Graduation Date

Summer 8-14-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pharmaceutical Sciences

First Advisor

Ram I. Mahato

Abstract

Melanoma is a lethal malignancy with limited treatment options for advanced metastatic stages. New targeted therapeutic options with discovery of BRAF and MEK inhibitors have shown significant survival benefit. Despite the recent progress, inefficient tumor accumulation and dose limiting systemic toxicity remains pressing challenges for treating metastatic melanoma and there is a need for drug delivery approach to improve therapeutic index of chemotherapeutics. Nanoparticle based drug delivery represents promising approach to enhance efficacy and reduce the dose limiting systemic toxicity. Nanoparticles can be formulated either by physical encapsulation of drugs or by covalent conjugation of drugs to the polymeric backbone. Nanoparticles based strategies for encapsulation and conjugation of drugs to the polymer was reviewed in Chapter 2 where we summarized non-covalent interactions between polymer backbone and drug for physical encapsulation, various polymeric backbones for drug conjugation and application of photodynamic therapy in melanoma.

Phototherapy, a light activated treatment modality is a potential therapeutic option for treatment of melanoma. Excitation of photosensitizer by light of specific wavelength can be clinically utilized for fluorescence assisted tumor surgery, photoacoustic imaging, photochemical internalization and phototherapy. Indocyanine green, water soluble FDA approved anionic tricarbocyanine with excellent safety profile and absorption in the near infrared (NIR) range is an excellent photosensitizer. However, short half-life (2-4 minutes) and limited extravascular distribution restricts PT application of ICG. In chapter 3, we have described ICG based phototherapy wherein plasma circulation and tumor accumulation of ICG was improved by designing its micelles formulation. ICG micelles were formulated by covalently conjugating ICG-NH2 to the pendant carboxyl groups of poly (ethylene glycol)-block-poly(2- methyl-2-carboxyl-propylene carbonate) (PEG-PCC) copolymer using carbodiimide coupling. ICG conjugated amphiphillic polymer self-assembled into micelles with particle size of 30-50 nm and high drug loading. These ICG conjugated micelles exhibited significant in vitro photodynamic cytotoxicity. Use of sodium azide and NIR radiation at 4° C revealed photodynamic and photothermal as primary mechanism of cytotoxicity of ICG solution and ICG conjugated micelles respectively. In vivo NIR imaging demonstrated ICG conjugated micelles prolonged ICG circulation and increased its tumor accumulation through enhanced permeability and retention effect Increase in tumor accumulation improved therapeutic efficacy with complete tumor regression in NIR irradiated ICG conjugated micelles compared to free ICG and control in A375 human melanoma tumor model in athymic nude mice. These results suggest that ICG conjugated micelles can be potentially utilized for phototherapy.

Clinical translation of tubulin inhibitors for treating melanoma is limited by multidrug efflux transporters, poor aqueous solubility, and dose-limiting peripheral toxicities. Tubulin inhibitors with efficacy in taxane-resistant cancers are promising drug candidates and can be used as single agent or in conjunction with other chemotherapy. In chapter 4, we describe synthesis of tubulin inhibitors with activity in taxane resistant cell lines with IC50 in nanomolar range for the treatment of metastatic melanoma. LY293, a 5 indole derivative analog, binds to colchicine binding site and does not exhibit clinically prevalent drug resistance mechanism such as multidrug resistance (MDR) protein, breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp). Since LY293 is poorly soluble in water, LY293 was formulated as polymeric nanoparticles for systemic therapy of melanoma. Methoxy polyethylene glycol-b-poly (carbonate-co-lactide) (mPEG-b-P (CB-co-LA)) random copolymer was synthesized and characterized by 1H NMR and gel permeation chromatography (GPC). Polymeric nanoparticles were formulated using o/w emulsification method with a mean particle size of 150 nm and loading efficiency of 7.40%. Treatment with LY293 loaded nanoparticles effectively inhibited the proliferation of melanoma cells in vitro and exhibited concentration dependent cell cycle arrest in G2/M phase. In vivo, LY293 loaded nanoparticles significantly inhibited the proliferation of highly aggressive metastasized melanoma in a syngeneic lung metastasis melanoma mouse model without toxicity to vital organs.

Islet transplantation has been performed in many patients especially undergoing kidney transplantation to treat Type I diabetes. Proportion of recipients who achieved insulin independence is low and is limited by long-term graft rejection and by primary non-function of islets. Primary non-function is characterized as the loss of islet viability and function caused by non-immune reasons, such as the disruption of islet microvasculature and apoptosis of islets due to production of inflammatory cytokines at the transplantation sites. In chapter 5, we studied the potential of human bone marrow derived mesenchymal stem cells (hBMSCs) as gene carriers for improving the outcome of human islet transplantation. hBMSCs were transduced with Adv-hVEGF-hIL-1Ra to overexpress human vascular endothelial growth factor (hVEGF) and human interleukin-1 receptor antagonist (hIL-1Ra). Viability of human islets co-cultured with hBMSCs was determined by membrane fluorescent method and glucose stimulation test. Transduced hBMSCs and human islets were co-transplanted under the kidney capsule of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ diabetic mice and blood glucose levels were measured over time to evaluate the efficacy of genetically modified hBMSCs. Our in vitro and in vivo results showed hBMSCs can be used as gene delivery vehicles to improve the outcome of islet transplantation without affecting their stemness and differentiation potential.

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