High Concentrations Of Adenosine Research Articles

Dual A2A/ A2B adenosine receptor antagonist M1069 counteracts immuno-suppressive mechanisms of adenosine and reduces tumor growth in vivo.

While A2A adenosine receptor (AR) was considered as a major contributor to adenosine-mediated immunosuppression, A2B, having the lowest affinity to adenosine, has also emerged as a potential contributor to tumor promotion. Therefore, in adenosine-rich tumor microenvironment (TME), where A2B could be complementary and/or compensatory to A2A, simultaneous targeting of A2A and A2B ARs can provide higher potential for cancer immunotherapy. We developed M1069 - a highly selective dual antagonist of the A2A and A2B AR. In assays with primary human and murine immune cells, M1069 rescued IL 2 production from T cells (A2A dependent) and inhibited VEGF production by myeloid cells (A2B dependent) in adenosine-high settings. M1069 also demonstrated superior suppression of secretion of pro tumorigenic cytokines CXCL1, CXCL5, and rescue of IL 12 secretion from adenosine differentiated dendritic cells compared to an A2A selective antagonist (A2Ai). In a one-way mixed lymphocyte reaction (MLR) assay, adenosine differentiated human and murine dendritic cells treated with M1069 demonstrated superior T cell stimulatory activity compared to dendritic cells differentiated in presence of A2Ai. In vivo, M1069 decreased tumor growth as a monotherapy and enhanced anti-tumor activity of bintrafusp alfa (BA) or cisplatin in syngeneic adenosinehi/CD73hi 4T1 breast tumor model, but not in the CD73 knockout (KO) 4T1 tumor model or in adenosinelow/CD73low MC38 murine colon carcinoma model. In summary, our dual A2A/A2B AR antagonist M1069 may counteract immune-suppressive mechanisms of high concentrations of adenosine in vitro and in vivo and enhance the anti-tumor activity of other agents, including BA and cisplatin.

A Tumor Environment-Activated Photosensitized Biomimetic Nanoplatform for Precise Photodynamic Immunotherapy of Colon Cancer.

Aggressive nature of colon cancer and current imprecise therapeutic scenarios simulate the development of precise and effective treatment strategies. To achieve this, a tumor environment-activated photosensitized biomimetic nanoplatform (PEG2000-SiNcTI-Ph/CpG-ZIF-8@CM) is fabricated by encapsulating metal-organic framework loaded with developed photosensitizer PEG2000-SiNcTI-Ph and immunoadjuvant CpG oligodeoxynucleotide within fusion cell membrane expressing programmed death protein 1 (PD-1) and cluster of differentiation 47 (CD47). By stumbling across, systematic evaluation, and deciphering with quantum chemical calculations, a unique attribute of tumor environment (low pH plus high concentrations of adenosine 5'-triphosphate (ATP))-activated photodynamic effect sensitized by long-wavelength photons is validated for PEG2000-SiNcTI-Ph/CpG-ZIF-8@CM, advancing the precision of cancer therapy. Moreover, PEG2000-SiNcTI-Ph/CpG-ZIF-8@CM evades immune surveillance to target CT26 colon tumors in mice mediated by CD47/signal regulatory proteins α (SIRPα) interaction and PD-1/programmed death ligand 1 (PD-L1) interaction, respectively. Tumor environment-activated photodynamic therapy realized by PEG2000-SiNcTI-Ph/CpG-ZIF-8@CM induces immunogenic cell death (ICD) to elicit anti-tumor immune response, which is empowered by enhanced dendritic cells (DC) uptake of CpG and PD-L1 blockade contributed by the nanoplatform. The photodynamic immunotherapy efficiently combats primary and distant CT26 tumors, and additionally generates immune memory to inhibit tumor recurrence and metastasis. The nanoplatform developed here provides insights for the development of precise cancer therapeutic strategies.

Choline chloride based natural deep eutectic solvents coupling with ultra-high performance liquid chromatography-tandem mass spectroscopy for effective extraction and rapid detection of adenosine and cordycepin in Cordyceps millitaris

In this study, a series of choline chloride-based natural deep eutectic solvents (NADESs) was successfully fabricated and combined with ultra-high performance liquid chromatography-tandem mass spectroscopy (UHPLC-MS/MS) to detect adenosine and cordycepin in Cordyceps militaris. The analysis validation produced precise results with outstanding parameters for analysing extraction samples. In particular, NADESs with a choline chloride to citric acid ratio of 1:2 exhibited exceptional performance under ultrasound-assisted conditions. Using response surface methodology, we optimised the extraction process, obtaining high concentrations of adenosine (2.67 ± 0.05 mg/g) and cordycepin (3.73 ± 0.11 mg/g) from C. militaris. Compared to conventional methods, the NADES-based extraction proved more efficient, delivering elevated levels of target compounds by 36.5-fold for adenosine and 1.5-fold for cordycepin. Additionally, a mechanistic insight was offered by analysing post-extraction samples. The analytical approach was utilised to assess the adenosine and cordycepin content in various C. militaris samples available in the Vietnamese market. The detected values ranged from 1.73 ± 0.07 to 2.78 ± 0.11 mg/g for adenosine and from 2.87 ± 0.04 to 4.52 ± 0.14 mg/g for cordycepin. This analytical method holds promise for accurately evaluating adenosine and cordycepin in C. militaris.

