UNIVERSITY of CALIFORNIA Los Angeles Lysozyme-Triggered
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UNIVERSITY OF CALIFORNIA Los Angeles Lysozyme-Triggered Nanodiamond Contact Lens for Glaucoma Treatment & Phenotypically-based Combinatorial Drug Optimization for Multiple Myeloma Treatment A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Oral Biology by Kangyi Zhang 2015 ABSTRACT OF THE DISSERTATION Lysozyme-Triggered Nanodiamond Contact Lens for Glaucoma Treatment & Phenotypically-based Combinatorial Drug Optimization for Multiple Myeloma Treatment by Kangyi Zhang Doctor of Philosophy in Oral Biology University of California, Los Angeles, 2015 Professor Dean Ho, Chair In glaucoma treatment, ocular devices such as drug-loaded contact lenses have recently emerged as preferred candidates over eyedrops. Timolol maleate (TM), a prevalent glaucoma drug, exhibits side effects when excess drug enters the systemic circulation. Unlike eyedrops and drug-soaked lenses, new designs should provide sustained release and minimize undesirable burst release. Current innovations have not addressed the important issue of drug elution from contact lens during wet storage and shipment. Here we present a nanodiamond (ND)-embedded contact lens capable of lysozyme-triggered release of TM for sustained therapy. We found that ND-nanogel containing lysozyme-cleavable polymers enabled the controlled and sustained release of TM in the presence of lysozyme. Nanodiamonds also improved the mechanical properties of the poly-HEMA lenses without compromising on water content, oxygen permeability and optical clarity. In vitro cell viability assays on primary trabecular meshwork cells revealed that the released TM retained their antioxidant activity – an indicator of timolol efficacy. ii This successful lysozyme activation of our functionalized ND nanogel can be easily applied to other diseases and drugs where localized triggered release is desired. Conventional combinatorial treatment for multiple myeloma and other cancers uses an additive approach of maximum tolerated doses from single-drug experiments. These doses do not reflect true drug behavior in combinatorial therapy. A rapid and robust platform is necessary to optimize multiple parameters to find the best drug combination for improved efficacy and safety. Here, we present Response Surface Optimization (RSO) where we rapidly narrowed down our top three myeloma drug candidates (Bortezomib-Panobinostat-Dexamethasone) from a starting list of fourteen drugs in a short span of five months. The mechanism-independent RSO platform screens drugs based on maximizing the therapeutic window – increased healthy cell viability and decreased cancer cell viability. After the 1 st iteration experiment, a linear regression experimental process (equation) is generated using MATLAB to reconcile the empirical data. Undesirable candidates are removed before performing a 2nd screening iteration. In the 3 rd iteration, the optimum drug concentrations of the best drug combinations are determined. Response surface maps for output based on multi-parameters are plotted and the optimum is determined. Interestingly, this three-drug combination was the focus of recent myeloma clinical trials for refractory and relapsed patients. While conventional drug screening approaches demand huge time and money investment, the RSO platform rapidly and accurately converges on prime solutions. The RSO is applicable to the clinic where inter-patient variability requires adaptive treatment interventions based on individual clinical outcomes. iii The dissertation of Kangyi Zhang is approved. Daniel T. Kamei Kang Ting Wenyuan Shi Dean Ho, Committee Chair University of California, Los Angeles 2015 iv TABLE OF CONTENTS Acknowledgements ix Biographical Sketch x Lysozyme-Triggered Nanodiamond Contact Lens for Glaucoma Treatment 1 - Background and Significance 2 - Methodology 5 - Results and Discussion 10 Phenotypically-based Combinatorial Drug Optimization for 17 Multiple Myeloma Treatment - Background and Significance 17 - Methodology 20 - Results and Discussion 23 Figures for “Lysozyme-Triggered Nanodiamond Contact Lens for Glaucoma 34 Treatment” (Details on page vi) Figures for “Phenotypically-based Combinatorial Drug Optimization 43 for Multiple Myeloma Treatment” (Details on page vi) Bibliography 58 v LIST OF FIGURES Figure A1: Schematic illustration of our lysozyme-activated drug eluting contact lens 35 Figure A2: Characterization of ND-nanogel during synthesis 36 Figure A3: FTIR spectra of chitosan and N-acetylated chitosan 37 Figure A4: Characterization of physical properties of contact lenses 38 Figure A5: SEM images of poly-HEMA lens and ND-nanogel lens 39 Figure A6: Enzyme-triggered drug release of ND-nanogel lens 40 Figure A7: Fluorescence