Recombinant Apyrase (AZD3366) Against Myocardial Reperfusion Injury.

PURPOSE: Recombinant apyrase (AZD3366) increases adenosine production and ticagrelor inhibits adenosine reuptake. We investigated whether intravenous AZD3366 before reperfusion reduces myocardial infarct size (IS) and whether AZD3366 and ticagrelor have additive effects. METHODS: Sprague-Dawley rats underwent 30 min ischemia. At 25 min of ischemia, animals received intravenous AZD3366 or vehicle. Additional animals received intravenous CGS15943 (an adenosine receptor blocker) or intraperitoneal ticagrelor. At 24 h reperfusion, IS was assessed by triphenyltetrazolium chloride. Other rats were subjected to 30 min ischemia followed by 1 h or 24 h reperfusion. Myocardial samples were assessed for adenosine levels, RT-PCR, and immunoblotting. RESULTS: AZD3366 and ticagrelor reduced IS. The protective effect was blocked by CGS15943. The effect of AZD3366 + ticagrelor was significantly greater than AZD3366. One hour after infarction, myocardial adenosine levels significantly increased with AZD3366, but not with ticagrelor. In contrast, 24 h after infarction, adenosine levels were equally increased by AZD3366 and ticagrelor, and levels were higher in the AZD3366 + ticagrelor group. One hour after reperfusion, AZD3366 and ticagrelor equally attenuated the increase in interleukin-15 (an early inflammatory marker after ischemic cell death) levels, and their combined effects were additive. AZD3366, but not ticagrelor, significantly attenuated the increase in RIP1, RIP3, and P-MLKL (markers of necroptosis) 1 h after reperfusion. AZD3366, but not ticagrelor, significantly attenuated the increase in IL-6 and GSDMD-N (markers of pyroptosis) 1 h after reperfusion. At 24 h of reperfusion, both agents equally attenuated the increase in these markers, and their effects were additive. CONCLUSIONS: AZD3366 attenuated inflammation, necrosis, necroptosis, and pyroptosis and limited IS. The effects of AZD3366 and ticagrelor were additive.

Inhibition of the prostaglandin D2-GPR44/DP2 axis improves human islet survival and function.

AIMS/HYPOTHESIS: Inflammatory signals and increased prostaglandin synthesis play a role during the development of diabetes. The prostaglandin D2 (PGD2) receptor, GPR44/DP2, is highly expressed in human islets and activation of the pathway results in impaired insulin secretion. The role of GPR44 activation on islet function and survival rate during chronic hyperglycaemic conditions is not known. In this study, we investigate GPR44 inhibition by using a selective GPR44 antagonist (AZ8154) in human islets both in vitro and in vivo in diabetic mice transplanted with human islets. METHODS: Human islets were exposed to PGD2 or proinflammatory cytokines in vitro to investigate the effect of GPR44 inhibition on islet survival rate. In addition, the molecular mechanisms of GPR44 inhibition were investigated in human islets exposed to high concentrations of glucose (HG) and to IL-1ß. For the in vivo part of the study, human islets were transplanted under the kidney capsule of immunodeficient diabetic mice and treated with 6, 60 or 100 mg/kg per day of a GPR44 antagonist starting from the transplantation day until day 4 (short-term study) or day 17 (long-term study) post transplantation. IVGTT was performed on mice at day 10 and day 15 post transplantation. After termination of the study, metabolic variables, circulating human proinflammatory cytokines, and hepatocyte growth factor (HGF) were analysed in the grafted human islets. RESULTS: PGD2 or proinflammatory cytokines induced apoptosis in human islets whereas GPR44 inhibition reversed this effect. GPR44 inhibition antagonised the reduction in glucose-stimulated insulin secretion induced by HG and IL-1ß in human islets. This was accompanied by activation of the Akt-glycogen synthase kinase 3ß signalling pathway together with phosphorylation and inactivation of forkhead box O-1and upregulation of pancreatic and duodenal homeobox-1 and HGF. Administration of the GPR44 antagonist for up to 17 days to diabetic mice transplanted with a marginal number of human islets resulted in reduced fasting blood glucose and lower glucose excursions during IVGTT. Improved glucose regulation was supported by increased human C-peptide levels compared with the vehicle group at day 4 and throughout the treatment period. GPR44 inhibition reduced plasma levels of TNF-α and growth-regulated oncogene-α/chemokine (C-X-C motif) ligand 1 and increased the levels of HGF in human islets. CONCLUSIONS/INTERPRETATION: Inhibition of GPR44 in human islets has the potential to improve islet function and survival rate under inflammatory and hyperglycaemic stress. This may have implications for better survival rate of islets following transplantation.