Suppression of P2X7R by Local Treatment Alleviates Acute Gouty Inflammation.

Gout is the most common inflammatory arthritis associated with interleukin-1β (IL-1β) accumulation during exacerbation. In this study, we aimed to clarify whether potassium channel antagonists attenuate local inflammation in mice with monosodium urate (MSU)-induced gout. We cultured human macrophage THP-1 cells and evaluated the molecular levels of both IL-1β and potassium channels stimulated with MSU and/or potassium channel antagonists. Acute gout models were generated in IL-1β luciferase transgenic male mice using synovium-like subcutaneous air pouches with MSU injection. Their luciferase activities were monitored following potassium channel blocker treatment using the IVIS Spectrum CT imaging system. The lavages and tissues were extracted from their air pouches, followed by cell counting and pathological analysis. MSU stimulation increased the gene expression levels of pro-IL-1β, P2x7r and Kv1.3, whereas the expression of Kcnq1 was decreased in phorbol 12-myristate 13-acetate-induced THP-1 cells. Both high and low concentrations of the P2x7 receptor inhibitor adenosine 5'-triphosphate (ATP) derivative periodate oxidized ATP (oATP) decreased the production of IL-1β in the supernatant of THP-1 cells. The sixth hour was the peak time of IL-1β luciferase activity after MSU intervention in vivo. oATP ameliorated the synovial IL-1β luciferase activity, reduced inflammatory cell infiltration and alleviated the erosive damage in the cartilage. The anti-inflammatory properties of potassium channel inhibitors, especially of oATP, might point to new strategies for local anti-inflammatory therapy for acute gout.

Immunogenetic Metabolomics Reveals Key Enzymes That Modulate CAR T-cell Metabolism and Function.

Immune evasion is a critical step of cancer progression that remains a major obstacle for current T cell-based immunotherapies. Hence, we investigated whether it is possible to genetically reprogram T cells to exploit a common tumor-intrinsic evasion mechanism whereby cancer cells suppress T-cell function by generating a metabolically unfavorable tumor microenvironment (TME). In an in silico screen, we identified ADA and PDK1 as metabolic regulators. We then showed that overexpression (OE) of these genes enhanced the cytolysis of CD19-specific chimeric antigen receptor (CAR) T cells against cognate leukemia cells, and conversely, ADA or PDK1 deficiency dampened this effect. ADA-OE in CAR T cells improved cancer cytolysis under high concentrations of adenosine, the ADA substrate, and an immunosuppressive metabolite in the TME. High-throughput transcriptomics and metabolomics analysis of these CAR T cells revealed alterations of global gene expression and metabolic signatures in both ADA- and PDK1-engineered CAR T cells. Functional and immunologic analyses demonstrated that ADA-OE increased proliferation and decreased exhaustion in CD19-specific and HER2-specific CAR T cells. ADA-OE improved tumor infiltration and clearance by HER2-specific CAR T cells in an in vivo colorectal cancer model. Collectively, these data unveil systematic knowledge of metabolic reprogramming directly in CAR T cells and reveal potential targets for improving CAR T-cell therapy.

Adenosine reduces sinoatrial node cell action potential firing rate by uncoupling its membrane and calcium clocks.

