Hepatocytes, either fresh or cryopreserved, have been used very successfully to determine hepatic metabolism of drug research compounds. Hepatocyte suspensions are very powerful tools to study drug metabolism. On the one side they can be used to determine intrinsic clearance, which can then be easily scaled to predict in vivo clearance in the species of interest. On the other side hepatocyte suspensions can also be used to elucidate metabolic routes. In recent years major progress has been made in refining cryo-preservation techniques so that highly metabolically competent cells are available from all relevant preclinical species and man. Inter-individual variability, especially in human hepatocytes, has been overcome by the use of pooled cell preparations from as many as 100 individual donors. Hepatocytes are often referred to as the ‘gold standard’ in vitro system for an accurate estimation of the in vivo hepatic clearance mainly for two reasons.
Hepatocytes contain all phase I and phase II drug metabolising enzymes and their corresponding co-factors and for compounds which show poor passive permeability but are efficiently taken up by transport proteins, cell-free systems like microsomes may lead to significant over-predictions of in vivo metabolic clearance.
However, it should be mentioned that clearance via biliary excretion cannot be assessed by hepatocyte CLint assays.
The XenoGesis hepatocyte stability protocol
||Cell suspension. 96 well with shaking
||Cryopreserved human; rat; dog; cynomolgus monkey; guinea pig; mouse hepatocytes (other species upon request)
|| 0.5 x 106/mL
|Test compound concentration
|Number of time points
|Recommended number of replicates
|Typical turnaround time
Note: Assay conditions can be tailored to client requirements.
To determine metabolic stability the test compound is mixed with a hepatocyte suspension. At set time points, small aliquots are withdrawn and the test compound concentration therein is measured by LC-MS/MS. The resulting concentration-time profile is then used to calculate intrinsic clearance (CLint) and half-life (t½).
The following example of dextromethorphan in a rat hepatocytes suspension shows how CLint and t½, are derived from the raw data.
Metabolic stability of dextromethorphan in rat hepatocytes. Data are the means of triplicate determination ± one standard deviation.
For substrate concentrations significantly below Km metabolism follows first order kinetics, which means that the compound concentration-time profile can be described by:
Where C(t) is the compound concentration at time t, C(0) is the starting concentration and k is the elimination rate constant.
Natural logarithmic transformation (ln) allows us to fit a linear regression.
ln(concentration) vs. time profile of dextromethorphan in rat hepatocyte suspension.
This ln(concentration) vs.time profile can be mathematically described by:
Where k represents the slope of the regression line.
Solving for k results in:
Once k is known, half-life (t½) can be calculated using:
Furthermore, CLint can be calculated by introducing the cell density ([cell]) in 106/mL: