Date of Award

5-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Molecular Therapeutics and Cell Signaling

Research Advisor

Clinton Stewart, Pharm.D.

Committee

Suzanne Baker, Ph.D. Shannon Matta, Ph.D. John C. Panetta, Ph.D. Cynthia Wetmore, M.D., Ph.D.

Keywords

CNS penetration, Crenolanib, Erlotinib, Microdialysis, Tyrosine Kinase Inhibitors

Abstract

For the past three decades, advances in the treatment of central nervous system (CNS) tumors such as malignant glioma have only been modest. One particular challenge facing treatment of brain tumors is the delivery of therapeutically effective concentrations of anti-cancer agents to the target site in the brain. The sanctuary of the brain is protected by several barrier systems such as the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). These barriers restrict the passage of anti-cancer drugs into the brain via several protective mechanisms.

In the present study, we used cerebral microdialysis sampling, a technique for sampling unbound molecules in brain extracellular fluid (ECF) via semi-permeable probe, to assess the role of murine ATP Binding Cassette (ABC) transporters Bcrp1, P-gp, and Mrp4 in CNS penetration of molecularly targeted agents under investigation for treatment of malignant glioma. We choose the specific inhibitor of epidermal growth factor receptor (EGFR), erlotinib (TarcevaTM), and the specific inhibitor of platelet-derived growth factor receptor (PDGFR), crenolanib, as examples of tyrosine kinase inhibitors currently tested for treatment of malignant glioma. Given the poor microdialysis probe recovery of these lipophilic molecules, we enhanced their recovery by including an affinity-based trapping agent, 10% hydroxypropylbetacyclodextrin (HPBCD), in the perfusate. Using this technique, we studied erlotinib and its major metabolite, OSI-420, penetration in control and transporter-deficient mice. We showed that Bcrp1 is the main efflux transporter preventing erlotinib and OSI-420 penetration in mouse brain. Intracellular accumulation studies confirmed the role of BCRP in erlotinib and OSI-420 transport. We also characterized the role of solute carrier transporters in erlotinib and OSI-420 brain accumulation. Our data show that erlotinib and OSI-420 are substrates for members of the SLC22A family of uptake transporters, OAT3 and OCT2.

We then sought to characterize the disposition of tyrosine kinase inhibitors in malignant glioma using cerebral microdialysis. We decided to use a transgenic mouse model that highly recapitulates several features of the human glioma including tumor histology and genetic profiles. However, the bregma commonly used as a reference point to place microdialysis cannula does not appear on images derived by magnetic resonance imaging (MRI), the imaging method used to identify the size and location of the spontaneously arising tumors. Thus, we realized that a new technique to implant the microdialysis guide cannula would be necessary. Using angiography studies of mouse brain vasculature and T2-weighted MRI, we identified the intersection of the midline suture and the rostral rhinal vein on the mouse brain surface as a reference point for implanting the microdialysis cannula. This point correlated with the intersection between the midline and the olfactory bulb/frontal lobe border visualized on T2- weighted MRI. Our method allowed for accurate placement of microdialysis cannula in tumors developing in several regions of the mouse brain.

While cerebral microdialysis is commonly used to monitor CNS disposition of single anti-cancer drug at a time, the feasibility of simultaneous sampling of multiple anti-cancer agents via cerebral microdialysis has not been reported. However, combining anti-cancer drugs represents a promising strategy for treatment of resistant CNS tumors, as malignant glioma. Given the role played by EGFR and PDGFR in providing multiple inputs for sustaining glioma cell survival and proliferation, combining inhibitors of EGFR (erlotinib) and PDGFR (crenolanib) represents a promising treatment strategy for these tumors. The goal of our last set of studies was to optimize microdialysis conditions to sample crenolanib and erlotinib as single agents or in combination from tumor ECF in a xenograft mouse model of glioma. By including 10% HPBCD in the perfusate, probe recovery of both erlotinib and crenolanib was significantly increased. To estimate probe recovery we used the zero-flow rate (ZFR) which estimated stock concentrations with 15% accuracy. The enhanced recovery achieved by including HPBCD coupled with sensitive analytical techniques allowed us to determine crenolanib and erlotinib penetration in tumor ECF under steady state conditions. No significant differences were observed in drug penetration between groups treated with single agent or those treated with both drugs.

In conclusion, we developed techniques to improve microdialysis probe recovery of lipophilic agents administered as single agents or in combination. We also developed an MRIguided method to implant microdialysis cannula in a spontaneous glioma murine model. These techniques enhance our ability to perform microdialysis studies on a large spectrum of anticancer agents in clinically relevant murine models. Using the developed techniques, we identified efflux and uptake transporters that regulate erlotinib CNS disposition. We evaluated the extent of erlotinib and crenolanib penetration in malignant glioma models. Our results shed more light on the extent of tumor penetration of two tyrosine kinase inhibitors currently being tested for treatment of malignant glioma.

DOI

10.21007/etd.cghs.2012.0083

Comments

Two year embargo expired May 2014

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