The spontaneous action potential (AP) firing rate of sinoatrial nodal cells (SANC) is regulated by a system of intracellular Ca2+ and membrane ion current clocks driven by Ca2+-calmodulin-activated adenylyl cyclase-protein kinase-A signaling. The mean AP-cycle length (APCL) and APCL variability inform on the effectiveness of clock coupling. Endogenous ATP metabolite adenosine binds to adenosine receptors (A1, A3) that couple to Gi protein-coupled receptors, reducing spontaneous AP firing rate via Gβγ signaling that activates IKAch,Ado. Adenosine also inhibits adenylyl cyclase activity via Gαi signaling, impacting cAMP-mediated protein kinase-A-dependent protein phosphorylation. We hypothesize that in addition to IKAch,Ado activation, adenosine impacts also Ca2+ via Gαi signaling and that both effects reduce AP firing rate by reducing the effectiveness of the Ca2+ and membrane clock coupling. To this end, we measured Ca2+ and membrane potential characteristics in enzymatically isolated single rabbit SANC. 10 µM adenosine substantially increased both the mean APCL (on average by 43%, n = 10) and AP beat-to-beat variability from 5.1 ± 1.7% to 7.2 ± 2.0% (n = 10) measured via membrane potential and 5.0 ± 2.2% to 10.6 ± 5.9% (n = 40) measured via Ca2+ (assessed as the coefficient of variability = SD/mean). These effects were mediated by hyperpolarization of the maximum diastolic membrane potential (membrane clock effect) and suppression of diastolic local Ca2+releases (LCRs) (Ca2+-clock effect): as LCR size distributions shifted to smaller values, the time of LCR occurrence during diastolic depolarization (LCR period) became prolonged, and the ensemble LCR signal became reduced. The tight linear relationship of coupling between LCR period to the APCL in the presence of adenosine "drifted" upward and leftward, i.e. for a given LCR period, APCL was prolonged, becoming non-linear indicating clock uncoupling. An extreme case of uncoupling occurred at higher adenosine concentrations (>100µM): small stochastic LCRs failed to self-organize and synchronize to the membrane clock, thus creating a failed attempt to generate an AP resulting in arrhythmia and cessation of AP firing. Thus, the effects of adenosine to activate Gβγ and IKACh,Ado and to activate Gαi, suppressing adenylyl cyclase activity, both contribute to the adenosine-induced increase in the mean APCL and APCL variability by reducing the fidelity of clock coupling and AP firing rate.

Bioscaffold developed with decellularized human amniotic membrane seeded with mesenchymal stromal cells: assessment of efficacy and safety profiles in a second-degree burn preclinical model

Therapies to deep burn injuries remain a global challenge. Human amniotic membrane (hAM) is a biomaterial that has been increasingly explored by the field of regenerative medicine. A decellularized hAM (DhAM) can be used as scaffold for mesenchymal stromal cells (MSCs) to grow without the loss of their stemness potential, allowing its application as cell therapy for wound healing. In this work, we associated DhAM with adipose-derived MSCs (DhAM + AD-MSCs), as a therapy strategy for second-degree burns in a preclinical model. Animals with induced second-degree burns were divided into four groups: control, which consists of a non-adherent gauze; a synthetic commercial dressing as the positive control (Control+); DhAM; and DhAM plus rat AD-MSCs (DhAM + AD-MSCs), followed by detailed and long term analysis (5 weeks). The macroscopical analysis showed the healing improvement in the wound area after the DhAM + AD-MSC treatment. Histological analysis also showed no alteration in the animal organs and a regular epithelial progression in comparison to the control. This observation was also confirmed by the analysis of suprabasal layers in the neoepidermis with CK10, showing a stratified and differentiated epithelium, when compared to Control and Control+. A strong CD73 (ecto-5′-nucleotidase) labeling was observed in the first 2 weeks postburn in dermis and epidermis. The expression in dermis was stronger in the second week in the middle of the wound, when comparing the Control+ with DhAM + AD-MSCs (p = 0.0238). In the epidermis the expression of CD73 was increased in all regions when compared to the control. This data suggests the involvement of this protein on wound healing. A low CD11b labeling was observed in DhAM + AD-MSCs treatment group mainly in the last treatment week, in comparison to Control and Control+ (p < 0.0001), which indicates a reduction in the inflammatory process. MSCs through CD73 can release high concentrations of adenosine, an immunosuppressive molecule, suggesting that this could be the mechanism by which the inflammation was better modulated in the DhAM + AD-MSCs group. The results obtained with this preclinical model confirm the effectiveness and safety of this low-cost and highly available dressing for future clinical application as a therapy for burn treatments.

Overcoming high level adenosine-mediated immunosuppression by DZD2269, a potent and selective A2aR antagonist

