GPR55 Antagonist CID16020046 Attenuates Obesity-Induced Airway Inflammation by Suppressing Chronic Low-Grade Inflammation in the Lungs.

GPR55 is a receptor for lysophosphatidylinositols (LPIs) in digestive metabolites. Overnutrition leads to obesity, insulin resistance, and increased LPI levels in the plasma. The involvement of LPIs and GPR55 in adiposity, hepatic steatosis, and atherosclerosis has been previously elucidated. However, the therapeutic efficacy of GPR55 antagonists against obesity-induced airway inflammation has not been studied. The present study investigated whether CID16020046, a selective antagonist of GPR55, could modulate obesity-induced airway inflammation caused by a high-fat diet (HFD) in C57BL/6 mice. Administration of CID16020046 (1 mg/kg) inhibits HFD-induced adiposity and glucose intolerance. Analysis of immune cells in BALF showed that CID16020046 inhibited HFD-induced increase in immune cell infiltration. Histological analysis revealed the HFD induced hypersecretion of mucus and extensive fibrosis in the lungs. CID16020046 inhibited these HFD-induced pathological features. qRT-PCR revealed the HFD-induced increase in the expression of Ifn-γ, Tnf-α, Il-6, Il-13, Il-17A, Il-1ß, Nlrp3, and Mpo mRNAs in the lungs. CID16020046 inhibited the HFD-induced increases in these genes. The expression levels of adipokines were regulated by the HFD and CID16020046. AdipoQ in the lungs and gonadal white adipose tissue was decreased by the HFD and reversed by CID16020046. In contrast, Lep was increased by the HFD and suppressed by CID16020046. The findings suggest the potential application of the GPR55 antagonist CID16020046 in obesity-induced airway inflammation.

GPR55 Antagonist CID16020046 Protects against Atherosclerosis Development in Mice by Inhibiting Monocyte Adhesion and Mac-1 Expression.

GPR55 recognizes several lipid molecules such as lysophosphatidylinositol. GPR55 expression was reported in human monocytes. However, its role in monocyte adhesion and atherosclerosis development has not been studied. The role of GPR55 in monocyte adhesion and atherosclerosis development was investigated in human THP-1 monocytes and ApoE-/- mice using O-1602 (a potent agonist of GPR55) and CID16020046 (a specific GPR55 antagonist). O-1602 treatment significantly increased monocyte adhesion to human umbilical vein endothelial cells, and the O-1602-induced adhesion was inhibited by treatment with CID16020046. O-1602 induced the expression of Mac-1 adhesion molecules, whereas CID16020046 inhibited this induction. Analysis of the promoter region of Mac-1 elucidated the binding sites of AP-1 and NF-κB between nucleotides -750 and -503 as GPR55 responsive elements. O-1602 induction of Mac-1 was found to be dependent on the signaling components of GPR55, that is, Gq protein, Ca2+, CaMKK, and PI3K. In Apo-/- mice, administration of CID16020046 ameliorated high-fat diet-induced atherosclerosis development. These results suggest that high-fat diet-induced GPR55 activation leads to the adhesion of monocytes to endothelial cells via induction of Mac-1, and CID16020046 blockage of GPR55 could suppress monocyte adhesion to vascular endothelial cells through suppression of Mac-1 expression, leading to protection against the development of atherosclerosis.

Structure-Activity Relationship of the GPR55 Antagonist, CID16020046.

