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Metabolic Stability - Microsomes

Background:
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).  A common system for measuring hepatic metabolism in early drug discovery, restricted to Phase I reactions, uses liver microsomes, a subcellular fraction containing major drug-metabolizing enzymes, including the cytochrome P450 (CYP) family and flavin monooxygenase (FMO).
 
Metabolic stability screening in microsomes alerts to potential liabilities of compounds, provides useful information to develop structure-metabolic stability relationships and helps to identify compounds with favourable pharmacokinetic properties.

This high throughput method is based on measuring the disappearance of parent compound. By using liver microsomes of different animal species and human, potential species related differences in the metabolism can also be detected (preliminary Met ID study).
   Assay description

   Species
     mouse, rat, dog, monkey, human 

   Compound concentration
       3µM (0.1% DMSO)

   Compound requirements
      50µl of 10mM solution or
      1-2 mg of dry matter

   Detection method
      LC-MS/MS with internal standard

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

 Assay controls
   positive control compounds: testosterone, verapamil and propranolol
   negative control: without co-factor
Figure 1. Mean intrinsic clearance (mean value from duplicate incubations within each run) in-house assay data
obtained for 12 commercial compounds in three separate experiments
Assay details adjustable to client’s and/or project specific requests
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