20.500.12556/RUL-173420 Določanje termodinamske in kinetične topnosti zaviralcev topoizomeraze IIα Determination of the thermodynamic and kinetic solubility of topoisomerase IIα inhibitors Topnost je ključna fizikalno-kemijska lastnost, ki se uporablja pri odkrivanju in razvoju zdravil. Po definiciji je topnost snovi največja količina snovi, ki se raztopi v določenem volumnu topila in se razlikuje glede na topilo in pogoje. V okviru diplomskega dela smo zaviralcem topoizomeraze IIα določali termodinamsko in kinetično topnost. Termodinamska oz. ravnotežna topnost je topnost spojine v ravnotežju, pri čemer je raztopljena spojina v ravnotežju z neraztopljeno snovjo. Kinetična topnost pa je največja topnost najhitreje precipitirajoče vrste spojine, ki se pogosto meri z uporabo koncentriranih raztopin v organskih topilih. Pri merjenju termodinamske topnosti dobimo bolj ponovljive in natančne podatke v primerjavi s podatki kinetične topnosti. Za merjenje topnosti smo uporabili metodo stresanja. Pri tem smo najprej pripravili umeritveno krivuljo v koncentracijskem območju od 1 do 100 μM. Za merjenje termodinamske topnosti smo pripravili vzorce tako, da smo fosfatni pufer dodali trdnemu vzorcu, za merjenje kinetične topnosti pa smo koncentrirano osnovno raztopino vzorca v DMSO redčili s fosfatnim pufrom. Vzorce smo nato stresali 24 ur pri 37 ˚C. Po 24 h smo vzorce centrifugirali 10 min pri 18000 rpm, supernatant nato redčili z diluentom in vzorce analizirali z UHPLC sklopljenim z UV/Vis detektorjem. Kakovost umeritvenih krivulj smo določili s koeficientom regresije, pri čemer smo določili prag R2 ≥ 0,999. Za preverjanje točnosti metode smo uporabili tudi kontrolne vzorce pri koncentracijah 2, 20 in 60 μM. Ugotovili smo, da so se največje napake pojavile pri najnižjih koncentracijah. Nekatere spojine so razpadle bodisi pri določanju termodinamske bodisi kinetične topnosti. Na določenih spojinah smo izvedli dodatne teste, v okviru katerih smo preverjali njihovo stabilnost vrazličnih časovnih točkah stresanja in z zamenjavo fosfatnega pufra s 0,1% trifluoroocetno kislino. Ugotovili smo, da čas stresanja ne vpliva na stabilnost spojin, so pa spojine bolj topne v kislem okolju. V fosfatnem pufru se spojina ni raztopila in smo zato videli samo razpadne produkte, z dodatkom trifluoroocetne kisline pa se spojina raztopi in zato vidimo tudi njen vrh. Za potrditev razpada smo na eni izmed spojin izvedli dodatne analize z napravama LC-MS in HRMS, pri čemer smo ugotovili, da spojina razpade v manjši meri, nastali razpadni produkti pa so vodotopni in jih je zato mogoče zanesljivo določiti v supernatantu. Solubility is a key physicochemical property used in drug discovery and development. By definition, the solubility of a substance is the maximum amount of the substance that dissolves in a given volume of solvent, and it varies depending on the solvent and conditions. In the framework of this thesis, we determined both thermodynamic and kinetic solubility of topoisomerase IIα inhibitors. Thermodynamic or equilibrium solubility refers to the solubility of a compound at equilibrium, where the dissolved compound is in balance with the undissolved substance. Kinetic solubility, on the other hand, is the maximum solubility of the most rapidly precipitating species of a compound, often measured using concentrated stock solutions in organic solvents. Measurements of thermodynamic solubility provide more reproducible and accurate data compared to kinetic solubility. To measure solubility, we used the shake-flask method. First, a calibration curve was prepared in the concentration range of 1 to 100 μM. For thermodynamic solubility measurements, phosphate buffer was added to the solid sample, while for kinetic solubility measurements, a concentrated stock solution of the sample in DMSO was diluted with phosphate buffer. The samples were then shaken for 24 hours at 37 ˚C. After 24 hours, the samples were centrifuged for 10 minutes at 18,000 rpm, the supernatant was diluted with diluent, and the samples were analyzed using UHPLC coupled with a UV/Vis detector. The quality of calibration curves was evaluated using the regression coefficient, with a threshold set at R² ≥ 0.999. To verify method accuracy, we also used control samples at concentrations of 2, 20, and 60 μM. We found that the largest errors occurred at the lowest concentrations. Some compounds degraded either during thermodynamic or kinetic solubility determination. For certain compounds, additional tests were carried out to assess their stability at different shaking time points and by replacing phosphate buffer with 0.1% trifluoroacetic acid. We found that shaking time did not affect compound stability, but compounds were more soluble in acidic medium. In phosphate buffer, the compound did not dissolve and only degradation products were observed, while with the addition of trifluoroacetic acid the compound dissolved, and its peak could therefore be detected. To confirm degradation, further analyses with LC-MS and HRMS were performed for one of the compounds, revealing that the compound degraded to a minor extent. The resulting degradation products were water-soluble and could therefore be reliably quantified in the supernatant. termodinamska in kinetična topnost metoda stresanja tekočinska kromatografija visoke zmogljivosti zaviralci topoizomeraze IIα thermodynamic and kinetic solubility shake-flask method high-performance liquid chromatography (HPLC) topoisomerase IIα inhibitors true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2025-09-17 08:45:15 2025-09-17 08:45:20 2025-11-09 03:39:20 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 118173 28886.pdf 28886.pdf 1 E2FAE1D77C87C7861F98C5DAA87178A7 0fe163d26569fcedf68c7e57735c64596ab491206c46ea9ad4ff708fe1724d9a b63902a6-9391-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=217830 Fakulteta za farmacijo 0 0 0