Development of a Prodrug of Camptothecin for Enhanced Treatment of Glioblastoma Multiforme

Abstract

A novel therapeutic approach for glioblastoma multiforme (GBM) therapy has been carried out through in vitro and in vivo testing by using the prodrug camptothecin-20-O-(5-aminolevulinate) (CPT-ALA). The incorporation of ALA to CPT may promote uptake of the cytotoxic molecule by glioblastoma cells where the heme synthesis pathway is active, improving the therapeutic action and reducing the side effects over healthy tissue. The antitumor properties of CPT-ALA have been tested on different GBM cell lines (U87, U251, and C6) as well as in an orthotopic GBM model in rat, where potential toxicity in central nervous system cells was analyzed. In vitro results indicated no significant differences in the cytotoxic effect over the different GBM cell lines for CPT and CPT-ALA, albeit cell mortality induced by CPT over normal cell lines was significantly higher than CPT-ALA. Moreover, intracranial GBM in rat was significantly reduced (30% volume) with 2 weeks of CPT-ALA treatment with no significant side effects or alterations to the well-being of the animals tested. 5-ALA moiety enhances CPT diffusion into tumors due to solubility improvement and its metabolic-based targeting, increasing the CPT cytotoxic effect on malignant cells while reducing CPT diffusion to other proliferative healthy tissue. We demonstrate that CPT-ALA blocks proliferation of GBM cells, reducing the infiltrative capacity of GBM and promoting the success of surgical removal, which improves life expectancy by reducing tumor recurrence.

Keywords: 5-aminolevulinic acid; blood−brain barrier; camptothecin; glioblastoma multiforme; targeting.

Figure 1

Synthesis scheme for CPT-ALA.

Figure 2

MTT cell viability assays in GBM cell lines and normal CNS cells after 24 and 72 h incubation. Drug concentration is referred to CPT equivalent (lower scale). Cell viability data are expressed as the mean ± SEM: (A) cortical cells (n = 10); (B) astrocytes (n = 10); (C) C6 cell line (n = 6); (D) U87 cell line (n = 7); (E) U252 cell line (n = 7).

Figure 3

Flow cytometry results after incubation of C6 cells for 24 h with increasing concentrations of CPT-ALA, CPT+5-ALA, and CPT. (A–D) Flow cytometry results after incubation of C6 cells for 24 h with CPT-ALA. X-axis: PI fluorescence (PE filter). Y-axis: FITC fluorescence. Representative scatter plots (Q1, early apoptotic cells; Q2, late apoptosis cells; Q3, healthy cells; Q4, necrotic cells) of cells with no treatment (A), 0.002 μg of CPTeq/mL (B), 0.4 μg of CPTeq/mL (C), and 1.6 μg of CPTeq/mL (D). (E) Percentage of viable, apoptotic, and necrotic cells treated with CPT-ALA, CPT+5-ALA, and CPT (n = 5). Untreated cells were used as negative controls. Data are expressed as the mean ± SD. Viable cells showed significant differences between CPT-ALA (***p < 0.001), CPT+5-ALA (*p < 0.05), and CPT (***p < 0.001) groups and the untreated group. Apoptotic cells showed significant differences between CPT-ALA (***p < 0.001), CPT+5-ALA (*p < 0.05), and CPT (*p < 0.05) groups and the untreated group.

Figure 4

Cell cycle phase study of C6 cells after incubation for 6 and 24 h with increasing concentrations of CPT-ALA, CPT+5-ALA, and CPT. (A–D) Cell cycle phase reports after 24 h incubation with CPT-ALA: control (A), 0.002 μg of CPTeq/mL (B), 0.4 μg of CPTeq/mL (C), and 1.6 μg of CPTeq/mL (D). (E, F) Percentage of cells in each phase of the cell cycle (G0/G1, S, and M) vs CPT-ALA, CPT+5-ALA, and CPT after 6 h incubation (E) and 24 h incubation (F) (n = 5). Legend: P4, phase G0/G1; P5, phase S; P6, phase M. Data are expressed as the mean ± SD. Statistical significance (***p < 0.001) compared to the untreated group for CPT-ALA group; statistical significance (***p < 0.001) compared to the untreated group for CPT+5-ALA group; statistical significance (***p < 0.001) compared to the untreated group for CPT group.

Figure 5

Hematoxylin and eosin staining of histological slices from CPT-ALA and control groups. The therapy was extended for 2 weeks (0.8 mg of CPTeq/kg, 4 doses): (a–d) heart; (e–h) kidney; (i–l) spleen; (m–p) liver; (q–t) lung.

Figure 6

Antitumor activity of CPT-ALA against glioblastoma in Wistar rats. (A–C) Brain slices tissue reconstruction of the control group (n = 3). (D–F) Brain slices tissue reconstruction of CPT-ALA group (0.8 mg of CPTeq/kg, 4 doses in 2 weeks) (n = 5). Nuclei were stained with HOECHST (blue) and astrocytes with GFAP (red). Scale bar: 1 mm. (G) Normalized tumor volume comparison: control group (n = 3); CPT-ALA group (n = 5). Data are expressed as the mean ± SEM: *p < 0.05. (H) 3D representation of normalized tumor volume. The blue outer sphere corresponds to the control group, whereas the green inner sphere refers to the CPT-ALA group.

Figure 7

Brain histology and immunohistochemistry after administration of CPT-ALA against glioblastoma in Wistar rats. (A–C) Cystic glioblastoma brain sample reconstruction in a Wistar rat specimen treated with CPT-ALA (0.8 mg of CPTeq/kg, 4 doses in 2 weeks). Scale bar 50 μm. (D–I) GFAP expression in glioblastoma and brain tissue in control group (n = 3) (D–F) and CPT-ALA group (n = 5) (G–I). Scale bar: 250 μm. (J) Percentage of proliferative cells inside tumor in control group (n = 3) and CPT-ALA group (n = 5). Data are expressed as the mean ± SEM: *p < 0.05. Nuclei were stained with HOECHST (blue) and astrocytes with GFAP (red) except (C) where nuclei were stained with hematoxylin and cytoplasm with eosin.

Figure 8

Apoptotic cells stained with TUNEL label (green) in brain slices of control and CPT-ALA treated animals. Nuclei were stained with HOECHST (blue).