PT  - JOURNAL ARTICLE
AU  - Polli, Joseph W.
AU  - Wring, Stephen A.
AU  - Humphreys, Joan E.
AU  - Huang, Liyue
AU  - Morgan, Jonathon B.
AU  - Webster, Lindsey O.
AU  - Serabjit-Singh, Cosette S.
TI  - Rational Use of in Vitro P-glycoprotein Assays in Drug Discovery
DP  - 2001 Nov 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 620--628
VI  - 299
IP  - 2
4099  - http://jpet.aspetjournals.org/content/299/2/620.short
4100  - http://jpet.aspetjournals.org/content/299/2/620.full
SO  - J Pharmacol Exp Ther2001 Nov 01; 299
AB  - P-glycoprotein (Pgp) affects the absorption, distribution, and clearance of a variety of compounds. Thus, identification of compounds that are Pgp substrates can aid drug candidate selection and optimization. Our goal was to evaluate three assays used to determine whether compounds are Pgp substrates. Sixty-six compounds were tested in monolayer efflux, ATPase, and calcein-AM assays. Assay results yielded two categories of compounds. Category I (n= 35) exhibited concordance across the assays. Category II (n = 31) revealed differences among the assays that related to the apparent permeability (Papp) of the compounds. Within category II, two groups were discerned based on the absence (group IIA, n = 10, nontransported substrates) or presence (group IIB, n = 21, transported substrates) of monolayer efflux. Detection of efflux (group IIB) was associated with compounds having low/moderate Pappvalues (mean = 16.6 nm/s), whereas inability to detect efflux (group IIA) was associated with compounds having high Pappvalues (mean = 535 nm/s). The calcein-AM and ATPase assays revealed Pgp interactions for highly permeable group IIA compounds but were less responsive than monolayer efflux for low/moderate Papp compounds of group IIB. All assays detected substrates across a broad range of Papp, but the efflux assay was more prone to fail at high Papp, whereas the calcein-AM and ATPase assays were more prone to fail at low Papp. When Papp is low, efflux is a greater factor in the disposition of Pgp substrates. The efflux assay is more reliable at low/moderate Papp and is the method of choice for evaluating drug candidates despite low throughput and reliance on liquid chromatography with tandem mass spectrometry. The American Society for Pharmacology and Experimental Therapeutics