Hypoxia is a common feature of many solid tumors, including colorectal, breast and ovarian carcinoma. Under hypoxia, tumor cells escape p53-mediated apoptotic cell death and instead adopt a HIF-mediated pro-survival and pro-metastatic pathway. CA IX is a membrane bound carbonic anhydrase isozyme over-expressed in hypoxic tumor cells as a HIF-mediated adaptive response to tumor hypoxia. CA IX-mediated intracellular pH homeostasis and extracellular pH drop have been implicated in survival, invasion, metastasis, and resistance of hypoxic tumor cells to weakly basic chemotherapeutic drugs, e.g., doxorubicin. Inhibition of CA IX by small molecule inhibitors as well as monoclonal antibodies (mAbs) has been shown to be an effective approach to regress the tumor growth. Given its high expression level and the functional importance in hypoxic tumors, inhibition of CA IX catalytic activity by small molecule CA IX inhibitors has the potential to rescue, potentiate, or even synergize with the efficacy of doxorubicin against CA IX expressing hypoxic tumors. Building upon this hypothesis, in this dissertation, we focused our efforts towards evaluating combinations of CA IX inhibitors together with doxorubicin and map for synergistic cytotoxicity. In Chapter 2, we established the cellular models we used throughout this dissertation: colorectal cancer cell model HT-29, triple negative breast cancer cell model MDA-MB-231 and ovarian cancer cell model SKOV-3, previously known to express CA IX. In Chapter 3, we explored and established a relationship between CA IX expression and the efficacy of weakly basic drug doxorubicin against tumor cells taking a set of 8 high grade serous ovarian carcinoma cell models (representing ovarian cancer of different origin and subtypes). In Chapter 4, we screened a series of ureido sulfonamide CA IX inhibitors to select leads to use in combination with doxorubicin against the same cancer cell lines. We used a plethora of in vitro and in silico screening techniques to shortlist the best performing compounds for efficient tumor killing. Subsequently, in Chapter 5, we developed and validated LC-MS/MS methods for simultaneous quantitation of doxorubicin and ureido sulfonamide CAIs and successfully applied them to identify and quantitate these drugs/drug combinations in SKOV-3 cell lysates and external cell growth media.
In Chapter 6, the best performing CAIs selected in Chapter 4 were initially tested in combination with doxorubicin 2D under normoxia and hypoxia. Cell viability upon treatment with drug combinations and the combination index values were calculated from the cell viability assay data to map for synergism between doxorubicin and CAIs. The 2D experiments allowed us to further select CAIs to be tested in combination with doxorubicin in 3D against tumor spheroids generated from the same cancer cells. As expected, we were able to identify CAI and doxorubicin combinations that synergized to kill the cancer cells in 3D tumor spheroids. Towards the end of this chapter, using SKOV-3 as the model cell line, we established the mechanism of synergism by LC-MS/MS as well as by live cell image analysis.
In Chapter 7, we optimized a PEG-PCL based polymeric drug delivery system (DDS) for co-delivery of doxorubicin and CAIs. Thorough pre-formulation and formulation studies allowed us to identify critical formulation and process parameters and optimize them to maximize drug loading and encapsulation efficiency. The optimized method was also tested for its reproducibility and for the stability of the final formulations. Stability of the final formulations was the final criteria used to further screen the compounds and combinations in the polymeric DDSs. A release study was conducted towards the end of this chapter to discern the release kinetics of the drug pairs co-loaded in the polymeric micelles.
In Chapter 8, lead formulations were tested for their efficacy against the cell models displaying the highest and lowest CA IX expression, namely SKOV-3 and MDA-MB-231 cell lines. Several control experiments were also conducted in parallel and free CAIs, free doxorubicin, micelle loaded with CAIs, micelle loaded with doxorubicin, liposome loaded with doxorubicin and liposome loaded with doxorubicin in presence of CAIs were included to test on the same tumor models. A thorough comparative analysis of these different delivery modalities was also made. Finally, a correlation between efficacy and drug release kinetics of co-loaded micelles were identified and discussed.
In conclusion, the synergistic cytotoxicity of ureido sulfonamide CA IX inhibitors were thoroughly investigated and synergistic dose combinations were identified. A PEG-PCL-based drug delivery system was optimized to co-load and deliver synergistic dose combinations in vitro in 2D and 3D cell models. / Pharmaceutical Sciences
| Identifer | oai:union.ndltd.org:TEMPLE/oai:scholarshare.temple.edu:20.500.12613/10216 |
| Date | 05 1900 |
| Creators | SUFIAN, MD ABU |
| Contributors | Ilies, Marc A., Fassihi, Reza, Lebo, David, Mesaros, A. Clementina |
| Publisher | Temple University. Libraries |
| Source Sets | Temple University |
| Language | English |
| Detected Language | English |
| Type | Thesis/Dissertation, Text |
| Format | 241 pages |
| Rights | IN COPYRIGHT- This Rights Statement can be used for an Item that is in copyright. Using this statement implies that the organization making this Item available has determined that the Item is in copyright and either is the rights-holder, has obtained permission from the rights-holder(s) to make their Work(s) available, or makes the Item available under an exception or limitation to copyright (including Fair Use) that entitles it to make the Item available., http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | http://dx.doi.org/10.34944/dspace/10178, Theses and Dissertations |
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