Comparison of in Vitro P-Glycoprotein Screening Assays:  Recommendations for Their Use in Drug Discovery

Abstract

The ATP-dependent drug efflux pump P-glycoprotein (P-gp) affects the absorption and disposition of many compounds. P-gp may also play role in clinically significant drug-drug interactions. Therefore, it is important to find potential substrates or inhibitors of P-gp early in the drug discovery process. To identify compounds that interact with this transporter, several P-gp assays were validated and compared by testing a set of 28 reference compounds, including inhibitors of cytochrome P450 3A4 (CYP3A4). The assays included in silico predictions, inhibition assays (based on cellular uptake of rhodamine-123 or calcein AM), and functional assays (ATPase activity assay and transcellular transport assay, the latter for a subset of compounds). In addition, species differences were studied in an indirect fluorescence indicator screening assay and test systems expressing porcine, mouse, or human P-gp. Our results suggest that several P-gp assays should be used in combination to classify compounds as substrates or inhibitors of P-gp. Recommendations are given on screening strategies which can be applied to different phases of the drug discovery and development process.

Role of P-glycoprotein in transplacental transfer of methadone

“…A human cDNA-P-gp expressed in a commercially available membrane preparation was utilized to determine whether methadone, and other opiates, would stimulate the ATPase activity coupled to the binding of a ligand to the efflux transporter. An earlier investigation, utilizing the same method, determined that the morphine-stimulated ATPase activity was 3.7 nmol mg −1 min −1 [37], in agreement with our data ( Table 1). The rates for P-gp-coupled ATP hydrolysis stimulated by the interaction of methadone, LAAM, and BUP are shown in Table 1.…”

Unravelling the complex drug–drug interactions of the cardiovascular drugs, verapamil and digoxin, with P-glycoprotein

“…In Caco-2 cells containing human Pgp, verapamil had a higher permeability ratio with Pgp at low opposed to higher verapamil concentrations [23]. Also, human Pgp overexpressed in LLC-PK1 cells had higher efflux ratios at 350 nM than 5 μM verapamil [21,25]. Therefore, we propose that verapamil occupancy at the H site .…”

“…From results of in vitro studies, the drug is known to activate Pgp-coupled ATP-hydrolysis [20]. This drug manifests a spectrum of characteristics, ranging from being a good substrate to a non-substrate for the transporter, which depends on the cell type being evaluated in in vitro cell studies [21][22][23][24][25][26] or host tissue type in in vivo studies [27][28][29]. Although the actual molecular details of these interactions are currently unknown, the drug has been shown to inhibit the ATPase activity of a second drug by competitive, non-competitive and allosteric mechanisms in an in vitro study [30].…”

Effects of a combretastatin A4 analogous chalcone and its Pt-complex on cancer cells: A comparative study of uptake, cell cycle and damage to cellular compartments

“…4a). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 14 of the clinically established chemosensitiser verapamil [35,36] ( (Figure 4 here)) …”

“…P-gp overexpressing KBv1 +Vbl cervix carcinoma cells [35] alongside their wild type parent cells were treated with chalcones 1 and 2 in the presence of calcein-AM (Fig. 4a).…”