Graduation Date

Fall 12-18-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pharmaceutical Sciences

First Advisor

Tatiana Bronich, Ph.D.

Abstract

Combination chemotherapy is commonly used to treat cancer, because such a therapy regimens usually involve sequential administration of multiple drugs and allow targeting different cell signaling pathway. The co-delivery of drug combination at a controlled ratio via the same vehicle is offering the advantages such as spatial-temporal synchronization of drug exposure, synergistic therapeutic effects and suppression of drug resistance. Undoubtedly, there are several molecular and pharmacological factors that determine the effectiveness of drug combinations. A rationally designed drug combination is required since certain drug ratios and the definitive exposure to the targets of interest can only be synergistic while others are additive or even antagonistic. Cisplatin (CDDP) and paclitaxel (PTX) combination has resulted in improvement in the ovarian cancer treatment compare to individual drug with limited side effects. Multiple clinical trials studied the overall efficacy of CDDP and PTX and found significant benefit over the pre-existing treatments. Since then, this combination has been the treatment of choice for both early stage as well as advanced cases of ovarian cancer. However, administration of two different agents comes with the inconvenience of repeated or extended duration of drug infusion in patients. Moreover, the most extensively used conventional formulation of paclitaxel, Taxol®, utilizes Cremophor EL (polyethoxylated castor oil) that has been linked to significant toxicities including allergic, hypersensitivity and anaphylactic reactions during infusion that require premedication and prolonged peripheral neuropathy. Combining such drugs in one delivery carrier is therefore a well-suited and convenient strategy for controlling the pharmacokinetics and co-delivery of the desired drug ratio in vivo, to maximize the therapeutic potency and minimize drug-associated toxicities. In an attempt to develop such multidrug vehicle, we designed a functional biodegradable and biocompatible polypeptide-based polymeric nanogels. Triblock copolymers containing the blocks of ethylene glycol, glutamic acid and phenylalanine (PEG–PGlu–PPhe) were successfully synthesized via NCA-based ring-opening copolymerization and their composition was confirmed by 1H NMR. Self-assembly behavior of PEG–PGlu90–PPhe25 was utilized for the synthesis of hybrid micelles with PPhe hydrophobic core, cross-linked ionic PGlu intermediate shell layer, and PEG corona. Cross-linked micelles (nanogels, NG) were about 90 nm in diameter (ξ-potential = − 20 mV), uniform (narrow size distribution), and exhibited nanogels-like behavior. Degradation of NG was observed in the presence of proteolytic enzymes (cathepsin B). The resulting NG can incorporate the combination of drugs with very different physical properties such as CDDP (15 w/w% loading) and PTX (9 w/w% loading). Binary drug combination in NG exhibited synergistic cytotoxicity against human ovarian A2780 cancer cells and exerted a superior antitumor activity by comparison to individual drug-loaded NGs or free cisplatin in cancer xenograft model in vivo. However, this system relies solely on the enhanced permeation and retention (EPR) effect to facilitate the delivery of the drug combination to the tumor site. Regardless of the importance and popularity of EPR effect-based drug delivery, this strategy has some limitations related to the inter- and intra-tumor heterogeneity, variations in the density as well as permeability of the tumor vasculature that can affect the accumulation of nanocarriers. One of the popular approaches to circumvent these problems is by surface-functionalization of the drug carrier with ligands that can target receptors with differential expression on the cancer cell surface, which helps in increasing the mean residence time of the delivery system at the tumor site and improving target cell uptake.

