Donald L. Melchior, The Fluorosome Company, Worcester, MA 01605, USA; Frances J. Sharom, Department of Molecular and Cellular Biology, University of Guelph, Guelph ON Canada N1G 2W1; E.J. Dell, BMG LABTECH, 13000 Weston Parkway, Cary NC 27513, USA, 04/2010
P-glycoprotein (Pgp; ABCB1), a member of the ATP-binding cassette (ABC) superfamily, exports structurally diverse hydrophobic compounds from the cell driven by ATP hydrolysis. (1) Pgp expression has been linked to the efflux of chemotherapeutic drugs in human cancers leading to multidrug resistance. Pgp activity can also result in low oral absorption and poor brain penetration. Interaction of drugs with the Pgp may also cause an increase in toxicity of co-administered compounds.
Interaction of drugs with active transporters such as Pgp is of increasing interest to the pharmaceutical industry based, in large part, on new draft FDA guidelines requiring knowledge of whether a drug candidate is a substrate and/or inhibitor of Pgp.
The Fluorosome Company’s Fluorosome®-trans-pgp assay, together with BMG LABTECH’s FLUOstar Omega, provides a rapid, sensitive and specific reconstituted Pgp liposome assay system for identification of compounds that interact with the transporter. The assay measures the ability of a compound to compete with a Pgp substrate for transport, and determines the IC50 value. The FLUOstar Omega’s ability to inject at the point of measurement assures that no data is lost. Up to 50 readings per second taken concurrently upon injection, provides ample data for calculation of first order transport rates. Single concentration inhibition measurements can be made very rapidly, and an IC50 determination carried out in only 10 minutes thereby.
The Pgp is a 170 kDa intrinsic membrane protein that effluxes a wide range of drugs from the cell. Pgp is also an outwardly directed flippase for fluorescent phospholipid and glycosphingolipid derivatives, which suggests that it may also translocate drug molecules from the inner to the outer membrane leaflet. The membrane lipid bilayer plays and important role in Pgp function and may regulate both binding and transport of drugs.
Fig. 2: The Pgp Efflux Pump
Isolation and Purification of Pgp
Purified Pgp (>90%) is isolated from Pgp-overexpressing CHRB30 cells by detergent (CHAPS) extraction followed by affinity chromatography on Con A-Sepharose. (2) Micellar Pgp is reconstituted into lipid bilayers by mixing in solution with CHAPS-solubilized micellar phospholipid, and the detergent is removed by gel exclusion chromatography. The resulting reconstituted Pgp is aliquoted and stored frozen at -70°C.
Manufacture of Fluorosome-trans-pgp
Frozen reconstituted Pgp is thawed and mixed in buffer with fluorophore (BSA-fluorescein) then converted into Fluorosome-trans-pgp by extrusion. The resulting unilamellar vesicles are separated from unencapsulated fluorophore by gel exclusion chromatography. The fluorosome-trans-pgp is then subjected to Quality Control Criteria; size, fluorescence, passive permeability, and ATP-stimulated transport of a control Pgp substrate.
The Fluorosome-trans-pgp assay procedure.
A typical picture of fluorescence signals measured over time is given in Figure 3.
Fig. 3: Fluorescence kinetic signal curves for different samples
The slope for each well (Fig.3) was plotted against concentration of test compound, and IC50 values were calculated by using a robust fit for a single order decay (Fig. 4 and 5).
Fig. 4: Single order decay fit with Nicardipine as test compound
Fig. 5: Single order decay fit with Carvedilol as test compound
Correlation of Fluorosome-trans-pgp IC50 Data and Cell Monolayer IC50 Data
IC50 values are given as µM concentrations. Bidirectional transport inhibition studies were conducted on the same set of compounds as were measured with the Fluorosome-trans-pgp assay, examples of which are shown in Fig. 6.
Bidirectional transport inhibition studies employed LLC-MDR1 cell monolayers and [3H]digoxin (0.1 µM) as the substrate essentially as described in. (3)
Fig. 6: Cell monolayer IC50 values reported as Efflux Ratio, i.e. changes in net transport Papp B-A - Papp A-B.
Results using the Fluorosome-trans-pgp assay are in excellent agreement with those obtained by cell culture methodology.
We report a novel assay specific for measuring inhibition of the P-glycoprotein multidrug transporter (Pgp; ABCB1) and an ideal instrument for its use.