Prediction of Metabolic Clearance for Low-Turnover Compounds Using Plated Hepatocytes with Enzyme Activity Correction.
OBJECTIVE
Prediction of metabolic clearance has been a challenge for compounds exhibiting minimal turnover in typical in vitro stability experiments. The aim of the current study is to evaluate the utilization of plated human hepatocytes to predict intrinsic clearance of low-turnover compounds.


METHODS
The disappearance of test compounds was determined for up to 48 h while enzyme activities in plated hepatocytes were monitored concurrently in a complimentary experiment.


RESULTS
Consistent with literature reports, marked time-dependent loss of cytochrome P450 (CYP) enzyme activities was observed during the 48-h incubation period. To account for the loss of enzyme activities, a term "fraction of activity remaining" was calculated based on area-under-the-curve derived from the average rate of activity loss (kavg), and then applied as a correction factor for intrinsic clearance determination. Twelve compounds were selected in this study to cover phase I and phase II biotransformation pathways, with in vivo intrinsic clearance values, representing metabolic clearance only, ranging from 0.66 to 47 ml/min/kg. Determination of in vitro intrinsic clearance using three individual preparations of hepatocytes revealed a reasonably good agreement (generally within threefold) between the predicted and the observed metabolic clearance for all 12 compounds tested.


CONCLUSIONS
The current results indicated that plated hepatocytes can be utilized to provide clearance predictions for compounds with low-turnover in humans when corrected for the loss in enzyme activities.
Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, West Point, PA, 19486, USA. bennett_ma@merck.com.
Bennett
B
Ma
Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, West Point, PA, 19486, USA.
Roy
R
Eisenhandler
Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, West Point, PA, 19486, USA.
Yuhsin
Y
Kuo
Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, West Point, PA, 19486, USA.
Paul
P
Rearden
Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, Kenilworth, NJ, USA.
Ying
Y
Li
Department of Medicinal Chemistry, Merck Research Laboratories, West Point, PA, USA.
Peter J
PJ
Manley
Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, West Point, PA, 19486, USA.
Sheri
S
Smith
Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, West Point, PA, 19486, USA.
Karsten
K
Menzel
D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D022781:Hepatocytes / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D008657:Metabolic Clearance Rate; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D013997:Time Factors
D004364:Pharmaceutical Preparations; D003577:Cytochrome P-450 Enzyme System