N-desmethyl-loperamide is selective for P-glycoprotein among three ATP-binding cassette transporters at the blood-brain barrier

Abstract

[(11)C]N-desmethyl-Loperamide ([(11)C]dLop) is used in positron emission tomography (PET) to measure the in vivo activity of efflux transporters that block the passage of drugs across the blood-brain barrier. The three most prevalent ATP-binding cassette efflux transporters at the blood-brain barrier are P-glycoprotein (P-gp), multidrug resistance protein 1 (Mrp1), and breast cancer resistance protein (BCRP). We sought to measure the selectivity of dLop among these three transporters. The selectivity of dLop at low concentrations (< or =1 nM) was measured both as the accumulation of [(3)H]dLop in human cells that overexpress each transporter and as the uptake of [(11)C]dLop in brains of mice that lack genes encoding P-gp, Mrp1, or BCRP. The selectivity of dLop at high concentrations (> or =20 microM) was measured as the inhibition of uptake of a fluorescent substrate and the change in cytotoxicity of drugs effluxed at each transporter. Accumulation of [(3)H]dLop was lowest in cells overexpressing P-gp, and the uptake of [(11)C]dLop was highest in brains of mice lacking P-gp. At high concentrations, dLop selectively inhibited P-gp function and also decreased the resistance of only the P-gp-expressing cells to cytotoxic agents. dLop is selective for P-gp among these three transporters, but its activity is dependent on concentration. At low concentrations (< or =1 nM), dLop acts only as a substrate; at high concentrations (> or =20 microM), it acts as both a substrate and an inhibitor (i.e., a competitive substrate). Because low concentrations of radiotracer are used for PET imaging, [(11)C]dLop acts selectively and only as a substrate for P-gp.

Fig. 1.

Expression of P-gp, Mrp1, and BCRP in three pairs of cell lines as determined by Western blot analysis. The three pairs are (listed as parental and resistant, respectively) as follows: MCF-7 and MCF-7/VP16 (ABCC1); KB-3-1 and KB-8-5-11 (ABCB1); and H460 and H460/MX20 (ABCG2). A protein ladder was loaded with each Western blot to mark the bands for Mrp1 (190 kDa), P-gp (160 kDa), BCRP (70 kDa), and GAPDH (40 kDa). Cell lysates were probed for GAPDH as a control for the amount of protein loaded. A nonspecific band appears in all the lanes when probed against BCRP.

Fig. 2.

Time-dependent accumulation of [³H]dLop in parental cells of the ABCB1 line. Cells were incubated with [³H]dLop (1 nM), lysed at the times indicated, and assayed for radioactivity. Data represent means ± S.D. of three observations.

Fig. 3.

Accumulation of [³H]dLop by three pairs of parental (□) and drug-resistant (■) cells. Accumulation was significantly different between the parental and resistant cells of only the ABCB1 pair. The amount of [³H]dLop in cells was assayed 45 min after the addition of [³H]dLop (1 nM). Data represent means ± S.D. of three observations. ***, P < 0.001 by unpaired Student's two-tailed t test (α = 0.05).

Fig. 4.

Concentration of radioactivity (% SUV) measured by PET in brains of four strains of mice after injection of [¹¹C]dLop. Three strains of mice were selectively knocked out for the gene that encodes either P-gp (○), Mrp1 (□), or BCRP (■); the fourth strain was wild-type (●). Concentration of radioactivity in abcb1a/b-knockout mice was at least 2.5 times higher (P < 0.0001 by two-way ANOVA) than that in the other three strains of mice. Symbols represent mean ± S.D. values from four mice per strain. For clarity, the concentration of radioactivity taken up into brain in the first 3 min is shown as an inset.

Fig. 5.

The ability of dLop and Lop to inhibit (as competitive substrates) the function of P-gp, Mrp1, and BCRP. Both dLop and Lop selectively inhibited only P-gp function. The cellular accumulation of transporter-specific fluorescent substrate is shown as a bar that represents the mean fluorescence intensity normalized to accumulation of untreated parental cells for a total of 10,000 cells. For each cell line pair, accumulation is shown for untreated parental and resistant cells (white bar), inhibitor-treated resistant cells (black bar), and dLop- or Lop-treated resistant cells (shade-graduated bars). Fluorescent substrates with activity for each transporter were used: rhodamine 123 (Rh123) for P-gp, calcein-AM (Ca-AM) for Mrp1, and mitoxantrone (MTX) for BCRP. In addition to substrate, an inhibitor was used for each ABC transporter: cyclosporin A (CsA; 10 μM) for P-gp, MK-571 (50 μM) for Mrp1, and fumitremorgin C (FTC; 5 μM) for BCRP. Bars represent the mean ± S.D. of three observations. Some error bars are smaller than the line thickness of the bars and, therefore, are not visible.