannabinoids present in the Cannabis plant, some, such as O-1602 and abnormal cannabidiol, have no or only little affinity to the CB1 or CB2 cannabinoid receptors and instead exert their effects through other receptors. Here, we investigated whether the synthetic regioisomers of cannabidiol, abnormal cannabidiol, and a closely related compound, O-1602, display antitumorigenic effects in cellular models of breast cancer and whether it could reduce tumorigenesis in vivo. Several studies have shown the effects of cannabinoids on chemotherapy-sensitive breast cancer cell lines, but less is known about the antitumorigenic effects of cannabinoids in chemotherapy-resistant cell lines. Paclitaxel-resistant MDA-MB-231 and MCF-7 breast cancer cell lines were used to study the effect of O-1602 and abnormal cannabidiol on viability, apoptosis, and migration. The effects of O-1602 and abnormal cannabidiol on cell viability were completely blocked by the combination of GPR55 and GPR18-specific siRNAs. Both O-1602 and abnormal cannabidiol decreased viability in paclitaxel-resistant breast cancer cells in a concentration-dependent manner through induction of apoptosis. The effect of these cannabinoids on tumor growth in vivo was studied in a zebrafish xenograft model. In this model, treatment with O-1602 and abnormal cannabidiol (2 µM) significantly reduced tumor growth. Our results suggest that atypical cannabinoids, like O-1602 and abnormal cannabidiol, exert antitumorigenic effects on paclitaxel-resistant breast cancer cells. Due to their lack of central sedation and psychoactive effects, these atypical cannabinoids could represent new leads for the development of additional anticancer treatments when resistance to conventional chemotherapy occurs during the treatment of breast and possibly other cancers.

G-protein coupled receptor 55 agonists increase insulin secretion through inositol trisphosphate-mediated calcium release in pancreatic β-cells

G-protein coupled receptor 55 (GPR55) is an orphan G-protein coupled receptor, which is activated by endocannabinoids and lipid transmitters. Recently, GPR55 was shown to play a role in glucose and energy homeostasis, and insulin secretion is essential to maintain glucose homeostasis in the body. In Type 2 Diabetes Mellitus (T2DM), chronic insulin resistance and a progressive decline in β-cell function result in β-cell dysfunction, this leads to defect in insulin secretion, which is the key process in the development and progression of T2DM. GPR55 agonists were shown to increase insulin secretion, however the underlying mechanisms were not fully understood. Therefore the aim of the present study was to examine the effects of potent GPR55 agonists, O-1602 and abnormal cannabidiol (Abn-CBD), on glucose-induced insulin secretion in a mouse pancreatic β-cell line, MIN6, and the underlying mechanisms with a focus on intracellular calcium (Ca2+). Our results demonstrated that O-1602 and Abn-CBD increased glucose-induced insulin secretion in MIN6 cells, which was abolished by a PLC inhibitor, U73122. Glucose-induced Ca2+ transients were enhanced by O-1602 and Abn-CBD, and this was significantly reduced by U73122 and inositol trisphosphate (IP3) receptor inhibitors, 2-aminoethoxydiphenyl borate (2-APB) and xestospongin C, as well as by Y-27632, a Rho-associated protein kinase (ROCK) inhibitor. Interestingly, O-1602 and Abn-CBD could directly induce intracellular Ca2+ transients through IP3-mediated Ca2+ release. In conclusion, GPR55 agonists increased insulin secretion through calcium mobilisation from IP3-sensitive ER stores in β-cells.

Abnormal Cannabidiol Modulates Vitamin A Metabolism by Acting as a Competitive Inhibitor of CRBP1.

Cellular retinol-binding proteins (CRBPs) facilitate the uptake and intracellular transport of vitamin A. They integrate retinoid metabolism, playing an important role in regulating the synthesis of bioactive vitamin A metabolites. Thus, CRBPs constitute potential pharmacological targets to modulate cellular retinoid status that in turn may have applications in the treatment of certain immunological, metabolic, and ocular disorders. Here we identify abnormal cannabidiol (abn-CBD) as a nonretinoid inhibitor of cellular retinol-binding protein 1 (CRBP1). X-ray crystal structures of CRBP1 in complex with abn-CBD and its derivatives revealed a distinctive mode of protein-ligand interaction and provided a molecular basis for the high affinity and selectivity of this compound. We demonstrated that abn-CBD modulates the flux of retinoids via the retinoid cycle in vivo. Furthermore, the biological activity of abn-CBD was evidenced by its ability to protect against light-induced retinal damage in Balb/cJ mice. Altogether, our findings indicate that targeting selected CRBPs with a small-molecule inhibitor can potentially lead to the development of new therapeutic agents to counteract diseases with etiologies involving imbalance in retinoid metabolism or signaling.

