Tyrosine kinase inhibitor gefitinib enhances topotecan penetration of gliomas

Abstract

Gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, increases brain parenchymal extracellular fluid (ECF) accumulation of topotecan, a substrate of the ATP-binding cassette (ABC) transporters P-glycoprotein (Pgp/MDR-1) and breast cancer resistance protein (BCRP/ABCG2). The effect of modulating these transporters on topotecan penetration in gliomas has not been thoroughly studied. Thus, we performed intracerebral microdialysis on mice bearing orthotopic human gliomas (U87 and MT330) and assessed topotecan tumor ECF (tECF) penetration and the effect of gefitinib on topotecan tECF penetration and intratumor topotecan distribution. We found that topotecan penetration (P(tumor)) of U87 was 0.96 +/- 0.25 (n = 7) compared with that of contralateral brain (P(contralateral), 0.42 +/- 0.11, n = 5; P = 0.001). In MT330 tumors, P(tumor) (0.78 +/- 0.26, n = 6) and P(contralateral) (0.42 +/- 0.11, n = 5) also differed significantly (P = 0.013). Because both tumor models had disrupted blood-brain barriers and similar P(tumor) values, we used U87 and a steady-state drug administration approach to characterize the effect of gefitinib on topotecan P(tumor). At equivalent plasma topotecan exposures, we found that P(tumor) after gefitinib administration was lower. In a separate cohort of animals, we determined the volume of distribution of unbound topotecan in tumor (V(u,tumor)) and found that it was significantly higher in groups receiving gefitinib, implying that gefitinib administration leads to a greater proportion of intracellular topotecan. Our results provide crucial insights into the role that transporters play in central nervous system drug penetration and provide a better understanding of the effect of coadministration of transporter modulators on anticancer drug distribution within a tumor.

Figure 1

Morphology and growth of U87 and MT330 tumor models. A, Histologic appearance of the xenografts (hematoxylin-eosin stain). Dotted yellow lines indicate the tumor margin. B, Tumor bioluminescence signal versus time. Mean ± SD of 5-15 values and representative bioluminescence pictures are shown.

Figure 2

Topotecan penetration of tumor and contralateral brain ECF after a 4-mg/kg i.v. dose in U87 and MT330 models. A, Comparison of P_tissue means (SD), calculated as AUC_u,tissueECF/AUC_u,plasma topotecan ratios in tumor and in contralateral brain tissue. **P = 0.001; *P = 0.013; t test, as compared to contralateral brain. B, Representative concentration-time graphs (experimental data and model-fitted curves) of topotecan (unbound lactone) in tECF and plasma. Histologic images confirming probe location are shown (yellow dots limit tumor margins, blue dashes limit cannula track, and red dashes limit probe track; lines were drawn with Adobe Photoshop V11.0).

Figure 3

Expression of ABC transporters in U87 and MT330 tumor models. A, Western blots of cells cultured in vitro. B, Immunostaining of tumor xenografts. Negative controls (tissue slides incubated with nonspecific rat IgG) are shown for comparison.

Figure 4

Effect of gefitinib on topotecan ECF concentration in U87 tumors at the steady-state. A, Plasma concentration-time profile of topotecan (unbound lactone) in U87 tumor-bearing mice after insertion of pumps releasing 25 μg topotecan/h and before and after an oral dose of gefitinib (200 mg/kg, 14 h after pump insertion) (n=22; 1-4 plasma samples obtained from each; individual data and model-fitted curve are shown). B, Representative microdialysis data from a U87 tumor-bearing mouse implanted with a pump releasing 25 μg topotecan/h. Topotecan (unbound lactone) concentration in tECF dialysate and plasma are shown before and after administration of gefitinib (200 mg/kg) at 14 h. C, Tumor penetration (P_tumor), as C_ss,tECF/C_ss,plasma ratios calculated from microdialysis-derived tECF and plasma topotecan (unbound lactone) concentrations at the steady-state in mice receiving 25 μg/h topotecan, before (TPT25) and after (TPT25-GEF) receiving gefitinib (200 mg/kg at h 14 of topotecan) and in a group (TPT12-GEF) receiving 12.5 μg/h topotecan and 200 mg/kg gefitinib 0.5 h before topotecan. Individual values and mean ± SD are shown. * P = 0.017, Mann-Whitney test; **P = 0.009 paired t test.

Figure 5

Effect of gefitinib on topotecan accumulation in tumor xenografts in vivo and in tumor cells in vitro. A, V_u,tumor values at steady-state in U87 tumors from mice receiving topotecan 25 μg/h (TPT25), topotecan 25 μg/h and gefitinib 200 mg/kg (TPT25-GEF), or topotecan 12.5 μg/h and gefitinib 200 mg/kg (TPT12-GEF; plasma exposure equivalent to that in the TPT25 group). Individual values and mean ± SD are shown. *P = 0.044, **P = 0.002; one-way ANOVA, post-hoc t-test with Bonferroni correction. B, Topotecan accumulation in tumor cells in vitro in the presence and absence of gefitinib. Values are the percentage of the maximum accumulation (mean ± SD; n=3) in control cells (topotecan 0.1 μM, no gefitinib) at each time point. *P < 0.05, **P < 0.01, as compared to accumulation in control cells at the same time point; one-way ANOVA, post-hoc t-test with Bonferroni correction.

Figure 6

Sensitivity of tumor cells to topotecan (TPT; 100-0.001 μM), gefitinib (GEF; 50-0.05 μM), and combinations of topotecan (100-0.001 μM) and gefitinib (1 and 10 μM). Values are the mean ± SD percentage of growth in untreated cells (n=5).