Breast cancer as photodynamic therapy target: Enhanced therapeutic efficiency by overview of tumor complexity

Photodynamic therapy is a minimally invasive and clinically approved procedure for eliminating selected malignant cells with specific light activation of a photosensitizer agent. Whereas interstitial and intra-operative approaches have been investigated for the ablation of a broad range of superficial or bulky solid tumors such as breast cancer, the majority of approved photodynamic therapy protocols are for the treatment of superficial lesions of skin and luminal organs. This review article will discuss recent progress in research focused mainly on assessing the efficacies of various photosensitizers used in photodynamic therapy, as well as the combinatory strategies of various therapeutic modalities for improving treatments of parenchymal and/or stromal tissues of breast cancer solid tumors. Cytotoxic agents are used in cancer treatments for their effect on rapidly proliferating cancer cells. However, such therapeutics often lack specificity, which can lead to toxicity and undesirable side effects. Many approaches are designed to target tumors. Selective therapies can be established by focusing on distinctive intracellular (receptors, apoptotic pathways, multidrug resistance system, nitric oxide-mediated stress) and environmental (glucose, pH) differences between tumor and healthy tissue. A rational design of effective combination regimens for breast cancer treatment involves a better understanding of the mechanisms and molecular interactions of cytotoxic agents that underlie drug resistance and sensitivity.Fil: Lamberti, María Julia. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Biología Molecular. Sección Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rumie Vittar, Natalia Belen. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Biología Molecular. Sección Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rivarola, Viviana. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Biología Molecular. Sección Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Anti-cancer Prodrugs-Three Decades of Design

The conventional old treatment method for cancer therapy is associated with severe side effects along with several limitations. Therefore, searching and developing new methods for cancer became crucial. This mini review was devoted on the design and synthesis of prodrugs for cancer treatment. The methods discussed include targeted prodrugs which are depending on the presence of unique cellular conditions at the desired target, especially the availability of certain enzymes and transporters at these target sites, antibody directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT) which is considered one of the important strategies for the treatment of cancer and prodrugs based on enzyme models that have been advocated to understand enzyme catalysis. In this approach, a design of prodrugs is accomplished using computational calculations based on molecular orbital and molecular mechanics methods. Correlations between experimental and calculated rate values for some intramolecular processes provided a tool to predict thermodynamic and kinetic parameters for intramolecular processes that can be utilized as prodrugs linkers. This approach does not require any enzyme to catalyze the prodrug interconversion. The interconversion rate is solely dependent on the factors govern the limiting step of the intramolecular process

Emerging targeted strategies for the treatment of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a widespread genetic disease that leads to renal failure in the majority of patients. The very first pharmacological treatment, tolvaptan, received Food and Drug Administration approval in 2018 after previous approval in Europe and other countries. However, tolvaptan is moderately effective and may negatively impact a patient's quality of life due to potentially significant side effects. Additional and improved therapies are still urgently needed, and several clinical trials are underway, which are discussed in the companion paper Müller and Benzing (Management of autosomal-dominant polycystic kidney disease-state-of-the-art) Clin Kidney J 2018; 11: i2-i13. Here, we discuss new therapeutic avenues that are currently being investigated at the preclinical stage. We focus on mammalian target of rapamycin and dual kinase inhibitors, compounds that target inflammation and histone deacetylases, RNA-targeted therapeutic strategies, glucosylceramide synthase inhibitors, compounds that affect the metabolism of renal cysts and dietary restriction. We discuss tissue targeting to renal cysts of small molecules via the folate receptor, and of monoclonal antibodies via the polymeric immunoglobulin receptor. A general problem with potential pharmacological approaches is that the many molecular targets that have been implicated in ADPKD are all widely expressed and carry out important functions in many organs and tissues. Because ADPKD is a slowly progressing, chronic disease, it is likely that any therapy will have to continue over years and decades. Therefore, systemically distributed drugs are likely to lead to potentially prohibitive extra-renal side effects during extended treatment. Tissue targeting to renal cysts of such drugs is one potential way around this problem. The use of dietary, instead of pharmacological, interventions is another

