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. 2009 Aug 15;69(16):6573-80.
doi: 10.1158/0008-5472.CAN-09-1075. Epub 2009 Aug 4.

Small-molecule multidrug resistance-associated protein 1 inhibitor reversan increases the therapeutic index of chemotherapy in mouse models of neuroblastoma

Catherine A Burkhart  1 , Fujiko Watt, Jayne Murray, Marina Pajic, Anatoly Prokvolit, Chengyuan Xue, Claudia Flemming, Janice Smith, Andrei Purmal, Nadezhda Isachenko, Pavel G Komarov, Katerina V Gurova, Alan C Sartorelli, Glenn M Marshall, Murray D Norris, Andrei V Gudkov, Michelle Haber
Affiliations

Small-molecule multidrug resistance-associated protein 1 inhibitor reversan increases the therapeutic index of chemotherapy in mouse models of neuroblastoma

Catherine A Burkhart et al. Cancer Res. .

Abstract

The multidrug resistance-associated protein 1 (MRP1) has been closely linked to poor treatment response in several cancers, most notably neuroblastoma. Homozygous deletion of the MRP1 gene in primary murine neuroblastoma tumors resulted in increased sensitivity to MRP1 substrate drugs (vincristine, etoposide, and doxorubicin) compared with tumors containing both copies of wild-type MRP1, indicating that MRP1 plays a significant role in the drug resistance in this tumor type and defining this multidrug transporter as a target for pharmacologic suppression. A cell-based readout system was created to functionally determine intracellular accumulation of MRP1 substrates using a p53-responsive reporter as an indicator of drug-induced DNA damage. Screening of small-molecule libraries in this readout system revealed pyrazolopyrimidines as a prominent structural class of potent MRP1 inhibitors. Reversan, the lead compound of this class, increased the efficacy of both vincristine and etoposide in murine models of neuroblastoma (syngeneic and human xenografts). As opposed to the majority of inhibitors of multidrug transporters, Reversan was not toxic by itself nor did it increase the toxicity of chemotherapeutic drug exposure in mice. Therefore, Reversan represents a new class of nontoxic MRP1 inhibitor, which may be clinically useful for the treatment of neuroblastoma and other MRP1-overexpressing drug-refractory tumors by increasing their sensitivity to conventional chemotherapy.

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Figures

Figure 1
Figure 1
MRP1 expression in MRP+/+ or MRP−/− murine neuroblastoma tumor cells from MRP+/+/hMYCN or MRP−/−/hMYCN transgenic mice. A, Neuroblastoma tumors were harvested from hMYCN transgenic mice that were MRP1+/+ or MRP1−/−. Membrane proteins were purified from tumor cells and MRP1 levels determined by western blot. Representative samples from each MRP1 genotype are shown. MCF7/VP cells, which overexpress MRP1, were used as a positive control. B, Effect of MRP1 status on the sensitivity of neuroblastoma cells to chemotherapeutic drugs. MRP+/+ or MRP−/− murine neuroblastoma allografts were established in nude mice. Cytotoxic drug treatment vincristine (0.2mg/kg), etoposide (12mg/kg)) commenced when allografts were approximately ~100–125 mg in size and continued for 5 consecutive days. The growth delay factor is the growth delay calculated from 2.5X the tumor starting volume and normalized by subtracting the growth delay of the saline controls. The number of mice per treatment group is as follows: vincristine-MRP1+/+ n=8 and MRP1−/− n=7; etoposide -MRP1+/+ n=20 and MRP1−/− n=17. Error bars indicate standard error of the mean.
Figure 2
Figure 2
Fold accumulation of daunorubicin in parental MCF7 or MRP1-overexpressing MCF7/VP human breast cancer cells in the presence of hit compounds (structures of the pyrazolopyrimidine class of MRP1 inhibitors are shown). Data are presented relative to verapamil controls. Basal accumulation in the absence of any modulator is indicated by the dotted line. Compounds indicated by the black bars are representative hits from screening A. Compounds indicated with the gray bars represent the best compounds identified in the screening B. A representative experiment is presented.
Figure 3
Figure 3
Effects of pyrazolopyrimidines on MRP1-expressing tumor cells. A, Total cell lysates (20 μg) were isolated from each tumor cell line and separated by SDS-PAGE. Western blots were probed with a MRP1 specific antibody and anti-rat secondary antibody. MCF7/VP (2 μg) was included as a positive control. B, Tumor cell lines were treated with a range of concentrations of doxorubicin, vincristine, or etoposide in the presence or absence of 10 μM pyrazolopyrimidines. Verapamil and MK571 served as control transporter modulators. Data are presented as fold sensitization (IC50 drug alone/IC50 drug + modulator).
Figure 4
Figure 4
Efficacy and safety of Reversan. Upon development of a tumor, hMYCN transgenic mice were treated with conventional chemotherapeutic agents (0.2 mg/kg vincristine (A) or 6 mg/kg etoposide (B)) in the presence or absence of 10 mg/kg Reversan daily for 5 consecutive days. Mice were followed until signs of tumor progression were evident. C, 7-week old female BALB/c mice (n=5/dose/treatment group except for 0.4 mg/kg vincristine ±10 mg/kg Reversan, where n=18/treatment group) were treated ip with 0.05–0.4 mg/kg vincristine for 5 consecutive days in the presence or absence of 10 mg/kg Reversan or 10 mg/kg cyclosporin A and observed for signs of toxicity for up to 14 days. Mouse survival per vincristine dose is presented. D, Blood was collected from BE(2)-C tumor bearing nude mice following treatment with 9 mg/kg etoposide ± 10 mg/kg Reversan and analyzed for blood cell parameters, including total white blood cells (WBC), neutrophils (NEUT), lymphocytes (LYM), monocytes (MONO) and eosinophils (EOS).
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