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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2014-12-31.

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Permanent Link: http://ufdc.ufl.edu/UFE0043692/00001

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Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2014-12-31.
Physical Description: Book
Language: english
Creator: Fueth, Matthias
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: Pharmaceutics -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
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Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Matthias Fueth.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Song, Sihong.
Electronic Access: INACCESSIBLE UNTIL 2014-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043692:00001

Permanent Link: http://ufdc.ufl.edu/UFE0043692/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2014-12-31.
Physical Description: Book
Language: english
Creator: Fueth, Matthias
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: Pharmaceutics -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Matthias Fueth.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Song, Sihong.
Electronic Access: INACCESSIBLE UNTIL 2014-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043692:00001


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1 IN VIVO AND IN VITRO PROTECTIVE EFFECT S OF ALPHA 1 ANTITRYPSIN ON DOXORUBICIN INDUCED CYTOTOXICITY By MATTHIAS FUETH A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011

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2 2011 MATTHIAS FUETH

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3 To my m other a nd f ather

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4 ACKNOWLEDGEMENTS I would like to gratefully and sincerely thank my supervisor, Dr. Sihong Song for his conti nuous support, intelligent guidance understanding, patience, and most importantly, his friendship throughout my graduate program. I have been amazingly fortunate to have an advisor who gave me the freedom to explore on my own and at the same time the guid ance to recover when my steps faltered. D r. Song taught me how to question thoughts and express ideas. His mentorship was outstanding in providing a well rounded experience consistent my long term career goals. He encouraged me to not only grow as an exper imentalist and a scientist but also as an instructor and an independent thinker His patience and support helped me overcome many crisis situations and finish this dissertation. I hope that one day I would become as good an advisor as D r. Song has been to me. me, Dr. Song I thank you M y gratitude is extended to Dr. Hartmut Derendorf, who I was fortunate to have on my supervisory committee I highly appreciate h is assistance and guidance in getting my graduate career started by allowing me to complete an internship in his department six years ago and providing me with the foundation for becoming a pharmaceutical scientist. Besides his academic and professional encouragement, I am thankful for his friendship and en trust ing over one hundred international students to me. For his support and help in many personal situations, I cannot thank him enough. I believe that his actions provided me with the unique opportunity to gain a wider breadth of experience while still a graduate stud ent I would also like to express my gratitude to my supervisory committee member Dr. Michael J. Katovich for his insightful comments and constructive criticisms at different

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5 stages of my research which helped me understand and enrich my ideas I am indebt ed to Dr. Katovich for his expert advice and providing equipment and practical advice allowing me to do up to date and high standard research. Furthermore, I greatly appreciate my supervisory committee member Dr. Anthony Palmieri for his construct ive guida nce and availability. I deeply thank him for entrusting the organization of classes and seminars to me. The trust he put in me helped me grow as a graduate student and beyond that I will miss his experienced and valuable opinions in our daily conversation s about research, sports and life in general. I also thank all the faculties, post doc fellows and graduate students in the College of Pharmacy fo r their friendship and support. A special gratefulness is extended to Dr. Yuanquin Lu and Dr. Christian Grimst ein who helped me during the most challenging moments of my experiments. Their professional advices and constant availability made my research possible. I also want to express my appreciation to Dr. Yan Ren, Huong T Le, Mong jen Chen and Erin Bruce for the ir assistance with my experiments and constructive suggestions during lab meetings Th e i r friendship, incredible knowledge and availability when I needed the most will never be forgotten A big thank you goes to the office staff of the Department of Pharma ceutics, Patricia J. Khan, Robin Keirnan Sanchez, Sarah Scheckner and Kim berly Howell for their kindly support on administrative issues. I am also grateful to all former interns with special thanks to Katja Giersch, Godelieve Ponjee, Lonneke Erkelens, Yue Zheng and Laura Herlan for th eir invaluable support and assistance throughout my experiments

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6 Finally, I would like to thank my family and friends, especially my mother and my father for their love, encouragement and never ending support. I am also very thankful to my girl friend, Eva, for her love, patience, encouragement and help during the course of my work here at the University of Florida Though only my name appears on the cover of this dissertation, a great many people have contributed to its produ ction. I owe my gratitude to all those people who have made this dissertation possible and because of whom my graduate experience has been one that I will cherish forever.

