Among breast cancer subtypes, triple negative breast cancer (TNBC) represents around 15-20% of newly diagnosed cancer cases. Together with heterogeneous behavior among single patients, TNBC presents with a complex molecular landscape, low detection rate, and an aggressive and highly proliferative profile and these characteristics provide for the poor prognosis of TNBC patients. The lack of hormonal receptor (ER, PR, and HER2) expression impedes the administration of targeted therapies, and for this reason, the development of novel therapies for TNBC represents a primary clinical need.
Research into the development of polymer conjugates for the treatment of TNBC falls into four main areas:
i) The detailed study and comprehension of relevant preclinical mouse models that faithfully mimic the clinical scenario as a nanomedicine evaluation platform.
ii) The development of new polymer-drug combination conjugates with optimized drug ratio and stimuli-responsive polymer-drug linker(s) in order to achieve adequate drug(s) pharmacokinetics at the site of action.
iii) The exhaustive physicochemical characterization that allowed the establishment of structure-activity relationships (SAR). Additionally, the integration of -omics in order to identify pharmacological biomarkers to better design and monitor the activity of selected nanoconjugates.
We now address these main points in this Thesis, which aimed to design polymer-drug combination conjugates for the treatment of TNBC. Drug combination therapies have emerged as a valid option for the treatment of breast cancer, as this approach permits synergistic drug targeting of multiple pathways.
Studies employing animal models of disease allow for the understanding of disease development and progression, the search for therapeutic targets, and the validation of therapeutic strategies; however, the lack of accurately characterized research models that faithfully mimic the pathological features of human TNBC frequently hampers research aims. We now present the broad and detailed characterization of preclinically relevant spontaneously metastatic TNBC murine models that faithfully
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reproduce the human clinical scenario. Our comparisons have uncovered important descriptors (some coming from –omics analysis) regarding the interconnected tissular/molecular processes driving disease progression towards metastasis. These descriptors include metastatic spread via the lymphatic route, immune system remodeling, cancer-associated adipocytes, and crucial metabolomic alterations.
As part of the development of a polymer-based therapy for TNBC, we present a versatile and straightforward methodology for the preparation of well-defined polyglutamate-based drug combination conjugates based on the well-established properties of the poly-glutamic acid (PGA) as a multivalent and biodegradable polymer carrier. We synthesized and characterized a family of conjugates containing amino acid-based proteolytic drug linkers including Gly, Gly-Gly, Gly-Phe-Leu-Gly as key drivers in the final macromolecule solution conformation (which drives the biological fate). These new drug delivery systems incorporate both chemotherapy (doxorubicin) and endocrine therapy (the aromatase inhibitor aminoglutethimide) as a synergistic combination. Overall, we demonstrate how the presence of a small flexible Gly linker can drastically modify the spatial conformation of the entire polymer–drug macromolecule, promoting the synergistic release of both drugs and significantly improving the biological activity.
The release of drug combinations conjugated to the polymer backbone via protease-cleavable drug linkers, such as those noted above, relies on the heterogeneous expression levels (at both the patient and tumor level) of various proteolytic enzymes some of them not found within the tumor microenvironment, target we wish to reach to be able to modulate metastatic processes. To improve drug release intracellularly but also at the tumor microenvironment, we developed a new family of conjugates incorporating the pH-sensitive hydrazone linker. These conjugates provided improved antitumor and antimetastatic activity (supported by histology and transcriptomic analysis); however, we noted that the pH-sensitive drug-linker length significantly influences the cell death mechanism involved.
Finally, we applied the knowledge acquired from the development of the previous polymer-drug combination conjugates, together with the selection of a more powerful combination of synergistic drugs for TNBC treatment (including a tyrosine-kinase inhibitor, TKI and a topoisomerase inhibitor, TI) for the development of an even more
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effective family of polymer-drug conjugates. This novel system demonstrated enhanced antitumor activity in a human TNBC mice model, leading to a 50% primary tumor growth reduction and the almost complete remission of lung metastasis.
Overall, the findings exhibited throughout this Thesis highlight the need for a deeper understanding of polymer-drug conjugates at supramolecular level, including the need for a complete physicochemical characterization to allow the design of more effective polymer-drug conjugates. Additionally, we highlight the importance of the full characterization of the animal models employed and the parameters driving primary tumor development and the pathways modulating metastatic spread and tumor-side pathologies
