In vitro screening of metabolic clearance using two concentration points.
For high throughput screens, the quickest methodology possible is desirable, but a substantial amount of potentially useful information is lacking since most screens for metabolic stability are conducted at one concentration, and sometimes at one time point. Information that would benefit projects during the discovery phase are to know the metabolic rate linearity (K(m) value) and projected hepatic clearance (CL(h) value), which is possible by the addition of one more concentration. This study used the FDA-preferred probe cytochrome P450 substrates to determine K(m), V(max), and CL(int) values. The results showed that compounds with relatively high metabolic rates produced more accurate and reproducible results that match well with predicted K(m) values according to the FDA. On the other hand, compounds with relatively low metabolic rates yielded more variable results. Thus, the use of two substrate concentrations should be useful with screening assays for assessing the kinetic values for other compounds.
Williams
Eric T
ET
DMPK-Andover, Eisai Inc., 4 Corporate Drive, Andover, MA 01810, USA.
Farah
Nadia
N
Pelletier
Robert D
RD
Lai
W George
WG
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D055808:Drug Discovery; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D008657:Metabolic Clearance Rate; D008954:Models, Biological; D015335:Molecular Probes / Q000378:metabolism*; D015203:Reproducibility of Results; D013379:Substrate Specificity
D015335:Molecular Probes; D003577:Cytochrome P-450 Enzyme System