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ADME PK

Hepatocyte stability assay

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.

Measurement of in vitro intrinsic clearance using hepatocytes

  • The liver is the most important site of drug metabolism in the body. Approximately 60% of marketed compounds are cleared by hepatic CYP-mediated metabolism2.
  • Hepatocytes contain the full complement of hepatic drug metabolizing enzymes (both phase I and phase II) maintained within the intact cell.
  • Hepatocytes can be used to determine the in vitro intrinsic clearance of a compound.
  • The use of species-specific cryopreserved hepatocytes can be used to enable an understanding of interspecies differences.
  • Hepatocytes can be used to profile for metabolites formed by both phase I and phase II enzymes.
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

Protocol

Hepatocyte stability assay protocol

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.

Data

Data from Cyprotex's Hepatocyte Stability assay

 
human hepatocyte stability data
Figure 1
Cyprotex's human hepatocyte stability data and literature values3,4,5,6 were scaled to in vivo intrinsic clearance (predicted CLint) and compared to observed values of intrinsic clearance in 19 compounds.

Human hepatocyte CLint (μL/min/106 cells) from the Cyprotex assay and from literature3,4,5,6 were scaled to in vivo CLint (mL/min/kg) using a hepatocellularity of 99 x 106 cells/g liver and a human liver weight of 21.4 g liver/kg. CLint predictions were assessed for the predicted error (difference between the predicted and observed in vivo value). The bias of CLint prediction was assessed from the geometric mean of the ratio of predicted and observed value and the fold under-prediction calculated. The data from the Cyprotex assay showed greater predictive capability when compared with data from the literature. Using literature values, the fold under-prediction was 2.7. Using Cyprotex values, the fold under-prediction was 1.5.
Figure 2
Graph illustrating intrinsic clearance data for 19 compounds generated in Cyprotex's hepatocyte stability assay. The data show the mean ± standard deviation of 3 separate incubations.

The graph shows consistency of data between separate runs of the assay. Pooled hepatocytes typically from 5 different donors are used for the human hepatocyte stability assay to reduce the problems associated with inter-individual variability.

Q&A

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.

Test compound disappearance with time in the presence of hepatocytes

Figure 3
Graph shows test compound disappearance with time in the presence of hepatocytes.

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 CategoryIntrinsic Clearance (µL/min/106 cells)
HumanMonkeyDogRatMouse
Low <3.5 <5.2 <1.9 <5.1 <3.3
High >19.0 >28.3 >10.5 >27.5 >17.8
Table 1
Classification bands typically used for categorizing compounds into low, medium or high clearance.

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.

Comparison of the metabolic stability of ethoxycoumarin, 7-hydroxycoumarin, and testosterone
Figure 4
Comparison of the metabolic stability of ethoxycoumarin, 7-hydroxycoumarin, and testosterone in the presence of freshly isolated and cryopreserved human hepatocytes.

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.

References

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

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