In Vitro and in Vivo Reversal of P-Glycoprotein-mediated Multidrug Resistance by a Novel Potent Modulator, XR9576

The effect of modulators on the accumulation of[ ³H]daunorubicin by EMT6/AR1.0 cells. The cells were incubated with various concentrations of XR9576 (•), GG918 (▪), or 100 μm Vpm plus [³H]daunorubicin for 1 h before measuring cell-associated radioactivity as described in“ Materials and Methods.” Results are expressed as a percentage of that seen in the presence of 100 μm Vpm. Data plotted, the mean ± SD of quadruplicate data points.

Inhibition of Rh123 efflux from 2780AD cells by XR9576 and CsA. After a 1-h loading period in the presence of Rh123 and XR9576 at 300 nm (▪), 200 nm (▴), 100 nm (▾), and 50 nm (♦) or CsA at 20 μm (•), the cells were washed and incubated in fresh medium for the indicated times (efflux period). At the end of the efflux periods, the cell-associated Rh123 was measured as described in “Materials and Methods.”

Effect of XR9576 on efflux of[ ³H]daunorubicin from EMT6/AR1.0 cells. EMT6/AR1.0 cells were loaded with [³H]daunorubicin in the presence or absence of the indicated concentrations of XR9576. Cells were then washed and allowed to efflux in the presence (—–) or absence (- - - -) of the modulator for the indicated time periods. Data are plotted relative to cells in the presence of XR9576 at T₀. Each point, the mean of quadruplicate measurements.

Persistence of activity in EMT6/AR1.0 cells after removal of modulators from the medium. EMT6/AR1.0 cells were exposed to the modulator for 1 h, washed, and incubated in modulator-free medium for the indicated periods before the addition of[ ³H]daunorubicin and further incubation for 1 h. Cell-associated [³H]daunorubicin was measured along with the cell number as detailed in the “Materials and Methods” section. Time points, incubation period between removal of the modulator and the addition of [³H]daunorubicin for the accumulation assay. The values represent the mean of triplicate determinations.

Inhibition of [³H]azidopine labeling of P-gp by XR9576, CsA, and Vpm. Crude H69/LX4 membrane extracts were incubated with modulator and [³H]azidopine prior to UV cross-linking as described in “Materials and Methods.”[ ³H]azidopine binding to P-gp was visualized by SDS-PAGE followed by autoradiography.

A and B, effect of p.o. administration of XR9576 and GG918 on the antitumor activity of doxorubicin against the MC26 mouse colon carcinoma. Tumor slurry was implanted s.c., and 24 h later, the animals were randomized and treated once only. The modulator was administered p.o. 1 h before doxorubicin (5 mg/kg) i.v. Animals were weighed twice a week, and the tumor weight was determined on day 14 after implantation as detailed in“ Materials and Methods.” The mean tumor weights ± SE, from groups of 15–18 animals are shown. The Ps refer to comparison of doxorubicin-alone group with the combination-schedule groups. Vehicle, 5% dextrose; Dox, doxorubicin. C and D, body weights for the various treatment groups after tumor implantation.

Effect of p.o. administration of XR9576 on the antitumor activity of paclitaxel in mice bearing the sensitive A2780 ovarian carcinoma xenografts. Nude mice bearing established s.c. A2780 tumors were treated on day 0, 2, and 4 (arrows) as described in“ Materials and Methods.” The modulator was administered 2 h before the cytotoxic agent. The values represent mean ± SE of six animals per group. The growth rate of the tumor was significantly reduced (P < 0.01) by administration of paclitaxel and by paclitaxel in combination with XR9576. There was no difference in the response between these two groups.

Restoration of paclitaxel antitumor effect on MDR 2780AD ovarian carcinoma xenografts by XR9576. Effects of p.o. (A) and i.v. (B) administration of XR9576 in combination with paclitaxel on the growth rate of the tumors. Animals bearing s.c. tumors were treated three times at 2-day intervals. Arrows, days of treatment. The modulator was administered p.o. 2 h before and i.v. 1 h before paclitaxel. The values represent mean ± SE of six animals per group. The growth rate of the tumor was significantly reduced (P < 0.01) by coadministration of XR9576 with paclitaxel compared with drug-alone or vehicle-treated groups.

Sensitization of etoposide antitumor effect on H69/LX4 SCLC xenografts by XR9576. Effects of p.o. (A) and i.v. (B) administration of XR9576 in combination with etoposide on the growth rate of tumors. Animals bearing s.c. tumors were treated three times at 5-day intervals. Arrows, the days of treatment. The modulator was administered p.o. 2 h before and i.v. at the same time as etoposide. Each group consisted of eight animals, and the values represent mean ± SD. The growth rate of the tumors was significantly (P < 0.01, Mann-Whitney U test) reduced by coadministration of XR9576, either p.o. or i.v. with etoposide compared with drug-alone or vehicle-treated groups.

Effect of XR9576 administered p.o. on the activity of vincristine in mice bearing H69/LX4 SCLC xenografts. Animals bearing s.c. tumors were treated 3 times at 6-day intervals. Arrows, the days of treatment. The modulator was administered 2 h before the cytotoxic agent. Each group consisted of five to seven animals. The growth rate of the tumors was significantly (P < 0.01, Mann-Whitney U test) reduced by administration of XR9576 with vincristine compared with cytotoxic-drug-alone or vehicle-treated groups.