AZD8797 is an allosteric non-competitive modulator of the human CX3CR1 receptor.

The chemokine receptor CX3CR1 has been implicated as an attractive therapeutic target in several diseases, including atherosclerosis and diabetes. However, there has been a lack of non-peptide CX3CR1 inhibitors to substantiate these findings. A selective small-molecule inhibitor of CX3CR1, AZD8797, was recently reported and we present here an in-depth in vitro characterization of that molecule. In a flow adhesion assay, AZD8797 antagonized the natural ligand, fractalkine (CX3CL1), in both human whole blood (hWB) and in a B-lymphocyte cell line with IC50 values of 300 and 6 nM respectively. AZD8797 also prevented G-protein activation in a [(35)S]GTPγS (guanosine 5'-[γ-thio]triphosphate) accumulation assay. In contrast, dynamic mass redistribution (DMR) experiments revealed a weak Gαi-dependent AZD8797 agonism. Additionally, AZD8797 positively modulated the CX3CL1 response at sub-micromolar concentrations in a ß-arrestin recruitment assay. In equilibrium saturation binding experiments, AZD8797 reduced the maximal binding of (125)I-CX3CL1 without affecting Kd. Kinetic experiments, determining the kon and koff of AZD8797, demonstrated that this was not an artefact of irreversible or insurmountable binding, thus a true non-competitive mechanism. Finally we show that both AZD8797 and GTPγS increase the rate with which CX3CL1 dissociates from CX3CR1 in a similar manner, indicating a connection between AZD8797 and the CX3CR1-bound G-protein. Collectively, these data show that AZD8797 is a non-competitive allosteric modulator of CX3CL1, binding CX3CR1 and effecting G-protein signalling and ß-arrestin recruitment in a biased way.

Discovery of Clinical Candidate AZD5462, a Selective Oral Allosteric RXFP1 Agonist for Treatment of Heart Failure.

Optimization of the highly potent and selective, yet metabolically unstable and poorly soluble hRXFP1 agonist AZ7976 led to the identification of the clinical candidate, AZD5462. Assessment of RXFP1-dependent cell signaling demonstrated that AZD5462 activates a highly similar panel of downstream pathways as relaxin H2 but does not modulate relaxin H2-mediated cAMP second messenger responsiveness. The therapeutic potential of AZD5462 was assessed in a translatable cynomolgus monkey heart failure model. Following 8 weeks of treatment with AZD5462, robust improvements in functional cardiac parameters including LVEF were observed at weeks 9, 13, and 17 without changes in heart rate or mean arterial blood pressure. AZD5462 was well tolerated in both rat and cynomolgus monkey and has successfully completed phase I studies in healthy volunteers. In summary, AZD5462 is a small molecule pharmacological mimetic of relaxin H2 signaling at RXFP1 and holds promise as a potential therapeutic approach to treat heart failure patients.

The putative cannabinoid receptor GPR55 affects osteoclast function in vitro and bone mass in vivo.

GPR55 is a G protein-coupled receptor recently shown to be activated by certain cannabinoids and by lysophosphatidylinositol (LPI). However, the physiological role of GPR55 remains unknown. Given the recent finding that the cannabinoid receptors CB(1) and CB(2) affect bone metabolism, we examined the role of GPR55 in bone biology. GPR55 was expressed in human and mouse osteoclasts and osteoblasts; expression was higher in human osteoclasts than in macrophage progenitors. Although the GPR55 agonists O-1602 and LPI inhibited mouse osteoclast formation in vitro, these ligands stimulated mouse and human osteoclast polarization and resorption in vitro and caused activation of Rho and ERK1/2. These stimulatory effects on osteoclast function were attenuated in osteoclasts generated from GPR55(-/-) macrophages and by the GPR55 antagonist cannabidiol (CBD). Furthermore, treatment of mice with this non-psychoactive constituent of cannabis significantly reduced bone resorption in vivo. Consistent with the ability of GPR55 to suppress osteoclast formation but stimulate osteoclast function, histomorphometric and microcomputed tomographic analysis of the long bones from male GPR55(-/-) mice revealed increased numbers of morphologically inactive osteoclasts but a significant increase in the volume and thickness of trabecular bone and the presence of unresorbed cartilage. These data reveal a role of GPR55 in bone physiology by regulating osteoclast number and function. In addition, this study also brings to light an effect of both the endogenous ligand, LPI, on osteoclasts and of the cannabis constituent, CBD, on osteoclasts and bone turnover in vivo.

