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Hi All out there, not sure if I'm in the right place but here goes all
the same. Essentially my questions relate to the enzymatic cleavage of
prodrugs (in the main esters).
As a chemist we hope to perform our own in-house (academic not
industrial) enzymatic cleavage studies, and as we will essentially be
leaving our comfort zone I thought it may be a good idea to consult
those with with a better knowledge of the subject; though believe me
it is not for the want of trying, I have a stack of literature
reaching knee high but to date there appears to be no definitive
procedure for such a task.
Q1. We wish to assess our prodrug in serum (80mg/ml protein), general
consensus appears to be to use serum diluted to 80% with buffer. We
are happy with this but why is this relatively standard when the serum
only constitutes a little over 50% in terms of blood volume, why not
dilute the serum down to replicate whole blood, ie nearer 55% as
apposed to 80%?
Q2. To test our cmpds against liver esterases we intend to purchase S9
liver fractions for such a task, as we do not have access to liver
homogenates. Many papers appear to use liver homogenate diluted to 10%
but do not quote protein concentration. We plan to work with S9 liver
fraction at 20mg/ml protein but have no feel for this value or at what
dilution we should work? Also is it standard to work with systems at
different protein concentrations, ie 80% serum in the region of 50 mg/
ml protein, and liver homogenate at around 1 mg/ml?
Q3. Overall drug concentrations in serum/liver homogenate 'cleavage
assays' vary from anything between 1 micromolar to 1 millimolar, in
the main. Our aim is to carry out the experiment under pseudo-first
order kinetics, so I understand starting concentration will have no
impact, but from experience is there a preferred drug concentration to
That's all for now so I would just like to say thank you in advance
for any assistance which you may be able to provide.
Warmest regards, David Rennison
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The following message was posted to: PharmPK
> Q1. We wish to assess our prodrug in serum (80mg/ml protein), general
> consensus appears to be to use serum diluted to 80% with buffer. We
> are happy with this but why is this relatively standard when the
> only constitutes a little over 50% in terms of blood volume, why not
> dilute the serum down to replicate whole blood, ie nearer 55% as
> apposed to 80%?
Serum is essentially the same as plasma (minus some clotting factors).
If it were me I'd use plasma in case those clotting factors somehow
modified your results.
Whole blood is a mixture of undiluted plasma plus cells (mainly red with
some white and platelets). While it is true that plasma typically
represents 55% of whole blood the blood cells can be considered as just
a nuisance from the perspective of looking at plasma esterase activity.
Of course, after you've done your plasma experiments you might want to
look at whole blood in order to include the esterases in the cellular
component of blood.
I dont see any point in diluting plasma (or serum). Undiluted it will
have esterase concentrations similar to those in vivo.
Nick Holford, Dept Pharmacology & Clinical Pharmacology
University of Auckland, 85 Park Rd, Private Bag 92019, Auckland, New
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