One such receptor of interest is the folate receptor (FR). Its natural ligand, FA, comes with the advantages of high binding affinity, stability and a simple chemical structure together with ease of availability, making it a suitable targeting ligand for ovarian cancer therapy. FA can thus be successfully conjugated to macromolecular systems without loss of binding affinity to its receptor. Our group has previously demonstrated a tumor-specific delivery and improved anti-cancer effect in vivo of CDDP-loaded NGs decorated with FA targeting groups. In the next part of our study, we designed FA-linked NGs incorporating platinum-taxane combination, and examined whether FR-targeted concurrent delivery of synergistic combination of CDDP and PTX can lead to enhanced therapeutic efficacy compared to nontargeted NG system. FA-decorated NGs significantly suppressed the growth of intraperitoneal ovarian tumor xenografts outperforming their nontargeted counterparts without extending their cytotoxicity to the normal tissues. We also confirmed that synchronized co-delivery of the platinum-taxane drug combination via single carrier to the same targeted cells is more advantageous than a combination of targeted single drug formulations administered at the same drug ratio. Lastly, we demonstrated that the same platform can also be used for localized chemotherapy. Our data indicate that intraperitoneal administration can be more effective in the context of targeted combination therapy. Our findings suggest that multifunctional NGs are promising drug delivery carriers for improvement of current treatment for ovarian cancer.

Combination chemotherapy is also very common in the treatment of breast cancer. Trastuzumab (Herceptin™), in combination with chemotherapy is currently used for treatment of ErbB2-overexpressing breast cancers, following surgical removal of the primary tumor. However, patients either do not respond to Trastuzumab based therapies or relapse during the course of the treatment, necessitating the development of newer therapeutics. ErbB2 depends on heat shock protein 90 (HSP90) association for stability and, among client proteins of the chaperone, ErbB2 is perhaps the most sensitive to HSP90 inhibition. HSP90 inhibitors (such as the ansamycin antibiotic, Geldanamycin and related molecules like 17-AAG) have shown significant promise in pre-clinical models of ErbB2-driven breast cancer as well as initial phase I/II clinical trials. The mechanism involves attenuation of oncogenic signaling via degradation of ErbB2 as well as other critical downstream signaling mediators in the pathway, which include phospho-Akt and c-Raf. In a cancer cell, these signaling molecules are strongly dependent on HSP90 to maintain their stability. In a recently completed phase II study of 17-AAG and trastuzumab, an overall clinical benefit (including stable disease) was seen in 57% of the patients with ErbB2-positive metastatic breast cancer progressing on trastuzumab. We therefore hypothesized that HSP90-inhibition will enhance ErbB2-targeted drug delivery by promoting the endocytic uptake of ErbB2-bound nano-encapsulated cargo and facilitating its re-routing from a recycling pathway to the lysosomes. Using Trastuzumab-conjugated NGs (Trast-NG) encapsulating the DNA-damaging drug Doxorubicin (DOX) as a model chemotherapeutic, we demonstrate through both in vitro and in vivo studies that HSP90-inhibition can indeed lead to an enhancement of targeted delivery of DOX specifically into ErbB2-overexpressing breast cancer cells. As a consequence, a sub-therapeutic and non-toxic dose of the HSP90 inhibitor 17AAG markedly improves the efficacy of ErbB2-targetd nanogels in vivo. In a follow up study, we attempted to co-encapsulate DOX and 17-AAG since combining drugs in one delivery carrier is a well-suited strategy for controlling the pharmacokinetics and co-delivery of the desired drug ratio in vivo. However, co-incorporation of drug molecules with different physicochemical properties, such as hydrophilic DOX and hydrophobic 17-AAG, has been challenging. We have recently described biodegradable polymeric nanogels (NGs) based on poly(ethylene glycol)-b-poly(L-glutamic acid) (PEG-b-PGA) with pendant phenylalnine functionalities [23]. Such NGs have multiple hydrophobic domains formed by phenylalanine moieties within the cross-linked PGA polyion cores surrounded by a hydrophilic PEG shell. Herein, we explored these novel NGs for co-encapsulation of 17-AAG and DOX. The potency of this co-delivery system was evaluated in a panel of human breast cancer lines and in an ErbB2-driven orthotopic xenograft model. We demonstrate that NGs-based co-delivery of synergistic combination of 17-AAG and DOX exhibited superior antitumor efficacy compared to a combination of free drugs. The implications of our results may support a new platform for delivery of combinations of HSP90 inhibitors with cytotoxic agents for treatment of various types of cancers.

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