Date of Award

5-2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Interdisciplinary Program

Research Advisor

Clinton Stewart, Pharm.D.

Committee

Shannon G. Matta, Ph.D. Bernd Meibohm, Ph.D. Alex Sparreboom, Ph.D. Christopher M. Waters, Ph.D.

Abstract

Camptothecin analogs such as topotecan are currently tested in clinical trials for brain tumors. However the clinical outcome is far below the expectations, which are derived from the promising preclinical studies. This discrepancy could be partially attributed to the presence of two barrier systems in the brain, the blood brain barrier (BBB) and blood cerebrospinal fluid (CSF) barrier (BCB). Further investigations have demonstrated multiple ATP-binding cassette (ABC) transporters are present at the two barriers. Camptothecin analogs are well known substrates for several of the transporters. This work defined the role of two ABC transporters, Bcrp1 and P-gp in determination of topotecan CNS penetration in mouse models by using cerebral microdialysis. Microdialysis sampling of drugs in ventricular cerebrospinal fluid (vCSF) provides insight into drug penetration into the brain across the blood CSF barrier (BCB); however, this method has been reported primarily in larger animal species. The initial studies were designed to improve our existing surgical technique to implant a microdialysis cannula into the lateral ventricle to sample vCSF. The modifications done in these studies consisted of changes in the stereotaxic coordinates and insertion of the cannula at a 20° angle. Exact placement of the probe was confirmed using ultrasound (US), micro-computed tomography (CT), and histologic review of serial paraffin sections. Studies of topotecan CSF penetration in the FVB mouse using this modified approach determined that the vCSF to plasma AUC ratio of unbound topotecan lactone was greater than that previously reported using previous surgical technique. Results of studies from this lab have shown that topotecan has differential CNS penetration, and that vCSF topotecan penetration is greater than penetration into brain parenchymal extracellular fluid (ECF). We hypothesize Bcrp1 and P-gp together play a major role in regulation of topotecan CNS penetration. To further characterize the topotecan CNS transport mechanisms, topotecan penetration in two CNS compartments, vCSF and brain parenchymal ECF were determined in several knockout mouse models (Bcrp1(-/-), Mdr1a/b(-/-) and Bcrp1/Mdr1a/b(-/-) by adapting an intracerebral microdialysis technique. After an intravenous (i.v.) bolus dose of 4 mg/kg topotecan, unbound topotecan lactone vCSF penetration was defined by a vCSF to plasma area under the curve ratio (AUCu,vCSF/AUCu,plasma). The topotecan vCSF penetration (AUCu,ECF/AUCu,plasma) was significantly decreased in all three knockout models compared with wild type. The penetration into the brain parenchyma, defined by the ECF to plasma AUC ratio, was not changed in Bcrp1(-/-) but significantly increased in Bcrp1/Mdr1a/b(-/-). Pre-treatment with the EGFR tyrosine kinase inhibitor gefitinib (200 mg/kg) to inhibit Bcrp1 and P-gp transporters significantly decreased topotecan vCSF penetration but increased ECF penetration in FVB wild type. The topotecan lactone to carboxylate AUC ratio in vCSF (AUClactone,CSF/AUCcarboxylate,CSF) was calculated in FVB wild type and three knockout models. Interestingly, a preferential disposition of topotecan lactone over carboxylate was apparent in vCSF of wild type, Bcrp1(-/-) and Mdr1a/b(-/-) but not in Bcrp1/Mdr1a/b(-/-). These studies are the first to carefully assess the contribution of efflux transporters to topotecan penetration into brain ECF and vCSF, and to assess the importance of lactone compared with carboxylate. Whereas these studies have all been conducted in a non-tumor bearing model, we evaluated the effect of the presence of a primary CNS tumor on topotecan CNS penetration. An orthotopic human glioblastoma (luciferase labeled U-87) mouse model was constructed. Preliminary intratumoral microdialysis studies have not found different topotecan disposition in tumor from normal brain. However, the model might be biased due to the position of tumor inoculation and will require further study for clarification. In conclusion, our findings suggest ABC transporters, Bcrp1 and P-gp, play a major role in topotecan CNS penetration by enhancing topotecan penetration into vCSF and restricting penetration into ECF. This insight can be translated in clinical studies to improve topotecan CNS penetration.

DOI

10.21007/etd.cghs.2008.0288

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Neurosciences Commons

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