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We are conducting a first in human study for one of our compounds and
in addition to doing plasma PK, we are also collecting one sample per
subject to evaluate the protein binding. I know that the bioanalysis
of the plasma samples has to be done using a validated GLP method.
What about plasma protein binding. Is it necessary to have a validated
GLP method to do that, especially since there could be serious
implications on the cost. Any suggestions regarding a good contract
lab that can do this using equilibrium dialysis?
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The following message was posted to: PharmPK
Martin,
I have some experience in measuring free drug concentrations in plasma.
Just a couple of points:
1. Equilibrium dialysis results in changing the actual concentration of
the drug in the plasma, so you would need to establish that the protein
binding is not concentration dependent before using equilibrium dialysis
to measure individua samples.
2. Based on my experience however I would use ultrafiltration and not eq
dialysis to determine free drug concentrations, for a range of technical
reasons. This involves one additional step in the assay (ie
ultrafiltration - after appropriate validation) and we would usually use
the same assay as for total drug concentrations. This results in an
additional cost of around $A7-$A10 per sample (plus validation costs).
Regards,
Ross Norris,
Associate Professor. PhD, MAppSc, BAppSc.
Australian Centre for Paediatric Pharmacokinetics & Therapeutic Advisory
Service, Mater Pharmacy Services, Raymond Terrace, South Brisbane, Q
4101, Australia.
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The following message was posted to: PharmPK
Great to see you collecting in vivo PB data
NB May be affected by other parameters e.g. temperature, pH
Cheers
Matt
Dr Matt Doogue
Clinical Pharmacologist / Endocrinologist
Flinders Medical Centre, Flinders University
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Hi Martin,
I would think for ex vivo PB samples mostly the separation of free
drug is not done under GLP, whereas the corresponding determination of
drug levels are often done with the GLP method. If free levels are
expected to beradiolabeled drug. A possible concentration dependency of PB as well
as a suitable method for free drug separation (eq. Dial, UF, etc.)
should be tested in vitro before hand. For the methods you have to
make sure they are suitable for your drug, e.g for UF: free drug
permeates with no relevant retention through the membrane and for eq.
dial.: you get an equilibrium during the incubation time.
Regards,
Markus
H Markus Weiss, PhD
Novartis Institutes for BioMedical Research
Translational Sciences - DMPK
CHBS, WSJ-210.4.25
Novartis Pharma AG, Werk St. Johann
CH-4057 Basel, Switzerland
Email : markus.weiss.-a-.novartis.com
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Are you doing the dog's protein binding in vitro? If so run the human
plasma over the concentration range you expect. Once you have done
this will know if the binding is concentration dependent, then you
proceed with the in vivo with confidence. You will have to establish a
calibration curve for the buffer. I prefer equilibrium dialysis. I
used the 96 well plates from Harvard and experienced no non-specific
binding which I used to find with ultrafiltration.
Stanley Cotler
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