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Metabolic Stability - Hepatocytes

Drugs are eliminated from the body either as unchanged parent or as metabolite.  Metabolic stability plays a major role in drug clearance, with the liver being the primary site for drug biotransformation via two major enzymatic reactions: Phase I (modifications to the molecular structure itself) and Phase II reactions (conjugation reactions). The metabolic stability of compounds is commonly investigated early on in the drug discovery process using various in vitro test systems, in order to guide project teams on potential metabolic liabilities.

Hepatocytes represent an independent in vitro cellular test system, containing both Phase I and Phase II enzymes, cofactors and drug transporters, used to assess the metabolic stability of compounds and for early identification of species specific differences. 

In addition to metabolic stability studies, hepatocytes are used to identify and compare metabolite profiles (MetID) among different species.
   Assay description

      cryopreserved hepatocytes
      0.5x106 cells/ml

      mouse, rat , dog, minipig, rabbit, monkey, human

   Compound concentration
      metabolic stability: 1µM (0.03% DMSO)
      MetID: 10µM (0.03% DMSO)

   Compound requirements
      50µl of 10mM stock solution or
      1-2 mg of dry matter (preferred for metID)

   Detection method
      LC-MS/MS with internal standard

      %remaining, half-life, in vitro clearance,
      predicted in vivo hepatic clearance and %LBF (liver blood flow)

   Assay controls
     - reference compounds:
         testosterone and umbelliferone
    -  stability in KHB buffer and/or inactivated hepatocytes

Figure 1.

Clearance values obtained for  reference compounds in 5 different species

Assay details adjustable to client’s and/or project specific requests

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