Development of immobilized enzyme reactors based on human recombinant cytochrome P450 enzymes for phase I drug metabolism studies.
Two cytochrome P450 (CYP)-based immobilized enzyme reactors (IMERs) were developed to perform automated on-line phase I drug metabolism studies. For this purpose, biotinylated recombinant CYP2D6 or CYP3A4 reconstituted systems were anchored to the surface of two monolithic mini-columns (2 mm x 6 mm I.D.), which had been covalently grafted with NeutrAvidin. After optimization of immobilization conditions, the obtained IMERs were integrated on-line into a LC hyphenated to an electrospray ionization MS/MS system. Studies with probe substrates and a known competitive inhibitor were performed, showing the potential of CYP-based IMERs in drug metabolism. In the optimized conditions, ca. 15 experiments were carried out with each bioreactor.
Nicoli
R
R
Laboratory of Pharmaceutical Analytical Chemistry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 20 Boulevard d'Yvoy, 1211 Geneva 4, Switzerland.
Bartolini
M
M
Rudaz
S
S
Andrisano
V
V
Veuthey
J-L
JL
D001360:Avidin; D019149:Bioreactors; D019921:Biotinylation; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D019389:Cytochrome P-450 CYP2D6 / Q000378:metabolism*; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D065692:Cytochrome P-450 CYP3A Inhibitors; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*
D065690:Cytochrome P-450 CYP2D6 Inhibitors; D065692:Cytochrome P-450 CYP3A Inhibitors; D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; C093483:neutravidin; D001360:Avidin; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human