PSMA-1-DOXORUBICIN CONJUGATES FOR TARGETED THERAPY OF PROSTATE CANCER by NATALIE WALKER Submitted in partial fulfillment of the requirements for the degree of Master of Science Biomedical Engineering CASE WESTERN RESERVE UNIVERSITY May, 2019 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis of Natalie Walker candidate for the degree of Master of Science. Committee Chair Efstathios Karathanasis, PhD Committee Members James Basilion, PhD, Research Advisor Christopher Hoimes, DO Xinning Wang, PhD Date of Defense 14 January 2019 *We also certify that written approval has been obtained for any proprietary material contained therin. Contents List of Tables ..................................................................................................................... iv List of Figures ..................................................................................................................... v Abstract ............................................................................................................................... 1 Introduction ......................................................................................................................... 2 Table 1: Review of PSMA Expression in Nonprostate Malignancies. ................... 5 Figure 1: Outline of Structures of Three PSMA-1-Doxorubicin Prodrug Conjugates............................................................................................................... 8 Figure 2: Proposed Mechanism for Prodrug Release of Free Doxorubicin ............ 8 Methods............................................................................................................................... 9 Materials ......................................................................................................................... 9 General Comments.......................................................................................................... 9 Synthesis of PSMA-1(Cys) (Glu-CO-Glu’-Amc-Ahx-Glu-Glu-Glu-Cys-NH2)............ 9 Synthesis of PSMA-1-Doxorubicin Conjugates ........................................................... 11 Synthesis of PSMA-1-SMCC-Dox ........................................................................... 11 Synthesis of PSMA-1-MC-Val-Cit-PABC-Dox ....................................................... 12 Synthesis of PSMA-1-MMCCH-Dox ....................................................................... 13 Cell Culture ................................................................................................................... 13 In Vitro Cellular Uptake Studies ................................................................................... 13 i Cytotoxicity Assay ........................................................................................................ 14 Mouse Tumor Xenograft Studies .................................................................................. 15 Statistical Analysis ........................................................................................................ 15 Results ............................................................................................................................... 16 Chemistry ...................................................................................................................... 16 Figure 3: Structure and Characterization of PSMA-1(Cys) .................................. 17 Figure 4: Synthesis and Characterization of SMCC-Dox ..................................... 18 Figure 5: Synthesis and Characterization of Non-cleavable PSMA-1-SMCC-Dox ............................................................................................................................... 19 Figure 6: Synthesis and Characterization of MC-Val-Cit-PABC-Dox................. 20 Figure 7: Synthesis and Characterization of Cathepsin-Cleavable PSMA-1-MC- Val-Cit-PABC-Dox............................................................................................... 21 Figure 8: Synthesis and Characterization of MMCCH-Dox................................. 23 Figure 9: Synthesis and Characterization of Acid-Labile PSMA-1-MMCCH-Dox ............................................................................................................................... 24 Figure 10: Absorption Spectra of Free Doxorubicin and PSMA-Targeted Conjugates............................................................................................................. 25 In Vitro Cellular Uptake Studies ................................................................................... 25 Figure 11: In Vitro Drug Uptake and Cellular Localization by Fluorescence Microscopy ........................................................................................................... 26 ii In Vitro Cytotoxicity ..................................................................................................... 27 Figure 12: 48 Hour Cytotoxicity of Free and PSMA-Targeted Doxorubicin ....... 28 Figure 13: Cytotoxicity of Dox and PSMA-MMCCH-Dox, Alternate Conditions ............................................................................................................................... 29 Mouse Tumor Xenograft Response .............................................................................. 30 Figure 14: Tumor Response and Mouse Weight .................................................. 30 Discussion ......................................................................................................................... 31 Figure 15: Test of PSMA-1-MMCCH-Dox Cleavage in Cell Culture Media ...... 36 Acknowledgements ........................................................................................................... 41 Bibliography ..................................................................................................................... 42 iii List of Tables Table 1: Review of PSMA Expression in Nonprostate Malignancies. ............................... 5 iv List of Figures Figure 1: Outline of Structures of Three PSMA-1-Doxorubicin Prodrug Conjugates ....... 8 Figure 2: Proposed Mechanism for Prodrug Release of Free Doxorubicin ........................ 8 Figure 3: Structure and Characterization of PSMA-1(Cys) .............................................. 17 Figure 4: Synthesis and Characterization of SMCC-Dox ................................................. 18 Figure 5: Synthesis and Characterization of Non-cleavable PSMA-1-SMCC-Dox ......... 19 Figure 6: Synthesis and Characterization of MC-Val-Cit-PABC-Dox............................. 20 Figure 7: Synthesis and Characterization of Cathepsin-Cleavable PSMA-1-MC-Val-Cit- PABC-Dox ........................................................................................................................ 21 Figure 8: Synthesis and Characterization of MMCCH-Dox............................................. 23 Figure 9: Synthesis and Characterization of Acid-Labile PSMA-1-MMCCH-Dox ......... 24 Figure 10: Absorption Spectra of Free Doxorubicin and PSMA-Targeted Conjugates ... 25 Figure 11: In Vitro Drug Uptake and Cellular Localization by Fluorescence Microscopy ........................................................................................................................................... 26 Figure 12: 48 Hour Cytotoxicity of Free and PSMA-Targeted Doxorubicin ................... 28 Figure 13: Cytotoxicity of Dox and PSMA-MMCCH-Dox, Alternate Conditions .......... 29 Figure 14: Tumor Response and Mouse Weight .............................................................. 30 Figure 15: Test of PSMA-1-MMCCH-Dox Cleavage in Cell Culture Media .................. 36 v PSMA-1-Doxorubicin Conjugates for Targeted Therapy of Prostate Cancer Abstract by NATALIE WALKER Metastatic-castration resistant prostate cancer poses a serious clinical problem with poor outcomes, and targeted treatments that would widen the therapeutic window are desired. Prostate-specific membrane antigen (PSMA) is a membrane-bound glycoprotein that is overexpressed in prostate cancer and found in the neovasculature of many other tumors. Our previously developed ligand, PSMA-1, has affinity in the nM range. The aim of this study is to synthesize and evaluate three PSMA-1-Doxorubicin conjugates using different chemical linkers: non-cleavable SMCC, protease-cleavable MC-Val-Cit-PABC-PNP, and the acid-labile hydrazone MMCCH. PSMA-1-Doxorubicin conjugates are selective for PSMA(+) cell lines by fluorescence microscopy, but only PSMA-1-MMCCH-Dox showed evidence of linker cleavage, doxorubicin release to the nucleus of the cell, and cytotoxic activity. It was also more effective and less toxic than free doxorubicin in a flank tumor mouse model. In the future, these targeted agents may aid in treatment of patients with prostate cancer or other PSMA-expressing tumors. 1 Introduction Prostate cancer is the third most common non-skin cancer in the United States, after breast and lung cancers. In 2018, there were an estimated 164,690 new cases diagnosed and 29,430 deaths, up from 161,360 and 26,730 respectively in 2017 (1). Screening with serum prostate specific antigen (PSA) allows for 78% of prostate cancers to be diagnosed at the localized stage, facilitating therapy with radical prostatectomy or radiation therapy (1) (2) (3). Patients with metastatic disease, however, may see initial response to suppression of gonadal testosterone, but the vast majority of them progress to metastatic castration-resistant prostate