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I am working with a new compound which is practicaly insoluble in
water. It is soluble only in DMF(dimethyl formamide). I wish to
determine its inhibition potential (IC50) using liver microsomes and
purified CYPs. The compound precipitates when its solution in DMF is
added to buffer and NADPH regenerating system (Total DMF < 2%V/V) at
concentrations greater than 5µM . Does anyone have any experience in
conducting inhibition studies with such insoluble compounds up to 200µM
concentration? Any suggestions will be greatly appreciated.
Sonali Kakar
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Dear Sonali,
This does bring up the question of the relevance of the IC50 you are
measuring. In addition, even if you do manage to get it in solution, the
added solvent may be perturb the enzyme and therefore you get an
incorrect
figure.
An alternative would be to allow the microsomal or purified CYP system
to
solubilise the compound directly, for example from an aliquot dried onto
the wall of the tube, then transfer the solution to a fresh tube before
starting the reaction with the regenerating system. However, to
determine
the actual concentration then requires a bioanalytical method as you
cannot
rely on knowledge of the amount of added compound. This then brings up
the
question of which concentration you use, the total in the system (free +
bound) or just the free. We normally think of the free concentration as
being related to the plasma free levels (at equilibrium anyway). Liver
total drug can be many fold higher or lower than plasma (and have
different
kinetics). There are many reports of poor correlation of plasma levels
with
CYP activity when plasma fu is taken into account. However, people often
forget that there is another fu to consider, that in the tissue, which
will
normally be different from the plasma fu.
Best regards, Phil.
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Dear Sonali,
from a very pragmatic point of view you might consider to perform your
inhibition experiments in a concentration range limited by the
solubility of your compound (under physiologically relevant
conditions). Even provided that there is a trick to solubilize the
compound at 200 uM what would be the relevance of a Ki value determined
under these conditions for the in vivo situation. Your interpretation
would most likely be such that any inhibition seen at higher
concentrations is irrelevant in vivo because of the limited solubility
of the inhibitor.
Best regards
Alex
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Dear Sonali,
You can add the drug (drug dissolved in any solvent, etc) in clean glass
tubes and evaporate the solvent in the tube when you perform the
metabolic
assay. Then you can add the buffer, NADPH generating system etc and
vortex
it nicely before you do the assay.
By this, the solvent used to dissolve the drug may not interfere with
the
metabolic assay as well as the drug will not precipitate.
Hope this helps.
Regards
Savithiri shivakumar
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Hi everyone,
I have few questions to Dr. Savithri's suggestion.
1. How to find out how much drug was actually in
solution to be available for enzymes (initial
concentration)since solubility is a limitation. Even
if vortexed vigorously,drug will be in suspension at
higher conc. Moreover, as the soluble drug will be
metabolized over time, the insoluble drug will go into
solution and the product formed will keep on
increasing with time. I wonder if the results obtained
under this condition would be realistic.
Dr. Sunil bajad (Patil)
Research Associate
Department of Medical Sciences
School of Vet. Medicine
University of Wisconsin-Madison,
2015 Linden Drive west,
Madison 53706, WI,USA
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Sunil,
I completely agree with you.
If you do as suggested you can not ensure that all your drug will
solubilize
in the buffer.
Data may not be taken into account if you are not confident with the
initial
concentration.
Regards,
Frederic DOC
PFIZER Global R&D
Fresnes Laboratories
FRANCE
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