Doctor of Philosophy

dissertationDrug-free macromolecular therapeutics are a new paradigm in polymer-based nanomedicines. Instead of carrying cytotoxic small molecular weight drugs, drug-free macromolecular therapeutics crosslink proteins in the cell membrane through hybridization of oligonucleotides to initiate apoptosis signaling. However, the mechanism of the nanomedicines was not fully understood. To study the mechanism and to better understand the interactions between the therapeutics and the cell membrane, super-resolution optical microscopy was used. Super-resolution imaging was performed on Raji B cells treated with the drug-free conjugates. The clustering of CD20 and lipid rafts was quantified. Lipid raft cluster size increased after treatment with drug-free conjugates. Drug-free conjugates induced apoptosis in a lipid raft-dependent mechanism where stable lipid rafts are needed for proper initiation of apoptosis. Direct stochastic optical reconstruction microscopy revealed nanoscale differences in membrane distribution of CD20 and lipid rafts. Pair-correlation analysis of super-resolution images showed lipid raft sizes of ~200 nm in cells treated with drug-free conjugates. General applicability of direct stochastic optical reconstruction microscopy to studying drug-delivery systems was also demonstrated. Two conceptually different polymer-based therapeutics were labeled with 4 different synthetic fluorophores, and three-dimensional (3D) direct stochastic optical reconstruction microscopy was conducted at different time points to track localization of the therapeutic components. An internalized polymer conjugate was localized in clusters at 4 h, but after 24 h, the polymer released into the cytosol a fluorophore attached via an enzymatically degradable peptide. Pair-correlation functions of the dye attached to the polymer and the released dye showed changes in their decay lengths between 4 h and 24 h. The pair-correlation function of the released dye showed random distribution after 24 h. Using reversible addition?fragmentation chain-transfer (RAFT) polymerization, branched and star polymers were synthesized to study the effect of architecture on apoptosis induction in Raji B cells. A new chain transfer monomer was synthesized in order to produce controlled branched polymers in RAFT polymerization. A degradable tetra-functional chain transfer agent was also synthesized. The star chain transfer agent produced degradable star polymers of high molecular weight (~170 kDa). Drug-free conjugates were synthesized to produce linear, branched, and star polymer-MORF2 conjugates. Apoptosis in Raji B cells was measured but the three different architectures induced the same levels of apoptosis as measure by annexin V and caspase 3

Engineering mesothelin-binding proteins as targeted cancer diagnostics and therapeutics

Cancer is a significant global health concern; and traditional therapies, including chemotherapeutics, are often simultaneously toxic yet ineffective. There is a critical need to develop targeted cancer therapeutics which specifically inhibit molecules or molecular pathways essential for tumor growth and maintenance. Furthermore, a targeted therapy is only effective when a patient\u27s tumor expresses the molecular target; therefore, companion diagnostics, including molecular imaging agents, are a necessary counterpart of targeted therapies. Mesothelin (MSLN) is a cell surface protein overexpressed in numerous cancers, including triple-negative breast, pancreatic, ovarian, liver, and lung, with limited expression in normal tissues. Aberrant MSLN expression promotes tumor progression, metastasis, and therapeutic resistance, and is correlated with poor prognoses. Promising results from pre-clinical and clinical trials to target MSLN with antibodies, antibody derivatives, immunotoxins, and antibody-drug conjugates for therapy demonstrate the promise of MSLN-targeting methods; however, none are currently approved for routine clinical use, and limitations are emerging for targeting agents under development. New targeted diagnostic and therapeutic approaches for MSLN-positive tumors have potential for substantial impact in the clinic. In this work, I used yeast-surface display and directed evolution to engineer novel proteins based on the non-antibody fibronectin (Fn3) scaffold that bind to MSLN with high affinity and specificity. Soluble engineered proteins were expressed and purified to high yields from a bacterial system. In vitro cytotoxicity and apoptosis assays demonstrated the potential of the proteins as targeted cancer therapeutics for MSLN-expressing tumors, and the engineered proteins enhanced cancer cell sensitivity to chemotherapy. Towards the goal of using engineered Fn3 variants for targeted drug delivery applications, in collaboration with our collaborators, we synthesized and validated a novel protein-polymer conjugate drug delivery system. Finally, I established and validated appropriate in vivo tumor models to evaluate our engineered proteins as molecular imaging diagnostic agents. My work has provided a candidate set of engineered proteins that can be used as molecular targeted therapeutics, drug-delivery vehicles, and molecular imaging diagnostics for MSLN-positive tumors, towards the ultimate goal of providing targeted treatment and diagnostic options for cancer patients where no such treatments currently exist

Spleen tyrosine kinase-mediated autophagy is required for epithelial-mesenchymal plasticity and metastasis in breast cancer

The ability of breast cancer cells to transiently transition between epithelial and mesenchymal states contributes to their metastatic potential. Therefore, driving tumor cells into a stable mesenchymal state, as opposed to complete tumor cell eradication, presents an opportunity to pharmacologically limit disease progression by promoting an asymptomatic state of dormancy. Here we compare a reversible model of epithelial-mesenchymal transition (EMT) induced by TGF-β to a stable mesenchymal phenotype induced by chronic exposure to the ErbB kinase inhibitor lapatinib. Only cells capable of returning to an epithelial phenotype resulted in skeletal metastasis. Gene expression analyses of the two mesenchymal states indicated similar transition expression profiles. A potently downregulated gene in both datasets was spleen tyrosine kinase (SYK). In contrast to this similar diminution in mRNA, kinome analyses using a peptide array and DNA-conjugated peptide substrates showed a robust increase in SYK activity upon TGF-β-induced EMT only. SYK was present in cytoplasmic RNA processing depots known as P-bodies formed during the onset of EMT, and SYK activity was required for autophagy-mediated clearance of P-bodies during mesenchymal-epithelial transition (MET). Genetic knockout of autophagy related 7 (ATG7) or pharmacological inhibition of SYK activity with fostamatib, a clinically approved inhibitor of SYK, prevented P-body clearance and MET, inhibiting metastatic tumor outgrowth. Overall, the current study suggests assessment of SYK activity as a biomarker for metastatic disease and the use of fostamatinib as a means to stabilize the latency of disseminated tumor cells

