A2B Receptor Research Articles

Simvastatin attenuates acute lung injury by activation of A2B adenosine receptor

To explore the protective mechanism of simvastatin in acute lung injury (ALI), the lipopolysaccharide (LPS) induced (5 mg/kg) ALI rat model was used to examine the effects of simvastatin. Following simvastatin treatment, the histopathological evaluation of lung tissues was made using hematoxylin and eosin (H&E) staining. Also, myeloperoxidase (MPO) activity and the levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-10 were determined by ELISA. Blood gas analyses of arterial blood samples were performed to assess the pulmonary gas exchange. Moreover, the neutrophil count and total protein content were determined in the bronchoalveolar lavage (BAL) fluid. The ratio of wet lung to dry lung (W/D) and the alveolar fluid clearance (AFC) were calculated to estimate the severity of edema. Lastly, the levels of A2BAR, CFTR, claudin4, and claudin18 were also measured by qRT-PCR and Western blotting. Simvastatin treatment, in a dose-related manner, markedly improved the lung histological injury and decreased the levels of TNF-α, IL-1β, and increased IL-10 in LPS induced ALI. Also, pulmonary neutrophil count was alleviated. Besides, a decreased ratio of W/D lung also confirmed the simvastatin intervention. Notably, simvastatin reduced the levels of A2BAR, CFTR, and claudin18 but upregulated claudin4 in lung tissues. Additionally, treatment with PSB1115, an antagonist of A2BAR, countered the protective effect of simvastatin in ALI. Our study demonstrates that simvastatin has a protective effect against LPS-induced ALI by activating A2BAR and should be exploited as a novel therapeutic target for the treatment of ALI.

Activation of central adenosine A2B receptors mediate brain ghrelin-induced improvement of intestinal barrier function through the vagus nerve in rats

Leaky gut that is a condition reflecting intestinal barrier dysfunction has been attracting attention for its relations with many diseases such as irritable bowel syndrome or Alzheimer dementia. We have recently demonstrated that ghrelin acts in the brain to improve leaky gut via the vagus nerve. In the present study, we tried to clarify the precise central mechanisms by which ghrelin improves intestinal barrier function through the vagus nerve. Colonic permeability was estimated in vivo by quantifying the absorbed Evans blue in colonic tissue in rats. Adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), blocked the intracisternal ghrelin-induced improvement of intestinal hyperpermeability while dopamine, cannabinoid or opioid receptor antagonist failed to prevent it. Since DPCPX can block adenosine A1 and adenosine A2B receptors, we examined which subtype is involved in the mechanism. Intracisternal injection of adenosine A2B agonist but not adenosine A1 agonist improved colonic hyperpermeability, while peripheral injection of adenosine A2B agonist failed to improve it. Intracisternal adenosine A2B agonist-induced improvement of colonic hyperpermeability was blocked by vagotomy. Adenosine A2B specific antagonist, alloxazine blocked the ghrelin- or central vagal stimulation by 2-deoxy-d-glucose-induced improvement of intestinal hyperpermeability. These results suggest that activation of adenosine A2B receptors in the central nervous system is capable of improving intestinal barrier function through the vagal pathway, and the adenosine A2B receptors may mediate the ghrelin-induced improvement of leaky gut in a vagal dependent fashion. These findings may help us understand the pathophysiology in not only gastrointestinal diseases but also non-gastrointestinal diseases associated with the altered intestinal permeability.

BAY 60-6583 Enhances the Antitumor Function of Chimeric Antigen Receptor-Modified T Cells Independent of the Adenosine A2b Receptor.

