Graduation Date

Spring 5-6-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pharmaceutical Sciences

First Advisor

Dr. Tatiana K. Bronich

Abstract

Combination therapy is preferred over monotherapy to treat cancer as it can show better therapeutic outcomes and also delay the onset of resistance by targeting multiple cell-survival pathways in cancer cells. Rationally developed combinations with monoclonal antibodies and small molecule drugs in the form of antibody-drug conjugates or antibody nanoparticle conjugates allow us to take advantage of the cellular targeting of the potent cytotoxic agents, thereby widening the scope for dose reduction while maintaining the required therapeutic response. This can in turn improve patient tolerability by reducing the off target toxicities. For the therapy of ErbB2 positive breast cancer, the monoclonal antibody, Trastuzumab, is the FDA approved therapy. The receptor tyrosine kinase, ErbB2 is a viable target in 20-25 % breast cancer patients due to its overexpression. Its degradation is associated with slower progression of the disease and increased survival times. While the monoclonal antibody Trastuzumab (Herceptin™) is the first line therapy in such patients, monotherapy with Trastuzumab has shown little benefit and therefore must be given with chemotherapeutic agents. Such combinations also help in delaying the development of resistance to Trastuzumab, since multiple cellular pathways can be targeted simultaneously. ErbB2 is a client protein of heat shock protein 90 and 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) is a potent inhibitor of HSP90. Previous work in our lab has demonstrated strong synergy of action between 17-AAG and a model cytotoxic agent doxorubicin. In order to further improve the efficacy of the therapy, our goal was to replace doxorubicin with a more potent, clinically relevant agent paclitaxel (PTX), which has been shown to have strong synergistic antitumor effect with 17-AAG in ErbB2-driven breast cancers. Since synergy of such therapy is often sequence and dose ratio specific, co-delivery of the drugs via the same vehicle is desirable as well as beneficial. For this purpose, polymeric micelles prepared from a biodegradable block copolymer were chose. Polypeptides have an inherent property to assemble into supramolecular structures in solution. The formation of supramolecular structures is a controlled and organized process that depends by and large on the nature of the polypeptide and conditions of the solvent it is exposed to. Formation of amphiphilic copolymers based on such polypeptides can allow for tailoring the assembly process to a predefined nanoscale supramolecular structure, which can then be used as drug delivery vehicles. The overall process of self-assembly of such amphiphilic copolymers can then be regarded as a complex phenomenon of structural organization that is governed by the nature of constituent hydrophilic and hydrophobic blocks, their relative lengths, as well as properties of the solvent-phobic block that is the driving force for self-assembly. The inherent biocompatibility and biodegradability of polypeptides is of additional advantage for their biological applications. For the purpose of the current study, amphiphilic block copolymer with following composition was chosen: polyethylene glycol (PEG) as the hydrophilic, stealth imparting block and polyleucine (PLeu) as the hydrophobic part and the initiator of micelle formation in aqueous environment. The variables explored in the current study were altering the ratio of lengths of constituent blocks as well as chirality of PLeu block and the temperature of solvent used for preparation of micelles via the film rehydration method. The impact of all these variables on the thermodynamic stability as well as type of secondary structures formed and the influence of these attributes on the ability of the micelles to encapsulate a combination of hydrophobic drugs into their core are also described. Dual drug-loaded micelles thus prepared could load 17-AAG and PTX in a ratio 2:1 by weight. The formulation showed a high level of synergy on BT-474 cells that express a high amount of ErbB2 while the synergy was negligible in ErbB2low MCF-7 cells. The strong synergy also observed when the formulation was tested in an orthotopic breast cancer mouse model developed using ErbB2 overexpressing BT-474 cells, and an arrest in the growth of tumors in animals treated with dual drug-loaded micelles was observed, while both 17-AAG and PTX were used at sub therapeutic doses of 10 mg and 5 mg equivalents per kg body weight. The lower doses also helped avoid toxicity associated with the therapy. We also show the importance of simultaneously delivering the two drugs via a single carrier system as opposed to cocktail of individual drug-loaded micelles administered at equivalent doses, which has a better therapeutic outcome than the cocktail therapy. These combination drug-loaded micelles were developed as a platform for chemotherapy with Trast. The triple therapeutic system of Trast with combination drug-loaded micelles containing 17-AAG and PTX exhibited an even stronger anticancer effect, with complete regression of tumors at the end of treatment, which reached a palpable size again after day 45 with much slower progression than other treatment controls.

