PharmPK Discussion - Microsome stability assay - low recovery for the control group without NADPH

• On 24 Oct 2013 at 15:14:41, Yu L. Ho sent the message
  

  
  Hi,
  
  We are conducting a liver microsome stability assay in different species and found quite low parents
  remaining in the negative control group without the NADPH cofactor. Could this be due to some other
  metabolism independent of NADPH? Any ideas or suggestion for some related reference?
  
  Thanks,
  
  Yu
  
  

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• On 24 Oct 2013 at 17:10:40, Lea Huang (LHuang.-at-.intermune.com) sent the message
  

  
  Hi Yu,
  
  Yes, NADPH independent metabolism are very possible. You can search online, I am sure there will be
  a few references show up.
  
  Cheers,
  
  Lea Huang
  DMPK, Preclinical Development
  
  

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• On 24 Oct 2013 at 17:11:29, Xiaodong Shen (shenxiaodong11.aaa.yahoo.com) sent the message
  

  
  How about ester hydrolysis?
  
  

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• On 24 Oct 2013 at 17:12:25, Manish Issar (missar.aaa.westernu.edu) sent the message
  

  
  Well, one of the purpose of the negative control is to access whether the drug is legitimately being
  converted to its metabolite(s).  In absence of a negative control one could unknowingly be
  addressing chemical or a combination of chemical and enzymatic conversion rather than purely
  enzymatic conversion of drug to its metabolite(s) hence could be misleading.  Some drugs could be
  chemically unstable in aqueous buffers while they could also be metabolized by the microsomal
  enzymes at the same time.  I am not sure if this case is happening in your case but it would be
  important to check for chemical degradation before you make any unusual conclusions.
  
  Hope that helps
  
  Manish
  
  Manish Issar, Ph.D
  Assistant Professor of Pharmacology
  Western University of Health Sciences
  College of Osteopathic Medicine of the Pacific
  Pomona, CA 91766
  Email: missar.-at-.westernu.edu
  
  

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• On 24 Oct 2013 at 17:13:26, Rana Rais (rrais2.-at-.jhmi.edu) sent the message
  

  
  If the compound is being hydrolysed by liver esterases  it will show low recovery in negative
  control without cofactor. Also one test you can do is to  do chemical stability in buffer without
  cofactors or microsomes.
  
  

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• On 25 Oct 2013 at 09:56:38, Yu L. Ho (yuliu.ho.at.gmail.com) sent the message
  

  
  Thanks all for all the great feedback. Other than esterase hydrolysis and testing buffer (PBS)
  stability, any other investigational plans suggested?
  
  Yu
  
  

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• On 25 Oct 2013 at 10:12:51, McGraw, Joseph E (Joseph.McGraw.-a-.cuw.edu) sent the message
  

  
  I would recommend checking nonspecific and specific binding to microsomes by incubating for a very
  short time (essentially just mixing) with no nadph then extracting. Use intact microsomes and
  deactivated/denatured microsomes. These experiments may provide some insight.
  Regards
  Joe
  
  

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• On 25 Oct 2013 at 13:42:11, Chen, XiaoHui (xiaohui.chen.-at-.novartis.com) sent the message
  

  
  You may also want to check the compound's solubility in the media. Compounds can crash out of
  "solution" due to poor solubility to generate a false positive degradation profile. It is especially
  suspicious when you get same hockey-stick like stability profile in different species. Another thing
  I may ask is how much you know about your negative control compound and how you chose it as a
  negative control. Do you have historical data that you can compare with? Hope it will help.
  
  Xiaohui Chen
  Novartis Institutes for BioMedical Research, Inc.
  250 Massachusetts Avenue
  Cambridge, MA 02139 USA
  
  

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• On 26 Oct 2013 at 08:19:35, Ravi (bravikanth.-a-.gmail.com) sent the message
  

  
  Dear Yu,
  
  Just wondering whether your compound has an amide bond in its structure.
  
  Amide bond hydrolysis does not require a cofactor.
  
  Regards,
  
  Ravi
  
  

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