Study of Two Dose Regimens of Ticagrelor Compared With Clopidogrel in Patients Undergoing Percutaneous Coronary Intervention for Stable Coronary Artery Disease

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Influences of genetic variation on effects of clopidogrel and ticagrelor
Genetic variation in the activity of key cytochrome P450 (CYP) enzymes partly explains limited efficacy of clopidogrel in some individuals, including loss-of-function alleles in CYP2C19 that have been associated with reduced clopidogrel active metabolite formation and increased risk of stent thrombosis. [1][2][3][4][5] Genetic variants affecting ticagrelor and AR-C124910XX levels are uncommon and have limited effect on the levels as well as no detectable impact on efficacy or safety of ticagrelor. 6

Inclusion criteria
For inclusion in the study, subjects should fulfill the following criteria: 1. Provision of informed consent prior to any study specific procedures 2. Male or female aged greater than 18 years 3. Previous invasive coronary angiography with plan for PCI with coronary stent implantation for stable coronary artery disease

Exclusion criteria
Subjects should not enter the study if any of the following exclusion criteria are fulfilled: 1. Requirement for a chronic total occlusion to be crossed in order for any stent implantation to proceed 2. Plan for coronary angiography with a view to PCI if appropriate (i.e. current coronary anatomy not known) 3. Intention to use platelet function tests or genotyping to guide antiplatelet therapy 4. Known allergy to or intolerance of aspirin, clopidogrel or ticagrelor 5. Treatment with antiplatelet medication apart from aspirin or clopidogrel that cannot be stopped 10 days prior to PCI (e.g. ticagrelor, prasugrel, dipyridamole, ticlopidine, abciximab, tirofiban), for example because of continuing indication 6. Planned treatment or consideration of treatment with oral antiplatelet medication other than aspirin or clopidogrel following PCI

High-performance liquid chromatography (HPLC) methods
Samples with stop solution were centrifuged at 1500g and the supernatant deproteinised by the addition of ice-cold 70% perchloric acid before further centrifugation at 13000g and storage of the supernatant at -80°C. HPLC was performed using a Waters 2695 HPLC analyser and a Waters 2487 Ultra-Violet/Visible (UV/Vis) detector with wavelength 258 nm. Data was collected by DataApex Clarity software. The column was a Waters Xbridge C18 5um (250mm x 4.6mm id) at a temperature of 23°C. The mobile phase was 2% acetonitrile in aqueous ammonium hydroxide (0.1%) for 35 mins ramping to 98% acetonitrile after 36 mins until 40 mins (1mL/min). The injection volume was 100uL. The lower limit of quantification for adenosine was 0.01 µmol/L.

Genetic analysis methods
DNA was extracted from whole blood samples using Chemagen Chemagic 10k kits (Perkin Elmer, Baesweiler, Germany) followed by elution in Tris-EDTA buffer. The DNA was quantified using Quantifluor® dsDNA System (Promega, Madison, WI, USA).
Genotyping was done using Taqman assays (Applied Biosystems, Life Technologies, Pleasanton, CA, USA) using an Applied Biosystems 7900HT Real-Time PCR System.

Excluded patient on strong CYP3A inducer
One patient randomized to ticagrelor 60mg bid was subsequently found to have been taking a strong CYP3A inducer throughout the study and was, therefore, included in error. Their pharmacodynamic and pharmacokinetic data were excluded from the main analyses to avoid misleading comparison of the groups. It was confirmed that no other patients in the study received excluded medication. The patient was informed of this error and agreed for their individual data to be presented anonymously in view of the scientific interest. After ticagrelor 180-mg loading dose, their VerifyNow P2Y12 assay showed PRU 220 and percentage inhibition 21%. Pre-and post-maintenance dose of ticagrelor 60mg at 1 month, these values were 223 and 4% pre-dose and 208 and 21% post-dose, respectively. LTA results were consistent with these values. Corresponding to these low levels of platelet P2Y 12 inhibition, plasma levels of ticagrelor and AR-C124910XX were also low, indicating ultra-rapid metabolism of ticagrelor and its active metabolite: following ticagrelor 180-mg loading dose, levels were 55 and 105 ng/mL, respectively; pre-maintenance dose, levels were 7.5 and 36.4 ng/mL, respectively, and post-maintenance dose, levels were 18.6 and 60 ng/mL, respectively.
These findings illustrate the importance of checking on relevant CYP3A-mediated drug interactions when using ticagrelor and avoiding the use of ticagrelor in patients receiving strong CYP3A inducers.

Supplementary genetic analyses
The presence of a gain-of-function allele for CYP2C19 did not influence the relationship between clopidogrel and either of the ticagrelor doses (Supplementary