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Dear all
It would be of great help if you can clarify my doubts about following?
1. According to US FDA guidance, 20 permeability markers should be
studied for Caco-2 permeability study. Is it required to study all the
permeability markers during assessment of permeability of a drug
compound and also during establishing the Caco-2 permeability study
setup at the start? If not then, how many markers should be selected
so that US FDA accepts the Caco-2 permeability data? How many no of
samples should be analyzed for a compound?
2. USFDA recommends PEG as zero permeability marker? Can we use
Lucifer yellow as monolayer integrity marker? Does US FDA accepts this
data?
3. What should be the optimum seeding density and TEER value for 24
well plate? What is the effect of less seeding density on Papp and
TEER value as irrespective of no of cells per inserts they are going
to form monolayer covering whole area of insert?
4. Some of the low permeability drugs (like Furosemide) have similar
Caco-2 permeability as Zero permeability markers (like Lucifer
yellow). So, Is this significant to study another integrity marker
apart from such a low permeability marker?
Thanks in anticipation of a positive response.
Regards
Sachin Ramrao Patil
Deptt of Pharm.Tech Formulations
NIPER, SAS Nagar
Mohali, Punjab-160062
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Hi, Caco-2 permeability assay can be done using 2 high, 2 low and two
Pgp substrates validated in-house. This kind of validation is enough
in a Discovery setup. However, there are situtations where in-house
data for all the 20 compounds are required if one is filing a drug for
a Bio-Waiver. If filing for USFDA three different concentrations and 3
different time points (Cumulative concentrations) are usually
required. For In-house data Lucifer Yellow should be fine. If filing
for FDA PEG might become a mandate. Usually a seeding density of
50-60,000 cells is appropriate for a 24-well plate. If one is able to
achieve the required TEER and %LY yellow with low seeding density, i
think one can go ahead. Lucifer Yellow measurements are made to test
post-experiment monolayer integrity. I think there is no strict yes or
no for this question. I hope this helps. Regards, Ravindra Varma Alluri
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The following message was posted to: PharmPK
Hi Sachin
1. According to US FDA guidance, 20 permeability markers should be
studied for Caco-2 permeability study. Is it required to study all the
permeability markers during assessment of permeability of a drug
compound and also during establishing the Caco-2 permeability study
setup at the start? If not then, how many markers should be selected
so that US FDA accepts the Caco-2 permeability data? How many no of
samples should be analyzed for a compound?
I'm NOT a FDA representative, but here are my common sense comments:
Basically you need to prove that your in-vitro Caco-2 transport assay
is not only reproducible, but also robust. Fact is that many
laboratories experience a shift of the Caco-2 permeability data
measured and the low permeable compound are normally more affected
than the higher permeable compounds. This in turn leads to a change of
the in vitro-in vivo curve and thus to a change for the prediction of
your compound XYZ, estimated based on this relationship. Assuming that
you obtained a suitable curve for your 20 initial permeability markers
a year ago and you run during your screening always one high, moderate
and low marker (maybe even change these quality control compounds by
time to time), then you can check if the marker still accurately fit
the original curve. If not ...you have a problem, since your assay
seems not robust enough and you need a new set of measurement.
Take one sample more than you think you need in order to obtain an
accurate Papp, that should do!
2. USFDA recommends PEG as zero permeability marker? Can we use
Lucifer yellow as monolayer integrity marker? Does US FDA accepts this
data?
No idea, if the FDA accepts this, but fact is the different PEGs will
give you different permeabilities: All low, but different. Lucifer
yellow can be tricky too, if you want to compare your A2B data for
instance later with B2A experiments, so be careful what you wish for.
However, I don't believe that the FDA would complain if you give them
several other low permeable (maybe even in-house) compounds and argue
for your choice of compounds.
3. What should be the optimum seeding density and TEER value for 24
well plate? What is the effect of less seeding density on Papp and
TEER value as irrespective of no of cells per inserts they are going
to form monolayer covering whole area of insert?
The seeding density will affect your TEER, but the observed effect
depends already on the seeding density in the flask and the culture
conditions in the flask. In my point of view, TEER should be low,
reported as Ohm*cm2, stable over the 'days in culture' (i.e. no
extreme fluctuation on the plates after day 7-9 seeded on the plate)
and measured before and after the experiment! I have used plates with
TER values of 280 +/- 40 Ohm*cm2 (21 days), and these gave me nice and
reproducible results over years, while other Caco-2 clones that I had
used with TER over 600 up to 1500 Ohm*cm2 were not so reliable in
their performance.
Later on you will affect the cell density and hence TER also by for
instance 'the first time of changing the medium on the plates after
seeding' and ' days in culture' etc.
4. Some of the low permeability drugs (like Furosemide) have similar
Caco-2 permeability as Zero permeability markers (like Lucifer
yellow). So, Is this significant to study another integrity marker
apart from such a low permeability marker?
Why don't you try to measure compounds similar to the compounds you
want to estimate the permeability for? Trying to predict a low
permeable weakly acidic compounds with values measured for high
permeable weakly basic compounds, or to predict transcellular
permeation based on paracellular data is maybe not the smartest idea!
Sibylle
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Dear Sachin,
Regarding the seeding density and the TEER values for 24 well format
there is a lot of literature available.Many of which suggest a seeding
density of 100,000 (One lack cells) per cm2(centimeter square), the 24
well plates has a surface area of 0.33 cm2 so you can try optimizing
with 33,000 cells/well. There are other publications which suggest a
seeding density of 50-60,000 cells/well. With my personal experience I
can suggest you to try with 33,000 cells/ well and standardize the no
of cells which can be seeded. Literature values for TEER are around
400 Ohm*cm2 (for 1cm2 surface area). You can try and standardize the
no. of cells to be seeded so that you will be getting a resistance of
around 250-300 Ohm*cm2 which has worked for me.
Hope this helps
--Naveed Shaik Abdul
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