Chimeric antigen receptor (CAR) T cells are powerful in eradicating hematological malignancies, but their efficacy is limited in treating solid tumors. One of the barriers is the immunosuppressive response induced by immunomodulatory signaling pathways. Pharmacological targeting of these immunosuppressive pathways may be a simple way to improve the efficacy of CAR T cells. In this study, anti-CD133 and anti-HER2 CAR T cells were generated from healthy donors, and combination therapy using CAR T cells and small molecules targeting adenosine receptors was performed in vitro and in vivo with the goal of probing for potential synergistic antitumor activities. The adenosine A2b receptor agonist, BAY 60-6583, was found to significantly increase cytokine secretion of CD133-or HER2-specific CAR T cells when co-cultured with the respective target tumor cells. The in vitro cytotoxicity and proliferation of CAR T cells were also enhanced when supplied with BAY 60-6583. Furthermore, the combination with this small molecule facilitated the anti-HER2 CAR T cell-mediated elimination of tumor cells in a xenograft mouse model. However, the enhanced antitumor activities could not be suppressed by knockout of the adenosine A2b receptor in CAR T cells. Furthermore, mass spectrometry and computational methods were used to predict several potential alternative targets. Four potential targets (pyruvate kinase M (PKM), Talin-1, Plastin-2, and lamina-associated polypeptide 2) were captured by a photo-affinity probe, of which PKM and Talin-1 were predicted to interact with BAY 60-6583. Overall, our data suggest that BAY 60-6583 upregulates T cell functions through a mechanism independent of the adenosine A2b receptor.

Impact of Adenosine A2 Receptor Ligands on BCL2 Expression in Skeletal Muscle Cells

Background: Adenosine plays the role of regulating cell differentiation, proliferation, and apoptosis in various kinds of cells through the B-cell lymphoma 2 (BCL2) pathway. Objectives: Since anti-apoptotic (BCL2) expression plays a role in controlling apoptosis in some cell lines, this study was designed to investigate whether adenosine analogue, NECA (non-selective adenosine receptors agonist), selective adenosine A2B receptor antagonist, PSB 603, and a selective adenosine A2A receptor agonist, CG21680, affect BCL2-gene expression in the skeletal muscle cells of rats. The purpose of this investigation was to test the hypothesis that CG21680 treatment would significantly intensify BCL2 gene expression in rat skeletal muscle. Methods: Flasks measuring 25 cm2 were employed in culturing the rat L6 skeletal muscle cells. After treating these differential cells, the relative mRNA expression level for the BCL2 gene, at varying conditions of treatment, was measured using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Results: From the qRT-PCR analysis results, it was concluded that BCL2 expression was markedly amplified after selective adenosine A2A receptor agonist, CGS21680 (p < 0.01) treatment. More prospective validation for the adenosine receptors’ contribution in modulating apoptosis by NECA was delivered by the outcomes from the combined pre-treatment of the cells with NECA and PSB 603. These outcomes show that when starved skeletal muscle cells are treated with a combination of NECA and 100 nM PSB 603, there was a substantial decrease in comparison to either treatment used on its own. Conclusions: This study’s results showed, for the first time, an increase in BCL2 gene expression within skeletal muscle after CGS21680 treatment. Hence, the prospective escalation in BCL2 protein expression might have a protective role to play against apoptosis and avert damage to the skeletal muscle.

Molecular Mechanisms of Adenosine Stress T1 Mapping.

Adenosine stress T1 mapping is an emerging magnetic resonance imaging method to investigate coronary vascular function and myocardial ischemia without application of a contrast agent. Using gene-modified mice and 2 vasodilators, we elucidated and compared the mechanisms of adenosine myocardial perfusion imaging and adenosine T1 mapping. Wild-type (WT), A2AAR-/- (adenosine A2A receptor knockout), A2BAR-/- (adenosine A2B receptor knockout), A3AR-/- (adenosine A3 receptor knockout), and eNOS-/- (endothelial nitric oxide synthase knockout) mice underwent rest and stress perfusion magnetic resonance imaging (n=8) and T1 mapping (n=10) using either adenosine, regadenoson (a selective A2AAR agonist), or saline. Myocardial blood flow and T1 were computed from perfusion imaging and T1 mapping, respectively, at rest and stress to assess myocardial perfusion reserve and T1 reactivity (ΔT1). Changes in heart rate for each stress agent were also calculated. Two-way ANOVA was used to detect differences in each parameter between the different groups of mice. Myocardial perfusion reserve was significantly reduced only in A2AAR-/- compared to WT mice using adenosine (1.06±0.16 versus 2.03±0.52, P<0.05) and regadenoson (0.98±026 versus 2.13±0.75, P<0.05). In contrast, adenosine ΔT1 was reduced compared with WT mice (3.88±1.58) in both A2AAR-/- (1.63±1.32, P<0.05) and A2BAR-/- (1.55±1.35, P<0.05). Furthermore, adenosine ΔT1 was halved in eNOS-/- (1.76±1.46, P<0.05) versus WT mice. Regadenoson ΔT1 was approximately half of adenosine ΔT1 in WT mice (1.97±1.50, P<0.05), and additionally, it was significantly reduced in eNOS-/- mice (-0.22±1.46, P<0.05). Lastly, changes in heart rate was 2× greater using regadenoson versus adenosine in all groups except A2AAR-/-, where heart rate remained constant. The major findings are that (1) although adenosine myocardial perfusion reserve is mediated through the A2A receptor, adenosine ΔT1 is mediated through the A2A and A2B receptors, (2) adenosine myocardial perfusion reserve is endothelial independent while adenosine ΔT1 is partially endothelial dependent, and (3) ΔT1 mediated through the A2A receptor is endothelial dependent while ΔT1 mediated through the A2B receptor is endothelial independent.