Novel thioamide derivatives as neutral CB1 receptor antagonists.

A novel class of cannabinoid-1 (CB1) receptor antagonists for the treatment of obesity is presented. The carboxamide linker in a set of 5,6-diaryl-pyrazine-2-amide derivatives was transformed into the corresponding thioamide, by using a one-pot synthesis. The structural series of thioamides not only showed retained CB1 potency (below 10nM), but also showed improved solubility. In addition, the neutral antagonist 2c significantly reduced body weight in cafeteria diet obese mice.

l-α-Lysophosphatidylinositol (LPI) aggravates myocardial ischemia/reperfusion injury via a GPR55/ROCK-dependent pathway.

The phospholipid l-α-lysophosphatidylinositol (LPI), an endogenous ligand for GPR55, is elevated in patients with acute coronary syndrome, and a GPR55 antagonist cannabidiol (CBD) reduces experimental ischemia/reperfusion (I/R) injury. While LPI activates multiple signaling pathways, little is known about which ones are important in cardiomyocytes. In this study we explored whether activation of the Rho kinase/ROCK/p38 MAPK pathway is responsible for LPI-induced extension of I/R injury. Using a high-throughput screening method (dynamic mass redistribution; DMR), mouse- and human-induced pluripotent stem cell (iPSC) cardiomyocytes exposed to LPI were shown to exhibit a rapid, sustained, and concentration-dependent (1 nmol L-1-30 µmol L-1) cellular response. Y-27632 (ROCK inhibitor; 10 & 50 µmol L-1) and CBD (1 µmol L-1) both abolished the DMR response to LPI (10 µmol L-1). In murine iPSC cardiomyocytes, LPI-induced ROCK and p38 MAPK phosphorylation, both of which were prevented by Y-27632 and CBD, but did not induce JNK activation or cleavage of caspase-3. In hearts isolated from wild type (WT) mice subjected to 30 minutes global I/R, LPI (10 µmol L-1) administered via the coronary circulation increased infarct size when applied prior to ischemia onset, but not when given at the time of reperfusion. The exacerbation of tissue injury by LPI was not seen in hearts from GPR55-/- mice or in the presence of Y-27632, confirming that injury is mediated via the GPR55/ROCK/p38 MAPK pathway. These findings suggest that raised levels of LPI in the vicinity of a developing infarct may worsen the outcome of AMI.

Plasminogen binding inhibitors demonstrate unwanted activities on GABAA and glycine receptors in human iPSC derived neurons.

Plasminogen binding inhibitors (PBIs) reduce the risk of bleeding in hemorrhagic conditions. However, generic PBIs are also associated with an increased risk of seizures, an adverse effect linked to unwanted activities towards inhibitory neuronal receptors. Development of novel PBIs serve to remove compounds with such properties, but progress is limited by a lack of higher throughput methods with human translatability. Herein we apply human induced pluripotent stem cell (hiPSC) derived neurons in combination with dynamic mass redistribution (DMR) technology to demonstrate robust and reproducible modulation of both GABAA and glycine receptors. These cells respond to GABA (EC50 0.33 ±â€¯0.18 µM), glycine (EC50 11.0 ±â€¯3.7 µM) and additional ligands in line with previous reports from patch clamp technologies. Additionally, we identify and characterize a competitive antagonistic behavior of the prototype inhibitor and drug tranexamic acid (TXA). Finally, we demonstrate proof of concept for effective counter-screening of lead series compounds towards unwanted GABAA receptor activities. No activity was observed for a previously identified PBI candidate drug, AZD6564, whereas a discontinued analog, AZ13267257, could be characterized as a potent GABAA receptor agonist.

Exploring the insulin secretory properties of the PGD2-GPR44/DP2 axis in vitro and in a randomized phase-1 trial of type 2 diabetes patients.

