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Dear All:
I recently read a paper about the protein binding measurement in liver micrsomes, in which described that the incubation mixture (containing microsomes and phosphate buffer, etc) should be left at room temperature overnight to ensure loss in microsome viability before adding test compound and conducting ultrafiltration. Is it necessary for this kind of protein binding study?
Any response is appreciated! Thanks!
Jian
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Dear Jian
To scale up In-Vitro intrinsic clearance to In-Vivo clearance, microsomal binding is required.It has been observed that plasma protein binding and microsomal non specific binding values are not the same for molecules.Therefore microsomal binding experiment has to be conducted to determine binding.
Nonspecific binding factor is very much required in In-Vitro clearance.
Hope this will help
Rahul Vats
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The following message was posted to: PharmPK
Dear Jian, dear All,
I think the question regarded the need to leave the microsomes overnight at
RT before using them in a binding experiment, not the actual experiment-
right?
Here is my (home-made!) explanation, hope it is the right one!
If one used "fresh" microsomes, then theoretically, if the compound under
investigation in the microsomal binding assay were also a substrate of
microsomal CYPs, and the turnover were reasonably high, one might not see
the true extent of microsomal binding of compound- because some of it would
have been (specifically) metabolised and therefore unavailable for the
(non-specific) binding.
On the other hand, you want to look at binding to the natural microsomal
protein, so any treatment that inactivates CYPs by gross protein denaturing
would also introduce problems, as would any CYP inhibitory chemical that
might itself bind non-specifically.
Hence, the easiest way to hopefully look at non-specific binding only is to
just leave the microsomes sitting around until they have lost catalytic
activity without grossly interfering with protein structure, for example
leaving out overnight at RT.
Best,
Constance
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The following message was posted to: PharmPK
Dear Jian
Cofactors such as NADPH are usually added to initiate metabolic reactions with microsomal preparations so if they are not present when doing ED then you would expect no metabolism to occur - see eg Obach 1997 DMD 25 2512 ....
Also binding may generally speaking be dominated by binding to/partition into the phospholipid component of the microsomal preparations; eg see Margolis JM and Obach RS (2003) Drug Metab Dispos 31:606-611 but there are many (much) older papers that support this view. However, others, eg see some of John Miners publications, have described saturable binding models ...
regards
David Turner
Principal Scientist
Simcyp Limited.
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