NDLI: Cell-Free Microfluidic Determination of P-glycoprotein Interactions with Substrates and Inhibitors

Content Provider	Springer Nature Link
Author	Eyer, Klaus Herger, Michael Krämer, Stefanie D. Dittrich, Petra S.
Copyright Year	2014
Abstract	The membrane protein P-glycoprotein (P-gp) plays key roles in the oral bioavailability of drugs, their blood brain barrier passage as well as in multidrug resistance. For new drug candidates it is mandatory to study their interaction with P-gp, according to FDA and EMA regulations. The vast majority of these tests are performed using confluent cell layers of P-gp overexpressing cell lines that render these tests laborious. In this study, we introduce a cell-free microfluidic assay for the rapid testing of drug- P-gp interactions. Cell-derived vesicles are prepared from MDCKII-MDR1 overexpressing cells and immobilized on the surface of a planar microfluidic device. The drug is delivered continuously to the vesicles and calcein accumulation is monitored by means of a fluorescence assay and total internal reflection fluorescence (TIRF) microscopy. Only small amounts of compounds (~10 μl) are required in concentrations of 5, 25 and 50 μM for a test that provides within 5 min information on the apparent dissociation constant of the drug and P-gp. We tested 10 drugs on-chip, 9 of which are inhibitors or substrates of P-glycoprotein and one negative control. We benchmarked the measured apparent dissociation constants against an alternative assay on a plate reader and reference data from FDA. These comparisons revealed good correlations between the logarithmic apparent dissociation constants (R$^{2}$ = 0.95 with ATPase assay, R$^{2}$ = 0.93 with FDA data) and show the reliability of the rapid on-chip test. The herein presented assay has an excellent screening window factor (Z’-factor) of 0.8, and is suitable for high-throughput testing.
Starting Page	3415
Ending Page	3425
Page Count	11
File Format	PDF
ISSN	07248741
Journal	Pharmaceutical Research
Volume Number	31
Issue Number	12
e-ISSN	1573904X
Language	English
Publisher	Springer US
Publisher Date	2014-06-14
Publisher Place	Boston
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Pharmacology/Toxicology Pharmacy Biochemistry Medical Law Biomedical Engineering
Content Type	Text
Resource Type	Article
Subject	Organic Chemistry Pharmacology Molecular Medicine Pharmacology (medical) Biotechnology Pharmaceutical Science

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4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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