BACKGROUND/AIMS: CID16020046 blocks the effect of the lipid lysophosphatidylinositol (LPI) at its receptor, GPR55. CID16020046 and another antagonist, ML193, have been used to investigate GPR55-mediated effects of LPI on cells, tissues, and in vivo. Here we describe the structure-activity relationship of CID16020046. METHODS: Yeast or human cells were engineered to express GPR55 or control receptors. Cells were pretreated with a test agent before agonist challenge. Functional responses were quantified by yeast gene-reporter or calcium imaging. RESULTS: Three substituents around the central pyrazololactam core of CID16020046 are each tolerant to substitution without abolishing GPR55 activity. Analogues of CID16020046 with potency at GPR55 ranging >1,000-fold are described, including several lacking activity up to the top concentration tested. One analogue, compound 1 (GSK875734A), has approximately 50-fold greater potency than CID16020046 in an inverse agonist assay. CID16020046, ML193 and 2 further antagonists (ML191 and ML192) all block the effect of a surrogate agonist at human GPR55. ML193, CID16020046 and several other examples of the pyrazololactam chemotype were also shown to antagonise rat GPR55. CONCLUSION: These data confirm the utility of CID16020046 and ML193 as tools to investigate the physiological role of GPR55, and offer starting points for GPR55 antagonists with optimised pharmacokinetic or other properties.

Scotopic vision in the monkey is modulated by the G protein-coupled receptor 55.

The endogenous cannabinoid system plays important roles in the retina of mice and monkeys via their classic CB1 and CB2 receptors. We have previously reported that the G protein-coupled receptor 55 (GPR55), a putative cannabinoid receptor, is exclusively expressed in rod photoreceptors in the monkey retina, suggesting its possible role in scotopic vision. To test this hypothesis, we recorded full-field electroretinograms (ERGs) after the intravitreal injection of the GPR55 agonist lysophosphatidylglucoside (LPG) or the selective GPR55 antagonist CID16020046 (CID), under light- and dark-adapted conditions. Thirteen vervet monkeys (Chlorocebus sabaeus) were used in this study: four controls (injected with the vehicle dimethyl sulfoxide, DMSO), four injected with LPG and five with CID. We analyzed amplitudes and latencies of the a-wave (photoreceptor responses) and the b-wave (rod and cone system responses) of the ERG. Our results showed that after injection of LPG, the amplitude of the scotopic b-wave was significantly higher, whereas after the injection of CID, it was significantly decreased, compared to the vehicle (DMSO). On the other hand, the a-wave amplitude, and the a-wave and b-wave latencies, of the scotopic ERG responses were not significantly affected by the injection of either compound. Furthermore, the photopic ERG waveforms were not affected by either drug. These results support the hypothesis that GPR55 plays an instrumental role in mediating scotopic vision.

GPR55 antagonist CID16020046 suppresses DNCB-induced atopic dermatitis-like symptoms by suppressing Th1/Th2/Th17 populations in mice.

G protein-coupled receptor 55 (GPR55) is a lipid-sensing receptor that plays a role as an immune mediator and is primarily upregulated during immune cell activation. There is a lack of knowledge about the role of GPR55 in allergic inflammatory diseases such as atopic dermatitis. The purpose of this study was to investigate the role of GPR55 through the use of its antagonist, CID16020046, in an atopic dermatitis mouse model. It was found that BALB/c mice develop lesions similar to those associated with atopic dermatitis following sensitization and repeated exposure to 1-chloro-2,4-dinitrobenzene (DNCB). It was found that CID16020046 (1 mg/kg, i.p.) alleviated the atopic dermatitis-like symptoms as well as immune dysregulation caused by DNCB. Based on histopathological analysis, CID16020046 reduced ear thickening and mast cell counts in the dermis. CID16020046 decreased DNCB-induced increases in serum IgE levels, as measured using enzyme-linked immunosorbent assays. A significant reduction in lymph node hypertrophy was also observed with CID16020046 as well as significant reductions in CD4+ T helper 1 (Th1), Th2, and Th17 cells in the lymph nodes. As a result of the administration of CID16020046, cytokines of Th1 (IFN-γ), Th2 (IL-4 and IL-13), and Th17 (IL-17A) types were also reduced in the skin and lymph nodes. In conclusion, blocking GPR55 alleviates DNCB-induced atopic dermatitis-like symptoms, suggesting that GPR55 is a potential therapeutic target for allergic inflammatory diseases via immunoregulation.

Gpr55 deficiency crucially alters cardiomyocyte homeostasis and counteracts angiotensin II induced maladaption in female mice.

