title: Determination of unbound ticagrelor and its active metabolite (AR-C124910XX) in human plasma by equilibrium dialysis and LC-MS/MS.
abstract: Ticagrelor is the first direct acting reversibly binding oral platelet P2Y(12) receptor antagonist. The parent molecule and the main metabolite (AR-C124910XX) are both able to block adenosine diphosphate-induced receptor signaling with similar potency. Drug binding to plasma proteins reduces free drug available for pharmacologic activity. Therefore, assessing unbound drug is important for interpretation of pharmacokinetic/pharmacodynamic findings. This paper describes the development and validation of an equilibrium dialysis/LC-MS/MS method for measuring unbound ticagrelor and AR-C124910XX in human plasma. Plasma samples (200μl) were dialysed against phosphate buffered saline (37 °C, 24h) in 96-well dialysis plates to separate unbound analytes. Drug-protein binding alterations during dialysis were minimized by maintaining physiologic conditions (pH 7.4, 37 °C). Ticagrelor and AR-C124910XX were quantified in dialysates (unbound fraction), retentates and plasma (total concentration) using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methods. Calibration curves were established for the retentate and plasma (total concentration) in the ranges 5-5000 ng/ml (ticagrelor) and 2.5-2500 ng/ml (AR-C124910XX), and for the dialysate in the range 0.25-100 ng/ml (both analytes). Both ticagrelor and AR-C124910XX were highly protein bound (>99.8%), i.e. unbound fraction <0.2%. Yet, the methodology was successfully applied to determine unbound concentrations of ticagrelor and AR-C124910XX in clinical samples.
authors: AstraZeneca R&D, Mölndal, S-431 83 Mölndal, Sweden. Henrik.Sillen@astrazeneca.com
authors: Henrik
authors: H
authors: Sillén
authors: Melanie
authors: M
authors: Cook
authors: Patty
authors: P
authors: Davis
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000097:blood / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D003956:Dialysis / Q000379:methods*; D006801:Humans; D011485:Protein Binding; D058921:Purinergic P2Y Receptor Antagonists / Q000097:blood* / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D000077486:Ticagrelor
chemical_list: D058921:Purinergic P2Y Receptor Antagonists; D000077486:Ticagrelor; D000241:Adenosine