Purpose Ellagitannins are high molecular weight polyphenols present in high quantities in various food products. They are metabolized by human and animal gut microbiota to postbiotic metabolites-urolithins, bioavailable molecules of a low molecular weight. Following absorption in the gut, urolithins rapidly undergo phase II metabolism. Thus, to fully evaluate the mechanisms of their biological activity, the in vitro studies should be conducted for their phase II conjugates, mainly glucuronides. The aim of the study was to comparatively determine the influence of urolithin A, iso-urolithin A, and urolithin B together with their respective glucuronides on processes associated with the inflammatory response. Methods The urolithins obtained by chemical synthesis or isolation from microbiota cultures were tested with their respective glucuronides isolated from human urine towards modulation of inflammatory response in THP-1-derived macrophages, RAW 264.7 macrophages, PBMCs-derived macrophages, and primary neutrophils. Results Urolithin A was confirmed to be the most active metabolite in terms of LPS-induced inflammatory response inhibition (TNF-α attenuation, IL-10 induction). The observed strong induction of ERK1/2 phosphorylation has been postulated as the mechanism of its action. None of the tested glucuronide conjugates was active in terms of pro-inflammatory TNF-α inhibition and anti-inflammatory IL-10 and TGF-β1 induction. Conclusion Comparative studies of the most abundant urolithins and their phase II conjugates conducted on human and murine immune cells unambiguously confirmed urolithin A to be the most active metabolite in terms of inhibition of the inflammatory response. Phase II metabolism was shown to result in the loss of urolithins’ pharmacological properties.

Adenosine is the key immunometabolite responsible for immune tolerance in tumors. It is present in normal tissue in low concentrations, having various physiological functions. In the tumor, its concentration increases rapidly, as a result of overexpression of enzymes producing adenosine, additionally enhanced by hypoxia and inflammation. Adenosine inhibits the biological functions of T lymphocytes infiltrating the cancer tissue by binding to the A2A receptor. The affinity to A2B receptor is believed to attenuate the response of dendritic cells and other parts of innate system. Thus blocking simultaneously the effects mediated by both receptor subtypes with dual inhibitor seems to be a viable approach to a single agent cancer immunotherapy or a combination with current immunotherapeutic agents. We have discovered a novel series of potent and selective A2A/A2B inhibitors. Best compounds present subnanomolar activity in both in vitro recombinant cellular model and in primary human cells. Our antagonists dose-dependently restore the adenosine agonist-impaired functional activity of CD4+ and CD8+ human T-lymphocytes (cytokine release assays). We observe also the relieve of adenosine-related suppression in NK cells cytotoxicity. Most importantly tested compounds present improved primary pharmacological profile in comparison to A2A inhibitors currently tested in clinical trials. Further validation in in vivo models showed efficacy standalone and in combination with checkpoint inhibitors. Citation Format: Michal Galezowski, Paulina Wegrzyn, Aneta Bobowska, Claude Commandeur, Katarzyna Dziedzic, Marcin Nowogrodzki, Alicja Obara, Joanna Szeremeta-Spisak, Anna Dzielak, Iwona Lozinska, Marcelina Dudek, Anita Janiga, Jacek Reus, Marek Wronowski, Mateusz Swirski, Adam Radzimierski, Magdalena Ziembik, Joanna Mierzwicka, Katarzyna Wojcik-Jaszczynska, Elzbieta Gocek, Karolina Grycuk, Aniela Golas, Olga Pierzchala, Julian Zachmann, Mateusz Nowak. Characterization of novel dual A2A/A2B adenosine receptor antagonists for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3770.

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Adenosine is important messenger molecule that is involved in signal transmission in central nervous, cardiovascular and immunological systems. The immunosuppressive role of adenosine attract attention of many researchers recently and reversal of that effect is addressed by several different approaches i.e.: inhibition of enzymes that are producing the adenosine in tumor microenvironment like CD39 and CD73 but also the antagonization of adenosine receptors mainly A2A and A2B subtypes. Here we present a novel series of unsurmountable, dual A2A/A2B antagonists that retain its nanomolar potency in tumor-like adenosine-rich environment. This advantageous profile comes mainly from long residence time in adenosine receptors which transfers into high receptor occupancy even after full clearance of the compound from plasma. We are demonstrating how our inhibitors restore adenosine depleted immune functionality in the series of functional in vitro assays in primary cells. This includes the cytokine release by activated CD4+ and CD8+ human T-lymphocytes (driven by A2A) and dendritic cells (driven by A2B). Most importantly presented compounds show improved in vivo pharmacological profile in comparison to adenosine inhibitors currently tested in clinical trials manifested by high inhibition of CREB phosphorylation in mouse model. Citation Format: Michal Galezowski, Paulina Węgrzyn, Aneta Bobowska, Katarzyna Dziedzic, Joanna Szeremeta-Spisak, Marcin Nowogrodzki, Grzegorz Satala, Alicja Obara, Iwona Lozinska-Raj, Marcelina Dudek, Anita Janiga, Jacek Reus, Marek Wronowski, Magdalena Zastawna, Grzegorz Statkiewicz, Maciej Rogacki, Magdalena Ziembik, Karolina Grycuk, Foteini Soukou, Kinga Michalik, Agnieszka Adamus, Karolina Wiatrowska, Natalia Lewandowska, Aniela Golas, Olga Haberkiewicz, Rod Porter, Krzysztof Brzozka, Mateusz Nowak. Novel dual A2A A2B adenosine receptor antagonists for cancer immunotherapy: in vitro and in vivo characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4135.

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