Abstract PS12-12: Efficacy and safety of AB928 plus pegylated liposomal doxorubicin (PLD) with or without IPI-549 in participants with metastatic ovarian and triple negative breast cancer

Abstract Background: Adenosine, derived from ATP released by dying cancer cells in response to chemotherapy, activates the A2a and A2b receptors (R) on immune cells, resulting in an ineffective anti-tumor immune response. Adenosine receptor blockade may improve the efficacy of chemotherapy, notably anthracycline regimens, by enhancing immunogenicity within the tumor microenvironment. AB928, the first clinical-stage small molecule dual A2aR/A2bR antagonist, is highly potent and well tolerated in combination with chemo/immunotherapy. Novel combinations of AB928 with PLD +/- IPI-549, a PI3Kgamma inhibitor believed to shift tumor associated macrophages toward an anti-tumor phenotype, may offer potential clinical benefit for tumors previously considered immunotherapy-resistant. Methods: ARC-2 (NCT03719326) is an ongoing Phase (Ph) 1/1b, open-label study in patients (pts) with metastatic or locally advanced, unresectable ovarian or triple negative breast (TNBC) cancer. Eligible pts must have ECOG performance status 0-1 and at least one measurable lesion. In dose escalation, AB928 (75 or 150 mg) administered orally once daily was given with standard PLD in a 3+3 design. Once a tolerable doublet regimen was established, escalating doses of oral IPI-549 (30 or 40 mg once daily) were added. Two expansion cohorts, the doublet of AB928 150 mg + standard PLD and the triplet (+ IPI549 40 mg), are ongoing in pts with TNBC. Results: As of 19Jun2020, 29 pts received AB928 as part of the doublet or triplet regimen (Table 1). The number of prior therapies for escalation and expansion range from 0-11 (median=2) and 0-5 (median=1), respectively. Across both regimens, most treatment emergent AEs (TEAEs) were Grade (Gr) 1 or 2. The most common TEAEs (&amp;gt;6 pts) were fatigue, anemia, constipation, stomatitis, cough, and nausea. Four pts reported 6 Gr ≥3 SAEs; none were related to AB928 or IPI-549 and one event (pleural effusion) was at least possibly related to PLD. One pt discontinued study treatment for an AE (Gr 3 peripheral neuropathy) related to the triplet regimen; this pt had a complete response (CR) at the time of discontinuation. Of those pts treated with AB928 + PLD, 16 pts had at least one on-study disease evaluation, 2 pts (1 TNBC, 1 ovarian) achieved a partial response (PR), 8 stable disease (SD) and 6 progressive disease (PD) as best response. The addition of IPI-549 (n=11) resulted in 1 complete response (CR; ovarian), 3 PR (2 ovarian, 1 TNBC), 4 SD, and 3 PD. Nine pts received prior immunotherapy as part of an anticancer regimen; 2/9 achieved a PR on study treatment (1 confirmed, 1 pending). Thirteen pts had prior anthracycline; 6/13 achieved SD as best overall response, including 1 pt who received doublet therapy &amp;gt;1 year. Conclusions: AB928 and PLD +/- IPI-549 is tolerable in pts with advanced ovarian cancer and TNBC. Doublet and triplet combination treatment were associated with clinical benefit, including responses in those with progression after prior immunotherapy. The promising observation that late-line pts, including those previously treated with an anthracycline and/or immunotherapy, can experience clinical benefit with AB928 combination therapy warrants further investigation. Table 1. ARC-2 Cohort Enrollment and AB928 +/- IPI-549 Dose ReceivedStudy Part:Phase 1Phase 1bRegimen:DoubletTripletDoubletTripletAB928 dose:75 mg150 mg150 mg150 mg150 mgIPI-549 dose:––30 mg40 mg–40 mgEnrolled, nOvarian3431––TNBC020286 Citation Format: Jasgit C. Sachdev, Amy Prawira, Arvind Chaudhry, Vinod Ganju, Caroline Trudeau, Jennifer Scott, Rachel Woloski, Melissa Paoloni, Halle Zhang, Olivia Gardner. Efficacy and safety of AB928 plus pegylated liposomal doxorubicin (PLD) with or without IPI-549 in participants with metastatic ovarian and triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS12-12.

