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Dear all,
I have a little doubt related to protein binding.
In a experiment i got 99%plasma protein binding and 98.6% microsomal binding of our in house compound.Now in another set of experiment we found that it is also a inhibitor of CYP 3A4. In a such case where we are injecting this compound to rat then what is the possibility of CYP inhibition.
As per my knowledge high extent of protein binding will prevent the drug to move towards liver but as the same time because of high microsomal binding (almost equal binding) it will bind to CYP and can show inhibitory effect on it.
Now lets assume that 1PPM is fraction unbound drug which is free to move to liver tissue and will get distributed there in microsome or liver tissues till equillibrium achieve. So will it be continue till liver concentration achieve same concentration of fraction unbound which is present in plasma i.e 1PPM (considering partition of this drug in plasma :tissue is 50:50). Now in this case 98% of this 1ppm will get bind to tissue? and will change the equillibrium and act as drivng force ?or tissue binding is not a major factor for eqillibrium shift?
Apart from this (98.6% binding) microsomal binding of drug is showing inhibition or only free drug is responsible for inhibitory effect of drug on CYP 3A4 in this case?
So in secound situation my effective concentration of drug at 3A4 site will be only unbound fraction i.e 10ng (1000ng*1/100).
and as defined by FDA it will not be a potent inhibitor ({I}/IC50>1) which is my major concern if {I} is effective free concentration at active site as in this case at 3A4 site (microsomes)
Eagerly waiting for your reply
Regards,
Rahul Vats
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Dear Rahul
You are right that it is free drug that you need to consider. However, in my opinion:
Non-specific microsomal binding as normally measured is an in vitro property used to correct the apparent in vitro CLint (Km). As such the extent of binding is dependent upon (or at least correlated with*) the in vitro microsomal protein concentration - see for example the scaling procedures described by Austin et al 2002 DMD.
In order to apply a similar correction in vivo you would need
i) assume the binding is via the same mechanism in vivo (see below!), and
ii) even if i) is true you would need to know the protein concentration in vivo.
Point i) is unlikely to be true for at least 2 reasons:
a) there are components other than "microsomal" tissue (see next point) that a drug might bind to
in vivo, and
b) microsomes are not present in vivo - they are vesicle-like structures formed when endoplasmic reticulum is spun down from tisue extracts.
c) there may be other components in vitro not present in vivo (buffer etc, apparatus surfaces etc)
Additionally, there is usually high uncertainty / issues with the experimental determination of the extent of binding of such highly bound drugs - I guess your drug is highly lipophilic in common
with many new candidate compounds these days?
There are lots of papers upon Ki determination if you look in DMD for example.
regards
David
*There are various studies suggesting that binding is predominantly to the phospholipid
component of microsmal preparations rather than to protein itself.
--
David Turner, PhD Principal Scientist - Modelling and Simulation
Simcyp Limited Blades Enterprise Centre, John Street, Sheffield, S2 4SU, UK
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Dear Rahul,
In the case of compounds with high apparent protein biding, the experimental method used to determine binding can influence the outcome.
If you are using a method like micro-dialysis or ultra-filtration to determine free fraction for a compound with high non-specific binding, you could be generating artificially low free fraction values. This is due to loss of unbound compound to the membrane used to separate free from bound compound. If you are concerned this might be the case for your compound, you might try an ultra-centrifugation method to determine free fraction in vivo or in vitro.
Best regards,
Ian
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Dear Ian,
Maybe you can use the HARVARD apparatus for micro dialysis, which made of Teflon, the NSB rate is low enough to do the PB assay!
I just using it , it looks good to some of the compounds.
regards,
Chang
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The following message was posted to: PharmPK
Hi,
To avoid non specific binding, micro-dialysis is the best choice among the three methods.
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As long as the compound or the protein the compound binds to does not bind to the dialysis membrane.
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The following message was posted to: PharmPK
Having a compound bind to membranes is not necessarily a problem. As long as there is equilibrium (and the compound does not degrade in the meantime), and one quantitates drug on both sides of the membrane, protein binding can be determined.
Kind regards, Jan
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