Progress in Aptamer-Mediated Drug Delivery Vehicles for Cancer Targeting and Its Implications in Addressing Chemotherapeutic Challenges

Aptamers are novel oligonucleotides with flexible three-dimensional configurations that recognize and bind to their cognate targets, including tumor surface receptors, in a high-affinity and highly specific manner. Because of their unique intrinsic properties, a variety of aptamer-mediated nanovehicles have been developed to directionally transport anti-cancer drugs to tumor sites to minimize systemic cytotoxicity and to enhance permeation by these tumoricidal agents. Despite advances in the selection and synthesis of aptamers and in the conjugation and self-assembly of nanotechnologies, current chemotherapy and drug delivery systems face great challenges. These challenges are due to the limitations of aptamers and vehicles and because of complicated tumor mechanisms, including heterogeneity, anti-cancer drug resistance, and hypoxia-induced aberrances. In this review, we will summarize current approaches utilizing tumor surface hallmarks and aptamers and their roles and mechanisms in therapeutic nanovehicles targeting tumors. Delivery forms include nanoparticles, nanotubes, nanogels, aptamer-drug conjugates, and novel molecular trains. Moreover, the obstacles posed by the aforementioned issues will be highlighted, and possible solutions will be acknowledged. Furthermore, future perspectives will be presented, including cutting-edge integration with RNA interference nanotechnology and personalized chemotherapy, which will facilitate innovative approaches to aptamer-based therapeutics

Doctor of Philosophy

dissertationSpecific delivery of chemotherapeutic agents to cancerous tissues can potentially result in increased safety and decreased toxicity. For example, nanomedicines, including polymer-drug conjugates, can potentially accumulate in solid tumors via the "enhanced permeability and retention" (EPR) effect. Further increases in delivery can be achieved via active targeting strategies, wherein cancer-specific targeting moieties enhance cellular binding and internalization. This dissertation describes a strategy which attempts to increase the tumor accumulation and efficiency of active targeting using a combination of tumor hyperthermia and heat shock protein (HSP) targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-drug conjugates. Following exposure to hyperthermia, increases in cellular stress results in initiation of the heat shock response, which includes the synthesis and migration of certain heat shock proteins to the cell surface. It is hypothesized that the induced expression of these HSPs can serve as specific molecular targets for the delivery of anticancer macromolecular chemotherapeutics. HPMA copolymer-drug conjugates bearing peptides targeting cell surface expressed glucose regulated protein 78 kDa (GRP78), a member of the HSP70 family of proteins, were synthesized, characterized, and evaluated for activity in prostate cancer models. Following exposure to hyperthermia, the cell surface expression of GRP78 was also evaluated and correlated to increases in cellular delivery and cytotoxicity of GRP78 targeted HPMA copolymeriv drug conjugates. Combination index analyses indicated that a combination of hyperthermia and HSP targeted drug therapy resulted in combined synergistic effects. In vivo, gold nanorod-mediated plasmonic photothermal therapy was utilized to induce tumor hyperthermia in a human prostate cancer animal model to enhance the delivery and efficacy of GRP78 targeted HPMA copolymer-drug conjugates. Results demonstrate that a combination of tumor hyperthermia and HSP targeting can increase tumor accumulation and cellular delivery of macromolecular chemotherapeutics, enabling safer and more effective therapies

New and emerging therapeutic options for malignant pleural mesothelioma: review of early clinical trials.

Malignant pleural mesothelioma (MPM) is a rare tumor that is challenging to control. Despite some benefit from using the multimodality-approach (surgery, combination chemotherapy and radiation), survival remains poor. However, current research produced a list of potential therapies. Here, we summarize significant new preclinical and early clinical developments in treatment of MPM, which include mesothelin specific antibody and toxin therapies, interleukin-4 (IL-4) receptor toxins, dendritic cell vaccines, immune checkpoint inhibitors, and gene-based therapies. In addition, several local modalities such as photodynamic therapy, postoperative lavage using betadine, and cryotherapy for local recurrence, have also shown to be effective for local control of disease

Polymeric Nanocarriers for Treatment of Melanoma and Genetically Modified Mesenchymal Stem Cells to Improve Outcome of Islet Transplantation

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