Apixaban

Ticagrelor [purity 99.64%, high performance liquid chromatography (HPLC)] and ticagrelor-d7 (Purity: 96%, HPLC) were procured from MuseChem, Fairfield, NJ, USA. Apixaban (purity 98%, HPLC) and rivaroxaban (purity 99.7%, HPLC) were obtained from Toronto Research Chemicals, Toronto, Ontario, Canada and AK Scientific Inc, Union City, CA, USA, respectively. All other reagents were of standard analytical grade. The test and control circuit components were procured from McMaster–Carr, Elmhurst, IL, USA and Molded Products Inc., Harlan, IA, USA. Bovine whole blood (Lampire Biological Inc., Pipersville, PA, USA) with 4.5 U/mL Na-Heparin and total protein ranging between 6.6–8.8 g/dL, was used for all runs. Maxi–Therm Pediatric Blankets 56436-I (Cincinnati Sub Zero LLC, Cincinnati, OH, USA) connected with Polystat standard 3–6 L heat/cool bath (Cole–Parmer, Vernon Hills IL, USA) were used to maintain the temperature of the blood pool at 37°C throughout the runs. All tested DrugSorb-ATR devices were gamma sterilized and used the same filling, finishing and sterilization processes as commercially available devices. All instruments used for the measurement of run parameters were calibrated using instrumentation and standards from the United States National Institute of Standards and Technology (NIST) and are compliant with ISO-10 012:2003 and ANSI/NCSL Z540-3-2006.

Apixaban and Apixaban-d3 (internal standard, IS) were obtained from Toronto Research Chemicals (Ontario, Canada). Methanol, ammonium formate, and formic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA) and Duksan (Korea). All reagents used in this study were either LC grade or extra-pure grade. Millipore Milli-Q system at 18.2 MΩ (Billerica, MA, USA) was used to obtain deionized water. Drug-free blank plasma containing ethylenediaminetetraacetic acid (EDTA)-K3 was bought from Biochemed (Winchester, VA, USA).

For the first part of the study, we obtained whole blood samples from 20 healthy volunteers who had not taken any medications (12 males and eight females; aged 29 - 54 years). All blood samples were numbered in order of registration. To confirm the in vitro antithrombotic effects of Rivaroxaban and apixaban at the expected clinical peak drug level, blood samples were prepared with drugs as follows. Rivaroxaban and apixaban were obtained from Toronto Research Chemicals (Toronto, Ontario, Canada). Rivaroxaban and apixaban were each dissolved in 100% dimethylsulfoxide to yield stock solutions with a concentration of 5 mM. Stock solutions were then diluted in blood samples at the time of assay (first 16 samples in the Rivaroxaban group and the rest of four samples in the apixaban group). The target final concentration of Rivaroxaban in this experiment was 800 μM, which corresponds to the expected peak concentration (C_max-equivalent) of Rivaroxaban in blood after a dose of 15 mg/day [12 (link)]. Similarly, blood samples were spiked with apixaban stock solutions. The final concentration of apixaban was 450 μM, which corresponded to the peak concentration of apixaban in blood after a dose of 10 mg/day clinically [13 (link), 14 (link)]. We immediately evaluated the antithrombotic effects of these spiked blood samples in comparison with thrombus formation in control blood by the T-TAS^®.