Metabolic Stability Assays

The metabolic stability assays provide a means to measure the rate of disappearance of a test compound over time in either microsomal or hepatocyte incubations, and these data are used to calculate intrinsic clearance. Microsomal assays primarily assess metabolism by the cytochrome P450 system (phase I enzymes) while hepatocyte assays more broadly assess the overall cellular metabolism of the test compound (phase I and phase II enzyme pathways). We provide metabolic stability assays for all small molecule formulations such as pharmaceuticals, industrial chemicals and consumer products.

Our hepatocyte stability assays use a genetically modified version of the immortalized human liver cell line HepaRG™, created with our proprietary CompoZr® zinc finger nuclease (ZFN) technology. Apart from fresh human hepatocytes, HepaRG cells are the most metabolically active liver cell line described to date and have the potential for use as a viable surrogate in many functional liver assays, including metabolism and clearance testing, with none of the drawbacks of limited availability and donor-to-donor variation.

Liver metabolism is the major route for elimination for many drugs in humans and animals. The native ability of hepatic enzymes to metabolize a drug is commonly referred to as “intrinsic clearance” and is used to determine overall hepatic clearance, which takes into account additional factors such as hepatic blood flow and drug/protein binding.

Data from metabolic screening assays allow customers to identify metabolic liabilities early on and focus on the improvement of drug candidates through structure activity relationships.
 

 Microsomal Stability Assay Protocol

 
Test System Human liver microsomes (pooled)
Test Compound Concentration 3 µM (or custom)
Microsome Concentration 0.5 mg/mL
Time Points 0, 5, 15, 30, 45, 60 minutes
Cofactor NADPH
Number of Replicates 3
Negative Control Vehicle (0.1% DMSO)
Heat inactivated microsomes
Positive Controls Verapamil (rapid clearance)
Diazepam (low clearance)
Compound Requirements 50 µL of 10 mM solution
Analysis Method LC-MS/MS
Data Delivery Intrinsic clearance
Half-life

 

 Hepatocyte Stability Assay Protocol

 
Test System HepaRG liver cell line, clone 5F
Test Compound Concentration 3 µM (or custom)
Time Points 0, 5, 15, 30, 45, 60, 90, 120 minutes
Number of Replicates 3
Negative Control Vehicle (0.1% DMSO)
Positive Controls Testosterone
Ethoxycoumarin
Compound Requirements 50 µL of 10 mM solution
Analysis Method LC-MS/MS
Data Delivery Intrinsic clearance
Half-life

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References

  • Lubberstedt M et al. (2010) HepaRG human hepatic cell line utility as a surrogate for primary human hepatocytes in drug metabolism assessment in vitro, J Pharmacol Toxicol Methods 63, 59-68
  • Obach RS (1999) Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: An examination of in vitro half-life approach and nonspecific binding to microsomes, Drub Metab Dispos 27, 1350-1359
  • Zanelli U et al. (2012) Comparison of cryopreserved HepaRG cells with cryopreserved human hepatocytes for prediction of clearance for 26 drugs, Drug Metab Dispos 40, 104-110