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7 TABLE OF CONTENTS page ACKNOWLEDGEMENTS ................................ ................................ ............................... 4 LIST OF TABLES ................................ ................................ ................................ .......... 10 LIST OF FIGURES ................................ ................................ ................................ ........ 11 LIST OF ABBREVIATIONS ................................ ................................ ........................... 14 ABSTRACT ................................ ................................ ................................ ................... 15 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 17 Rationale ................................ ................................ ................................ ................. 17 Doxorubicin (DOX, Adriamycin ) ................................ ................................ ........ 17 Cardiomyopathy ................................ ................................ ............................... 19 Human Alpha 1 Antitrypsin (hAAT) ................................ ................................ .. 21 Objectives ................................ ................................ ................................ ............... 23 Objective 1: Evaluation Of The Protective Effect Of h AAT In Vitro .................. 24 Objective 1a: Determination of doxorubicin dose and exposure time ........ 24 Objective 1b: Determination of doxorubicin's binding effect to hAAT ......... 24 Objective 1c: Evaluation of the protective effect of hAAT ........................... 25 Objective 2: Evaluation Of The Therapeutic Effect Of hAAT Therapy On Doxorubicin Induced Cardiom yopathy In Mouse Models. ............................. 25 Objective 2a: Evaluation of hAAT therapy in C57BL/6 mice. ..................... 25 Objective 2b: Examination of hAAT in NO D hAATtg mice. ........................ 26 Objective 3: Examination Of The Therapeutic Potential Of hAAT Therapy On Doxorubicin Induced Cardiomyopathy In Rats. ................................ ....... 26 Objective 3a: Examination of hAAT therapy in SD rats using IP injection .. 27 Objective 3b: Examination of hAAT therapy in young S D rats using IV injections ................................ ................................ ................................ 27 Objective 3c: Examination of hAAT therapy in old SD rats using IV injections ................................ ................................ ................................ 27 2 EVALUATION OF THE PROTECTIVE EFFECT OF HUMAN ALPHA 1 ANTITRYPSIN THERAP Y IN VITRO ................................ ................................ ...... 29 Rationale ................................ ................................ ................................ ................. 29 Materials And Methods ................................ ................................ ........................... 30 Cell Prepara tion ................................ ................................ ................................ 30 Evaluation Of Cell Viability ................................ ................................ ............... 30 Data Analysis ................................ ................................ ................................ ... 31 Experim ental Designs ................................ ................................ ............................. 31

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8 Determination Of Doxorubicin Dose And Exposure Time ................................ 31 Acellular Method Validation ................................ ................................ .............. 31 ........................ 32 .......................... 32 Evaluation Of Therapy Strategies To Prevent D OX Induced Cytotoxicity ........ 33 Results And Discussion ................................ ................................ .......................... 33 Determination Of Doxorubicin Dose And Exposure Time In C2C12 C ells. ....... 33 Determination Of Doxorubicin's Binding Effect To hAAT In Vitro ..................... 34 The Protective Effect Of hAAT In C2C12 Cells And Cardiomyocytes. ............. 35 3 E VALUATION OF THE THERAPEUTIC EFFECT OF HUMAN ALPHA 1 ANTITRYPSIN THERAPY ON DOXORUBICIN INDUCED CARDI OMYOPATHY IN MOUSE MODELS ................................ ................................ .............................. 50 Rationale ................................ ................................ ................................ ................. 50 Materials And Methods ................................ ................................ ........................... 51 Electrocardiography (ECG) ................................ ................................ .............. 51 Human Alpha 1 Antitrypsin ELISA ................................ ................................ .... 52 Mouse Cardiac Troponin I ................................ ................................ ................ 52 Data analysis ................................ ................................ ................................ .... 53 Experimental Designs ................................ ................................ ............................. 53 Evaluation Of hAAT Therapy in C57BL/6 Mice. ................................ ................ 53 Animals ................................ ................................ ................................ ...... 54 Examination Of hAAT Therapy in NOD hAATtg Mice. ................................ ...... 54 Animals ................................ ................................ ................................ ...... 55 Results And Discussion ................................ ................................ .......................... 56 Evaluation Of hAAT Therapy On Doxorubicin Induced Cardiomyopathy Using C57BL/6 Mice. ................................ ................................ .................... 56 Examination Of The Therapeutic Potential Of hAAT On Doxorubicin Induced Cardiomyopathy Using NOD hAATtg Mice. ................................ ..... 57 4 EXAMINATION OF THE THERAPEU TIC POTENTIAL OF HUMAN ALPHA 1 ANTITRYPSIN (HAAT) THERAPY ON DOXORUBICIN INDUCED CARDIOMYOPATHY IN RAT MODELS ................................ ................................ 81 Rationale ................................ ................................ ................................ ................. 81 Ma terials And Methods ................................ ................................ ........................... 81 Animals ................................ ................................ ................................ ............. 81 Echocardiopgraphy (ECHO) ................................ ................................ ............. 82 El ectrocardiography (ECG) ................................ ................................ .............. 82 Human Alpha 1 Antitrypsin ELISA ................................ ................................ .... 83 Human Alpha 1 Antitrypsin Antibody ELISA ................................ ..................... 83 Data analysis ................................ ................................ ................................ .... 83 Experimental Designs ................................ ................................ ............................. 84 Examination Of The Therapeutic Potential Of h AAT Therapy In SD Rats Using An I P Administration Route. ................................ ................................ 84