BackgroundAdenosine is a potent immunosuppressant whose levels in the tumor microenvironment (TME) are often much higher than those in normal tissues. Binding of adenosine to its receptor A2aR activates a cascade of genes and leads to immunosuppression. In addition, immune checkpoint blockage markedly increases A2aR expression in T cells, which could dampen their anti-tumor response. Several A2aR antagonists are under clinical development, but with limited clinical benefit reported so far. These A2aR antagonists showed much diminished activity at high adenosine levels found in TME, which may explain their clinical underperformance. We report the discovery and early clinical development of DZD2269, a novel A2aR antagonist which can fully block A2aR mediated immunosuppression commonly found in TME. Adenosine stimulates phosphorylation of cyclic AMP response element binding protein (CREB) in T cells and inhibits anti-tumor cytokine secretion in PBMCs in a dose-dependent manner. DZD2269 was able to reverse the immunosuppression induced by high concentrations of adenosine, as demonstrated by inhibiting CREB phosphorylation in T cells, restoring Th1 cytokine secretion in PBMCs, and stimulating dendritic cells (DCs) maturation. As a single agent, DZD2269 showed anti-tumor growth in multiple syngeneic mouse tumor models, and more profound anti-tumor effects were observed when DZD2269 was in combination with immune checkpoint inhibitors, radiotherapy, or chemotherapy. A good PK/PD relationship was observed in these animal models. In the phase 1 clinical study, downregulation of pCREB was detected in human T cells, consistent with preclinical prediction. Our data support further clinical development of DZD2269 in patients with cancer.MethodsThe selectivity of DZD2269 for adenosine receptors was tested in engineered cell lines, and its efficacy in blocking A2aR signaling and reversing adenosine-mediated immunosuppression was assessed in human T cells and peripheral blood mononuclear cells (PBMCs). The anti-tumor effects of DZD2269 were evaluated in multiple syngeneic mouse models as a single agent as well as in combination with chemotherapy, radiotherapy, or immune checkpoint inhibitors. A phase 1 study in healthy volunteers (NCT04932005) has been initiated to assess safety, pharmacokinetics (PK) and pharmacodynamics (PD) of DZD2269.ResultsAdenosine stimulates phosphorylation of cyclic AMP response element binding protein (CREB) in T cells and inhibits anti-tumor cytokine secretion in PBMCs in a dose-dependent manner. DZD2269 was able to reverse the immunosuppression induced by high concentrations of adenosine, as demonstrated by inhibiting CREB phosphorylation in T cells, restoring Th1 cytokine secretion in PBMCs, and stimulating dendritic cells (DCs) maturation. As a single agent, DZD2269 showed anti-tumor growth in multiple syngeneic mouse tumor models, and more profound anti-tumor effects were observed when DZD2269 was in combination with immune checkpoint inhibitors, radiotherapy, or chemotherapy. A good PK/PD relationship was observed in these animal models. In the phase 1 clinical study, downregulation of pCREB was detected in human T cells, consistent with preclinical prediction.ConclusionDZD2269 is a novel A2aR antagonist which can fully block A2aR mediated immunosuppression commonly found in TME. Clinical development of DZD2269 in patients with cancer is warranted (NCT04634344).

Abstract 3499: M1069 as dual A2A/A2B adenosine receptor antagonist counteracts immune-suppressive mechanisms of adenosine and reduces tumor growth in vivo

Abstract Under physiological conditions, the extracellular concentrations of adenosine are low, however, levels dramatically increase under metabolically stressful conditions, including inflammation and cancer. The regulatory functions of adenosine are mediated through four members of the adenosine receptor family: A1, A2A, A2B, and A3. While A2A was considered the major contributor to adenosine-mediated suppression of T cell, natural killer cell, and myeloid cell functions, A2B has also recently emerged as a potential modulator of these processes. Both A2A and A2B signal through the same Gs-mediated activation of adenylate cyclase, which can allow the lower affinity A2B receptor to compensate for the inhibition of A2A in an adenosine-rich tumor microenvironment (TME). A2B receptors are also reported to support tumor growth independent of A2A through the Gq subunit of G-protein-coupled receptor-mediated production of vascular endothelial growth factor (VEGF) by myeloid and tumor cells. Therefore, simultaneous targeting of both the A2A and A2B receptors may provide a higher potential for cancer immunotherapy in an adenosine-rich TME, where A2B can act in compensatory or complementary means to A2A. M1069 is a small-molecule, dual antagonist of the A2A and A2B adenosine receptors with a selectivity of &amp;gt;100 fold against the A1 and A3 receptors. In assays with primary human T cells, M1069 caused a dose-dependent suppression of 5′-N-ethylcarboxamide adenosine (stable analog of adenosine)-stimulated cyclic adenosine monophosphate (cAMP) and phosphorylated cAMP- response element binding protein (pCREB) induction and rescue of interleukin (IL)-2 production (A2A readout). M1069 also suppressed VEGF production from human macrophages (A2B readout) in adenosine-rich settings. M1069 exhibited superior suppression of protumorigenic cytokine secretion, including CXCL1, CXCL5 and granulocyte-colony stimulating factor, and the rescue of IL12 secretion from adenosine-differentiated dendritic cells, as compared to an A2Aselective antagonist. In addition, in a one-way mixed lymphocyte reaction assay, adenosine-differentiated dendritic cells treated with M1069 demonstrated superior T cell activation compared to adenosine-differentiated dendritic cells treated with an A2A-selective antagonist. These findings were further corroborated with the results from in vivo studies in a murine CD73hi/adenosine-rich 4T1 syngeneic breast tumor model, in which M1069, but not an A2A-selective antagonist, reduced tumor growth as a monotherapy and enhanced anti-tumor activity with chemotherapeutic agents. In summary, M1069 is a potent, dual A2A/A2B adenosine receptor antagonist, which is expected to counteract immune-suppressive mechanisms in the presence of high concentrations of adenosine and enhance the anti-tumor activity of chemotherapies. Citation Format: Rinat Zaynagetdinov, Kai Schiemann, Kalyan Nallaparaju, Natalya Belousova, Armine Matevossian, Zhouxiang Chen, Giorgio Kradjian, Meghana Pandya, Nemisha Dawra, Eva-Maria Krauel, Elissaveta Petrova, Oliver Poeschke, David Fischer, Marc Lecomte, Andree Blaukat, Bayard Huck, Jacques Moisan. M1069 as dual A2A/A2B adenosine receptor antagonist counteracts immune-suppressive mechanisms of adenosine and reduces tumor growth in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3499.

