Kompeticijsko određivanje konstanti vezanja dipiridamola i varfarina za humani serumski albumin polarizacijskom fluorescencijom

master's thesis

• Title (english): Competitive binding fluorescence polarization assay for determination of association constants of dipyridamole and warfarin to human serum albumin
• Author: Jan Radanović
• Mentor: Mario Gabričević (mentor)
• Committee member: Mario Gabričević (predsjednik povjerenstva)
• Committee member: Erim Bešić (član povjerenstva)
• Committee member: Tin Weitner (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of general and inorganic chemistry); Zagreb
• Defense date and country: 2023-07-06, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Cilj ovog rada bio je odrediti konstante ravnoteže reakcija vezanja dipiridamola i varfarina na humani serumski albumin
(HSA). Dobro poznavanje vezanja lijekova za proteine krvne plazme, posebice za HSA koji je među njima
najzastupljeniji, od velike je važnosti za predviđanje farmakokinetičkih svojstava lijekova koji se za njega vežu. S
obzirom na to da je samo nevezani udio lijeka farmakološki aktivan, učinak lijeka uvelike ovisi o svojstvima vezanja pa
stoga kompeticija za vezna mjesta na proteinu može biti uzrok interakcija među lijekovima. Metoda korištena u ovom
radu za proučavanje afiniteta vezanja lijekova za HSA naziva se polarizacija fluorescencije. Zasniva se na svojstvu malih
fluorescentnih molekula, među koje spada dipiridamol, da u slobodnom obliku rotiraju velikom brzinom i zahvaljujući
tome smanjuju polarizaciju fluorescencije. Prilikom vezanja u kompleks s velikom molekulom, u ovom slučaju HSA,
brzina rotacije se značajno smanjuje što dovodi do veće polarizacije fluorescencije. Dodatkom varfarina koji s
dipiridamolom ulazi u kompeticiju za vezno mjesto dipiridamol se iz kompleksa oslobađa u otopinu gdje ponovno rotira
velikom brzinom i smanjuje polarizaciju fluorescencije. Mjereći polarizaciju fluorescencije bilo je moguće kvantificirati
udio vezanog dipiridamola i izračunati konstante ravnoteže reakcija dipiridamola i varfarina za HSA na temelju mjerenja
dobivenim prvo titracijom otopine dipiridamola otopinom HSA, a zatim otopine HSA i dipiridamola otopinom varfarina.
Izračun je izveden u programu SPECFIT i razmotrena su dva modela vezanja. Pokazalo se da model s dva vezna mjesta
za dipiridamol na HSA bolje opisuje eksperimentalne podatke te da je polarizacija fluorescencije pogodna metoda za
proučavanje afiniteta vezanja lijekova za HSA.

Abstract (english)

The main goal of this work was to determine the binding equilibrium constants of dipyridamole and warfarin to human
serum albumin (HSA). A good understanding of blood plasma protein's drug binding properties, especially of HSA's
which is the most abundant among them, is of great importance for the prediction of the pharmacokinetic properties of
drugs bound by it. Since only the free fraction of the drug can be pharmacologically active, the drug's effect is greatly
dependent on its binding properties and because of that the drug's competition for the protein's binding sites can be a
cause of drug interactions. The method used in this work to study HSA's drug binding is called fluorescence polarization.
It is based upon the property of small fluorescent molecules, such as dipyridamole, to rotate at great speed when unbound
and because of that to reduce fluorescence polarization. By binding to a larger molecule, in this case HSA, dipyridamole's
rotation speed is greatly reduced, and fluorescence polarization is increased. With the addition of warfarin,
dipyridamole's binding site competitor, dipyridamole is released from HSA allowing it to rotate at great speed once
again and reduce fluorescence polarization. By measuring fluorescence polarization, it was possible to quantify the
bound fraction of dipyridamole and to calculate the binding equilibrium constants based on the measurements obtained
first by a titration of a dipyridamole solution with a HSA solution and then by a titration of both dipyridamole and HSA
solution with a warfarin solution. The calculations were performed in SPECFIT and two binding models were
considered. It was shown that the model in which HSA has two binding sites for dipyridamole better describes the
experimental data and that fluorescence polarization is a convenient method for studying HSA's drug binding properties.

• Keywords: polarizacija fluorescencije; konstanta ravnoteže; humani serumski albumin; dipiridamol; varfarin
• Keywords (english): fluorescence polarization; equilibrium constant; human serum albumin; dipyridamole; warfarin
• Language: croatian
• URN:NBN: urn:nbn:hr:163:100951
• Study programme: Title: Pharmacy; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2023-11-21 14:32:47