BACKGROUND AND PURPOSE: Cannabis stimulates several G-protein-coupled-receptors and causes bradycardia and hypotension upon sustained consumption. Moreover, in vitro studies suggest an interference of cannabinoid-signalling with cardiomyocyte contractility and hypertrophy. We aimed at revealing a functional contribution of the cannabinoid-sensitive receptor GPR55 to cardiomyocyte homeostasis and neurohumorally induced hypertrophy in vivo. EXPERIMENTAL APPROACH: Gpr55-/- and wild-type (WT) mice were characterized after 28-day angiotensin II (AngII; 1·µg·kg-1 min-1) or vehicle infusion. In isolated adult Gpr55-/- and WT cardiomyocytes, mitochondrial function was assessed under naïve conditions, while cytosolic Ca2+ handling was additionally determined following application of the selective GPR55 antagonist CID16020046. KEY RESULTS: Gpr55 deficiency did not affect angiotensin II (AngII) mediated hypertrophic growth, yet, especially in females, it alleviated maladaptive pro-hypertrophic and -inflammatory gene expression and improved inotropy and adrenergic responsiveness compared to WT. In-depth analyses implied increased cytosolic Ca2+ concentrations and transient amplitudes, and accelerated sarcomere contraction kinetics in Gpr55-/- myocytes, which could be mimicked by GPR55 blockade with CID16020046 in female WT cells. Moreover, Gpr55 deficiency up-regulated factors involved in glucose and fatty acid transport independent of the AngII challenge, accelerated basal mitochondrial respiration and reduced basal protein kinase (PK) A, G and C activity and phospholemman (PLM) phosphorylation. CONCLUSIONS AND IMPLICATIONS: Our study suggests GPR55 as crucial regulator of cardiomyocyte hypertrophy and homeostasis presumably by regulating PKC/PKA-PLM and PKG signalling, and identifies the receptor as potential target to counteract maladaptation, adrenergic desensitization and metabolic shifts as unfavourable features of the hypertrophied heart in females.

CID16020046, a GPR55 antagonist, attenuates sepsis­induced acute kidney injury.

Acute kidney injury (AKI) is the most common and serious complication of sepsis, and it is also the main cause of mortality in patients with sepsis. The G protein­coupled receptor 55 (GPR55) inhibitor CID16020046 was found to suppress the inflammatory response in sepsis models in mice. The aim of the present study was to investigate the effect of CID16020046 on AKI in sepsis mouse models and elucidate the possible underlying mechanisms. A sepsis model in mice was established by cecal ligation/perforation (CLP). The expression levels of GPR55 in the serum of patients with sepsis and the renal tissues of septic mice were determined via reverse transcription­quantitative PCR and western blot analyses, respectively. The pathological injury of renal tissue was evaluated using H&E and periodic acid­Schiff staining. ELISA was performed to detect the levels of renal injury­related factors, including blood urea nitrogen (BUN), creatinine (Cre), kidney injury molecule 1 (KIM1) and neutrophil gelatinase­associated lipocalin (NGAL) in septic mice. Moreover, the levels of pro­inflammatory cytokines (TNF­α, IL­6 and IL­1ß) were detected via ELISA and western blotting. Apoptosis was determined using TUNEL staining and western blotting. The expression levels of Rho­associated protein kinase (ROCK) pathway­related proteins (Ras homolog family member A, ROCK1 and ROCK2) was measured via western blotting. Finally, H&E staining was used to evaluate the effect of CID16020046 on various organs in mice. Compared with the control subjects, the expression level of GPR55 in the serum of patients with sepsis was significantly increased. Compared with the sham group, CID16020046 (20 mg/kg) significantly decreased the levels of BUN and Cre in the serum, as well as the contents of KIM1 and NGAL in the urine. Furthermore, CID16020046 significantly decreased the contents of TNF­α, IL­6 and IL­1ß in the serum and renal tissue of septic mice, and reduced cell apoptosis. In addition, CID16020046 effectively suppressed the expression levels of ROCK pathway­related proteins, and H&E staining revealed that CID16020046 (20 mg/kg) had no toxic effect on the heart, liver, spleen or lung in normal mice. In conclusion, CID16020046 may prove useful for the development of drugs for the treatment of sepsis­induced AKI.