Targeting the purinergic pathway in breast cancer and its therapeutic applications.

Breast cancer (BC) is the most frequent cause of death among women, representing a global public health problem. Here, we aimed to discuss the correlation between the purinergic system and BC, recognizing therapeutic targets. For this, we analyzed the interaction of extracellular nucleotides and nucleosides with the purinergic receptors P1 and P2, as well as the influence of ectonucleotidase enzymes (CD39 and CD73) on tumor progression. A comprehensive bibliographic search was carried out. The relevant articles for this review were found in the PubMed, Scielo, Lilacs, and ScienceDirect databases. It was observed that among the P1 receptors, the A1, A2A, and A2B receptors are involved in the proliferation and invasion of BC, while the A3 receptor is related to the inhibition of tumor growth. Among the P2 receptors, the P2X7 has a dual function. When activated for a short time, it promotes metastasis, but when activated for long periods, it is related to BC cell death. P2Y2 and P2Y6 receptors are related to BC proliferation and invasiveness. Also, the high expression of CD39 and CD73 in BC is strongly related to a worse prognosis. The receptors and ectonucleotidases involved with BC become possible therapeutic targets. Several purinergic pathways have been found to be involved in BC cell survival and progression. In this review, in addition to analyzing the pathways involved, we reviewed the therapeutic interventions already studied for BC related to the purinergic system, as well as to other possible therapeutic targets.

Protective Effect of Adenosine A2B Receptor Agonist, BAY60-6583, Against Transient Focal Brain Ischemia in Rat.

Cerebral ischemia is a multifactorial pathology characterized first by an acute injury, due to excitotoxicity, followed by a secondary brain injury that develops hours to days after ischemia. During ischemia, adenosine acts as an endogenous neuroprotectant. Few studies have investigated the role of A2B receptor in brain ischemia because of the low potency of adenosine for it and the few selective ligands developed so far. A2B receptors are scarcely but widely distributed in the brain on neurons, glial and endothelial cells and on hematopoietic cells, lymphocytes and neutrophils, where they exert mainly anti-inflammatory effects, inhibiting vascular adhesion and inflammatory cells migration. Aim of this work was to verify whether chronic administration of the A2B agonist, BAY60-6583 (0.1 mg/kg i.p., twice/day), starting 4 h after focal ischemia induced by transient (1 h) Middle Cerebral Artery occlusion (tMCAo) in the rat, was protective after the ischemic insult. BAY60-6583 improved the neurological deficit up to 7 days after tMCAo. Seven days after ischemia BAY60-6583 reduced significantly the ischemic brain damage in cortex and striatum, counteracted ischemia-induced neuronal death, reduced microglia activation and astrocytes alteration. Moreover, it decreased the expression of TNF-α and increased that of IL-10 in peripheral plasma. Two days after ischemia BAY60-6583 reduced blood cell infiltration in the ischemic cortex. The present study indicates that A2B receptors stimulation can attenuate the neuroinflammation that develops after ischemia, suggesting that A2B receptors may represent a new interesting pharmacological target to protect from degeneration after brain ischemia.

Involvement of CD73 and A2B Receptor in Radiation-Induced DNA Damage Response and Cell Migration in Human Glioblastoma A172 Cells.