PSB0788 ameliorates maternal inflammation‐induced periventricular leukomalacia-like injury

ABSTRACT Studies have shown that periventricular leukomalacia (PVL) is a distinctive form of cerebral white matter injury that pertains to myelination disturbances. Maternal inflammation is a main cause of white matter injury. Intrauterine inflammation cellular will be propagated to the developing brain by the entire maternal–placental–fetal axis, and triggers neural immune injury. As a low-affinity receptor, adenosine A2B receptor (A2BAR) requires high concentrations of adenosine to be significantly activated in pathological conditions. We hypothesized that in the maternal inflammation‐induced PVL model, a selective A2BAR antagonist PSB0788 had the potential to prevent the injury. In this work, a total of 18 SD pregnant rats were divided into three groups, and treated with intraperitoneal injection of phosphate buffered saline (PBS), lipopolysaccharide (LPS), or LPS+PSB0788. Placental infection was determined by H&E staining and the inflammatory condition was determined by ELISA. Change of MBP, NG2 and CC-1 in the brain of the rats’ offspring were detected by western blot and immunohistochemistry. Furthermore, LPS-induced maternal inflammation reduced the expression of MBP, which related to the decrease in the numbers of OPCs and mature oligodendrocytes in neonate rats. After treatment with PSB0788, the levels of MBP proteins increased in the rats’ offspring, improved the remyelination. In conclusion, our study shows that the selective A2BAR antagonist PSB0788 plays an important role in promoting the normal development of OPCs in vivo by the maternal inflammation‐induced PVL model. Future studies will focus on the mechanism of PSB0788 in this model.

Discovery and optimization of betulinic acid derivatives as novel potent CD73 inhibitors

The vast research and clinical result have verified the success of cancer immunotherapy. However, there is also facing the enormous challenges such as lack of precise pre-clinical models, optimal combined therapy regimen and acquired resistance to immunotherapy. Adenosine is a potent immune-modulating molecule and overexpression of CD73 on tumor leads to the high concentration of adenosine. Blockade of the key adenosine-generating enzyme CD73 can be a promising strategy for cancer immunotherapy. Here, we report the discovery of betulinic acid as a novel CD73 inhibitor lead compound by a hit-based substructure search strategy. Subsequent optimization led to the discovery of betulinic acid carbamate derivative ZM514 with 5.2-fold increased potency compared to lead compound. Simultaneously, study has showed that compound ZM514 was not a cytotoxic agent while betulinic acid showed modest antiproliferative activity. The present result provides a valuable inhibitor against the promising immuno-oncology target for further development.

Article Review: Autosomal Dominant Polycystic Kidney Disease: Renal Physiology Diagnosis, Treatment and Novel Therapies

In the case of Autosomal dominant polycystic kidney disease, patients are usually cured with Vasopressin (V2) receptor antagonists, which delay the ongoing growth of cyst formation and slow the pace of AD disease progression. Before we know more, it is uncertain if the increase in vasopressin amide levels that was detected during V2RAT treatment impacts the production of glucose in the intestines. Cell growth and fluid secretion are aided by high intracellular concentrations of adenosine 3',5;-cyclic monophosphate (cAMP), which leads to cyst development. SST, a hormone implicated in a variety of cell activities, has the potential to block the generation of intracellular cAMP. Nevertheless, since Somatostatin is quickly removed in vivo, it has little therapeutic promise. As a result, analogues with a longer half-life have been established, which might be potential medicines in the therapy of ADPKD. This review covers the complicated physiological consequences of Somatostatin, especially on the kidneys, as well as the possible therapeutic use of SST analogues in ADPKD.

