Rapid simultaneous determination of metabolic clearance of multiple compounds catalyzed in vitro by recombinant human UDP-glucuronosyltransferases.
The purpose of this study was to test the applicability of n-in-one (cocktail) incubations in the determination of intrinsic clearance (Cl(int)) as the slope of the linear portion of the Michaelis-Menten curve (velocity V vs. substrate concentration [S]) where substrate concentrations were low. A rapid, sensitive, and selective liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed for the analysis of samples produced by single-substrate and n-in-one (seven substrates: entacapone, 17beta-estriol, umbelliferone, 4-methylumbelliferone, tolcapone, hydroxyquinoline, and paracetamol) incubations conducted in 96-well plates with different recombinant UDP-glucuronosyltransferases (UGTs). The Cl(int) values obtained with n-in-one incubations were compared with those obtained in single-compound incubations and with V(max)/K(m) values determined by estimating the enzyme kinetic parameters V(max) and K(m) from the Michaelis-Menten curve. When substrate concentrations were well below their K(m) values, Cl(int) values determined as the slope of the linear part of the Michaelis-Menten fitting correlated well with the values determined as V(max)/K(m) ratios from the Michaelis-Menten curve. The correlation between Cl(int) values determined in single-substrate and n-in-one incubations was high as well. Together, the n-in-one incubations, the determination of Cl(int) values as the slope of the linear part of the Michaelis-Menten fitting, and LC/MS/MS as an analytical method proved to be effective approaches for increasing throughput in the first-phase screening of metabolic properties.
Hakala
Kati S
KS
Viikki Drug Discovery Technology Center (DDTC), Faculty of Pharmacy, University of Helsinki, 00014 Helsinki, Finland.
Suchanova
Bohumila
B
Luukkanen
Leena
L
Ketola
Raimo A
RA
Finel
Moshe
M
Kostiainen
Risto
R
D002396:Catechols; D002853:Chromatography, Liquid; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives; D007700:Kinetics; D013058:Mass Spectrometry; D009570:Nitriles; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity
D002396:Catechols; D009570:Nitriles; D011994:Recombinant Proteins; D006923:Hymecromone; C071192:entacapone; D014453:Glucuronosyltransferase