Določanje distribucijskega koeficienta in celične permeabilnosti protirakavih učinkovin s tekočinsko kromatografijo, sklopljeno s tandemsko masno spektrometrijo

Saksida, Teja (author), Ilaš, Janez (mentor)

Topoizomeraza IIα je encim, ki katalizira geometrijske spremembe v molekuli DNA in ima vlogo pri delitvi celic ter razvoju rakavih obolenj. Njeni zaviralci preprečujejo celično delitev, zato se danes uporabljajo kot protirakave učinkovine. Na njihovo farmakološko delovanje vpliva sposobnost prehajanja celične membrane, ki je tesno povezana z lipofilnostjo spojine. V magistrski nalogi smo preučevali povezavo med kemijsko strukturo zaviralcev topoizomeraze IIα, njihovo lipofilnostjo in prehajanjem celične membrane. Za analizo vzorcev smo razvili metodo tekočinske kromatografije zelo visoke ločljivosti, sklopljeno s tandemsko masno spektrometrijo. Uporabili smo trojni kvadrupol in ionizacijo z elektrorazprševanjem za določitev koncentracij analitov v vzorcih. Koncentracije spojin v vzorcu smo določili na osnovi umeritvene krivulje, pripravljene volumetrično v območju od 0,5 do 2000 nmol/L. Linearnost smo preverjali s koeficientom določitve oz. determinacije, katerega ciljana vrednost je bila vsaj 0,999. O lipofilnosti zaviralcev smo sklepali na osnovi določenih vrednosti distribucijskega koeficienta osmih spojin. Slednjega smo določili z miniaturno metodo stresanja pri dveh različnih volumskih razmerjih vodne in organske faze. Izkazalo se je, da je metoda primerna za analizo aminov, ne pa karboksilnih kislin. Dobljene vrednosti distribucijskega koeficienta so bile med 1 in 3,5, kar teoretično pomeni ustrezno lipofilnost in vodotopnost za prehajanje celičnih membran. S šestimi spojinami smo izvedli testiranje permeabilnosti na celični liniji MCF‑7 z zbiranjem vzorcev supernatanta in celičnega lizata. Eksperimentalni rezultati na celičnih vzorcih so potrdili prehajanje membran za eno izmed testiranih spojin z distribucijskim koeficientom 3,5. Rezultati so potrdili, da je distribucijski koeficient koristen parameter za napovedovanje osnovnih porazdelitvenih lastnosti spojin, vendar moramo za boljše razumevanje celične permeabilnosti upoštevati tudi druge lastnosti spojine. V nadaljevanju bi bilo smiselno še dodatno optimizirati analitsko metodo, predvsem za določitev najnižjih koncentracij, razširiti nabor spojin in izvesti dodatne meritve na celicah v daljših časovnih presledkih za boljši vpogled v prehajanje celičnih membran.

• Language: Slovenian
• Keywords: distribucijski koeficient, permeabilnost, tandemska masna spektrometrija, tekočinska kromatografija zelo visoke ločljivosti, zaviralci topoizomeraze IIα
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2025
• PID: 20.500.12556/RUL-173718
• Publication date in RUL: 20.09.2025

Secondary language

• Language: English
• Title: Determination of the distribution coefficient and cellular permeability of antitumor agents using liquid chromatography coupled with tandem mass spectrometry

Abstract

Topoisomerase IIα is an enzyme that catalyzes geometric changes in the DNA molecule and plays an essential role in cell division and cancer development. Its inhibitors prevent cell proliferation and are therefore used as antitumor agents. Their pharmacological effect depends on their ability to cross the cell membrane, which is closely linked to the lipophilicity of the compound. In this master's thesis, we examined the relationship between the chemical structure of topoisomerase IIα inhibitors, their lipophilicity, and their ability to permeate the cell membrane. For sample analysis, we developed a method based on ultra‑high‑performance liquid chromatography coupled with tandem mass spectrometry. A triple quadrupole mass analyzer with electrospray ionization was used to determine analyte concentrations in samples. Compound concentrations were determined using a calibration curve prepared volumetrically in the range from 0,5 to 2000 nmol/L. Linearity was verified by the coefficient of determination, with the target value of at least 0,999. Lipophilicity of inhibitors was assessed from distribution coefficient values of eight compounds, obtained using a miniaturized shake-flask method at two different volumetric ratios of aqueous and organic phase. The method proved suitable for amines, but not for carboxylic acids. The obtained distribution coefficient values ranged from 1 to 3,5, indicating theoretically appropriate lipophilicity and water solubility for crossing the cell membrane. Permeability was assessed for six compounds using the MCF‑7 cell line by collecting samples of supernatant and cell lysate. Experimental results confirmed membrane penetration for one of the tested compounds with a distribution coefficient of 3,5. The results confirmed that the distribution coefficient is a useful parameter for predicting the basic distribution properties of compounds, but a more comprehensive understanding of cellular permeability requires consideration of additional physicochemical properties. Follow-up experiments should include additional optimization of the analytical method, particularly for the determination of the lowest concentrations, as well as expanding the set of compounds and performing additional cell-based measurements at longer time intervals to obtain a more complete picture of membrane permeability.

• Keywords: distribution coefficient, permeability, tandem mass spectrometry, topoisomerase IIα inhibitors, ultra‑high‑performance liquid chromatography