spectral scan of Alexa Fluor ® 488-labeled NDs in water 41 and eluted solution from ND-nanogel lens Figure A8: Evaluation of the antioxidant capacity of timolol using primary human 42 trabecular meshwork cells Figure B1: Flowchart of the three stages of the Response Surface Optimization 44 (RSO) Figure B2: Schematic showing the background of the multiple myeloma 45 collaboration between National University of Singapore and University of California, Los Angeles Figure B3: An excerpt of the list of drug combinations in the second iteration 46 vi Figure B4: Table showing the design of the three iterations 47 Figure B5: List of 14 drugs used for the 1 st iteration 48 Figure B6: MATLAB multi-drug optimization experimental assessment of first 49 iteration (first attempt) Figure B7: MATLAB multi-drug optimization experimental assessment of first 50 iteration (second attempt) Figure B8: Multi-drug optimization experimental assessment for the 2 nd iteration with 51 9 drug candidates Figure B9: Multi-drug optimization experimental assessment for the 3 rd iteration with 52 5 drug candidates Figure B10: Four diagnostic plots generated specifically for the third iteration 53 Figure B11: Excerpt of the top 20 prescribed combinations in a multi-drug 54 optimization experimental assessment. Figure B12: Excerpt of the top 10 and bottom 10 prescribed combinations consisting 55 of two or three drugs. vii Figure B13: Pearson correlation plots for prescribed outputs vs experimental 56 outputs. Figure B14: Examples of experimentally-derived response surface plots for the 57 second iteration with nine drugs. viii ACKNOWLEDGEMENTS This thesis contains a version of a published journal article in ACS Nano. Ho Joong Kim, Kangyi Zhang, Laura Moore, and Dean Ho, “Diamond Nanogel-Embedded Contact Lenses Mediate Lysozyme-Dependent Therapeutic Release.” ACS Nano 8, no. 3 (2014): 2998–3005. doi:10.1021/nn5002968 Ho Joong Kim and Kangyi Zhang are co-authors. HJ Kim conceived of the design of the nanodiamond contact lenses and all chemical synthesis. Both HJ Kim and K Zhang evaluated the properties of the ND-nanogel and contact lenses. Both authors did the drug elution study. K Zhang performed the biological assays to check for drug efficacy. Research is made possible thanks to the National Science Foundation CAREER Award (CMMI- 0846323), Center for Scalable and Integrated Nano-Manufacturing (DMI-0327077), CMMI- 0856492, DMR-1105060, V Foundation for Cancer Research Scholars Award, Wallace H. Coulter Foundation Translational Research Award, Society for Laboratory Automation and Screening (SLAS) Endowed Fellowship, Beckman Coulter, and National Cancer Institute Grant U54CA151880. K. Zhang gratefully acknowledges individual fellowship support provided by Agency for Science, Technology and Research (A*STAR). K Zhang would like to thank Prof. Ho, Prof. Ting, Prof. Kamei, Prof. Shi, his parents and fellow laboratory members for their support throughout his PhD. ix BIOGRAPHICAL SKETCH 2009 - Brown University, BS, Biomedical Engineering 2012 - Northwestern University, MS, Biomedical Engineering Kangyi Zhang is a recipient of the Agency for Science, Technology and Research (A*STAR) BS-PhD scholarships. He is a member of the following honorary societies: Tau Beta Pi, Sigma Xi, Phi Beta Kappa and American Chemistry Society. Publications Ni M, Teo JC, Ibrahim MS, Zhang K, Tasnim F, Chow PY, Zink D, Ying JY, Characterization of membrane materials and membrane coatings for bioreactor units of bioartificial kidneys, Biomaterials . 2011 Feb;32(6):1465-76 Man HB, Zhang K, Robinson E, Chow EK, Ho D, Engineering Nanoparticulate Diamond for Applications in Nanomedicine and Biology, Chapter 15 Ultrananocrystalline Diamond 2nd Edition 2012 Shenderova, Elsevier Kim HJ, Zhang K, Moore L, Ho D, Diamond Nanogel-Embedded Contact Lenses Mediate Lysozyme-Dependent Therapeutic Release, ACS Nano 2014 8 (3) ; 2998–3005 Wang H, Lee DK, Chen KY, Chen JY, Zhang K, Silva A, Ho CM, Ho D, Mechanism-Independent Optimization of Combinatorial Nanodiamond and Unmodified Drug Delivery Using a Phenotypically-Driven Platform Technology, ACS Nano 2015 9 (3) ; 3332-3344D Conferences Zhang K, Kim HJ, Gatica M, Moore L, Chow EK, Ho D, Nanodiamond Therapeutic Delivery Vehicles for Cancer Treatment and Regenerative Medicine, International Association for Dental Research (20-23th Mar 2013) Seattle WA Zhang K, Kim HJ, Daneshgaran G, Yen A, Ho D, Nanodiamond-embedded chitosan hydrogel for localized, sustained, and targeted delivery of celecoxib against oral cancer, Materials Research Society (21-25 th April 2014), San Francisco, CA Cao YP, Kaplan A, Zhang K, Fei F, Youn LY, Lux R, Ho D, Kang M, In Vitro Effect of Nanodiamond on Oral Bacterial Biofilm, American Association of Endodontists (30th April-3rd May 2014) Washington DC