Pancreatic cancer (PC) is one of the most lethal malignancies, due to aggressive tumorigenicity, early metastasis and development of drug resistance to standard care chemotherapy. Since its approval in 1997, Gemcitabine (Gem) has been the first-line treatment for advanced disease. However, there is no standard second-line therapy after Gem failure. FOLFIRINOX, a combination of four agents, folinic acid, fluorouracil, irinotecan and oxaliplatin was approved by the FDA in 2010. The rationale for this combination was based on these drugs having a different mechanism of action, and, more importantly, non-overlapping toxicities. In cases that could tolerate FOLFIRINOX, an overall improvement in the survival times as well as quality of life was noted. However, even the toxicities are non-overlapping, the cumulative toxicity profile for FOLFIRINOX can become the dose limiting factor. In the first trial itself, 50.8% of the patients needed dose adjustment. The common toxicities observed with FOLFIRINOX include Febrile neutropenia, Thrombocytopenic bleeding, ≥ grade 3 platelets, Grade 2 persistent neurotoxicity, Grade 3 persistent neurotoxicity OR Grade 4 neurotoxicity and many more non-hematological toxicities. Most of the toxicities are severe enough to require discontinuation of the treatment or switching to lower doses or alternative agents. The combination of Gem with Cisplatin (CDDP) has been explored in clinical trials for metastatic disease. As a part of FOLFIRINOX, platinum compounds showed significant efficacy. Cisplatin acts by damaging the DNA. It is known to first get converted into the aqua form within the cell, which happens by the replacement of the labile chloro groups with water molecules. This active form is then able to form covalently linked adducts with the DNA. This initial assault then goes on to activate a series of signaling pathways that ultimately lead to apoptosis and cell death. The DNA adducts thus formed can cause distortion of the DNA and subsequent recognition by various cellular proteins. This leads to problems in DNA synthesis and replication and is reported to cause a prolonged G2 cell-cycle phase arrest. However, the exact mechanism of activation of the apoptotic pathways remains unclear. On the other hand, gemcitabine is a deoxycytidine analog. Its mechanism of activation involves conversion into its triphosphate form, which can then be incorporated into the DNA as a false nucleotide. Usually, one more deoxynucleotide can be incorporated into the DNA before the synthesis stops. Another minor mechanism of action of gemcitabine is its ability to inhibit ribonucleotide reductase, which plays a key role in the repair mechanism of the DNA. Many studies report the benefit of administration of gemcitabine prior to that of cisplatin; the reason cited for this is the increase in the formation of Pt-DNA adducts when the DNA had already been damaged and exposed due to the incorporation of deoxycytidine or active gemcitabine. The gemcitabine in turn inhibits the repair of the formed Pt-DNA adducts as well as reduces the efficacy of nucleotide excision repair by its ability to inhibit the action of ribonucleotide reductase. On the other hand, when Pt compounds are administered prior to gemcitabine, the formed Pt-DNA adducts can no longer allow for the incorporation of gemcitabine and that leaves no scope for gemcitabine to act. Our preliminary in vitro studies with the free drugs on T3M4 Simple Cells (COSMC deleted cells) showed that synergy of the combination is schedule-dependent, and Gem administration followed by CDDP showed the most potent cytotoxic activity. However, this combination proved to be only marginally effective in actual practice due to combined increased toxicity of both the agents. We have shown that encapsulation of CDDP in polymeric nanogels with cross-linked ionic cores enhanced its tumor accumulation and improved its safety profile. Additionally, sustained release profile of CDDP from nanogels allows for the administration of free Gem and CDDP loaded nanogels in a single injection while still retaining schedule-dependent synergy of the combination. Pancreatic ductal adenocarcinoma cells are known to express truncated O-glycans (Tn and STn antigens) and it was shown that decorating the nanogels with an antibody directed against this antigen further enhanced their uptake by tumor cells while reducing off-target accumulation in an in vivo pancreatic cancer model.

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