The treatment of malignant brain tumors, especially of high-grade gliomas, poses one of the major challenges in cancer therapy. Despite seminal advancements in imaging techniques for the early diagnosis of CNS neoplasms as well as amendments in surgery and radiooncology, the prognosis of patients with glioma is very poor. The administration of temozolomide adjuvant to irradiation after resection of the tumor - the current “gold standard” in the treatment of glioblastoma - has only marginally improved median survival by ca. 3 months. Hence, there is an urgent need for novel approaches to the treatment of malignant brain tumors. To investigate the efficiency of such regimens in vivo, predictive preclinical animal models are indispensible.
In this thesis, three subject areas were dealt with:
- evaluation of doxorubicin-loaded poly(butylcyanoacrylate) (PBCA) nanoparticle (NP) formulations as drug-carriers,
- modulation of the ATP-binding cassette transporter ABCG2, which is expressed in brain capillaries, by newly synthesized inhibitors, as a strategy to overcome the blood-brain barrier (BBB) with chemotherapeutics, which are substrates of this efflux pump, and
- refinement of orthotopic glioblastoma models in nude mice to allow noninvasive optical imaging of intracerebral tumors.
Human glioblastoma cells U-87 MG, U-118 MG and U-373 MG were incubated with free doxorubicin and doxorubicin loaded onto PBCA NPs. As the glioblastoma cells do neither express ABCB1 nor ABCG2, Kb-V1 and MCF-7/Topo cells were included, to study effects on both efflux pumps. In the crystal-violet chemosensitivity assay, cytotoxicities of the doxorubicin-loaded NPs and of the dissolved cytostatic were similar against all glioblastoma cells. By contrast, a higher efficacy of the NP formulations was observed on ABCB1-overexpressing Kb-V1, but not on ABCG2-expressing MCF-7/Topo cells, indicating an interaction of the NPs with the ABCB1 efflux pump. Therefore, increased drug concentrations in the brain after administration of such drug-loaded NPs to animals, as reported in literature, might be due interactions of the NPs with ABCB1 at the BBB.
The modulation of ABC transporters at the BBB represents an attractive strategy to increase intracerebral levels of drugs, which are known to be substrates of such efflux pumps.
For this reason, the identification of potent efflux pump inhibitors is a promising approach to improve the chemotherapy of brain tumors. A series of analogs, derived from the original ABCB1 inhibitor tariquidar, was investigated for potency against ABCG2 and selectivity vs. ABCB1. To expand methodology and to by-pass problems associated with the flow cytometric (FACS) mitoxantrone-efflux assay, two 96-well plate assays, using the fluorescent ABCG2 substrates Hoechst 33342 and pheophorbide a, were developed. Interestingly, slight structural modifications of tariquidar analogs yielded potent and selective inhibitors of ABCG2. The most potent modulator showed an IC50 value of 60 nM in the FACS assay and 65 and 126 nM in the microtiter plate assays. In view of future in vivo studies, the most potent inhibitors were investigated for stability in plasma by the means of HPLC-MS analysis. Unfortunately, these compounds are prone to rapid enzymatic cleavage at the central benzamide moiety. Therefore, compounds with improved drug-like properties are required. Since a regulation of ABCG2 by estrogens is discussed in literature, MCF-7/Topo cells were treated with estradiol and subjected to treatment with topotecan. No differences in the antiproliferative effect between estradiol-treated and non-treated controls were observed, indicating, at best, a therapeutically irrelevant regulation of the ABCG2-level by estradiol.
To investigate the therapeutic value of new concepts, e.g. the combined administration of selective inhibitors of ABC-transporters with appropriate cytostatics, the main focus of this work was the refinement of in vivo xenograft models. Indeed, orthotopic brain tumor models, allowing the monitoring of tumor progression by noninvasive optical imaging, were established in nude mice. For this purpose, human U-87 MG glioblastoma cells were transfected with the genes encoding luciferase2 (Luc2) and the recently discovered far-red fluorescent protein Katushka (Kat), respectively. Numerous clones were characterized in vitro with respect to bioluminescence / fluorescence, growth kinetics and chemosensitivity. As the transfectants were tumorigenic subcutaneously (s.c.), and Luc2 as well as Kat expression persisted in the s.c. model, the human glioblastoma variants were injected into the brains of nude mice, where reproducibly growing tumors developed. Most important is the fact that these intracerebral xenografts were accessible to both, bioluminescence and fluorescence imaging. Moreover, there was a direct correlation between the optical signals and the tumor load, confirmed by histology
