| Abstract [eng] |
Cardiovascular disease (CVD) is associated with atherosclerosis, which develops due to inflammatory processes in the blood vessels and loss of endothelial function (dysfunction). Recent studies have shown that anticoagulant rivaroxaban and two-component combination of sacubitril / valsartan used for the treatment of heart failure may also have an endothelial-affecting activity in addition to their direct indications. The mechanism of action of these drugs is still not clear. Based on previous research carried out in the Laboratory of Molecular Cardiology, it has been suggested that the activity of CYP4F2 enzyme may be related to the efficacy of these drugs. The aim of this pilot study was to investigate the metabolism of rivaroxaban and sacubitril by evaluating the metabolites in HUVEC and HepG2 cells treated with different concentrations of drugs and to evaluate the effect of sacubitril / valsartan and inhibitor of CYP4F2 on the activity of CYP4F2 enzyme in endothelial cell cultures. The human umbilical vein endothelial cells (HUVEC) used in this study were cultivated in a six-well plate. Cells were treated with seven different concentrations of rivaroxaban (0,1 μM, 0,2 μM, 0,5 μM, 1 μM, 2 μM, 5 μM, 10 μM) and sacubitril / valsartan (0,05 μM, 0,1 μM, 0,25 μM, 0,5 μM, 1 μM, 2,5 μM, 5 μM). Rivaroxaban solution was prepared by dissolving the drug in bovine serum albumin. Sacubitril / valsartan solutions were obtained by dissolving the drug in aqueous 0.9 % NaCl solution. Screening for sacubitril metabolites was performed in HUVEC cell lysates. Lysates were obtained by treating cells with lysis buffer. Cell transfection was performed by treating the cells with a miRNA-liposome complex (1:1). Cells were treated with the drug after 24 hours. Transfection with hsa-miR-24-3p mimic was performed to suppress the expression of CYP4F2 gene encoding the CYP4F2 enzyme and to evaluate the effect of this enzyme on sacubitril metabolism. Metabolites in the collected cell media were identified using liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) method. Quantification of the formed compounds of in cell media and HUVEC cell lysates was performed by ultra-efficient liquid chromatography-triple quadrupole mass spectrometry (UPLC-TQD-MS) technique. Metabolites M-1, M-2, M-5, M-8, M-10, M 11, M-18 of rivaroxaban and the M1 metabolite of sacubitril / valsartan have been identified in HUVEC cells exposed to different drugs concentrations. A new compound (Z)-doco-13-enamide was identified, which concentration varied with the concentration of rivaroxaban. The obtained data showed that sacubitril is not completely metabolized to the active metabolite M1 (sacubitrilat) when HUVECs are exposed to concentrations greater than 0.1 μM. CYP4F2 enzyme concentrations increase 2.6-fold with 5 μM sacubitril / valsartan treatment in HUVECs, and CYP4F2 protein concentration decreased in HepG2 cells with increasing drug concentrations. The hsa-miR-24-3p mimic inhibited the biotransformation of sacubitril to the active metabolite M1 in HUVECs. |
