Montelukast is a potent and durable inhibitor of multidrug resistance protein 2-mediated efflux of taxol and saquinavir

Abstract

The ATP binding cassette (ABC)-transporters are energy dependent efflux pumps which regulate the pharmacokinetics of both anti-cancer chemotherapeutic agents, e.g. taxol, and of human immunodeficiency virus-1 (HIV-1) protease inhibitors (HPIs), e.g. saquinavir. Increased expression of several ABC-transporters, especially P-glycoprotein (P-gp) and multidrug resistance protein 2 (MRP2), are observed in multidrug resistant (MDR) tumor cells and on HIV-1 infected lymphocytes. In addition, due to their apical expression on vascular endothelial barriers, both P-gp and MRP2 are of crucial importance towards dictating drug access into sequestered tissues. However, although a number of P-gp inhibitors are currently in clinical trials, possible inhibitors of MRP2 are not being thoroughly investigated. The experimental leukotriene receptor antagonist (LTRA), MK-571 is known to be a potent inhibitor of MRP transporters. Using the MRP2 over-expressing Madin-Darby canine kidney cell line, MDCKII-MRP2, we evaluated whether the clinically approved LTRAs, e.g. montelukast (Singulair) and zafirlukast (Accolate), can similarly suppress MRP2-mediated efflux. We compared the efficacy of increasing concentrations (20-100 microM) of MK-571, montelukast, and zafirlukast, in suppressing the efflux of calcein-AM, a fluorescent MRP substrate, and the radiolabeled [(3)H-] drugs, taxol and saquinavir. Montelukast was the most potent inhibitor (p<0.01) of MRP2-mediated efflux of all three substrates. Montelukast also increased (p<0.01) the duration of intracellular retention of both taxol and saquinavir. More than 50% of the drugs were retained in cells even after 90 min post removal of montelukast from the medium. Our findings implicate that montelukast, a relatively safe anti-asthmatic agent, may be used as an adjunct therapy to suppress the efflux of taxol and saquinavir from MRP2 overexpressing cells.

Figure-1

MRP2 gene expression and protein levels in MDCKII and MDCKII-MRP2 cells. In (A), RT-PCR amplification of mRNAs isolated from MDCKII and MDCKII-MRP2 cells were carried out. The PCR products were separated in an agarose gel containing ethidium bromide. A representative gel picture (n=4) showing the molecular weight markers (M), and bands for human MRP2 (450 bp) and GAPDH (225 bp) products, are depicted. In (B), immunofluorescence microscopy (IFM) of human MRP2 was carried out in both MDCKII and MDCKII-MRP2 cells by using an Alexa Fluor-tagged anti-MRP2. A representative photograph (n=3) showing MRP2 localization is depicted. Dapi stained nuclei are shown in blue and MRP2 staining is shown in green. The white bars represent 10 micro meter, and the red arrows are depicting perinuclear staining of MRP2. The MDCKII-MRP2 cells show constitutively higher expression of MRP-2 as compared to the parental MDCKII cells.

Figure-2

Suppression of MRP2-mediated calcein-AM efflux by the MRP inhibitors. The fluorescent MRP substrate, calcein-AM was used to measure efflux function in both MDCKII and MDCKII-MRP2 cell lines. Intracellular calcein retention is presented as mean fluorescent units (MFU) per μg of protein in cell extracts. Changes in intracellular fluorescence, in the absence (lane-1) or presence of the inhibitors (100 μM each), MK-571 (lane-2), montelukast (lane-3), zafirlukast (lane-4) or probenecid (lane-5), is shown. Bar graphs represent data from 4-5 separate experiments carried out in quadruplicate cultures, and error bars depict the standard error of means (±SEM). The symbols, * represent P<0.05 and ** represent P<0.01. Montelukast showed the highest MRP2 inhibitory potential.

Figure-3

Suppression of MRP2-mediated taxol and saquinavir efflux by the MRP inhibitors. Intracellular accumulation of ³H-taxol (A) or ³H-saquinavir (B) were measured, in the absence (lane-1) or presence of MK-571 (lane-2), montelukast (lane-3), zafirlukast (lane-4), or probenecid (lane-5). Cells were pre-exposed to the inhibitors (100 μM) for 15 mins followed by addition of the substrates and further incubation for 2 hrs. Plates were washed and cell extracts were obtained to monitor intracellular taxol or saquinavir retention, represented as radioactivity (CPM) per μg of protein. In each panel, bar graphs represent data from 3-4 independent experiments carried out in triplicates and error bars depict ±SEM. The symbols, * and ** represent P<0.05 and P<0.01, respectively. Montelukast was found to inhibit the MRP2-mediated efflux of both taxol and saquinavir.

Figure-4

Comparing the MRP2 inhibitory potencies of MK-571 and montelukast. The concentration dependent effect of MK-571 or montelukast (20-100 μM) on inhibition of calcein (A), taxol (B) or saquinavir (C) efflux from the MDCKII-MRP2 cells was assessed. Data obtained from three independent experiments (n=3) are shown. In each bar graph, data is presented as fold change as compared to controls (no inhibitor added). Error bars depict ±SEM and symbols, * and ** represent P<0.05 and P<0.01, respectively. At equimolar concentrations of each inhibitor, significant differences between MK-571 and montelukast are shown as P-values above the bar graphs. Montelukast is a more potent inhibitor of MRP2-mediated efflux of taxol than MK-571, and is similar in potency to MK-571 with respect to the inhibition of saquinavir efflux.

Figure-5

Montelukast is a durable inhibitor of MRP2-mediated taxol and saquinavir efflux. We wanted to measure the duration of MRP2 inhibition by MK571 or montelukast. The MDCKII-MRP2 cells were exposed to the inhibitors (100 μM each) for 30 mins prior to the incubation of cells with taxol (A) or saquinavir (B) for 2 hrs. Following the loading step in the presence of the inhibitors, cells were thoroughly washed and cultured in the absence of inhibitors. Intracellular radioactivities (CPM/μg protein) were determined at different time points (15-90 mins) post wash. Data presented in the line graphs depict fold change in radioactivity, as compared to the control values obtained at the 0-time point. These studies showed the peak-loading and temporal-efflux of taxol and saquinavir. Error bars depict the ±SEM of values, and significant differences are depicted as P-values (*, P<0.05 and **, P<0.01).

Figure-6

Structures of LTRAs, MK571, montelukast, zafirlukast and pranlukast. The structure of leukotriene D4 (LTD₄) is compared to the synthetic LTRAs. The first LTRA, MK-571 was developed by a dithioacetal linkage incorporation and replacement of carboxylic acids by an amide moiety. A quinolone-containing structure generated from MK-571 was developed to first yield verlukast (MK679) and the enantiomers were resolved to generate montelukast (Singulair®). Initial SAR analyses led to the fabrication of an indole-containing compound to yield zafirlukast and lastly, by introducing a tetrazole and a lipid backbone, the newly approved LTRA, pranlukast (ONO-1078) (Ultair®) was generated.