Date of Award

12-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Research Advisor

Ram I. Mahato, Ph.D.

Committee

Sarka Beranova-Giorgianni, Ph.D. Tomoko Fujiwara, Ph.D. Duane D. Miller, Ph.D. Xin A. Zhang, Ph.D.

Abstract

Current prostate cancer treatment remains ineffective primarily due to ineffectual therapeutic strategies and numerous tumor-associated physiological barriers which hinder efficacy of anticancer agents. Therefore, the focus of this study was to investigate a new combination therapy approach for treating prostate cancer and develop polymeric nanocarriers to facilitate anticancer drug and nucleic acid delivery.

It was hypothesized that simultaneously targeting androgen-androgen receptor (AR) and X-linked inhibitor of apoptosis protein (XIAP) signaling pathways would be effective in treating prostate cancer. The effect of bicalutamide (antiandrogen) and embelin (XIAP inhibitor) on the growth of prostate cancer cells in vitro and in vivo was first examined. Embelin induced caspase 3 and 9 activation in LNCaP and C4-2 cells by decreasing XIAP expression and was more potent than bicalutamide in killing prostate tumor cells irrespective of their androgen status. Using a combination of MTT assay and isobologram analyses, combination of bicalutamide and embelin was observed to be cell line and schedule dependent. Since bicalutamide and embelin are extremely hydrophobic, polymeric micelles were fabricated using polyethylene glycol-bpolylactic acid (PEG-b-PLA) copolymer to improve drug solubility. Micellar formulations were found to result in at least 60-fold increase in the aqueous solubility of bicalutamide and embelin. Tumor growth was also effectively regressed upon treatment with bicalutamide, but the extent of tumor regression was significantly higher when bicalutamide was formulated in micelles.

To further improve bicalutamide aqueous solubility, a series of novel biodegradable copolymers for the systematic micellar delivery of bicalutamide was designed and synthesized. Flory-Huggins interaction parameter (χFH) was used to assess compatibility between bicalutamide and poly (L-lactide) or poly (carbonate-co-lactide) polymer pairs. Polyethylene glycol-b-poly (carbonate-co-lactide) [PEG-b-P(CB-co-LA)] copolymers were synthesized and characterized by NMR and gel permeation chromatography. Micelles formulated using these copolymers had average diameter of 100 nm and distinct spherical shape. Drug loading studies revealed that adding the carbonate monomer could increase bicalutamide loading. Among the series, drug loading of micelles formulated with PEG-b-P(CB-co-LA) copolymer containing 20 mol% carbonate was about four-fold higher than PEG-b-PLLA and aqueous solubility of bicalutamide increased from 5 to 4000 µg/mL. CMC values for PEG-b-P(CB-co-LA) copolymers was up to 10-fold lower than those of PEG-b-PLLA. Bicalutamide-loaded PEG-bP(CB-co-LA) micelles showed significant inhibition of LNCaP cell growth in a dose dependent manner which was similar to the methanol solution of free drug.

Bicalutamide tends to act as an agonist rather than an antagonist after prolonged treatment. Hence, a second generation antiandrogen ((S)-N-(4-cyano-3-(trifluoromethyl) phenyl)-3-((4-cyanophenyl)(methyl)amino)-2-hydroxy-2-methylpropanamide) (CBDIV17)) was synthesized and its effect in combination with XIAP inhibitors for treating advanced prostate cancer was determined. CBDIV17 was more potent than bicalutamide and inhibited proliferation of C4-2 and LNCaP cells. CBDIV17-induced apoptosis more effectively compared to bicalutamide and significantly inhibited DNA replication. Combination of CBDIV17 and embelin resulted in supra-additive antiproliferative and apoptotic effects. Embelin downregulated AR expression and decreased androgen-mediated AR phosphorylation at Ser81. These hydrophobic drugs were solubilized using micelles prepared using polyethylene glycol-b-poly (carbonate-co-lactide) (PEG-b-p(CB-co-PLA)) copolymer. Combination therapy inhibited prostate tumor growth more effectively compared to control or monotherapy in vivo.

Polymeric micelles tend to be unstable in vivo. To address this, lactic acid- and carbonate-based biodegradable core- and core-corona crosslinkable copolymers for anticancer drug delivery were synthesized and evaluated. Methoxy poly (ethylene glycol)-b-poly (carbonate-co-lactide-co-5-methyl-5-allyloxycarbonyl-1,3-dioxane-2-one) [mPEG-b-P(CB-coLA-co-MAC)] and methoxy poly (ethylene glycol)-b-poly(acryloyl carbonate)-b-poly (carbonate-co-lactide) [mPEG-b-PMAC-b-P(CB-co-LA)] copolymers were synthesized by ring opening polymerization of LA, CB and MAC using mPEG as an macroinitiator and 1,8- diazabicycloundec-7-ene (DBU) as a catalyst. These amphiphilic copolymers which exhibited low polydispersity (1.08) and CMC values (0.8-1 mg/L) were used to prepare micelles with or without drug and stabilized by crosslinking via radical polymerization of double bonds introduced in the core and interface to improve stability. 1 H NMR and IR spectroscopy confirmed successful crosslinking (crosslinking efficiency of ~70%) while light scattering and transmission electron microscopy were used to determine micelle size and morphology. Crosslinked micelles demonstrated enhanced stability against extensive dilution with aqueous solvents and in the presence of physiological simulating serum concentration.

Since nucleic acids are not susceptible to chemoresistance, it was determined whether simultaneous AR and XIAP gene silencing via RNA interference (RNAi) has the potential to treat hormone refractory prostate cancer. Small interfering RNAs (siRNAs) targeting three different regions of AR and XIAP were screened by transfecting LNCaP and C4-2 prostate cancer cells and their silencing effects determined at mRNA and protein levels by real time RTPCR, Western blot and ELISA. The most potent siRNAs against AR and XIAP were selected and their combination was more effective in inducing apoptosis and inhibiting C4-2 cell proliferation. N-(2-hydroxypropyl) methacylamide (HPMA) based copolymer containing polycationic, lipid and pka modulator moieties was developed for delivering the most potent siRNA and bipartite plasmid encoding two different shRNAs targeting AR and XIAP. HPMA based copolymer showed efficient cellular uptake in prostate cancer cells.

The findings reported in this work demonstrate the potential benefit of combination therapy targeting AR and XIAP pathways for treating prostate cancer using polymeric nanocarriers. Systematic chemical tailoring of polymers for improving drug loading as well as efforts made to improve micelle stability through crosslinking also generated insights which can be applied to other drugs and cancer types. Results reported in this study therefore offer a new way of addressing the time old problem of treating cancer.

DOI

10.21007/etd.cghs.2012.0062

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