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Dear all
I used the Caco-2 cell model to investigate the absorption mechanisms
of our compound to predit its intestinal absorption.
However, I found that the compound is significant effluxed from the
cell monolayers. In addition, the efflux could be inhibited by
verapamil and Cyclosporin A. Both drug can decrease the BL-to-AP
transport (70%) and increase the AP-to-BL transport.
However, the MRP-1 inhibitor indomethacin (164uM) and MRP-2 inhibitor
probenecid (1 mM) can only inhibit the BL-to-AP transport by ~50% but
not change the AP-to-BL transport (0%).
Thanks so much if anybody can give me some advice on how to explain
this effect.
--
Best Regards,
Alec H Yu
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The following message was posted to: PharmPK
Dear Alec,
Caco-2 cells are meant to help at prediction of intestinal
permeability provided you include a proper (ie known) control.
Using it for trying to understand efflux transporter involvement is
not so easy. Your data suggest MDR1 involvement in net movement across
the cell monolayer. The fact that both indomethacin and probenecid
inhibit only BL-to-AP directed transport is tricky. MRP1 is located
where in Caco-2 cells that you used and what was your control for the
MRP1 "interaction test"? Could it be you are not (or not only)
inhibiting transporters involved in efflux but also those potentially
involved in "uptake" into Caco-2 cells?
You may want to assess drug associated with your monolayer when doing
the inhibition studies they may suggest drug "accumulates" are does
not enter as much in presence of certain inhibitors and this may help
to shed some light.
May personal view to understand involvement of ABC transporters at
level of absorption is to first test in LLC-MDR1 or MDCK-MDR1 cells
(with all the important controls!!) if your drug is a MDR1 substrate
drug and take it from there. In your situation that may not help so
much but surely can help you understand in a much better controlled
setting what role MDR1 may have (qualitatively at least).
Hope it helps for a bit. Next time you test in Caco-2 cells maybe test
AP-to-BL (you really on want to know permeability) only and if the
team/projects want to know about MDR1 test in an appropriate cells
system
kind regards
Hans
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The following message was posted to: PharmPK
Hello Alec
Assuming that you have a good control of mass balance, UWL and sink
conditions in you experiments, I would suggest the following:
1) Get a proper passive permeability prediction for your compound
(A2B) at high donor concentration or in the present of total
inhibition of all involved transporters in Caco-2. You can also
compare your results to predictions from phys-chem properties etc.
2) Run your compound in MDCKII-MDR1 or LLC-PK1-MDR1 cells and confirm
your data from the Caco-2 experiments.
3) Try to obtain Km, Jmax values for you compound, if you want to
simulate the contribution of the apical efflux to the overall transport.
4) Check if your compound is a potent inhibitor of the B2A transport
of digoxin.
5) Check your results from the indomethacin experiments (particularly
the mass balance). Indomethacin should also work on the apically
localised MRP2. How active are the UGTs in you Caco-2?
6) Have you thought about basolaterally localised uptake transporter
in your Caco-2.
Some questions to your experimental set up:
How long was your preincubation time for the inhibitors? - if the time
for the MRP inhibitors was too short, you might obtain only a partial
inhibition.
Have you added the inhibitors apical, basolateral or on both sides? -
if your inhibitor cannot get into the cell, you will not see inhibition.
Have you investigated/accounted for the amount of compound trapped in
the cells? - if the mass balance is incorrect, you will over-/under
estimate your inhibition.
Regards
Sibylle
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