Intrapallidal injection of cannabidiol or a selective GPR55 antagonist decreases motor asymmetry and improves fine motor skills in hemiparkinsonian rats.

Cannabidiol (CBD) presents antiparkinsonian properties and neuromodulatory effects, possibly due to the pleiotropic activity caused at multiple molecular targets. Recently, the GPR55 receptor has emerged as a molecular target of CBD. Interestingly, GPR55 mRNA is expressed in the external globus pallidus (GPe) and striatum, hence, it has been suggested that its activity is linked to motor dysfunction in Parkinson's disease (PD). The present study aimed to evaluate the effect of the intrapallidal injection of both CBD and a selective GPR55 antagonist (CID16020046) on motor asymmetry, fine motor skills, and GAD-67 expression in hemiparkinsonian rats. The hemiparkinsonian animal model applied involved the induction of a lesion in male Wistar rats via the infusion of the neurotoxin 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle via stereotaxic surgery. After a period of twenty days, a second surgical procedure was performed to implant a guide cannula into the GPe. Seven days later, lysophosphatidylinositol (LPI), CBD, or CID16020046 were injected once a day for three consecutive days (from the 28th to the 30th day post-lesion). Amphetamine-induced turning behavior was evaluated on the 14th and 30th days post-injury. The staircase test and fine motor skills were evaluated as follows: the rats were subject to a ten-day training period prior to the 6-OHDA injury; from the 15th to the 19th days post-lesion, the motor skills alterations were evaluated under basal conditions; and, from the 28th to the 30th day post-lesion, the pharmacological effects of the drugs administered were evaluated. The results obtained show that the administration of LPI or CBD generated lower levels of motor asymmetry in the turning behavior of hemiparkinsonian rats. It was also found that the injection of CBD or CID16020046, but not LPI, in the hemiparkinsonian rats generated significantly superior performance in the staircase test, in terms of the use of the forelimb contralateral to the 6-OHDA-induced lesion, when evaluated from the 28th to the 30th day post-lesion. Similar results were also observed for superior fine motor skills performance for pronation, grasp, and supination. Finally, the immunoreactivity levels were found to decrease for the GAD-67 enzyme in the striatum and the ipsilateral GPe of the rats injected with CBD and CID16020046, in contrast with those lesioned with 6-OHDA. The results obtained suggest that the inhibitory effects of CBD and CID16020046 on GPR55 in the GPe could be related to GABAergic overactivation in hemiparkinsonism, thus opening new perspectives to explain, at a cellular level, the reversal of the motor impairment observed in PD models.

Antinociceptive Effects of the GPR55 Antagonist CID16020046 Injected into the Rat Anterior Cingulate Cortex.

The G-protein coupled receptor, GPR55, modulates nociceptive processing. Given the expression of GPR55 in the anterior cingulate cortex (ACC), a key brain region involved in the cognitive and affective dimensions of pain, the present study tested the hypothesis that GPR55 signalling in the ACC facilitates inflammatory pain behaviour in rats. The expression of GPR55 in the ACC was confirmed by both western blotting and immunostaining, with evidence for neuronal localisation. Microinjection of the selective GPR55 antagonist CID16020046 into the ACC of adult male Sprague-Dawley rats significantly reduced second phase formalin-evoked nociceptive behaviour compared with vehicle-treated controls. CID16020046 administration was associated with a reduction in phosphorylation of extracellular signal-regulated kinase (ERK), a downstream target of GPR55 activation, in the ACC. Intra-ACC administration of CID16020046 prevented the formalin-induced increases in expression of mRNA coding for the immediate early gene and marker of neuronal activity, c-Fos, in the ipsilateral dorsal horn of the spinal cord. Intra-plantar injection of formalin reduced tissue levels of the endogenous GPR55 ligand 2-arachidonoyl-sn-glycero-3-phosphoinositol (2-AGPI) in the ACC, likely reflecting its increased release/utilisation. These data suggest that endogenous activation of GPR55 signalling and increased ERK phosphorylation in the ACC facilitates inflammatory pain via top-down modulation of descending pain control.

