Quantification of unbound concentration of ticagrelor in plasma as a proof of mechanism biomarker of the reversal agent, MEDI2452

Ticagrelor, a P2Y(12) antagonist, is approved for prevention of thromboembolic events. MEDI2452 is a potential reversal agent for ticagrelor and ticagrelor active metabolite (TAM). The total plasma exposure of ticagrelor and TAM in patients are roughly 0.5 +/- 1 and 0.2 +/- 0.5 mu mol/L, respectively. Both have similar high potency vs. P2Y(12) (Ki 2 nmol/L) but are plasma protein-bound to 99.8% and only the 0.2% free fraction is able to inhibit the P2Y(12) receptor. Thus, for unbound concentration measurements to be a proof of mechanism bio-marker for MEDI2452 a very high sensitivity is required. Using established techniques as equilibrium dialysis and LC-MS/MS, made it possible to evaluate the efficacy of the reversal agent by measuring reduction of unbound concentration of ticagrelor in the presence of MEDI2452. With challenges such as ultra-low concentrations, small sample volumes, recovery issues and adsorption to plastic we managed to develop a highly sensitive assay for determining unbound concentration levels of ticagrelor and TAM in plasma with a quantification limit of 30 pmol/L and 45 pmol/L, respectively. With this method we were able to detect close to a 100-fold MEDI2452 mediated reduction in the unbound concentration of both ticagrelor and TAM. The assay provided proof of mechanism as MEDI2452 concentration-and dose-dependently eliminated unbound concentration of ticagrelor and reversed its antiplatelet activity in preclinical models and will support future development of MEDI2452.