Back to the Top
a brief question relevant to Pgp assay
performed in CACO2 cell lines.
In accordance to FDA guidelines the permeability experiments were done
in triplicates over 3 different days. However, as there are no rules
to define acceptance criteria for the replications, my question is the
following: do anyone know how is the maximum acceptable variation ( in
term of CV%) among experiments ? is a CV % of 25-30 acceptable,
considering that we are dealing with cells ?
Thank you very much
Cell Therapeutics Inc (Branch Europe)
Via Ariosto, 23
20091 Bresso (MI)
E-mail: cecilia .allievi.at.ctimilano.com
Back to the Top
The following message was posted to: PharmPK
Here is a not so brief answer:
A colleague sent me your query, since I have worked several years with
Caco-2 cell and performed some Transwell experiments.
In general, your intraday variability should be lower than the
interday variability of the same passage (from one batch), since also
after 21 days in culture the cells do change; they don't change in
their passive permeability, but in their relative expression of some
TRANSPORTER for instance.
Thus, if you have a compound that is purely passively transported,
than your CV should be low (lower than 30%!). However, if your
compound is actively transported by several transporter, your results
can easily vary and the CV is not reflecting the experimental
reproducibility, but rather the change in your biological system.
If you have used 3 different passages at the same day after seeding
(i.e., 16 or 21 days, or you used the fast growing 7 days cells), than
your interday and intraday variability should be comparable, when you
have standardised your assay.
You should compare your RECOVERY data from the different days, since
bad and variable recovery is often a reason for high variability in
the calculated permeation data.
In addition, the INITIAL DONOR CONCENTRATION is important too, because
variability in the initial donor concentration will also lead to
variability in the calculated permeation data.
My personal experience is that a CV of 10% is reasonable for Transwell
results, if the ANALYTIC is good (e.g. radioactive material was used,
e.g. digoxin) and the before mentioned parameter are covered. A CV
above 30% needs in my point of view an explanation. If the reason is
the 'biological system' than you should figure out what has changed in
the system to affect your compound.
Unfortunately, people do use CVs around 30%, but they are normally
screening many compounds and, thus, they are only interested in
categorising the compound. For this purpose, even a bad CV is good
enough; however, if you want to use your permeability value for
scaling to effective permeability in Humans, you should aim for better
and more consistent data.
The heading of your email says 'PKPharm Pgp assay' and I guess that
you aim to calculate efflux ratios from your transport data. Also for
efflux ratios a standard deviation and CVs should be calculated,
leading to the problem that high&bad CVs from the transport data will
be propagated to the errors in the efflux ratios and you can easily
end up with not so easy interpretable or even useless data! Thus, the
more information you would like to extract from your transport data
the more you need to know about your test system: Caco-2!
Want to post a follow-up message on this topic?
If this link does not work with your browser send a follow-up message to PharmPK@boomer.org with "Pgp assay" as the subject
Support PharmPK by using the
Copyright 1995-2011 David W. A. Bourne (firstname.lastname@example.org)