Rational use of in vitro P-glycoprotein assays in drug discovery.

By J. Polli, S. Wring, J. Humphreys, Liyue Huang, J. Morgan, Lindsey O. Webster, C. Serabjit-Singh. Journal of Pharmacology and Experimental Therapeutics, 2001

662 citations recorded by the source corpus.

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 (P(app)) 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 P(app) values (mean = 16.6 nm/s), whereas inability to detect efflux (group IIA) was associated with compounds having high P(app) values (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 P(app) compounds of group IIB. All assays detected substrates across a broad range of P(app), but the efflux assay was more prone to fail at high P(app), whereas the calcein-AM and ATPase assays were more prone to fail at low P(app). When P(app) is low, efflux is a greater factor in the disposition of Pgp substrates. The efflux assay is more reliable at low/moderate P(app) and is the method of choice for evaluating drug candidates despite low throughput and reliance on liquid chromatography with tandem mass spectrometry.

Subfields: Molecular Biology, Oncology, Pharmacology

View Paper: https://www.semanticscholar.org/paper/9fd3c41af926e681b2493a46161c3721aa647892

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Citations in the corpus

• Chapter 11 - Reconstitution of membrane proteins in phospholipid bilayer nanodiscs. (2009, methodology): “There are currently four in vitro systems routinely utilized to study P-gp: whole cells overexpressing P-gp (Adachi et al., 2001; Polli et al., 2001; Takano et al., 1998; Wang et al., 2002), membrane fractions from those cells (Loo and Clarke, 2005; Loo et al.”
• Modulation of notch processing by gamma-secretase inhibitors causes intestinal goblet cell metaplasia and induction of genes known to specify gut secretory lineage differentiation. (2004, methodology): “…gene MDR1 was assessed in triplicate at a single time point of 60 min, at 1 and/or 10 mM, and in the presence or absence of a potent and specific inhibitor (GF120918, 2 mM; gift from Glaxo SmithKline, Research Triangle Park, North Carolina) as described elsewhere (Polli et al., 2001).”
• The conduct of in vitro and in vivo drug-drug interaction studies: a Pharmaceutical Research and Manufacturers of America (PhRMA) perspective. (2003, background): “However, inconsistencies have been observed compared with cellular systems where transport of the NME is measured directly (Polli et al., 2001).”
• Elucidation of the Impact of P-glycoprotein and Breast Cancer Resistance Protein on the Brain Distribution of Catechol-O-Methyltransferase Inhibitors (2017, background): “…limitation of the bidirectional transport assay that may prevent compounds with a Papp,AP-BL value close to or higher than 30 1026 cm/s from being identified as efflux substrates (Polli et al., 2001; Gameiro et al., 2017), emphasizing the importance of performing in vivo studies for such compounds.”
• Passive Permeability and P-Glycoprotein-Mediated Efflux Differentiate Central Nervous System (CNS) and Non-CNS Marketed Drugs (2002, methodology): “Culturing of cells and transport studies were completed as previously described (Polli et al., 2001b).”
• PBPK models for the prediction of in vivo performance of oral dosage forms. (2014, background): “In vitro permeability assay development, impact of the cell line, surfactants and time points have been discussed in detail elsewhere (Avdeef et al., 2007; Chiu et al., 2003; Matsson et al., 2005; Polli et al., 2001; Thiel-Demby et al., 2009).”