Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate Mgr. Dimitrios Vagiannis Supervisor RNDr. Jakub Hofman, Ph.D. Title of Doctoral Thesis Study on the role of pharmacokinetic mechanisms of drug resistance in new anticancer drugs with focus on solid tumors Cancer chemotherapy is an important tool for the cure of cancer. Although the development of new anticancer drugs has been rapidly progressing, the phenomenon of multidrug resistance (MDR) continues to be a key issue leading to therapy failure in oncological patients. MDR is based on pharmacodynamic as well as pharmacokinetic mechanisms. Pharmacokinetic MDR includes drug efflux transporters and biotransformation enzymes that decrease the amount of (active form of) a drug in tumors. While the MDR role of transporters has been well understood, the participation of drug metabolizing enzymes is still unclear. This thesis investigates the role of cytochromes P450 (CYPs) in cytostatic resistance. Furthermore, it focuses on the modulation of pharmacokinetic MDR using pharmacokinetic drug-drug interactions of new targeted antitumor drugs. Finally, it aims to confirm the in vitro findings in ex vivo patient-derived tumor explants. In our latest publication, we demonstrate the significant role of...

Breast cancer affects over three million women in the United States, but this disease burden is not shared equally across all races. Black women, in particular, are diagnosed with more advanced cancer at a younger age and experience a disproportionately high mortality rate compared to white women. Factors that contribute to such disparity include socioeconomic status, tumor biology, age, insurance status, comorbidities, obesity, patients’ reproductive history and barriers to quality care. These factors alone, however, do not account for all the racial differences in mortality and outcomes experienced by black women. There is a growing body of literature that indicates black women are not receiving the same treatment and care as white women. Black women are less likely to receive surgery, radiation therapy, hormone therapy and targeted therapy than white women. Black women are also more likely to experience delays in the initiation of treatment, early discontinuation of treatment and overall guideline non-concordant care. The current literature has presented widespread racial disparities in the treatment of black women with breast cancer. Future research needs to focus on tangible interventions such as physician bias training and patient navigators to mitigate the inequity of care in the treatment of breast cancer.

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Tumour cell dissemination is a major issue with the treatment of cancer, thus new therapeutic strategies which can control this process are needed. Antagonism of integrins highly expressed in tumours is one potential strategy. The integrins are transmembrane glycoprotein adhesive receptors. Two of the integrins, αVβ3 and αIIbβ3, are highly expressed in a number of tumours and induce bi-directional signalling through their interaction with extracellular matrix proteins, and growth factor receptors. Through this signalling they play an important role in a number of cellular processes that are involved in tumour dissemination such as tumour growth, migration, invasion, metastasis and angiogenesis. Dual αIIbβ3 and αVβ3 integrin antagonism will have a direct effect on β3-expressing tumour cells that leads to the inhibition of cell migration and dissemination. Furthermore, through targeting tumour cell interaction with endothelial cells and platelets, this will also lead to inhibition of angiogenesis and metastasis. The aim of this project was to characterise the expression of αVβ3 and αIIbβ3 integrin in a panel of tumour cell lines and in human tumour xenograft samples, and to develop and utilise cell-based models to investigate potential novel β3 antagonists. The expression of αV and β3 subunits was detected in xenograft tissue using immunoblotting techniques. A panel of cell lines of different tumour types including melanoma, prostate, breast, colon and non small cell lung carcinoma was then characterised for αVβ3 and αIIbβ3 integrin expression using immunoblotting and immunocytochemistry. Melanoma cell lines demonstrated the strongest αVβ3 expression. No αIIbβ3 integrin expression was seen in any of the cell lines evaluated. A selection of cell lines with varying αVβ3 expression were then used to develop a functional test for cell migration, the scratch wound healing assay. Migration of tumour cells that expressed αVβ3 integrin was inhibited by the known β3 antagonists, cRGDfV peptide and LM609 antibody. A panel of 12 potential novel β3 integrin antagonists was screened for cytotoxicity and activity in the validated scratch assay. ICT9055 was the most effective antagonist in inhibition of M14 cell migration as determined by the scratch assay, with an IC50 of < 0.1 µM. Therefore the work presented in this thesis has established models and tools for evaluating potential novel β3 integrin antagonists, and identified a promising molecule to progress for further preclinical evaluation. / Public Authority for Applied Education and Training (PAAET)

The causality dilemma between dysbiosis and cancer has given rise to numerous studies both exploring the mechanisms behind cancer progression and the associative shifts in the microbiota upon carcinogenesis. Aside from the hallmark study of Dr. Barry Marshall in establishing the true causal relationship between Helicobacter pylori and gastric adenocarcinoma, studies have only been successful in adding associative links of carcinogenesis mediated by bacteria to the literature. The current field is limited in its ability to establish causative relationships, and further work is needed to construct a reference community whose physiological responses reflect global community responses. In this thesis, the organism Lactobacillus acidophilus was selected as a pilot strain for the development of a novel framework to establish the fitness and physiological changes that occur when bacteria engage the human epithelial environment. The pilot strain was revived from the American Type Culture Collection (ATCC), verified through 16S rRNA Sanger sequencing, and grown in its conventional culture medium and human tissue culture medium to establish baseline growth rates and gauge its physiological responses to an in vitro tumor microenvironment. A set of standard conditions was proposed for growth under human tissue culture conditions. Finally, a metabolic study and spot plate assay were performed to elucidate the anabolic deficits and viability of this strain in human tissue culture medium, respectively. This research was performed to better understand the environmental and metabolic requirements for this pilot strain to inhabit the human epithelial environment, and to establish a workflow that will set the foundation for an appropriate clinical study to demonstrate the causative relationship between dysbiosis and carcinogenesis.

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