Primjena prirodnog niskotemperaturnog eutektičkog otapala u razvoju formulacije reaktivatora acetilkolinesteraze

master's thesis

• Title (english): Application of natural deep eutectic solvent for development of acetylcholinesterase reactivator formulation
• Author: Marina Štokić
• Mentor: Kristina Radošević (mentor)
• Committee member: Ivana Radojčić Redovniković (član povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Cell Technology, Application and Biotransformations); Zagreb
• Defense date and country: 2021-04-09, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Prirodna niskotemperaturna eutektička otapala, nova zelena otapala, istražuju se kao alternativna otapala za otapanje djelatnih farmaceutskih tvari. Razvoj učinkovitog antidota za otrovanja organofosfornim (OP) spojevima, ireverzibilnim inhibitorima esencijalnog enzima acetilkolinesteraze (AChE), predmet je mnogih istraživanja. U središtu pozornosti su tzv. centralno-aktivni oksimski reaktivatori fosfilirane AChE, kao što je, u vodi slabo topljivi oksim RS-194B. U ovom radu je pomoću programa COSMOtherm predviđena, a zatim i eksperimentalno određena topljivost oksima RS-194B u prirodnim niskotemperaturnim eutektičkim otapalima tekućinskom kromatografijom visoke djelotvornosti. Najbolja topljivost postignuta je u otapalu limunska kiselina:glukoza=1:1 s 30 %-tnim udjelom vode. Praćena je stabilnost formulacije oksima RS-194B u odabranom i referentnom otapalu tijekom mjesec dana na temperaturama 4 °C i 25 °C pri čemu nije zapažena razlika u stabilnosti tih formulacija. Prije određivanja kinetičkih parametara reaktivacije AChE inhibirane OP spojevima sarinom i VX-om pomoću formulacije oksima RS-194B u odabranom otapalu, ispitan je utjecaj samog otapala na AChE. Pokazalo se da otapalo inhibira aktivnost AChE u potpunosti i njegov udio mora se održavati ispod 0,2 % kako bi enzim zadržao 90 % aktivnosti. Nadalje, afinitet AChE i fosfilirane AChE je veći za vezanje oksima otopljenog u odabranom otapalu nego u vodi. Oksim je, bez obzira na otapalo bolji reaktivator VX-om nego sarinom fosfilirane AChE. Međutim, brzina reaktivacije fosfilirane AChE je manja s RS-194B u odabranom otapalu nego s RS-194B otopljenim u vodi. Stoga, iako je primjenom odabranog otapala postignuta bolja topljivost oksima RS-194B u usporedbi s vodom kao referentnim otapalom i poboljšan je afinitet AChE za RS-194B, otapalo limunska kiselina:glukoza=1:1, 30 % vode, nije najbolji izbor za pripremu formulacije RS-194B oksima za in vivo upotrebu zbog inhibitornog učinka na enzim te viskoznosti i niske pH vrijednosti samog otapala.

Abstract (english)

Natural deep eutectic solvents, new green solvents, are being investigated as alternative solvents for dissolving active pharmaceutical ingredients. The development of an effective antidote for poisoning by organophosphates (OPs), irreversible inhibitors of the essential enzyme acetylcholinesterase (AChE), has been a subject of many research. The focus is on the so-called centrally active oxime reactivators of phosphylated AChE like poorly water-soluble oxime RS-194B. In this study, the solubility of RS-194B oxime in natural deep eutectic solvents has been predicted by the COSMOtherm program and experimentally determined using high efficient liquid chromatography. Natural deep eutectic solvent citric acid: glucose=1:1 with 30 % water content has resulted with best solubility of oxime RS-194B. The stability of oxime RS-194B formulation in the selected natural deep eutectic solvent has been monitored during one month period at temperatures of 4 °C and 25 °C where no difference in the stability of these formulations was observed. Before determining the effect of RS-194B oxime formulation in the selected solvent on the reactivation kinetics of AChE inhibited by OPs sarin and VX, the effect of the solvent itself on the AChE was determined. It was found that the selected solvent inhibited AChE completely, and in order for AChE to maintain more than 90 % activity, its content has to be kept below 0,2 %. The affinity of AChE and phosphylated AChE is higher for the binding of oxime dissolved in the selected solvent than in water. The oxime, regardless of the solvent, is a better reactivator of VX- than sarin-inhibited AChE. However, the maximal first-order reactivation rate of phosphylated AChE is lower with RS-194B in the selected solvent than with RS-194B dissolved in water. Therefore, although the use of the selected solvent resulted in better solubility of RS-194B oxime compared to water as the reference solvent and improved the affinity of AChE for RS-194B, the solvent citric acid: glucose=1:1, 30 % water, is not suitable for preparing the RS-194B formulation for in vivo application due to its inhibitory effect on the enzyme, viscosity and low pH.

• Keywords: acetilkolinesteraza; COSMOtherm; oksim; prirodna niskotemperaturna eutektička otapala; topljivost
• Keywords (english): acetycholinesterase; COSMOtherm; oxime; natural deep eutectic solvents; solubility
• Language: croatian
• URN:NBN: urn:nbn:hr:159:061524
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2021-06-08 11:37:22