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.
Method: 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;
1st relay). The procedures were repeated on the next days (72 h (2nd relay), 96 h (3rd relay), and 120 h
(4th 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.
Conclusion: PHRA represents a useful experimental system for the evaluation of the metabolic fates of low clearance
compounds in drug development..
Keywords: Cryopreserved hepatocytes, hepatic clearance, human hepatocytes, low clearance compounds, metabolic clearance,
relay assay, slowly metabolized compounds.
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