AIMS/HYPOTHESIS: GPR44 (DP2, PTGDR2, CRTh2) is the receptor for the pro-inflammatory mediator prostaglandin D2 (PGD2) and it is enriched in human islets. In rodent islets, PGD2 is produced in response to glucose, suggesting that the PGD2-GPR44/DP2 axis may play a role in human islet function during hyperglycemia. Consequently, the aim of this work was to elucidate the insulinotropic role of GPR44 antagonism in vitro in human beta-cells and in type 2 diabetes (T2DM) patients. METHODS: We determined the drive on PGD2 secretion by glucose and IL-1beta, as well as, the impact on insulin secretion by pharmacological GPR44/DP2 antagonism (AZD1981) in human islets and beta-cells in vitro. To test if metabolic control would be improved by antagonizing a hyperglycemia-driven increased PGD2 tone, we performed a proof-of-mechanism study in 20 T2DM patients (average 54 years, HbA1c 9.4%, BMI 31.6 kg/m2). The randomized, double-blind, placebo-controlled cross-over study consisted of two three-day treatment periods (AZD1981 or placebo) separated by a three-day wash-out period. Mixed meal tolerance test (MMTT) and intravenous graded glucose infusion (GGI) was performed at start and end of each treatment period. Assessment of AZD1981 pharmacokinetics, glucose, insulin, C-peptide, glucagon, GLP-1, and PGD2 pathway biomarkers were performed. RESULTS: We found (1) that PGD2 is produced in human islet in response to high glucose or IL-1beta, but likely by stellate cells rather than endocrine cells; (2) that PGD2 suppresses both glucose and GLP-1 induced insulin secretion in vitro; and (3) that the GPR44/DP2 antagonist (AZD1981) in human beta-cells normalizes insulin secretion. However, AZD1981 had no impact on neither glucose nor incretin dependent insulin secretion in humans (GGI AUC C-peptide 1-2h and MMTT AUC Glucose 0-4h LS mean ratios vs placebo of 0.94 (80% CI of 0.90-0.98, p = 0.12) and 0.99 (90% CI of 0.94-1.05, p = 0.45), despite reaching the expected antagonist exposure. CONCLUSION/INTERPRETATION: Pharmacological inhibition of the PGD2-GPR44/DP2 axis has no major impact on the modulation of acute insulin secretion in T2DM patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT02367066.

Deletion of Gpr55 Results in Subtle Effects on Energy Metabolism, Motor Activity and Thermal Pain Sensation.

The G-protein coupled receptor 55 (GPR55) is activated by cannabinoids and non-cannabinoid molecules and has been speculated to play a modulatory role in a large variety of physiological and pathological processes, including in metabolically perturbed states. We therefore generated male mice deficient in the gene coding for the cannabinoid/lysophosphatidylinositol (LPI) receptor Gpr55 and characterized them under normal dietary conditions as well as during high energy dense diet feeding followed by challenge with the CB1 receptor antagonist/GPR55 agonist rimonabant. Gpr55 deficient male mice (Gpr55 KO) were phenotypically indistinguishable from their wild type (WT) siblings for the most part. However, Gpr55 KO animals displayed an intriguing nocturnal pattern of motor activity and energy expenditure (EE). During the initial 6 hours of the night, motor activity was significantly elevated without any significant effect observed in EE. Interestingly, during the last 6 hours of the night motor activity was similar but EE was significantly decreased in the Gpr55 KO mice. No significant difference in motor activity was detected during daytime, but EE was lower in the Gpr55 KO compared to WT mice. The aforementioned patterns were not associated with alterations in energy intake, daytime core body temperature, body weight (BW) or composition, although a non-significant tendency to increased adiposity was seen in Gpr55 KO compared to WT mice. Detailed analyses of daytime activity in the Open Field paradigm unveiled lower horizontal activity and rearing time for the Gpr55 KO mice. Moreover, the Gpr55 KO mice displayed significantly faster reaction time in the tail flick test, indicative of thermal hyperalgesia. The BW-decreasing effect of rimonabant in mice on long-term cafeteria diet did not differ between Gpr55 KO and WT mice. In conclusion, Gpr55 deficiency is associated with subtle effects on diurnal/nocturnal EE and motor activity behaviours but does not appear per se critically required for overall metabolism or behaviours.

Identification and characterisation of a novel splice variant of the human CB1 receptor.