Bispecific Aptamer Sensor toward T-Cell Leukemia Detection in the Tumor Microenvironment.

The current detection methods of malignant cells are mainly based on the high expression levels of certain surface proteins on these cells. However, many of the same surface marker proteins are also expressed in normal cells. Growing evidence suggests that the molecular signatures of the tumor microenvironment (TME) are related to the biological state of a diseased cell. Exploiting the unique molecular signature of the TME, we have designed a molecular sensing agent consisting of a molecular switch that can sense the elevated concentration of a small molecule in the TME and promote precise recognition of a malignant cell. We accomplished this by designing and developing a bispecific aptamer that takes advantage of a high concentration of adenosine 5′-triphosphate in the TME. Thus, we report a prototype of a bispecific aptamer molecule, which serves as a dual detection platform and recognizes tumor cells only when a given metabolite concentration is elevated in the TME. This system overcomes hurdles in detecting tumor cells solely based on the elevated expression of cell surface markers, providing a universal platform for tumor targeting and sensing.

Abstract 1704: Discovery and characterization of a novel triple A1/A2a/A2b adenosine receptor antagonist for cancer immunotherapy

Abstract Introduction: The adenosine pathways via type A2a and A2b receptors are known to suppress immunity by regulating the functions of immune cells, such as macrophages, dendritic cells, MDSC, T cells and NK cells in tumor microenvironment. Also, the adenosine pathway through an A1 receptor induces tumor growth by promoting cancer cell proliferation. On the other hand, it has been recently reported that the downregulation of A1R induces expression of tumor PD-L1. Herein, we introduce a novel antagonist of adenosine receptor, HM87277, which has potent antagonistic activity against three types of adenosine receptors A1R, A2aR and A2bR, and present preclinical results that the triple antagonist HM87277 may be more effective in treating cancer patients through appropriate combination with immune checkpoint blockers (ICBs). Materials and Methods: Receptor binding affinities were assessed using human adenosine receptors of type A1, A2a, A2b and A3. Cellular functional activity tests such as cAMP production. To identify adenosine receptor downstream signaling in cancer cells, p-ERK and p-JNK were measured by western blotting in SK-BR3 cells. PD-L1 induction by adenosine receptor antagonist was determined by flow cytometry using Canto II (BD) in PD-L1 expressing SK-MEL-28 cells. For cytotoxicity assay of T cell, isolated human CD8+ T cells were co-cultured with A1R expressing cancer cells in the presence of a high concentration of adenosine. Antitumor activity of HM87277 alone and in combination with ICBs was assessed in syngeneic mouse models using murine cancer cell lines. Results: HM87277 showed strong binding affinity toward human adenosine receptors of type A1, A2a and A2b, and potently inhibited the NECA-mediated production of intracellular cAMP in HEK293-A2aR cells. HM87277 significantly increased the proliferation of effector T cells. HM87277 potently inhibited the p-ERK and p-JNK, and induced the expression of PD-L1 in A1R-expressing cancer cells. As well, the combination of HM87277 and anti-PD-L1 mAb was observed to have synergistic inhibitory effect on tumor cell growth in co-culture with SK-MEL-28 and CD8 + T cells. Furthermore, in the mouse syngeneic model using murine cancer cell lines, HM87277 effectively inhibited tumor growth alone or in combination with anti-PD-L1 mAb. Conclusion: These results indicate that a triple antagonist HM87277, which simultaneously antagonizes type A1, A2a and A2b adenosine receptors, may not only stimulate immune cells such as cytotoxic CD8+ T cell but also inhibit the growth of cancer cells themselves through inhibition of ERK and JNK pathway. In addition, it showed a strong combination effect by enhancing the sensitivity to ICBs through the increase of PD-L1 in tumor cells. Therefore, it is considered that the triple antagonist HM87277 is worth developing as a potential anticancer agent. Citation Format: Sunyoung Jang, Seunah Jun, Hosun Lee, Yongtaek Lee, Joo-Yun Byun, Junghwa Park, Yu-Yon Kim, Young Gil Ahn, YoungHoon Kim, Kwee Hyun Suh. Discovery and characterization of a novel triple A1/A2a/A2b adenosine receptor antagonist for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1704.

Two New Adenosine Derivatives and their Antiproliferative Properties: An In Vitro Evaluation.