Glioblastoma is the most common malignant tumor of the central nervous system and is treated with a combination of surgery, radiation and chemotherapy. However, the tumor often acquires radiation resistance, which is characterized by an increased DNA damage response (DDR). Here, we show that CD73, which generates extracellular adenosine from ATP, and A2B receptor, which is activated by adenosine, are involved in the γ-radiation-induced DDR and the enhanced migration ability of human glioblastoma cell line A172. To investigate DDR, we evaluated ataxia telangiectasia mutated (ATM) activation and focus formation of histone H2A isoform γ (γH2AX) and p53-binding protein 1 (53BP1) in the nucleus of A172 cells after γ-irradiation. Antagonists of A2B receptor and CD73, or knockdown with small interfering RNA (siRNA), suppressed γ-radiation-induced DDR and promoted γ-radiation-induced cell death, as well as suppressing γ-radiation-induced cell migration and actin remodeling. These results suggest that activation of A2B receptor by extracellular adenosine generated via CD73 promotes γ-radiation-induced DDR, leading to recovery from DNA damage, and also enhances cell migration and actin remodeling. The CD73-A2B receptor pathway may be a promising target for overcoming radiation resistance and the acquisition of malignant phenotypes during radiotherapy of glioblastoma.

Inhibition of adenosine kinase attenuates myocardial ischaemia/reperfusion injury.

Increased adenosine helps limit infarct size in ischaemia/reperfusion‐injured hearts. In cardiomyocytes, 90% of adenosine is catalysed by adenosine kinase (ADK) and ADK inhibition leads to higher concentrations of both intracellular adenosine and extracellular adenosine. However, the role of ADK inhibition in myocardial ischaemia/reperfusion (I/R) injury remains less obvious. We explored the role of ADK inhibition in myocardial I/R injury using mouse left anterior ligation model. To inhibit ADK, the inhibitor ABT‐702 was intraperitoneally injected or AAV9 (adeno‐associated virus)—ADK—shRNA was introduced via tail vein injection. H9c2 cells were exposed to hypoxia/reoxygenation (H/R) to elucidate the underlying mechanisms. ADK was transiently increased after myocardial I/R injury. Pharmacological or genetic ADK inhibition reduced infarct size, improved cardiac function and prevented cell apoptosis and necroptosis in I/R‐injured mouse hearts. In vitro, ADK inhibition also prevented cell apoptosis and cell necroptosis in H/R‐treated H9c2 cells. Cleaved caspase‐9, cleaved caspase‐8, cleaved caspase‐3, MLKL and the phosphorylation of MLKL and CaMKII were decreased by ADK inhibition in reperfusion‐injured cardiomyocytes. X‐linked inhibitor of apoptosis protein (XIAP), which is phosphorylated and stabilized via the adenosine receptors A2B and A1/Akt pathways, should play a central role in the effects of ADK inhibition on cell apoptosis and necroptosis. These data suggest that ADK plays an important role in myocardial I/R injury by regulating cell apoptosis and necroptosis.

Systematic deletion of adenosine receptors reveals novel roles in inflammation and pyroptosis in THP-1 macrophages

Macrophages perform the fundamental function of sensing cellular damage, initiating and mediating immune response and tissue repair. Adenine nucleotides are in relatively high abundance in cells and are released from cells during tissue damage that are converted to adenosine in the extracellular environment. The A1, A2A, A2B and A3 adenosine receptors serve to regulate immune function. Despite characterization of the adenosine receptors, a comprehensive examination of adenosine receptor signaling in THP-1 macrophage cells has not been done. Moreover, previous studies employed chemical agonists and antagonists that have the potential for off-target affects. Here we systematically knockdown each of the four known adenosine receptors in THP-1 macrophages using validated siRNA and investigated their function under LPS stimulation. We demonstrate that the A1 receptor is required for adenosine-stimulated IL-10 and IL-1β secretion indicating an important role of this receptor during resolution of inflammation and tissue repair in these cells. The A1 and A3 receptor were required for IL-6 and IL-1β secretion showing a net pro-inflammatory role for these receptors. Finally, we present the novel finding that THP-1 macrophages lacking the A2B receptor undergo pyroptosis when exposed to LPS, demonstrating a novel role of the A2B receptor in regulation of programmed cell death during inflammation. This work underscores the fundamental importance of adenosine signaling and provides insight into the independent roles of the adenosine receptors in modulating cytokine signaling.

Co-inhibition of CD73 and ADORA2B Improves Long-Term Cigarette Smoke Induced Lung Injury.

