Hepatocyte Clearance/Stability Assay

Metabolism is a major clearance mechanism that primarily occurs in the liver. Hepatocytes are liver cells that can be derived from a variety of animals and used to determine the half-life (t1/2) or in vitro intrinsic clearance of test compounds (CLint, app).  Both microsomal and hepatocyte clearance assays, offered by Domainex, provide CLint, app, however, the latter mimics the in vivo system much more closely. Unlike microsomes, which contain only phase I enzymes, hepatocytes are a whole cell system that have a full complement of phase I and phase II enzymes alongside the necessary co-factors. Measuring hepatocyte clearance not only assists in ranking/triaging compounds for in vivo studies but is also used to help predict in vivo hepatic clearance and scaling to predict a suitable human dose.

Domainex’s Standard Experimental Procedure:

Test compounds are incubated with a suspension of liver hepatocytes at 37 °C. The reaction mixture is sampled at allocated timepoints into a cold stop plate containing acetonitrile and internal standard. The samples are subsequently quantified by Ultra-High Performance Liquid Chromatography (UHPLC)-Mass Spectrometry (MS), monitoring the depletion of test compound. Two controls are also assessed in each run (one for Phase I and one for Phase II metabolism) to ensure intrinsic clearance values fall within the acceptance criteria. Example hepatic turnover data is shown in Figure 1 and Table 1.

Data Analysis:

Hepatocyte incubation extracts are analysed using Waters Acquity UPLC TQ-S, TQS-Micro or TQ-XS instruments. Triple quadrupole mass spectrometers, operated in multiple reaction monitoring (MRM) mode, provide accurate quantification with excellent sensitivity, selectivity and reproducibility.     

Plotting ln compound concentration against time allows determination of half-life and intrinsic clearance using the equations below:

elimination rate constant equeation

intrinsic clearance equeation

Hepatocyte clearance/stability table
hepatocyte graphs

Figure 1: Left: Depletion of test compounds is monitored following incubation with hepatocytes. Right: Natural log linear plot of compound turnover allows CLint,app and t1/2 values to be calculated. 

Table 1: Table showing calculated CLint, app for the same four compounds.

Deliverables:

The results are reported in Excel file format as CLint, app (µl/min/106 cells), t1/2 (mins) and parent remaining (%) including standard error of mean (SEM). Any relevant comments about compound stability/solubility/binding are also included in the report.

Turnaround time from receipt of the compounds to release of data is typically less than 2 weeks.