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In Caco-2 studies, based on the Papp values, what is
the basis to classify a drug to be showing low,
medium, or high absorption? Also, if the drug is
showing medium or low absorption, will a caco-2 study
in the presence of a PGP inhibitor (eg Verapamil) be
enough to proove whether the drug is a PGP substrate?
Any advise and references will be highly appreciated.
Thanks
Ananda
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The following message was posted to: PharmPK
Dear Ananda,
Changes in absorption in the presence of verapamil can 'suggest' the
role of P-gp but may not be conclusive. Verapamil may influence other
efflux/influx transporters as well (known or unknown). A P-gp
transfected cell line may be a better bet to 'prove' its a P-gp
substrate.
Regards
Nagdeep Giri
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Dear Amanda,
We classify compounds as:
Low permeability Papp from 0 to 100 nm/sec
Medium permeability Papp from 100 to 500 nm/sec
High permeability Papp > 500 nm/sec
Remember that, even if CaCo-2 permeability has been extensively used as
direct absorption predictor, a lot of concerns exist about it and
correct estimations require the combined use of a lot of compound
descriptors like LogD, Solubility, gastrointestinal environment
changes, administered amount (Dose), gastrointestinal transepithelial
permeability (estimated with CaCo-2 cells or other epithelial systems
like MDCK cells or Rabbit jejunum) etc.
The easiest way to compose these information is to use a dedicated
software like GastroPlus (Sumulation Plus) or iDEA PK-Express (Lion
Bioscience).
Abourt a potential PGP substrate:
In my experience it's better to compare the Papp from Apical to
basolaterl (A>B) and from basolateral to apical (B>A) side of CaCo-2
colture devices (i.e. Transwells).
This is the easiest way to evidence a PGP substrate: an increase on B>A
Papp when compared to A>B is a possible alternative to the use of a PGP
inhibitor like verapamil. In our experience the easiest way is to
measure the B>A/A>B ratio: when this ratio is greater than 3 times we
consider compound as possible PGP substrate (further experiments are
then required like the use of an inhibitor, etc.)
We had a lot of in house examples of the goodness of this approach. A
test compound you can use is Erythromycin.
[You have to consider additionally that the use of verapamil requires
the setup of an experimental system to find the correct concentration
of inhibitor able to inhibit PGP, etc. etc...]
Best regards
Stefano Porzio
Pharmacokinetic and Tox. Dept.
Inpharzam Ricerche SA - ZAMBON-GROUP
Taverne - Switzerland
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The following message was posted to: PharmPK
Dear Ananda,
- As far as I'm aware of this, classifying the compounds into low,
medium or high absorption is done using a calibration curve relating
the fraction absorbed in humans to Papp observed using Caco-2 cells.
From this curve (sigmoidal when log Papp is considered), a compound is
classified as High if predicted absorption is above 80%, Low if
predicted absorption is below 20%, Medium if between 20% and 80%.
Needless to say that prior to classifying an unknown drug, the
laboratory should carefully establish its own calibration curve using
at least 20 known marketed drugs (about 30 is better). These reference
drugs should be judiciously selected: mainly, they should be passively
absorbed, easy to detect by HPLC or LCMS to avoid artefacts. Also,
absorption rate in human within the set of reference drugs should be
well balanced between the low , the medium and the high absorption
range. Once obtained, the calibration curve should enable the ranking
of unknown compounds within 1 log unit at least, given both
inter-individual variability in humans (Y axis) and analytical errors
(X axis). For reference, see Pontier et al, J Pharm Sci, 2001, 90:1608.
- If the drug is showing low or medium rate of absorption, the use of
one P-gp inhibitor is likely not enough to prove whether the drug is a
P-gp substrate (See J. Pachot et al, J Pharm Pharmaceut Sci, 2003 or
Garrigos et al, Biochem. Bioph. Res. Com., 1998 or Orlowsky et al,
Biochem. Bioph Res. Com., 1998). On one hand, the specificity of the
inhibitor for Pgp with respect to other efflux transporters should be
established. On the other hand, because of its multiple sites of
interaction, complete inhibition of Pgp is usually not achieved with a
single inhibitor. Unless working within a known series of compounds, we
usually use here at Oroxcell a set of 3 P-gp modulators: Verapamil,
Nicardipine and Progesterone. Also, transport assays should be
performed with careful selection of drug concentration and P-gp
modulator concentration in the transport media.
Hope this helps.
Best regards,
Frederic Massiere
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