Adenosine (ADO) involvement in lung injury depends on the activation of its receptors. The ADO A2A receptor (ADORA2A) and A2B receptor (ADORA2B) are best described to have both tissue-protective and tissue-destructive processes. However, no approach has been effective in delineating the mechanism(s) involved with ADO shifting from its tissue-protective to tissue-destructive properties in chronic airway injury. Using cigarette smoke (CS) as our model of injury, we chronically exposed Nuli-1 cells to 5% CS extract (CSE) for 3 years establishing a long-term CSE exposure model (LTC). We found significant morphological changes, decreased proliferation, and migration resulting in impaired airway wound closure in LTC. Further investigations showed that long-term CSE exposure upregulates CD73 and ADORA2B expression, increases ADO production, inhibits PKC alpha activity and p-ERK signaling pathway. Knocking down ADORA2B and/or CD73 in LTC activates PKC alpha and increases p-ERK signaling. Knocking down both showed better improvement in wound repair than either alone. In vivo experiments also showed that double knockout CD73 and ADORA2B remarkably improved CS-induced lung injury by activating PKC alpha, reducing the inflammatory cell number in bronchoalveolar lavage fluid and the production of inflammatory mediator IL-6, inhibiting the fibrosis-like lesions and decreasing collagen deposition surrounding bronchioles. Collectively, long-term CSE exposure upregulates CD73 expression and increases ADO production, which promotes low affinity ADORA2B activation and subsequent diminution of PKC alpha activity and ERK signaling pathway, and inhibition of airway wound repair. Moreover, the data suggesting ADORA2B and CD73 as potential therapeutic targets may be more efficacious in improving chronic CS lung diseases and impaired wound repair.

ARC-9: Phase Ib/II study to evaluate etrumadenant (AB928)-based treatment combinations in patients with metastatic colorectal cancer (mCRC).

TPS150 Background: ATP release from dying cancer cells in response to platinum-based chemotherapy increases extracellular immunosuppressive adenosine, which binds and activates the A2a and A2b receptors on immune cells. Adenosine-mediated signaling impairs activation, proliferation, and cytotoxic activity of effector T cells, resulting in inhibition of antitumor activity. Concomitant adenosine receptor blockade may therefore enhance the therapeutic efficacy of chemo/immunotherapy regimens. Etrumadenant (AB928), the first clinical-stage, small-molecule, dual adenosine receptor antagonist, is highly potent, pharmacodynamically active, and has been well tolerated in dose escalation studies as monotherapy or combined with chemo/immunotherapy. Recently, results were reported for the ARC-3 phase 1/1b study of etrumadenant + modified 5-fluorouracil + oxaliplatin (mFOLFOX-6) in patients (pts) with mCRC. In this study, etrumadenant + mFOLFOX-6 was well tolerated without significant additive toxicity. Disease control was observed in patients with RAS/BRAF mutated mCRC, as well as 3L+ disease previously treated with FOLFOX and/or FOLFIRI (PR and/or SD &gt;4 mo). Due to deep responses in 1L-3L+ pts, 6 pts had the opportunity to pursue surgery and radiotherapy with curative intent. The encouraging results from ARC-3 warrant further evaluation of etrumadenant-based combination therapy for mCRC. Methods: ARC-9 is a phase 1b/2, multicohort, open-label, randomized platform study designed to evaluate safety and clinical activity of etrumadenant (150 mg orally once daily [QD]) in combination with standard-of-care (SOC) regimens or novel therapeutics in pts with mCRC (Table). Cohort eligibility is based on prior anticancer treatment history. Pts enrolled in Cohorts A and B will be randomized (2:1) into the experimental vs SOC arms. Cohort C consists of a single arm to allow inclusion of novel agents as they become available with built in early stopping rules for futility. Pts who progress on the SOC arm of Cohort A can enroll in Cohort B; pts who progress on the SOC arm of Cohort B can crossover to the experimental arm. Primary endpoints across cohorts are shown in Table. Safety monitoring will occur throughout the trial, disease assessments will occur every 8 weeks, and correlative study pre- and on-treatment biopsies will be performed. [Table: see text]

Discovery of Potent and Selective Methylenephosphonic Acid CD73 Inhibitors.

