Hepatocyte Facts
  • 144 compounds - Compound List 
  • 6 concentrations 
  • 4 time points/conc
  • All data from a single donor pool 
  • N=3  raw data available
  • Total 72 data points / compd

Human Hepatocyte Assay

 

Description 

The human cryopreserved hepatocyte (HCH) assay measures the decrease in concentration of the drug over time as it is metabolized.  Metabolic turnover data was collected for 144 compounds using human cryopreserved hepatocytes.  The protocol collected data for each compound at 6 different initial concentrations, 4 time points, in triplicate for four hours.   An analysis method was developed which incorporated non-linear least squares regression, individual concentration and joint concentration analyses, and a stochastic Monte-Carlo approach to estimate the KM and CLint.

 

Method

Human Cryopreserved Hepatocytes were acquired from In Vitro Technologies (IVT, Baltimore, MD). To minimize donor variation, a pool of 6 donors was used to collect metabolic turnover data of parent drugs.  Donors were selected using the published data from IVT by comparing listed metabolic rates for marker compounds.  Donors were selected as close as possible to be representative of the entire population of donors.  Our subsequent investigations determined that the substitution of donors with similar turnover characteristics does not significantly impact the results observed.

 

72 Point Data Collection 

Data was collected using human cryopreserved hepatocytes.  A comprehensive method qas used to ensure the most accurate calculation of Km and Vmax estimates.  Six initial concentrations were used (0.4, 2, 10, 50, 125, and 250 uM) for most compounds.  Some compounds were limited by solubility and therefore concentrations adjusted to achieve concentrations as high as possible.  Samples were collected at 4 time points for each initial concentration.  All data points were collected in triplicate.  A written protocol can be found by clicking here.  The protocol also includes information on hepatocyte pool selection.

 

How are the Km and Clint values calculated?

We developed an Hepatocyte Data Expert Tool (HDET)™ which applies a nonlinear optimization to obtain refined estimates for Km and CLint.  Based on Michaelis-Menten kinetics our Metabolism Model’s Full (4-concentration) HDET calculates values for CLint and Km from the assay-generated metabolism data. Using an iterative process, HDET uses a series of hypothesis tests, confidence interval estimations, and simulations to identify and eliminate outliers in the data and to estimate an initial value for Km and CLint. 

 

Raw data

Each data set is accompanied by the raw data collected during the assays. Click here for an example raw data file.  Since we supply the raw data with the dataset, you can use our calculated values of Km and Vmax, or use the raw data and calculation method of your choice to determine those values.

 

 

Compound List

A list of compounds used in this assay can be found here.