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We are working on a chemical class, the compounds show the biological activity only when the plasma
concentration reaches >900uM and maintained (900-1600uM) for 3-4 hours. These acidic compounds are
highly protein bound (10uM= 99.99%). I would assume the activity is due to saturation of plasma
proteins. The plasma albumin concentration in blood is approximately 600-650uM and looks like we are
saturating the loading capacity of albumin so that more free drug is available to engage target (a
key protein in blood). I tried to prove that it is free drug (fu) causing the effect by running a
plasma protein binding assay (HTDialysis and ultracentrifugation) at 100, 500 and 1000uM. The result
in fact surprised me >99.99%. I was expecting at 100 and 500uM the PPB close to 99.99% and for
1000uM definitely <99.99.
These results look too good to believe and perhaps an artefact of an in-vitro assay at such a high
conc. IMHO, I don’t think any of the plasma protein binding assay are reliable at such a high
concentration especially for a highly protein bound drug.
The spiked plasma PPB in-vitro experiment comes very close the actual physiological conditions in
terms of pH and concentration, however I feel it is still far from the actual (dynamic) conditions a
compound is subjected in vivo and the plasma proteins conformation might differ in –vitro compared
We tried running the actual PK samples in PPB (HTDialysis),the pH was variable (range 7.3-8.5 due to
storage and freeze -thaw) and the fu obtained was 0.1-7% (low to high concentration with some
outliers), this could have explained the in-vivo results however the problem was how to explain
these values in terms of physiological pH 7.4 in mouse. I didn’t adjust the pH of samples as this
would change the concentrations. Is there a better way to carry out an experiment to explain the
free drug driven in-vivo effect at such a high concentration.
I appreciate thoughts from the members of this group.
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Jagdish, you may want to look into plasma PB using the charcoal adsorption method.
am I right that your main question is how to align your in vitro PPB values of fu=0.01% to in vitro
fu=0.1 to 7%?
What method did you use to determine in vivo fu? As an aside: To stabilize the pH during the
experiment you can place your setup in an incubator with a stable CO2 atmosphere.
I reckon your compound binds at least to two, if not more, binding sites on albumin, otherwise you
would not be able to see such high fractions bound at the concentrations used. When using standard
techniques for plasma protein binding you also need to consider the solubility of your compound in
pure buffer as this may strongly affect your results. In particular considering the exorbitant
concentrations. If your compound precipitates e.g. beyond 5µM you’ll see the result that you
observed no matter what. This also depends on how you treat the samples in terms of dilution before
running the analytics to deal with the high concentrations. In case precipitation is an issue, you
should use other plasma protein binding assays that have a work-around for this.
Some thoughts on your problem
1) You may not necessarily see saturation of protein binding around 0.6 mM (concentration of
HSA) e.g. if the compound binds to more than one binding site on albumin or binds in addition to
other proteins such as lipoproteins. Otherwise your assumption that you get activity only after
saturation may be valid, but your assay is not good enough to differentiate.
2) A methods that is useful to see differences in case of very high protein binding is
equilibrium gel filtration: Equilibrium Gel Filtration to Measure Plasma Protein Binding of Very
Highly Bound Drugs, Weiss and Gatlik, JOURNAL OF PHARMACEUTICAL SCIENCES 103:752–759, 2014
3) How can you explain that in the HTDialysis you have > 10 fold differences in fu between in
vitro an ex vivo (PK) samples?
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