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Facing solubility issues with a series of compounds, we found that
adding 0.1% BSA or 1% FCS improved the readout of compound
concentration with the control (culture media +protein) over 2hours.
Do you see this as a No/No for Hepatocytes and/or Microsomes stability
evaluation? what would be your recommended maximum concentration of
BSA in theses assays?
As for the FDA/Guidance I didn't find any recommendation on the
addition or not of BSA or FCS? Have you any comments?
Thank you in advance,
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One of the first papers to show a BSA effect on microsomal metabolism
was [Ludden et. al. JPET 282:391-396, 1997], where a ~20-fold decrease
in Km was reported for the CYP2C9 substrate phenytoin in human LM when
2% BSA was added. The mechanism of this effect was not known (one
would not expect a decreased in Km upon addition of a binding protein
based on the free-drug hypothesis).
The mechanism for this effect was later proposed to be sequestration
of inhibitory fatty acids released during microsomal incubations [Zhou
et. al. Life Sciences 75:2145-2155, 2004]. More recently, it has been
shown that the commonly reported under-prediction of in vivo CL for
UGT substrates based on LM incubations can be corrected by addition of
BSA to the incubations 1) ["The Albumin Effect"- Rowland et. al. DMD
36:1056-1062, 2008] and 2) [Kilford et. al. DMD 37:82-89, 2009].
Therefore, I would and do add BSA to all microsomal incubations (and
have been for at least 8 years now). 2% seems to be the optimal
concentration in most reports, but lower concentrations (0.1 - 0.5%)
can improve in vivo predictions for many substrates.
As to the regulatory status, I do not believe that FDA/CDER have
stated a position on this yet (despite of the fact that Linda Ludden
was at CDER when the original 1997 paper was published), so I would
advise caution on using BSA in experiments for regulatory submission
at this time.
San Diego, CA
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