Solid tumors are often associated with high levels of extracellular ATP. Ectonucleotidases catalyze the sequential hydrolysis of ATP to adenosine, which potently suppresses T-cell and NK-cell functions via the adenosine receptors (A2a and A2b). The ectonucleotidase CD73 catalyzes the conversion of AMP to adenosine. Thus, increased CD73 enzymatic activity in the tumor microenvironment is a potential mechanism for tumor immune evasion and has been associated with poor prognosis in the clinic. CD73 inhibition is anticipated to restore immune function by skirting this major mechanism of adenosine generation. We have developed a series of potent and selective methylenephosphonic acid CD73 inhibitors via a structure-based design. Key binding interactions of the known inhibitor adenosine-5'-(α,β-methylene)diphosphate (AMPCP) with hCD73 provided the foundation for our early designs. The structure-activity relationship study guided by this structure-based design led to the discovery of 4a, which exhibits excellent potency against CD73, exquisite selectivity against related ectonucleotidases, and a favorable pharmacokinetic profile.

Novel curcumin analog (cis-trans curcumin) as ligand to adenosine receptors A2A and A2B: potential for therapeutics

All four of the adenosine receptor (AR) subtypes mediate pain and have been targeted by pharmacologists to generate new therapeutics for chronic pain. The vanilloid phytochemicals, which include curcumin, capsaicin, and gingerol, have been shown to alleviate pain. However, there is little to no literature on the interaction of vanilloid phytochemicals with ARs. In this study, photochemical methods were used to generate a novel isomer of curcumin (cis-trans curcumin or CTCUR), and the interactions of both curcumin and CTCUR with the two Gs-linked AR subtypes were studied. Competitive binding assays, docking analysis, and confocal fluorescence microscopy were performed to measure binding affinity; cell survival assays were used to measure toxicity; and cAMP assays were performed to measure receptor activation. Competitive binding results indicated that CTCUR binds to both AR A2A and AR A2B with Ki values of 5 μM and 7 μM, respectively, which is consistent with our docking results. Fluorescence microscopy data also shows binding for A2B and A2A. Cell survival results show that CTCUR and CUR are nontoxic at the tested concentrations in these cell lines. Overall, our results suggest that vanilloid phytochemicals may be slightly modified to increase interaction with Gs-ARs, and thereby can be further explored to provide a novel class of non-opioid antinociceptives.

Macrophage-derived netrin-1 contributes to endometriosis-associated pain.

BackgroundEndometriosis-associated pain can be considered a type of neuropathic pain. Netrin-1 is an axon guidance cue that regulates axonal attraction or rejection in neural injury and regeneration. However, whether netrin-1 plays a role in endometriosis-associated pain remains unclear. This study aimed to determine the role of netrin-1 in endometriosis-related pain.MethodsPeripheral blood, peritoneal fluid, and endometrial tissues were sampled from women with (n=37) and without endometriosis (n=23). Lipopolysaccharide (LPS) and interferon gamma (IFN-γ) were used to stimulate human monocytic cell lines (THP-1) and rat alveolar macrophage-derived cell lines (NR8383) to induce M1 phenotype macrophages. Serum netrin-1 concentrations, endometrial expression levels of netrin-1, and its receptors including deleted in colorectal cancer (DCC), A2B adenosine receptor (A2BAR), uncoordinated B receptor (UNC5B), uncoordinated C receptor (UNC5C) and Down’s syndrome cell adhesion molecule (DSCAM) were assessed. The polarization phenotypes of the peritoneal macrophages were identified by detecting the marker expression of M1/M2 macrophages via flow cytometry. The expression levels of M1 markers and netrin-1 in THP-1/NR8383 cells were determined.ResultsThe expression levels of netrin-1 in serum and endometriotic lesions were significantly higher in women with endometriosis, and were positively correlated with the severity of endometriosis-associated pain. Netrin-1 was co-expressed with CD68 (a macrophage marker) in endometriotic lesions and was synthesized and secreted by THP-1 and NR8383 cells in the process of M1 polarization. In women with endometriosis, peritoneal macrophages were polarized towards the M1 phenotype. In addition, increased expression of DCC and A2BAR, and decreased expression of UNC5B, UNC5C and DSCAM were found in endometriotic lesions.ConclusionsThese results suggest that netrin-1 production by macrophages in endometriotic lesions may play an important role in endometriosis-associated pain.

Oligodendrocyte precursor cell maturation: role of adenosine receptors.

