Questions and answers on hepatocyte stability
Please provide an overview of Cyprotex's hepatocyte stability assay.
The hepatocytes are incubated with the test compound at 37°C. Samples are removed at the appropriate time points into methanol containing internal standard to terminate the reaction. Following centrifugation, the supernatant is analyzed by LC-MS/MS. The disappearance of test compound is monitored over a 60 minute time period. An example of a typical depletion profile is shown in Figure 3.
The ln peak area ratio (compound peak area/internal standard peak area) is plotted against time and the gradient of the line determined.
What are the benefits of using hepatocytes for drug metabolism studies?
The liver is the main organ of drug metabolism in the body. Hepatocytes contain both phase I and phase II drug metabolizing enzymes, which are present in the intact cell, and provide a valuable in vitro model for predicting in vivo hepatic clearance.
How do I interpret the data from the hepatocyte stability assay?
One of the main uses is that compounds can be ranked in terms of their intrinsic clearance values. Unless the compound is a pro-drug, very highly cleared compounds are generally considered to be unfavorable as they are likely to be rapidly cleared in vivo, resulting in a short duration of action. Classification bands can be used to categorize compounds into low, medium or high clearance. These classification bands are calculated from a rearrangement of the well stirred model7 detailed in the following equation assuming extraction ratios of 0.3 and 0.7 (the fraction of drug which is eliminated from the blood by an organ) for the low and high boundaries respectively. This can be scaled to intrinsic clearance (µL/min/106 cells) using the relevant liver weights8 and hepatocellularity9,10. Due to lack of literature information the monkey hepatocellularity was assumed to be 120x106 cell/g liver.
CLint =
Where CLH = E x QH
QH = liver blood flow (mL/min/kg)7
E = Extraction Ratio
CLH = Hepatic Clearance (mL/min/kg)
fu = fraction unbound in plasma (assumed at 1)
| Clearance Category | Intrinsic Clearance (µL/min/106 cells) |
|---|
| Human | Monkey | Dog | Rat | Mouse |
|---|
| Low |
<3.5 |
<5.2 |
<1.9 |
<5.1 |
<3.3 |
| High |
>19.0 |
>28.3 |
>10.5 |
>27.5 |
>17.8 |
What are the benefits of using cryopreserved hepatocytes, and how does the activity of freshly isolated hepatocytes compare with cryopreserved hepatocytes?
Cryopreservation of hepatocytes enables the cells to be stored for long periods of time and ensures no supply problems or delays in screening. With advances in cryopreservation techniques, cell viability and activity have improved dramatically, and cryopreserved cells now provide a viable alternative to freshly isolated cells (figure 4). Cryopreserved hepatocytes provide a convenient way of investigating interspecies differences in drug metabolism.
