Date of Award

12-2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Research Advisor

George C Wood, Ph.D.

Committee

Atul J Shukla, Ph.D. M. Waleed Gaber, Ph.D. Ram I. Mahato, Ph.D. James R. Johnson, Ph.D.

Abstract

Primary brain tumors are a relatively common cause of cancer-related deaths. High-grade gliomas are the most common type of primary brain cancer, and the affected patients have a median survival of less than 1 year. Almost all malignant gliomas are incurable with the present standards of healthcare. Currently accepted therapeutic adjuvants to surgery, such as radiotherapy and chemotherapy, provide only a minor improvement in the disease course and life expectancy for patients diagnosed with malignant gliomas. Often, chemotherapy has failed to make any significant impact on the prognosis of disease because of significant local and systemic toxicity, problems with transport of the drug across the blood brain barrier (BBB), and a high degree of chemoresistance demonstrated by tumor cells. Newer targeted delivery systems with more specificity for gliomas, improved safety profiles, and an enhanced ability to permeate through the BBB are actively under development as the next generation glioma therapies.

Blood brain barrier and vascular endothelial cells in and around glioma brain tumors highly express certain receptors such as transferrin for iron transport into brain tumors respectively. To explore the potential of this tumor induced expression of transferrin receptors for targeting drug carriers, in this study, I have developed and characterized liposome carriers containing paclitaxel, for targeted delivery to the glioma brain tumors.

A liposome drug delivery system specifically aimed at glioma tumors was designed in this study. Liposomes composed of egg phosphotidylchole (EPC), hydrogenated soybean phosphatidylcholine (HSPC), cholesterol, distearoyl phosphoethanolamine-PEG-2000 conjugate (DSPE-PEG) and DSPE-PEG-biotin were prepared by the lipid film hydration and extrusion process. Transferrin (Tf) with affinity for transferrin receptors over-expressed on blood brain barrier and glioma tumor vasculature were coupled to the distal end of poly ethylene glycol coated long circulating liposomes. The liposome delivery system was characterized in terms of size, lamellarity, ligand density, and drug loading properties. The effect of lipid concentration and type in the formulation on paclitaxel loading in the liposomes was studied. Functional properties of the delivery system were evaluated for, i) in vivo blood circulation time using blood sampling method and also using a novel intravital microscopic method, ii) Selective tumor localization in both flank and intracranial glioma models, and iii) anti-tumor efficacy in mouse flank and intracranial glioma tumors. Further, in order to improve physical and chemical stability of the delivery system and hence enhance its shelf life, a lyophilized formulation and process were developed.

Light scattering and electron microscopic observations of the formulations revealed presence of small unilamellar liposomes of about 133 nm in diameter. High performance gel filtration chromatography determinations of ligand coupling to the liposome surface indicated that about 72% of the transferrins were conjugated with biotin groups on the liposome surface. Optimized liposome formulation with 100 mM lipid concentration, 1:33 drug-to-lipid ratio, 5 mol% cholesterol, 5 mol% DSPE-PEG, and 0.01 mol% DSPE-PEG-biotin content yielded 1.3 ± 0.2 mg/mL liposomal paclitaxel with 97.2 ± 3% of the drug being entrapped in the liposomes. These liposomes released no significant amount of the encapsulated drug over 72 hrs at 37°C. Targeted liposomes showed significantly higher rate and extent of tumor accumulation in glioma flank tumors in vivo compared to non-targeted liposomes. Targeted liposomes also possessed long circulating properties with a T1/2 of about 9 hrs in mice. This increased circulation longevity, attributed to steric stabilization effects of PEG, enhanced target accumulation. Near infrared fluorescence imaging demonstrated that these liposomes accumulated selectively in flank tumors with tumor targeting index of 10.59 ± 1.08. Paclitaxel incorporated into the targeted liposomes delayed tumor growth by 7.7 days in 5 doses of 2 mg/Kg body weight. However, no significant tumor growth retardation was observed when paclitaxel was administered in free form (Cremophor EL solubilized form) at similar dose. A process and formulation were developed for freeze-drying the targeted liposome delivery system. Liposome formulations stabilized with 15% sucrose outside the liposomes were able to maintain particle size distribution and drug loading close to initial upon freeze-drying and rehydration.

A stable and effective targeted liposome delivery system was developed for paclitaxel to take this drug selectively to glioma brain tumors. This targeted delivery system could potentially improve therapeutic benefits of anticancer drugs with and increase safety when compared to existing solution dosage forms.

DOI

10.21007/etd.cghs.2007.0073

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