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Understand the metabolism of your compounds by using our hepatocyte stability assay to measure in vitro intrinsic clearance or to identify metabolites formed.
The hepatocyte stability assay is one of our portfolio of in vitro ADME screening services. Cyprotex deliver consistent, high quality data with cost-efficiency that comes from a highly automated approach.
Human hepatocytes have become the "gold standard" for evaluating hepatic metabolism and toxicity of drugs and other xenobiotics in vitro.
1LeCluyse EL and Alexandre E (2010) Methods Mol Biol 640; 57-82
| Cells | Cryopreserved hepatocytes |
|---|---|
| Species | Human, rat, mouse, dog, primate, minipig, rabbit, guinea pig (other species available on request) |
| Test Compound Concentration | 3 µM (different concentrations available) |
| DMSO Concentration | 0.25% |
| Incubation Time | 0, 5, 10, 20, 40 and 60 min |
| Compounds Requirements | 50 μL of 10 mM solution |
| Analysis Method | LC-MS/MS quantification |
| Assay Control | Known substrates which undergo either phase I or phase II metabolism Vehicle control incubation |
| Data Delivery | Intrinsic clearance Standard error of intrinsic clearance Half life |
| Follow on metabolite profiling and structural elucidation |
|---|
|
Cyprotex's hepatocyte stability assay can be extended to profile the metabolites that are formed. Cyprotex’s biotransformation services are supported by high resolution, accurate mass spectrometry. These services can provide information on an individual species’ metabolite profile, or a cross-species comparison to identify potential differences in metabolism which could in turn help to interpret pharmacology and toxicity data. Structural elucidation can also be performed on the potential metabolites’ MS/MS fragmentation data. All biotransformation studies are performed by a dedicated team of experts. Please refer to our Metabolite Profiling and Identification section for further details. |
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.
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.
How do you overcome the problems with inter individual variability in humans?
Cyprotex's hepatocyte stability assay uses cells pooled from a minimum of three different individual donors both male and female. This reduces the problems associated with inter individual variation in drug metabolism.
What stage in the drug discovery process does the hepatocyte stability assay tend to be used?
Clients tend to use the microsomal stability assay as a primary screen early in the drug discovery process. The hepatocyte stability assay is then used as a secondary screen for the more favorable compounds from the primary screening.
What may cause differences in the rates of clearance obtained from hepatocyte and microsomal assays?
Compounds that do not readily permeate cell membranes or are subject to efflux may appear to be more stable in hepatocyte incubations than in microsomal incubations. Compounds that undergo Phase II metabolism may appear less stable in hepatocyte compared to microsomal incubations.
1 LeCluyse EL and Alexandre E (2010) Methods Mol Biol 640; 57-82
2 McGinnity DF et al. (2004) Drug Metab Dispos 32; 1247-1253
3 Soars MG et al. (2002) J Pharmacol Exp Ther 301(1); 382-90
4 Shibata Y et al. (2002) Drug Metab Dispos 30(8); 892-896
5 Lau YY et al. (2002) Drug Metab Dispos 30(12); 1446-1454
6 McGinnity DF and Riley RJ (2004) Drug Metab Rev 36 (S1); 211
7 Houston JB. (1994) Biochem Pharmacol 47(9); 1469-1479
8 Davies B. and Morris T. (1993), Pharma Res 10(7); 1093-1095
9 Barter ZE et al. (2007) Curr Drug Metab 8(1); 33-45
10 Sohlenius-Sternbeck AK (2006) Toxicol. In Vitro 20; 1582-1586
Learn more about drug metabolism in Chapter 3 of our popular Everything you need to know about ADME guide.
