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.
Laboratory of Pharmaceutical Analytical Chemistry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 20 Boulevard d'Yvoy, 1211 Geneva 4, Switzerland.
R
R
Nicoli
M
M
Bartolini
S
S
Rudaz
V
V
Andrisano
J-L
JL
Veuthey
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