title: A new standardized electrochemical array for drug metabolic profiling with human cytochromes P450.
abstract: Over the past two decades, a wealth of information on the human cytochrome P450 enzymes and their role in drug metabolism both in vitro and in vivo has been gathered. Our understanding of this area has progressed greatly, but our confidence in the development of quantitative projections of drug interactions, made from in vitro data, is somehow still shaky. There are therefore no doubts in the necessity for reliable and fast methodologies for P450 drug metabolism analysis, capable of providing accurate and precise in vitro data. This paper reports on the first integration of a P450-electrode into a microtiter plate format for the rapid determination of the affinity parameters (K(M)) for a set of known drugs. The most relevant human drug metabolizing cytochromes P450, isoforms 3A4, 2D6, and 2C9, have been covalently bound to a gold electrode via a 10-carboxydecanethiol and 8-hydroxyoctanethiol (1:1) self-assembled monolayer at the bottom of an eight-well microtiter plate. The electrochemical response of the P450-electrode and the performance of the platform have been validated using a set of 30 known drugs with K(M) values spanning from less than 1 to more than 100 μM. The K(M) values obtained using this platform show an excellent error, and their ranking is within the range of those present in the literature determined from conventional incubation experiments with cytochrome P450s 3A4, 2D6, and 2C9.
authors.affiliation: Division of Molecular Biosciences, Imperial College London, Biochemistry Building, South Kensington, London, SW7 2AY, United Kingdom.
authors.forename: Andrea
authors.initials: A
authors.lastname: Fantuzzi
authors.forename: Lok Hang
authors.initials: LH
authors.lastname: Mak
authors.forename: Ennio
authors.initials: E
authors.lastname: Capria
authors.forename: Vikash
authors.initials: V
authors.lastname: Dodhia
authors.forename: Paola
authors.initials: P
authors.lastname: Panicco
authors.forename: Stephen
authors.initials: S
authors.lastname: Collins
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Gilardi
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000378:metabolism; D065729:Cytochrome P-450 CYP2C9; D019389:Cytochrome P-450 CYP2D6 / Q000737:chemistry / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D055664:Electrochemical Techniques / Q000379:methods* / Q000592:standards; D004566:Electrodes; D006046:Gold / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D004364:Pharmaceutical Preparations / Q000378:metabolism* / Q000592:standards
chemical_list: D004364:Pharmaceutical Preparations; D006046:Gold; D003577:Cytochrome P-450 Enzyme System; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human