Novel protective effect of O-1602 and abnormal cannabidiol, GPR55 agonists, on ER stress-induced apoptosis in pancreatic β-cells

Insulin resistance and β-cell dysfunction are the main defects in Type 2 Diabetes Mellitus (T2DM), and β-cell dysfunction and apoptosis is the critical determinant in the progression of T2DM. G-protein coupled receptor 55 (GPR55) is an orphan G-protein coupled receptor, which is activated by endocannabinoids and lipid transmitters. Recently, GPR55 was shown to regulate glucose and energy homeostasis, however its role in β-cell apoptosis was not studied. Therefore, in this study, we investigated the novel effect of GPR55 agonists, O-1602 and abnormal cannabidiol (Abn-CBD), on endoplasmic reticulum (ER) stress-induced apoptosis in mouse pancreatic β-cell lines, MIN6 and Beta-TC-6, and its underlying mechanisms. Our results showed that O-1602 and Abn-CBD reduced ER stress-induced apoptosis in MIN6 and Beta-TC-6 cells. This was through the phosphorylation of 3′-5′-cyclic adenosine monophosphate response element-binding protein (CREB) in β-cells, hence activating CREB downstream anti-apoptotic genes, Bcl-2 and Bcl-xL. Moreover, O-1602 and Abn-CBD directly activated kinases, CaMKIV, Erk1/2 and PKA, to induce CREB phosphorylation. Therefore, our results indicated that GPR55 agonists protected from β-cell apoptosis through CREB activation, thus up-regulating anti-apoptotic genes. In conclusion, our study provided a novel protective effect of GPR55 agonists on ER stress-induced apoptosis in β-cells and its underlying mechanisms mediating this protection, therefore we suggested that GPR55 might be a therapeutic target for T2DM.

Cover Image, Volume 20, Issue 4

The cover image, by Inmaculada Ruz‐Maldonado et al., is based on the Original Article LH‐21 and abnormal cannabidiol improve β‐cell function in isolated human and mouse islets through GPR55‐dependent and ‐independent signalling, DOI: 10.1111/dom.13180. image

The quest for endothelial atypical cannabinoid receptor: BKCa channels act as cellular sensors for cannabinoids in in vitro and in situ endothelial cells

Endothelium-dependent component of cannabinoid-induced vasodilation has been postulated to require G-protein-coupled non-CB1/CB2 endothelial cannabinoid (eCB) receptor. GPR18 was proposed as a candidate for eCBR. To address the hypothesis that the effects attributed to eCBR are mediated by G-protein-coupled receptor (GPCR)-independent targets, we studied the electrical responses in endothelial cells, focusing on BKCa channels. In patches excised from endothelial-derived EA.hy926 cells, N-arachidonoyl glycine (NAGly) and abnormal cannabidiol (abn-cbd), prototypical agonists for eCB receptor, stimulate single BKCa activity in a concentration- and Ca2+-dependent manner. The postulated eCB receptor inhibitors rimonabant and AM251 were found to inhibit basal and stimulated by NAGly- and abn-cbd BKCa activity in cell-free patches. In isolated mice aortas, abn-cbd and NAGly produced endothelial cell hyperpolarization that was sensitive to paxilline, a selective BKCa inhibitor, but not to GPR18 antibody, and mimicked by NS1619, a direct BKCa opener. In excised patches from mice aortic endothelium, single channel activity with characteristics similar to BKCa was established by the addition of abn-cbd and NAGly. We conclude that the two cannabinoids abn-cbd and NAGly initiate a GPR18-independent activation of BKCa channels in mice aortic endothelial cells that might contribute to vasodilation to cannabinoids.

LH-21 and abnormal cannabidiol improve β-cell function in isolated human and mouse islets through GPR55-dependent and -independent signalling.

To examine the effects of Abn-CBD (GPR55 agonist) and LH-21 (CB1 antagonist) on human and mouse islet function, and to determine signalling via GPR55 using islets from GPR55-/- mice. Islets isolated from human organ donors and mice were incubated in the absence or presence of Abn-CBD or LH-21, and insulin secretion, [Ca2+ ]i, cAMP, apoptosis, β-cell proliferation and CREB and AKT phosphorylation were examined using standard techniques. Abn-CBD potentiated glucose-stimulated insulin secretion and elevated [Ca2+ ]i in human islets and islets from both GPR55+/+ and GPR55-/- mice. LH-21 also increased insulin secretion and [Ca2+ ]i in human islets and GPR55+/+ mouse islets, but concentrations of LH-21 up to 0.1 μM were ineffective in islets from GPR55-/- mice. Neither ligand affected basal insulin secretion or islet cAMP levels. Abn-CBD and LH-21 reduced cytokine-induced apoptosis in human islets and GPR55+/+ mouse islets, and these effects were suppressed after GPR55 deletion. They also increased β-cell proliferation: the effects of Abn-CBD were preserved in islets from GPR55-/- mice, while those of LH-21 were abolished. Abn-CBD and LH-21 increased AKT phosphorylation in mouse and human islets. This study showed that Abn-CBD and LH-21 improve human and mouse islet β-cell function and viability. Use of islets from GPR55-/- mice suggests that designation of Abn-CBD and LH-21 as a GPR55 agonist and a CB1 antagonist, should be revised.

