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I have observed during some of my protein binding experiments that for the
same drug binding varies significantly from one concentration to
another.Like at 10=B5g/ml I have seen values around 88% and for the same
compound at 2.5=B5g/ml binding is 62%.
Could anyone suggest the possible mechanism for this.
Yati Chugh
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Hello Yati,
Concentration dependent protein binding is described mathematically as:
Fu= 1/[1+ K*P]
Fu= fraction unbound
K= affinity constant for protein binding
P= concentration of free protein bnding sites
Molar Concentrations of Plasma Proteins
Protein Molar Concentrations
albumin 5-7.5 x 10-4
AAG 0.9-2.2 x 10-5
Concentration-dependent protein binding occurs with all protein bound
drugs, as the fraction unbound increases with increased drug concentration
according the above equation (i.e. P decreases). The therpeutic drug
concentrations
can be used to predict if concentration-dependent protein binding occurs in the
therapeutic range. For example, the 10-20mg/l phenytion therapeutic range
corresponds to a 0.4-0.8 x 10-4 molar concentration which is well below the
normal 6 x 10-4 molar concentration of albumin, and concentration-dependent
protein binding does not occur in the therapeutic range. However,
concentration-
dependent does occur with salicylate with a therapeutic range of 7-22 x 10-4
molar.
However,this increase in Fu is opposite the direction you report in
your protein protein binding experiments. This could mean the presence of a
competitive protein binder at lower concentrations, or multiple binding
sites with different affinities. At any rate, your data departs from the
classic concentration-dependent protein binding as described above
Reference
1) Benet, L. et al., Pharmacokinetic Basis for Drug Treatment, Raven Press 1984
Good luck!
Mike Leibold, PharmD, RPh
ML11439.aaa.goodnet.com
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Dear Dr. Chugh,
In your experiments the binding increases with increasing
concentration. Normally the reverse is found that binding decreases
as a function of concentration due to saturation.
One of the possibilities is that you have to do with some artefacts.
What is the reproducibility of your measurements? Did you adjust the
pH of your plasma samples to pH 7.4 as the binding is mostly
dependent on the pH and pH increase up to 8.3 after storage at -20C .
Another possibility could be competitive interactions with some
endogenous or exogenous compounds present in your plasma (e.g.
plasticizers)! Are you using plasma preserved in plastic bags?
Perhaps there are probably other causes!
Best regards,
F. Belpaire
**********************************
Prof. Dr. F. Belpaire
Heymans Institute of Pharmacology
University of Gent Medical School
De Pintelaan 185
9000 Gent
Belgium
tel: 32/9/2403355
fax: 32/9/2404988
frans.belpaire.-at-.rug.ac.be
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)