Compartment-specific roles of ATP-binding cassette transporters define differential topotecan distribution in brain parenchyma and cerebrospinal fluid

Abstract

Topotecan is a substrate of the ATP-binding cassette transporters P-glycoprotein (P-gp/MDR1) and breast cancer resistance protein (BCRP). To define the role of these transporters in topotecan penetration into the ventricular cerebrospinal fluid (vCSF) and brain parenchymal extracellular fluid (ECF) compartments, we performed intracerebral microdialysis on transporter-deficient mice after an intravenous dose of topotecan (4 mg/kg). vCSF penetration of unbound topotecan lactone was measured as the ratio of vCSF-to-plasma area under the concentration-time curves. The mean +/- SD ratios for wild-type, Mdr1a/b(-/-), Bcrp1(-/-), and Mdr1a/b(-/-)Bcrp1(-/-) mice were 3.07 +/- 0.09, 2.57 +/- 0.17, 1.63 +/- 0.12, and 0.86 +/- 0.05, respectively. In contrast, the ECF-to-plasma ratios for wild-type, Bcrp1(-/-), and Mdr1a/b(-/-)Bcrp1(-/-) mice were 0.36 +/- 0.06, 0.42 +/- 0.06, and 0.88 +/- 0.07. Topotecan lactone was below detectable limits in the ECF of Mdr1a/b(-/-) mice. When gefitinib (200 mg/kg) was preadministered to inhibit Bcrp1 and P-gp, the vCSF-to-plasma ratio decreased to 1.29 +/- 0.09 in wild-type mice and increased to 1.13 +/- 0.13 in Mdr1a/b(-/-)Bcrp1(-/-) mice, whereas the ECF-to-plasma ratio increased to 0.74 +/- 0.14 in wild-type and 1.07 +/- 0.03 in Mdr1a/b(-/-)Bcrp1(-/-) mice. Preferential active transport of topotecan lactone over topotecan carboxylate was shown in vivo by vCSF lactone-to-carboxylate area under the curve ratios for wild-type, Mdr1a/b(-/-), Bcrp1(-/-), and Mdr1a/b(-/-)Bcrp1(-/-) mice of 5.69 +/- 0.83, 3.85 +/- 0.64, 3.61 +/- 0.46, and 0.78 +/- 0.19, respectively. Our results suggest that Bcrp1 and P-gp transport topotecan into vCSF and out of brain parenchyma through the blood-brain barrier. These findings may help to improve pharmacologic strategies to treat brain tumors.

Figure 1. Topotecan lactone penetration of vCSF after a bolus i.v. dose of 4 mg/kg topotecan

(A) vCSF-to-plasma AUC ratio of unbound topotecan lactone (mean ± SD from 3–6 mice). p<0.001, one-way ANOVA; *p<0.001, post-hoc t-test with Bonferroni correction, of each knockout model compared with wild-type mice. (B) Representative concentration-time plots of unbound topotecan lactone (TPT) in vCSF (□) and plasma (■) in wild-type, Mdr1a/b^−/−, Bcrp1^−/−, and Mdr1a/b^−/−Bcrp1^−/− mice. Model-fitted curves are represented for plasma and brain pharmacokinetic data.

Figure 2. Topotecan lactone penetration into brain ECF after a bolus i.v. dose of 4 mg/kg topotecan

(A) Brain ECF-to-plasma AUC ratio of unbound topotecan lactone (mean ± SD of 3–5 mice). p<0.001, one-way ANOVA; *p<0.001, post-hoc t-test with Bonferroni correction, of each knockout model compared with wild-type mice. (B) Representative unbound topotecan lactone (TPT) concentration-time plots in ECF (△) and plasma (▲) in wild-type, Bcrp1^−/−, and Mdr1a/b^−/−Bcrp1^−/− mice. Model-fitted curves are represented for plasma and brain pharmacokinetic data.

Figure 3. Topotecan penetration into vCSF and brain ECF after 200 mg/kg gefitinib by oral gavage followed by a bolus i.v. dose of 4 mg/kg topotecan

(A) Brain vCSF-to-plasma and ECF-to-plasma AUC ratios of unbound topotecan lactone in wild-type and Mdr1a/b^−/−Bcrp1^−/− mice (mean ±SD from 3 mice). *p<0.05, Student’s t- test. (B) Representative concentration-time plots of unbound topotecan lactone (TPT) in vCSF (□), ECF (△), and plasma (■, ▲) of wild-type and Mdr1a/b^−/−Bcrp1^−/− mice. Model- fitted curves are represented for plasma and brain pharmacokinetic data.

Figure 4. Immunohistochemical staining of Bcrp1 in wild-type and Mdr1a/b^−/−mice

(A) Bcrp1 staining is observed in the brain capillaries of wild-type and Mdr1a/b^−/−mice, with stronger staining in the Mdr1a/b^−/− strain. Negative controls for wild-type and Mdr1a/b^−/− mice are also shown. (B) Processed images from A, showing the stained areas in red. (C) Quantification of the intensity and area of Bcrp1 staining. Data represent the mean ± SD of six different fields from two different sections normalized to the mean values quantified in negative control slides. *p=0.012, Student’s t-test.