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Dear,
I'm doing some tests with equilibrium dialysis to determine protein binding of a
drug. We use the high-throughput system of HTDialysis. I did some first
preliminary tests, with plasma spiked with known concentrations of the drug, to
investigate the time to equilibrium.
My question is: How can you be sure when the equilibrium is reached? Do you have
to do some statistical tests to proof it, or can you just decide when the
equilibrium is reached, based on the raw data and the graph of the evolution of
the concentration of the drug in the buffer and plasma compartment? To our
experience, it is hard to proof it with a statistical test, because of the small
concentrations and small differences between concentrations in the buffer and
plasma compartment.
For every timepoint in the experiment I have 2 repeated measures, so it is not
possible to use a t-test (normality of data cannot be proven). So we would use a
non-parametric Mann Witney U test, based on the percentages of protein binding
on the different timepoints.
Can you give me some suggestions about this? What is your experience whit these
kind of experiments?
Thanks in advance,
Kind Regards,
Kim Vanstraelen
Hospital Pharmacist at the University Hospitals of Leuven
PhD student Clinical Pharmacy
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The following message was posted to: PharmPK
Dear Kim,
There are several ways to check time to equilibrium.
One is to spike in one series the buffer side and in another the plasma side.
When after a certain time plamsa:buffer ratios are the same for the two setups
you have reached an equilibrium.
Alternatively you can perform a time course of dialyzing spiked buffer against
pure buffer. The time to identical concentration on both buffer sides should be
predictive for the time to equilibrium in your experiment with spiked plasma.
Markus
Markus Weiss, PhD
Novartis Pharma AG
Postfach
CH-4002 Basel
SWITZERLAND
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The following message was posted to: PharmPK
Dear Kim,
in my experience with HTS systems equilibrium can be reached in 4-5 hrs at
37*C for almost all drugs (my experience with free fraction ranging from
0.01% to 80%) You can refer, for examples, to the approach followed by S.J.
Summerfield http://jpet.aspetjournals.org/content/316/3/1282.full.pdf+html
Really I suggest to have a minimum of 5-6 replicates and co-incubation in
the same run (then in the same HTSapparatus) of 3 reference standards for
low, medium and high protein binding.
We used as reference compound Ketorolac, Diclofenac and Ciglitazone for low,
medium and high fraction bound, respectively. Results of reference compounds
can "validate" the run, like QCs for an analytical run.
Your approach to monitor the time-dependent concentration of plasma and
buffer is really an excellent approach, however, mainly with compounds
showing very low unbound fraction (<0.05%) analytical method must be
well-tuned to avoid undesired oscillations of concentration and time-points
have to be selected accurately to describe the curve, to fit results and
finally to have a good estimation of the asymptote (concentration at
equilibrium).
Again, the correction for Donnan effect is critical for estimation of the
fraction: this is crucial and take care that many different approaches are
reported in the literature.
Kind regards
Stefano Porzio
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