The GPR55 antagonist CID16020046 mitigates advanced glycation end products (AGEs)- induced chondrocyte activation.

Osteoarthritis (OA) is one of the most common degenerative joint diseases in aging people. The activation of chondrocytes and their dysregulation are closely related to the pathogenesis of OA. GPR55 is an unique orphan G-receptor which binds to cannabinoids. In this study, we explored the role of GPR55 in advanced glycation end productions (AGEs)- induced chondrocytes activation in cultured cells. We showed that AGEs dose dependently induced GPR55 expression in ATDC5 chondrocytes. The blockage of GPR55 by its newly discovered antagonist-CID16020046 mitigated AGEs- induced increase in cellular ROS and decrease in antioxidant NRF2. Moreover, CID16020046 showed a dose-response suppressive effect on AGEs- induced expression of the major inflammatory mediators, including COX-2 and iNOS, and the production of NO and PGE2. CID16020046 also dose responsively inhibited AGEs- induced key effectors of cartilage degradation such as MMP-3 and MMP-13. In consequence, CID16020046 showed robust inhibition on AGEs- induced type II collagen degradation. Mechanistically, our data demonstrated that CID16020046 mediated GPR55 blockage ameliorated AGEs- induced NF-κB activation as revealed by its inhibition on IκBα, nuclear p65 translocation and NF-κB promoter activity. Collectively, our study demonstrates that GPR55 signaling mediates AGEs- induced chondrocyte activation, and the targeted blockage of GPR55 pathway could be therapeutic choice in the treatment of osteoarthritis.

In vitro bioactivity approach of unripe genipap (Genipa americana L., Rubiaceae) fruit extract and its solid lipid microparticle.

Growing awareness in favor of innovative and healthier alternatives is creating a noticeable shift from synthetic colorants to natural additives. And, such a swing in the consumer market is growing slowly but noticeably. In this context, genipap (Genipa americana L.) fruit represents an emerging source of blue colorants in Latin America with extensive application possibilities. This is despite the fact that there are few studies concerning its toxicity predictive factors. In this early-stage study we propose to investigate safety issues around genipap extract (IBBP); we also attempt to identify fingerprint profiling of both IBBP extract and solid lipid microparticles containing IBBP extract (SLM-IBBP) using in vitro assays. The main compounds identified were genipin, and genipin 1-ß-gentiobioside. Results indicated that IBBP extract, at 25 µg/mL, was able to promote DNA damage in CHO-K1 cells, suggesting a genotoxic effect. On the other hand, the SLM-IBBP inhibited almost all cancer cell lines with GI50 ranging from 0.25 µg/mL to 43.5 µg/mL. Also, IBBP-SLM seems to exert a desirable apoptosis induction (at 25 µg/mL dosage). The next steps for our work, therefore, will focus on other nanoparticle formulation approaches, in particular with the use of natural Brazilian starch. An evaluation of the metabolism and distribution of microparticles, and their safety for food and pharmaceutical purposes, are also required.

Iridoid blue-based pigments of Genipa americana L. (Rubiaceae) extract: Influence of pH and temperature on color stability and antioxidant capacity during in vitro simulated digestion.

Iridoid blue-based pigments (IBBP) extract of Genipa americana L. represents a natural alternative additive for food applications and also exerts desirable biological effects on human health. In this study the iridoids present in the extract were identified, the influence of pH and temperature on color difference (ΔE) of IBBP was evaluated using a central composite design (CCD) and finally the antioxidant capacity was monitored before and after its in vitro digestion. Ten glucoside iridoids were detected and the main compounds identified were genipin, genipin 1-ß-gentiobioside and geniposide. It was also observed an increase of 17-18% of antioxidant capacity after the in vitro digestion, respectively. Among the conditions tested, the color of extract was more stable at 12-20 °C and low pH (3.0-4.0), suggesting that it is compatible for coloring acidic foods. Finally, the in vitro digestion also increased the antioxidant capacity (ORAC assay) by 39%.