Abnormal cannabidiol confers cardioprotection in diabetic rats independent of glycemic control

Chronic GPR18 activation by its agonist abnormal cannabidiol (trans-4-[3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol; abn-cbd) improves myocardial redox status and function in healthy rats. Here, we investigated the ability of abn-cbd to alleviate diabetes-evoked cardiovascular pathology and the contribution of GPR18 to this effect. Four weeks after diabetes induction by streptozotocin (STZ, 55mg/kg; i.p), male Wistar rats received abn-cbd, the GPR18 antagonist (1,3-dimethoxy-5-methyl-2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-,cyclohexen-1-yl]benzene;O-1918), their combination (100µg/kg/day, i.p, each) or their vehicle for 2 weeks. Abn-cbd had no effect on diabetes-evoked cardiac hypertrophy or impaired glycemic control (hyperglycemia and hypoinsulinemia), but alleviated the associated reductions in left ventricular (LV) contractility (dP/dtmax) and relaxation (dP/dtmin) indices, and the increases in LV end diastolic pressure (LVEDP) and cardiac vagal dominance. Abn-cbd also reversed myocardial oxidative stress by restoring circulating and cardiac nitric oxide (NO) and adiponectin (ADN) levels and enhancing GPR18 expression and phosphorylation of Akt, ERK1/2 and eNOS in diabetic rats’ hearts. Concurrent GPR18 blockade (O-1918) abrogated all favorable effects of abn-cbd in diabetic rats. Collectively, the current findings present evidence for abn-cbd alleviation of diabetes-evoked cardiovascular anomalies likely via GPR18 dependent restoration of cardiac adiponectin-Akt-eNOS signaling and the diminution of myocardial oxidative stress.

GPR55: A therapeutic target for Parkinson's disease?

The GPR55 receptor is expressed abundantly in the brain, especially in the striatum, suggesting it might fulfill a role in motor function. Indeed, motor behavior is impaired in mice lacking GPR55, which also display dampened inflammatory responses. Abnormal-cannabidiol (Abn-CBD), a synthetic cannabidiol (CBD) isomer, is a GPR55 agonist that may serve as a therapeutic agent in the treatment of inflammatory diseases. In this study, we explored whether modulating GPR55 could also represent a therapeutic approach for the treatment of Parkinson's disease (PD). The distribution of GPR55 mRNA was first analyzed by in situ hybridization, localizing GPR55 transcripts to neurons in brain nuclei related to movement control, striatum, globus pallidus, subthalamic nucleus, substantia nigra and cortex. Striatal expression of GPR55 was downregulated in parkinsonian conditions. When Abn-CBD and CBD (5 mg/kg) were chronically administered to mice treated over 5 weeks with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTPp), Abn-CBD but not CBD prevented MPTPp induced motor impairment. Although Abn-CBD protected dopaminergic cell bodies, it failed to prevent degeneration of the terminals or preserve dopamine levels in the striatum. Both compounds induced morphological changes in microglia that were compatible with an anti-inflammatory phenotype that did not correlate with a neuroprotective activity. The symptomatic relief of Abn-CBD was further studied in the haloperidol-induced catalepsy mouse model. Abn-CBD had an anti-cataleptic effect that was reversed by CBD and PSB1216, a newly synthesized GPR55 antagonist, and indeed, two other GPR55 agonists also displayed anti-cataleptic effects (CID1792197 and CID2440433). These results demonstrate for the first time that activation of GPR55 might be beneficial in combating PD.

The Effect of Chronic Activation of the Novel Endocannabinoid Receptor GPR18 on Myocardial Function and Blood Pressure in Conscious Rats.

Although acute activation of the novel endocannabinoid receptor GPR18 causes hypotension, there are no reports on GPR18 expression in the heart or its chronic modulation of cardiovascular function. In this study, after demonstrating GPR18 expression in the heart, we show that chronic (2 weeks) GPR18 activation with its agonist abnormal cannabidiol (abn-cbd; 100 µg·kg·d; i.p) produced hypotension, suppressed the cardiac sympathetic dominance, and improved left ventricular (LV) function (increased the contractility index dp/dtmax and reduced LV end-diastolic pressure, LVEDP) in conscious rats. Ex vivo studies revealed increased: (1) cardiac and plasma adiponectin (ADN) levels; (2) vascular (aortic) endothelial nitric oxide synthase (eNOS) expression, (3) vascular and serum nitric oxide (NO) levels; (4) myocardial and plasma cyclic guanosine monophosphate (cGMP) levels; (5) phosphorylation of myocardial protein kinase B (Akt) and extracellular signal regulated kinase 1/2 (ERK1/2) along with reduced myocardial reactive oxygen species (ROS) in abn-cbd treated rats. These biochemical responses contributed to the hemodynamic responses and were GPR18-mediated because concurrent treatment with the competitive GPR18 antagonist (O-1918) abrogated the abn-cbd-evoked hemodynamic and biochemical responses. The current findings present new evidence for a salutary cardiovascular role for GPR18, mediated, at least partly, via elevation in the levels of adiponectin.