PharmPK Discussion - Single point microsomal stability assay

• On 2 Mar 2009 at 17:35:00, Satya Jadhav (satya.jadhav.at.gmail.com) sent the message
  

  
  Hi all,
  Can anybody highlight and advice me on the experimental design of
  single point microsomal stability assay? I am doing it in three sets.
  
  Set 1. 0 min (NADPH) incubation (triplicate)
  Set 2. 15/30 min (NADPH) incubation (triplicate)
  Set 3. 15/30 min (No-NADPH) incubation (triplicate)
  
  Would it be prudent to include No-NADPH samples all the time? I have
  seen many reports that didnt include No-NADPH samples. However, I came
  across few compounds which are very unstable even without NADPH in it
  (unknown cause of degradataion, amidases/ esterases or simple chemical
  degradation??).
  
  What type of error parameter should I use while reporting the %
  remaining or T1/2 in microsomal stability assay?
  
  Thanks
  
  

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• On 6 Mar 2009 at 07:09:38, Satya Jadhav (satya.jadhav.at.gmail.com) sent the message
  

  
  Hi all,
  
  Can anybody highlight and advice me on the experimental design of
  single point microsomal stability assay? I am doing it in three sets.
  
  Set 1. 0 min (NADPH) incubation (triplicate)
  
  Set 2. 15/30 min (NADPH) incubation (triplicate)
  
  Set 3. 15/30 min (No-NADPH) incubation (triplicate)
  
  Would it be prudent to include No-NADPH samples all the time? I have
  seen many reports that didnt include No-NADPH samples. However, I came
  across few compounds which are very unstable even without NADPH in it
  (unknown cause of degradataion, amidases/ esterases or simple chemical
  degradation??).
  
  What type of error parameter should I use while reporting the %
  remaining or T1/2 in microsomal stability assay?
  
  Thanks
  
  [Sorry if this is a duplicate. I've had some mail server/rDNS issues
  recently. To help in the diagnosis I've sent a couple of test messages
  and repeats in the last couple of days. Some may have got through more
  often than I thought. It seems to be back to 'normal' and a few
  members may be receiving PharmPK messages for the first time in some
  time. If so, welcome back ;-) - db]
  
  

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• On 6 Mar 2009 at 07:09:38, Satya Jadhav (satya.jadhav.aaa.gmail.com) sent the message
  

  
  Hi all,
  
  Can anybody highlight and advice me on the experimental design of
  single point microsomal stability assay? I am doing it in three sets.
  
  Set 1. 0 min (NADPH) incubation (triplicate)
  
  Set 2. 15/30 min (NADPH) incubation (triplicate)
  
  Set 3. 15/30 min (No-NADPH) incubation (triplicate)
  
  Would it be prudent to include No-NADPH samples all the time? I have
  seen many reports that didnt include No-NADPH samples. However, I came
  across few compounds which are very unstable even without NADPH in it
  (unknown cause of degradataion, amidases/ esterases or simple chemical
  degradation??).
  
  What type of error parameter should I use while reporting the %
  remaining or T1/2 in microsomal stability assay?
  
  Thanks
  
  

  Back to the Top

• On 6 Mar 2009 at 11:37:14, Sanjeev Thohan (sarxconsult.aaa.gmail.com) sent the message
  

  
  Here are a couple of nice reference articles for your perusal.  It
  should be readily available from Drug Metabolism and Disposition online
  http://dmd.aspetjournals.org/
  
  DMD 29:1332-1336, 2001
  INFLUENCE OF MICROSOMAL CONCENTRATION ON APPARENT INTRINSIC
  CLEARANCE: IMPLICATIONS FOR SCALING IN VITRO DATA
  J. CORY KALVASS, DAVID A. TESS, CRAIG GIRAGOSSIAN, MICHAEL C.
  LINHARES, and TRISTAN S. MAURER
  Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Global
  Research and Development Groton, Connecticut
  
  DMD 33:1304-1311, 2005
  A UNIFIED MODEL FOR PREDICTING HUMAN HEPATIC, METABOLIC CLEARANCE
  FROM IN VITRO INTRINSIC CLEARANCE DATA IN HEPATOCYTES AND MICROSOMES
  Robert J. Riley, D. F. McGinnity, and R. P. Austin
  
  

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• On 6 Mar 2009 at 20:31:57, Parnali Chatterjee (parnalic.-a-.hotmail.com) sent the message
  

  
  Hello Satya:
  
  A few comments:
  
  1. The 0 min incubation can be carried out in buffer (without
  microsomal ptn and NADPH), which can act as a control to evaluate the
  stability of the NCE as well as the activity without cofactor
  2. Therefore only two incubations 0 (buffer) and 60 min (Ptn and
  NADPH) run in triplicate would suffice.
  
  For these exps, addition of FBS helps in solubility of the NCE.  I
  haven't seen anyone report error parameters, since these assays are
  for early discovery studies. Hope this helps.
  
  Regards
  
  Parnali Chatterjee
  
  

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• On 7 Mar 2009 at 00:40:21, Parnali Chatterjee (parnalic.-a-.hotmail.com) sent the message
  

  
  I should have added this to my previous comment..
  
  If your objective is to determine the CLint from the microsomal
  stability assay, then fub would be the correction factor you would add
  to the equation to determine CLint.
  
  There are many literature reports that have explored In Vitro-In Vivo
  Correlation and the use of fub in improving the correlation (a
  previous post has attached a few).
  
  But, if you are interested in calculating the % remaining from 2 pts
  and is an early discovery study, then it may not be necessary to
  determine the fub.
  
  

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