Topoisomerases are nuclear enzymes, which are responsible for cleaving and untangling the supercoiled DNA molecules. They are thus playing a key role in regulating its topological structure. Scientists recognized them as potential targets for anticancer agents. Since then, numerous potential inhibitors have been in research and development phases.
In this thesis, we analyzed six compounds that could potentially act as anticancer agents by inhibiting the group of topoisomerase II enzymes. We determined their lipophilicity and plasma protein binding, two critical parameters in the research and development of new active pharmaceutical ingredients. These two properties allow us to predict the behavior of compounds in the human body and select more suitable candidates for further drug development phases, as they provide insights into the pharmacokinetic, physicochemical and toxicological characteristics of the compounds. Lipophilicity was determined by measuring the distribution coefficient at pH 7.4 (LogD7.4) using a modified shake-flask method in HPLC vials. Plasma protein binding was assessed using equilibrium dialysis method with the Pierce RED Device (Thermo Fisher Scientific), which is specifically designed for rapid equilibrium dialysis and validated for determination of plasma protein binding. All samples were analyzed by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). A triple quadrupole detector was used, which allowed us to develop an appropriate analytical method. For sample quantification, we established a calibration curve method, which was verified using two control standards.
We determined that all investigated compounds exhibited LogD7.4 values between 2 and 4, along with extensive plasma protein binding (>99.3%). Based on the measured LogD7.4 values, we conclude that their lipophilicity is appropriate, and the compounds are suitable for further development. However, the plasma protein binding results indicate that their pharmacokinetic properties are not optimal. These findings suggest that other physicochemical properties of the compounds will play a more significant role than plasma protein binding in their successful delivery to target sites.
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