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Final Master‘s thesis by G. Jablonskytė, title „The cell culture research: metabolism of new generation anticoagulants“. Supervisor V. Tatarūnas; Laboratory of Molecular Cardiology at the Institute of Cardiology, Lithuanian University of Health Sciences. - Kaunas. The aim of the study: to determine the metabolites of rivaroxaban and new compounds, involved in the effect of rivaroxaban, and to investigate the influence of inhibitors of CYP4F2 enzyme on the metabolism of anticoagulant in endothelial cell cultures. Objectives: 1. To identify metabolites of rivaroxaban in the media of endothelial cells after the exposure of HUVECs to different drug concentrations. 2. To determine new compounds that are formed upon the exposure of HUVEC cells to different concentrations of rivaroxaban. 3. To determine the effect of different concentrations of rivaroxaban and inhibitors of CYP4F2 enzyme on the levels of CYP4F2 enzyme in endothelial cell lysates. 4. To determine CYP4F2 gene variants in endothelial cells. Object: commercial human umbilical vein endothelial cells (HUVECs). Methods: cell cultivation in flasks, genotyping by real-time polymerase chain reaction, methods of liquid chromatography and mass spectrometry, enzyme-linked immunosorbent assay ELISA. Results and conclusions: rivaroxaban and M-1, M-2, M-5, M-8, M-10, M-11, M-18 metabolites were identified from cultured HUVEC cells after exposure with rivaroxaban. In addition, it was determined that the concentration of a new compound, which previously has not been linked with the effect of rivaroxaban, decreased as the concentration of rivaroxaban increases. CYP4F2 levels decreased with increasing rivaroxaban concentrations. Pre-treatement with 0.5 μM of rivaroxaban solution decreased the levels of enzyme in cell cultures by 17.2 %, 1 μM rivaroxaban - by 81.3 %, and 2 μM of rivaroxaban by 85.5 %. Treatment with Hsa miR 24-3p reduced the levels of CYP4F2. Enzyme levels decreased by 11.9 % in HUVECs exposed to 120 nM hsa-miR-24-3p. The CYP4F2 rs2108622 C/C, rs1558139 G/G, rs2074902 T/T and the CYP4F2 rs3093135 A/T variants were detected in HUVEC`s cells.

Objective and tasks. To evaluate and to determine whether CYP2C9 *2, *3, VKORC1 *2, *3 and CYP4F2 G1347A genotypes and clinical factors contribute to warfarin dosage in patients after heart valve surgeries. Methods. During the initiation of warfarin therapy, 175 patients, who had heart valve surgeries, were included into the study. During the long term treatment, totally 113 patients were analyzed. Patient anthropometric, clinical data (biochemical tests, used medications, addictions) and patient INR were obtained from the case histories. Genomic DNA was extracted from peripheral blood. The real – time polymerase chain reaction was used to detect the CYP2C9 * 2 (rs1799853) and *3 (rs1057910), VKORC1 *2 (rs9934438), VKORC1 *3 (rs7294) and CYP4F2 G1347A (rs2108622) genotypes. Statistical analysis. Results are presented as a mean and standard deviation (mean ± SD). A p≤0,05 was taken as statistically significant. Frequency of genotypes and alleles in patient’s sample is presented as % (percent). Results. The patients, users of amiodarone, required lower dosages of warfarin during treatment initiation, as compared to the non-users. During the long – term treatment period, the users of torasemide, calcium channel blockers and benzodiazepines, required lower warfarin dosages as compared to non-users of these drugs. During the long term treatment, patients with elevated creatinine or elevated alkaline phosphatase activity, required lower dosages of warfarin as compared to the patients with normal levels of these enzymes. Patients with such addictions (smoking and alcohol use) required higher dosages of warfarin. Besides the clinical factors, warfarin dosage was also influenced by these genetic variants (both at initiation of the warfarin therapy and during the long term treatment): CYP2C9*1*3 heterozygotes used lower warfarin dosages as compared to the wild-type homozygotes, VKORC1 C1173C carriers required higher dosages of warfarin than comparing to C/T and T/T genotype carriers, VKORC1 G3730G carriers required lower dosages of warfarin compared to A/A genotype carriers. The carriers of CYP4F2 A1347A required higher warfarin dosages, as compared to CYP4F2 G/G cariers, but only at initiation of warfarin therapy. Conclusions: Dosage of warfarin is influenced by such a clinical factors as the activity of plasma serum creatinine, the activity of alkaline phosphatase, medications - amiodarone, torasemide, calcium channel blockers, benzodiazepines, patient addictions (smoking, alcohol consumption). The genotypes CYP2C9 * 1 * 3, VKORC1 C1173T, VKORC1 G3730A and CYP4F2 G1347A are also significant in determining warfarin dosage.