Cannabinoid ligands are implicated in many physiological processes and to date two receptors have been identified. However, a growing body of evidence exists that suggests the presence of additional receptors. Whilst cloning the previously described hCB1a, we have identified a novel variant that we call hCB1b. Characterising these two splice variants demonstrates that they have a unique pharmacological profile and that their RNA's are expressed at low levels in a variety of tissues.

Pharmacological profiling of the hemodynamic effects of cannabinoid ligands: a combined in vitro and in vivo approach.

The receptors mediating the hemodynamic responses to cannabinoids are not clearly defined due to the multifarious pharmacology of many commonly used cannabinoid ligands. While both CB1 and TRPV1 receptors are implicated, G protein-coupled receptor 55 (GPR55) may also mediate some of the hemodynamic effects of several atypical cannabinoid ligands. The present studies attempted to unravel the pharmacology underlying the in vivo hemodynamic responses to ACEA (CB1 agonist), O-1602 (GPR55 agonist), AM251 (CB1 antagonist), and cannabidiol (CBD; GPR55 antagonist). Agonist and antagonist profiles of each ligand were determined by ligand-induced GTPγS binding in membrane preparations expressing rat and mouse CB1 and GPR55 receptors. Blood pressure responses to ACEA and O-1602 were recorded in anesthetized and conscious mice (wild type, CB1 (-/-) and GPR55(-/-)) and rats in the absence and presence of AM251 and CBD. ACEA demonstrated GTPγS activation at both receptors, while O-1602 only activated GPR55. AM251 exhibited antagonist activity at CB1 and agonist activity at GPR55, while CBD demonstrated selective antagonist activity at GPR55. The depressor response to ACEA was blocked by AM251 and attenuated by CBD, while O-1602 did not induce a depressor response. AM251 caused a depressor response that was absent in GPR55(-/-) mice but enhanced by CBD, while CBD caused a small vasodepressor response that persisted in GPR55(-/-) mice. Our findings show that assessment of the pharmacological profile of receptor activation by cannabinoid ligands in in vitro studies alongside in vivo functional studies is essential to understand the role of cannabinoids in hemodynamic control.

Succinate receptor GPR91, a Gα(i) coupled receptor that increases intracellular calcium concentrations through PLCß.

Succinate has been reported as the endogenous ligand for GPR91. In this study, succinate was confirmed to activate GPR91 resulting in both 3'-5'-cyclic adenosine monophosphate (cAMP) inhibition and inositol phosphate formation in a pertussis toxin (PTX)-sensitive manner. GPR91 agonist-mediated effects detected using dynamic mass redistribution (DMR) were inhibited with PTX, edelfosine and U73122 demonstrating the importance of not only the Gαi pathway but also PLCß. These results show that GPR91 when expressed in HEK293s cells couples exclusively through the Gαi pathway and acts through Gαi not only to inhibit cAMP production but also to increase intracellular Ca(2+) in an inositol phosphate dependent mechanism via PLCß activation.

N-arachidonoyl-L-serine is neuroprotective after traumatic brain injury by reducing apoptosis.

N-arachidonoyl-L-serine (AraS) is a brain component structurally related to the endocannabinoid family. We investigated the neuroprotective effects of AraS following closed head injury induced by weight drop onto the exposed fronto-parietal skull and the mechanisms involved. A single injection of AraS following injury led to a significant improvement in functional outcome, and to reduced edema and lesion volume compared with vehicle. Specific antagonists to CB2 receptors, transient receptor potential vanilloid 1 (TRPV1) or large conductance calcium-activated potassium (BK) channels reversed these effects. Specific binding assays did not indicate binding of AraS to the GPR55 cannabinoid receptor. N-arachidonoyl-L-serine blocked the attenuation in phosphorylated extracellular-signal-regulated kinase 1/2 (ERK) levels and led to an increase in pAkt in both the ipsilateral and contralateral cortices. Increased levels of the prosurvival factor Bcl-xL were evident 24 hours after injury in AraS-treated mice, followed by a 30% reduction in caspase-3 activity, measured 3 days after injury. Treatment with a CB2 antagonist, but not with a CB1 antagonist, reversed this effect. Our results suggest that administration of AraS leads to neuroprotection via ERK and Akt phosphorylation and induction of their downstream antiapoptotic pathways. These protective effects are related mostly to indirect signaling via the CB2R and TRPV1 channels but not through CB1 or GPR55 receptors.