Oligodendrocyte-formed myelin sheaths allow fast synaptic transmission in the brain and their degeneration leads to demyelinating diseases such as multiple sclerosis. Remyelination requires the differentiation of oligodendrocyte progenitor cells into mature oligodendrocytes but, in chronic neurodegenerative disorders, remyelination fails due to adverse environment. Therefore, a strategy to prompt oligodendrocyte progenitor cell differentiation towards myelinating oligodendrocytes is required. The neuromodulator adenosine, and its receptors (A1, A2A, A2B and A3 receptors: A1R, A2AR, A2BR and A3R), are crucial mediators in remyelination processes. It is known that A1Rs facilitate oligodendrocyte progenitor cell maturation and migration whereas the A3Rs initiates apoptosis in oligodendrocyte progenitor cells. Our group of research contributed to the field by demonstrating that A2AR and A2BR inhibit oligodendrocyte progenitor cell maturation by reducing voltage-dependent K+ currents necessary for cell differentiation. The present review summarizes the possible role of adenosine receptor ligands as potential therapeutic targets in demyelinating pathologies such as multiple sclerosis.

Interactions Between Adenosine Receptors and Cordycepin (3'- Deoxyadenosine) from Cordyceps Militaris: Possible Pharmacological Mechanisms for Protection of the Brain and the Amelioration of Covid-19 Pneumonia

At present, the novel Covid-19 pneumonia is prevalent, affecting millions of people. Here, we summarized the pharmacological basis of adenosine, adenosine receptors, adenosine agonist cordycepin (3'-deoxyadenosine), and Cordyceps product in the brain protection and amelioration of pneumonia to provide useful information to cope with the global pandemic of novel coronavirus (COVID-19). Adenosine, a mediator of innate immunity, is abundantly secreted by the injured lung tissues during inflammation. Through the activation of adenosine receptors A1, A2A, A2B and A3, adenosine plays an important role in protecting against acute lung injury and brain injury. Cordycepin (3-deoxyadenosine) is an activator of adenosine receptors. It can enhance human immunity, promote anti-inflammatory processes, inhibit RNA virus reproduction, protect against brain, lung, liver, heart, and kidney damage, and ameliorate lung-fibrosis in clinical and animal models. Cordyceps and cordycepin products could be used as a potential medicinal adenosine receptor agonist that can play a beneficial role in the amelioration of Covid-19 pneumonia and protection of brain.

Hypoxia-inducible factor-1alpha protects the liver against ischemia-reperfusion injury by regulating the A2B adenosine receptor

ABSTRACT Hepatic ischemia-reperfusion injury (IRI) is an inevitable complication associated with liver surgical procedures, and its pathological process remains elusive. Therefore, the present study investigated the role and mechanism of hypoxia-inducible factor-1alpha (HIF-1α) in hepatic IRI. Here, we constructed rat models with hepatic IRI and BRL-3A cell models with hypoxia/reoxygenation (H/R) insult. The extent of liver injury was assayed by measuring serum ALT/AST levels and performing H&E staining; the levels of SOD, MDA, MPO, IL-6 and TNF-α were determined using commercial kits; apoptosis was detected using the TUNEL assay and flow cytometry; and the expression of HIF-1α/A2BAR signaling-related molecules and apoptosis-associated indicators was detected using Western blotting or qRT-PCR. The expression level of HIF-1α was significantly upregulated in the liver of rats subjected to IRI, as well as in BRL-3A cells treated with H/R. HIF-1α overexpression exerted a protective effect on hepatic IRI or H/R insult by reducing serum aminotransferase levels and hepatic necrosis, inhibiting inflammation and apoptosis of hepatocytes, and alleviating oxidative stress. In contrast, inhibition of HIF-1α expression exacerbated hepatic injury induced by IR or H/R. Mechanistically, the expression level of A2BAR was markedly increased during hepatic IRI or H/R insult. Moreover, A2BAR expression increased with HIF-1α upregulation and decreased with HIF-1α downregulation. Importantly, inhibition of A2BAR signaling abolished HIF-1α overexpression-mediated hepatoprotection. Taken together, HIF-1α exerts protective effects on hepatic IRI by attenuating liver necrosis, the inflammatory response, oxidative stress and apoptosis, and its mechanism may be related to the upregulation of A2BAR signaling.

Extracellular Adenosine Induces IL-6 Production through Activation of A2B Receptor and Epidermal Growth Factor Receptor in Human Keratinocyte HaCaT Cells

Extracellular Adenosine Induces IL-6 Production through Activation of A2B Receptor and Epidermal Growth Factor Receptor in Human Keratinocyte HaCaT Cells