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Dear all,
I'm now using the Caco-2 cell model to study the absorption mechanisms
of our compound. And the preliminary results indicated a significant P-
gp efflux. To further study the exact mechanism, I hope to use some P-
gp inhibitors.
I'm very grateful if anyone can give me some advices on how to select
the P-gp inhibitors and which concentration should be used.
Thank you very much!
--
Best Regards,
Alec H Yu
Department of Biochemistry,
Hong Kong University of Science & Technology, HKUST
Clear Water Bay
Kowloon, Hong Kong
yuhuayu.-at-.vip.sina.com
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I have used 5 uM of elacridar in in-vitro transport studies.
Cyclosporin A is also known to be a potent inhibitor, which inhibits
well at concentrations of approximately 10 uM.
Sincerely,
Rob ter Heine
--
Rob ter Heine, MSc, PharmD
Department of Pharmacology, Slotervaart Hospital
Amsterdam, The Netherlands
E: rob.terheine.aaa.slz.nl
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Dear Alec,
I would not assume that P-gp is responsible for your efflux. I believe
that you must characterise your Caco-2 cells, and in my experience,
Caco-2 cells can express BCRP and MRP-2 without expressing any P-gp!
You can get antibodies for these transporters. There are selective
inhibitors. See the introduction in "In Bok Paek et all, Rapid
Communications in Mass Spectrometry, 2006, 20(9)1457-62". They list PSC
833 (valdospar), VX-710 (biricodar) and GG-918, XR-9576 (tariquidar),
LY335979 (zosuquidar), R101933 (laniquidar) and ONT-093 plus there own
HM-30181.
Not all are specific to P-gp, and sourcing these inhibitors is not easy.
Regards.
Ted
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Dear Alec
Take a look at the FDA guidance on drug-drug interactions (draft
guidance, Sept 2006: http://www.fda.gov/cder/guidance/6695dft.pdf
Be aware, however, that the most interesting Pgp inhibitors listed are
drugs owned by industrial companies which are now reluctant to give
samples of compound (I have tried a couple of times and been
unsuccessful).
It is likely that FDA will have to remove these compounds which are
now almost unavailable, or urge the pharmaceutical companies to make
them available for the scientific community. The other compounds
listed should be interesting for you to show that the observed efflux
is possibly Pgp-mediated; however, it should be remembered that
selectivity for Pgp is not always demonstrated.
All the best
Frederic Massiere
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Dear Alec,
Maybe this review could help you to clarify some ideas. However, it is
from 2003 an probably new reviews could be available.
Thomas H, Coley HM. Overcoming multidrug resistance in cancer: an
update on the clinical strategy of inhibiting p-glycoprotein. Cancer
Control. 2003 Mar-Apr; 10(2):159-65.
Good luck
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P-gp inhibitors:
Verapamil and cyclosporine A
You may test them in your test condition to decide the concentration.
Jim Y. Wu, Ph.D.
Principal Scientist, Immunopharmacology
Synta Pharmaceuticals Corp.
45 Hartwell Ave, Lexington, MA 02421
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Dear Alec
The following:
"To determine the transport of substrates without the influence of
P-gp-mediated efflux activity, absorptive and secretory
transport was measured as described above in the presence of 1 uM GW918
(approx. 30-fold greater than the reported Ki for inhibition of
P-gp-mediated efflux activity; Ref. 37) added to incubation, donor, and
acceptor solutions."
is extracted from:
Troutman MD and Thakker DR (2003) Novel experimental parameters to
quantify
the modulation of absorptive and secretory transport of compounds by
P-glycoprotein in cell culture models of intestinal epithelium. Pharm
Res
20:1210-1224.
Hope you find this useful.
Regards
Masoud
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Hello Alec:
Insight into the literature reveals that the functional expression of
P-gp is consistent across the passages of 25-40 that can be utilized
for the determination of bi-directional apparent permeability (R.
ELSBY, et al., 2008. Validation and application of Caco-2 assays for
the in vitro evaluation of development candidate drugs as substrates
or inhibitors of P-glycoprotein to support regulatory submissions,
38(7-8): 1140-1164).
The criteria for the selection of in-vitro P-gp inhibitor:
Inhibit P-gp with low Ki IC50 values (e.g., IC50 <10uM) and selective
No significant metabolism of the inhibitor
May be used as an in vivo P-gp inhibitor
Its interesting to note that USFDA recently published both a
commentary paper (Zhang et al. 2006) and draft DDI guidelines (Drug
Interaction Studies -Study Design, Data Analysis, and Implications for
Dosing and Labeling, September 2006) on what information is likely to
be needed during the drug development programme for new drug
application (NDA) submissions in order to address the potential for
DDIs mediated by P-gp.
FDA recommends the following to test whether the compound is a P-gp
substrate:
Range of concentrations (e.g. 1, 10, 100 \0x00M)
Time dependency of efflux (e.g. 1, 2, 3, 4 hrs)
Include known P-gp substrate as a positive control (e.g. digoxin,
vinblastine)
Conduct bi-directional transport assays in absence and presence of
potent P-gp inhibitors
Optimize experiments to determine recovery of substrate to allow
estimation of metabolism and non-specific binding
The list of FDA acceptable P-gp inhibitor includes Cyclosporine A,
Ketoconazole, LY335979, Nelfinavir, Quinidine, Ritonavir, Saquinavir,
Tacrolimus, Valspodar (PSC833), Verapamil, Elacridar and Reserpine
Hope this helps.
With Best regards,
SYED MUSTAFA,
Clinical Candidate Optimization,
Advinus Therapeutics Pvt. Ltd.,
International Biotech Park,
Genesis Square, Bio Resource Centre,
2nd Floor, Phase II, Hinjewadi,
Pune- 411 057 INDIA
syed.mustafa.aaa.advinus.com
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