Adenosine is a natural nucleoside present in a variety of organs and tissues, where it acts as a modulator of diverse physiological and pathophysiological processes. These actions are mediated by at least four G protein-coupled receptors, which are widely and differentially expressed in tissues. Interestingly, high concentrations of adenosine have been reported in a variety of tumors. In this context, the final output of adenosine in tumorigenesis will likely depend on the constellation of adenosine receptors expressed by tumor and stromal cells. Notably, activation of the A3 receptor can reduce the proliferative capacity of various cancer cells. This study aimed to describe the anti-proliferative effects of two previously synthesized adenosine derivatives with A3 agonist action (compounds 2b and 2f) through in vitro assays. We used gastric and breast cancer cell lines expressing the A3 receptor as in vitro models and theoretical experiments for molecular dynamics and determination of ADME properties. The antiproliferative effects of adenosine derivatives (after determining IC50 values) were comparable or even higher than those described for IB-MECA, a commercially available A3 agonist. Among possible mechanisms involved, apoptosis was found to be induced in MCF-7 cells but not in AGS or MDA-MB-231 cells. Surprisingly, we were unable to observe cellular senescence induction upon treatment with compounds 2b and 2f in any of the cell lines studied, although we cannot rule out other forms of cell cycles exit at this point. Both adenosine derivatives showed antiproliferative effects on gastric and breast cancer cell lines, and were able to induce apoptosis, at least in the MCF-7 cell line. Further studies will be necessary to unveil receptor specificity and mechanisms accounting for the antiproliferative properties of these novel semi-synthetic compounds.

Phase 1 trial of the adenosine A2A receptor antagonist inupadenant (EOS-850): Update on tolerability, and antitumor activity potentially associated with the expression of the A2A receptor within the tumor.

2562 Background: Tumors produce high levels of extracellular adenosine which suppress anti-tumor immune responses. Blocking A2A receptors, predominantly expressed on tumor-infiltrating immune cells, can reverse the immunosuppressive effect of adenosine. Inupadenant is a non brain-penetrant, potent and highly selective small molecule antagonist of the A2A receptor that remains active even at the high adenosine concentrations found in tumors. Methods: This is the phase I portion of an ongoing first-in-human, clinical trial (NCT02740985) to evaluate safety/tolerability, pharmacokinetic, pharmacodynamic and anti-tumor activity of inupadenant in adult patients with solid tumors who have exhausted standard treatment options. In addition, tumor biomarkers, including adenosine-pathway markers by immunohistochemistry (IHC), are being evaluated. We present updated results of the dose escalation, new results from the monotherapy expansion and new analysis of tumor biomarkers. Results: Overall, 42 patients (21 patients in the dose escalation and an additional 21 patients in a monotherapy expansion) with a median of 3 prior regimens were treated as of the data cut off (DCO, 30Nov20).The dose levels investigated, along with the most frequent (&gt;20%) treatment-emergent adverse events (TEAEs) across all dose levels are presented in the table. 7 AEs led to discontinuation; 2 (atrial fibrillation and myocardial infarction) were considered possibly study drug-related by the investigator. No dose reductions were required. Two partial responses (PRs) were reported: melanoma (NRAS-mutant; received prior immunotherapy), and prostate cancer (received antiandrogen and chemotherapy). At the DCO, both PRs were ongoing with a duration of response &gt;230 days. 12 patients had stable disease (SD) as best response and SD &gt;6 months was observed in 3 patients. Response and stable disease were associated with a higher number of cells expressing the A2A receptor within the tumor at baseline, as measured by IHC. Conclusions: Inupadenant monotherapy was generally well-tolerated as of the DCO at a dose of 80 mg twice daily with initial evidence of clinical benefit, including 2 durable PRs in patients who have exhausted standard treatment options. Analysis of pre-treatment tumor biopsies has identified the A2A receptor as a biomarker which may be associated with clinical benefit. Clinical trial information: NCT03873883. [Table: see text]

Release of adenosine-induced immunosuppression: Comprehensive characterization of dual A2A/A2B receptor antagonist

Immunosuppression is one of the main mechanisms facilitating tumor expansion. It may be driven by immune checkpoint protein expression, anti-inflammatory cytokine secretion or enhanced metabolic enzyme production, leading to the subsequent build-up of metabolites such as adenosine. Under physiological conditions, adenosine prevents the development of tissue damage resulting from a prolonged immune response; the same mechanism might be employed by tumor tissue to promote immunosuppression. Immune cells expressing A2A and A2B adenosine receptors present in an adenosine-rich environment have suppressed effector functions, such as cytotoxicity, proinflammatory cytokine release, antigen presentation and others, making them inert to cancer cells. This study was designed to investigate the dual antagonist potential of SEL330-639 to abolish adenosine-driven immunosuppression. SEL330-639 has slow dissociation kinetics. It inhibits cAMP production in human CD4+ cells, CD8+ cells and moDCs, which leads to diminished CREB phosphorylation and restoration of antitumor cytokine production (IL-2, TNFα, IL-12) in multiple primary human immune cells. The aforementioned results were additionally validated by gene expression analysis and functional assays in which NK cell line cytotoxicity was recovered by SEL330-639. Adenosine-driven immunosuppression is believed to preclude the effectiveness of immune checkpoint inhibitor therapies. Hence, there is an urgent need to develop new immuno-oncological strategies. Here, we comprehensively characterize SEL330-639, a novel dual A2A/A2B receptor antagonist effective in both lymphoid and myeloid cell populations with nanomolar potency. Due to its tight binding to the A2A and A2B receptors, this binding is sustained even at high adenosine concentrations mimicking the upper limit of the range of adenosine levels observed in the tumor microenvironment.

