Microfluidic enzyme reactor with immobilized human intestinal microsomes

master's thesis

• Title (croatian): Mikrofluidički enzimatski reaktor s imobiliziranim humanim intestinalnim mikrosomima
• Author: Zrinka Duvnjak
• Mentor: Hrvoje Rimac (mentor)
• Committee member: Hrvoje Rimac (predsjednik povjerenstva)
• Committee member: Ana Budimir (član povjerenstva)
• Committee member: Zrinka Rajić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of medicinal chemistry); Zagreb
• Defense date and country: 2022-04-07, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Human intestinal metabolism is emerging to be more important than what was previously thought, especially in pharma industry when the inhibitory potential of a new drug candidate is being determined. It was shown that intestinal metabolism has a critical role in determining a need for clinical drug-drug interaction studies for weak CYP3A perpetrators.
On the other hand, conventional static in vitro enzymatic assays demand a high consumption of enzymes and other chemicals, the separation step is always needed before product detection, and without flow-through conditions, there are limitations in imitating conditions in human body.
In this work, the first in its kind immobilized enzyme microreactor (IMER) was developed by using human-derived intestinal microsomes and characterized in the terms of enzyme immobilization yield and stability of enzyme activity. The microreactor was made from off-stoichiometric thiol-enes and microsomes were immobilized by fusion with previously immobilized biotinylated fusogenic liposomes being held in place via biotin-streptavidin interaction.
A full potential of this platform was shown by enzyme inhibition mechanism determination, with performing only one experiment where feed solution containing different inhibitor concentrations in different time points was passing through the microreactor
In addition, it was shown that CYP1B1 could be more expressed in HIM than it was previously reported.

Abstract (croatian)

Intestinalni metabolizam u ljudi sve više pridobiva na važnosti, pogotovo u farmaceutskoj industriji kada se određuje inhibitorni potencijal novih lijekova kandidata. Pokazano je da intestinalni metabolizam ima ključnu ulogu u određivanju potrebe za kliničkim lijek-lijek interakcija studijama za slabe CYP3A inhibitore.
Konvencionalni statički in vitro enzimatski eksperimenti zahtijevaju velik utrošak kemikalija pa tako i skupih kofaktora i enzima, prije detekcije potreban je korak odijeljivanja produkta, a bez prisutnosti protoka značajnija imitacija uvjeta ljudskog organizma nije ostvarena.
U ovom radu razvijen je mikrofluidički imobilizirani enzimatski reaktor s imobiliziranim humanim intestinalnim mikrosomima te je okarakteriziran u pogledu stabilnosti enzimatske aktivnosti. Mikroreakor je napravljen on tiol-ena u nestehiometrijskom omjeru, a mikrosomi su imobilizirani fuzijom mikrosoma s prethodnom imobiliziranim liposomima, koji su vezani biotin-streptavidin interakcijom.
Puni potencijal ove platforme pokazan je određivanjem mehanizma enzimatske inhibicije samo jednim eksperimentom u kojem supstrati reakcije teku preko imobiliziranih enzima u različitim koncentracijama tijekom eksperimenta.
Također, pokazano je i da bi CYP1B1 mogao biti više eksprimiran u humanim intestinalnim mikrosomima nego što je ranije bilo pokazano.

• Keywords: IMER; microfluidics; immobilization; intestinal metabolism; CYP1B1
• Keywords (english): IMER; mikrofluidika; imobilizacija; intestinalni metabolizam; CYP1B1
• Language: english
• URN:NBN: urn:nbn:hr:163:782994
• 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; Embargo expiration date: 2024-04-22
• Created on: 2022-05-26 10:46:20