P3 Development of a quantitative bioanalytical method for the assessment of adenosine in pre-clinical and clinical tumor samples.

Kelly J Goodwin , AstraZeneca, Waltham, MA
Alexandra Borodovsky , AstraZeneca, Waltham, MA
Richard Woessner , AstraZeneca, Waltham, MA
Eric Gangl , AstraZeneca, Waltham, MA
Ujjal Sarkar , AstraZeneca, Waltham, MA
Petar Pop-Damkov , AstraZeneca, Waltham, MA
Natalie Jones , AstraZeneca, Waltham, MA
Adrian Fretland , AstraZeneca, Waltham, MA
Adenosine is an important signaling molecule in the immune system and is the primary agonist of the adenosine receptor, A2a. Physiologically, A2aR plays a role in tissue protection from the immune system though its interaction with adenosine. When tissue damage occurs, increased levels of extracellular adenosine are released from the cell interacting with A2aR on the surface creating an environment for immune evasion. Specifically within the tumor microenvironment, inflammation of the tissue produces increased levels of extracellular adenosine leading to this immunosuppressive effect, which with prolonged exposure can bring about the evasion of tumors from the immune system and the progression of cancer. The goal of this project is to determine the adenosine concentration in preclinical syngeneic tumors and clinical tumor samples. Additionally this assessment will allow for a more informed selection of pre-clinical models to help develop A2aR inhibitors to reverse immunosuppressive effects of adenosine as it has been shown that the potency of A2aR inhibitors are directly related to the levels of adenosine in tissue. There are challenges in the quantitative bioanalysis of adenosine in biological matrices including: very short half-life from enzymatic degradation, hydrophilicity, low molecular weight, and successful separation from other nucleosides. With these challenges in mind, a novel method for the quantification of adenosine in tissue samples has been developed using pre-column derivatization with LC-MS/MS quantification. This method employs dansyl chloride as a derivatization agent to improve adenosine properties for better chromatographic separation and ionization. Furthermore organic processing of frozen tissues helps reduce/eliminate adenosine degradation after harvesting. Experiments thus far have shown successful adenosine derivatization in plasma, brain, liver and tumor matrices and a linear quantification range of 0.37µM to 37µM. Further development of this assay will help understand adenosine concentrations in tumors and better aid in understanding the PK/PD efficacy relationship of adenosine inhibition.