P2X7 Receptors in Neurodegeneration: Potential Therapeutic Applications From Basic to Clinical Approaches.

Purinergic receptors play important roles in central nervous system (CNS), where the bulk of these receptors are implicated in neuroinflammatory responses and regulation of cellular function of neurons, microglial and astrocytes. Within the P2X receptor family, P2X7 receptor is generally known for its inactivity in normal conditions and activation by moderately high concentrations (>100 μM) of extracellular adenosine 5′-triphosphate (ATP) released from injured cells as a result of brain injury or pathological conditions. Activation of P2X7R contributes to the activation and proliferation of microglia and directly contribute to neurodegeneration by provoking microglia-mediated neuronal death, glutamate-mediated excitotoxicity, and NLRP3 inflammasome activation that results in initiation, maturity and release of the pro-inflammatory cytokines and generation of reactive oxygen and nitrogen species. These components of the inflammatory response play important roles in many neural pathologies and neurodegeneration disorders. In CNS, expression of P2X7R on microglia, astrocytes, and oligodendrocytes are upregulated under neuroinflammatory conditions. Several in vivo studies have demonstrated beneficial effects of the P2X7 receptor antagonists in animal model systems of neurodegenerative diseases. A number of specific and selective P2X7 receptor antagonists have been developed, but only few of them have shown efficient brain permeability. Finding potent and selective P2X7 receptor inhibitors which are also CNS penetrable and display acceptable pharmacokinetics (PK) has presented challenges for both academic researchers and pharmaceutical companies. In this review, we discuss the role of P2X7 receptor function in neurodegenerative diseases, the pharmacological inhibition of the receptor, and PET radiopharmaceuticals which permit non-invasive monitoring of the P2X7 receptor contribution to neuroinflammation associated with neurodegeneration.

Simultaneous inhibition of CD73 and IL-6 molecules by siRNA-loaded nanoparticles prevents the growth and spread of cancer

High concentrations of adenosine and interleukin (IL)-6 in the tumor microenvironment have been identified as one of the leading causes of cancer growth. Thus, we decided to inhibit the growth of cancer cells by inhibiting the production of adenosine and IL-6 in the tumor environment at the same time. For this purpose, we used chitosan-lactate-PEG-TAT (CLP-TAT) nanoparticles (NPs) loaded with siRNA molecules against CD73, an adenosine-producing enzyme, and IL-6. Proper physicochemical properties of the produced NPs led to high cell uptake and suppression of target molecules. Administration of these NPs to tumor-bearing mice (4T1 and CT26 models) greatly reduced the size of the tumor and increased the survival of the mice, which was accompanied by an increase in anti-tumor T lymphocyte responses. These findings suggest that combination therapy using siRNA-loaded CLP-TAT NPs against CD73 and IL-6 molecules could be an effective treatment strategy against cancer that needs further study.

Adenosine Signaling in Autoimmune Disorders.

The molecular components of the purinergic system (i.e., receptors, metabolizing enzymes and membrane transporters) are widely expressed in the cells of the immune system. Additionally, high concentrations of adenosine are generated from the hydrolysis of ATP in any “danger” condition, when oxygen and energy availability dramatically drops. Therefore, adenosine acts as a retaliatory metabolite to counteract the nucleotide-mediated boost of the immune reaction. Based on this observation, it can be foreseen that the recruitment with selective agonists of the receptors involved in the immunomodulatory effect of adenosine might represent an innovative anti-inflammatory approach with potential exploitation in autoimmune disorders. Quite surprisingly, pro-inflammatory activity exerted by some adenosine receptors has been also identified, thus paving the way for the hypothesis that at least some autoimmune disorders may be caused by a derailment of adenosine signaling. In this review article, we provide a general overview of the roles played by adenosine on immune cells with a specific focus on the development of adenosine-based therapies for autoimmune disorders, as demonstrated by the exciting data from concluded and ongoing clinical trials.