A Novel Plated Hepatocyte Relay Assay (PHRA) for In Vitro Evaluation of Hepatic Metabolic Clearance of Slowly Metabolized Compounds.
OBJECTIVE
Development and validation of a novel assay, the Plated Hepatocyte Relay Assay (PHRA), for the determination of the metabolic fates of slowly metabolized compounds.


METHODS
Cryopreserved human hepatocytes were cultured for 4 h followed by incubation with slowly metabolized compounds for 24 h (initial incubation). On the next day, the incubated media were collected and added to hepatocytes was similarly prepared on the day of incubation (48 h incubation; 1(st) relay). The procedures were repeated on the next days (72 h (2(nd) relay), 96 h (3rd relay), and 120 h (4(th) relay) incubations).


RESULTS
A proof-of-concept study with two low clearance compounds, diazepam and tolbutamide, and a validation study with 15 ultra-low clearance compounds (CLnon-renal < 1 mL/min/kg) and low clearance compounds (CLnon-renal 1- 5.1 mL/min/kg) were performed. Linear time-dependent disappearance of the parent compounds was observed for all compounds. Application of published free fraction values in combination with a correction factor with in vitro hepatic clearance results obtained with the PHRA accurately predicted in vivo hepatic clearance.


CONCLUSIONS
PHRA represents a useful experimental system for the evaluation of the metabolic fates of low clearance compounds in drug development.
Chi-Chi
CC
Peng
Utkarsh
U
Doshi
Chandra
C
Prakash
In Vitro ADMET Laboratories LLC, 9221 Rumsey Road, Suite 8, Columbia, MD 21045, USA. lialbert@invitroadmet.com.
Albert P
AP
Li
D000328:Adult; D001681:Biological Assay / Q000379:methods*; D001711:Biotransformation; D002478:Cells, Cultured; D015925:Cryopreservation; D003975:Diazepam / Q000378:metabolism; D005260:Female; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D016014:Linear Models; D008297:Male; D008875:Middle Aged; D008954:Models, Biological; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D015203:Reproducibility of Results; D014044:Tolbutamide / Q000378:metabolism; D055815:Young Adult
D004364:Pharmaceutical Preparations; D014044:Tolbutamide; D003975:Diazepam