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9 Examination Of The Therapeutic Potential Of hAAT Therapy In Young SD Rats Using An I V Administration Route. ................................ ........................ 85 Examination Of The Therapeutic Potential Of hAAT Therapy In Old SD Rats Using An I V Administration Route ................................ ................................ 86 Results And Discussion ................................ ................................ .......................... 87 Examination Of The Therapeutic Potential Of Human Alpha 1 Antitrypsin (hAAT) Therapy On doxorubicin Induced Cardiomyopathy In Sprague Dawley Rats Using An Intraperitoneal Administration Route ......................... 87 Examination Of The Therapeutic Potential Of Human Alpha 1 Antitrypsin (hAAT) Therapy On doxorubicin Induced Cardiomyopathy In Young Sprague Dawley Rats Using An Intravenous Administration Route .............. 90 Examination Of The Therapeutic Potential Of Human Alpha 1 Antitrypsin (hAAT) Therapy On doxorubicin Induced Cardiomyopathy In Old Sprague Dawley Rats Using An Intravenous Administration Route ............................. 92 5 CONCLUSION ................................ ................................ ................................ ...... 123 LIST OF REFERENCES ................................ ................................ ............................. 127 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 1 32

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10 LIST OF TABLES Table page 2 1 Complete list of materials and equipment for the in vitro studies ........................ 37 2 2 Determination of doxorubicin dose and exposure time on C2C12 cells .............. 38 2 3 Determination of h s protective effect on doxorubicin induced cell de ath ... 45 3 1 Complete list of materials and equipment for the in vivo studies with two different mouse models ................................ ................................ ...................... 60 4 1 Complete list of materials an d equipment for the in vivo studies with three different rat models ................................ ................................ ............................. 95 4 2 Distribution of Sprague Dawley rats in IP multiple injection experiment ............. 96 4 3 Distribution of young Sprague Dawley rats in IV multiple injection experiment ... 96 4 4 Distribution of old Sprague Dawley rats in IV multiple injection experiment ....... 96

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11 LIST OF FIGURES Figure page 2 1 Timeline for doxorubicin dose and exposure time experiment on C2C12 cells .. 39 2 2 Repres entative images of C2C12 cells exposed to doxorubicin ......................... 40 2 3 Results of the experimental conditions determination ................................ ....... 41 2 4 Relative units versus concentration standard curve for protein binding study .... 42 2 5 .......................... 43 2 6 C ............................ 44 2 7 Timeline for the three different hAAT treatment strate gies.. ............................... 46 2 8 Effect of hAAT on doxorubicin induced cytotoxicity in C2C12 cells. (1 hour) ...... 47 2 9 Effect of h AAT on doxorubicin induced cytotoxicity in C2C12 cells. (16 hours) .. 48 2 10 Effect of hAAT on DOX induced cyto toxicity in rat primary cardiomyocytes ....... 49 3 1 Body weight change over the time of the experiment with nine C57BL/6 mice. .. 61 3 2 P P intervals over the time of the initial experiment with nine C57BL/6 mice.. ... 62 3 3 Q T intervals over the time of the initial experiment with nine C57BL/6 mice. .... 63 3 4 Early time point summary of body weight change and al l heart function p arameters using ECG for the initial exp eriment with nine C57BL/6 mice. ......... 64 3 5 End point summary of body weight change and all heart function parameters using ECG for the ini tial experiment with nine C57BL/6 mice.. ........................... 65 3 6 Timeline for the experiment with twenty C57BL/6 mice. ................................ ..... 66 3 7 hAAT levels over the time of the experiment with twenty C57BL/6 mice. ........... 67 3 8 Body weight change over the time of the experiment with 20 C57BL/6 mice. .... 68 3 9 Heart function was monitored with ECG examinations with attention to R R intervals over the time of the experiment with twenty C57BL/6 mice. ................. 69 3 10 Heart function was monitored with ECG examinations with attention to Q T intervals over the time of the experiment with twenty C57BL/6 mice. ................. 70

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12 3 11 Final summary of body weight change and all heart function parameters using ECG for the experiment with twenty C57BL/6 mice. ................................ 71 3 12 Cardiac Troponin I levels of the experiment with 20 C57BL/6 mice. .................. 72 3 13 Timeline for the experiment with the NOD hAATtg mouse model. ..................... 73 3 14 hAAT levels at the beginning of the experiment with NOD hAATtg m ice .......... 74 3 15 Body weight change over the time of the experiment with NOD hAATtg mice l. 75 3 16 Heart function was monitored with ECG examinations with attention to R R inter vals over the time of the experiment with NOD hAATtg mouse model. ....... 76 3 17 Heart function was monitored with ECG examinations with attention to Q T intervals over the time of the experiment wi th NOD hAATtg mouse model. ....... 77 3 18 Final summary of body weight change and all heart function parameters using ECG for the experiment with NOD hAATtg mouse model. ........................ 78 3 19 Survival curve over the time of the experiment with NOD hAATtg mice ............. 79 3 20 Representative histology of organs harvested at the endpoint of the ex periment with NOD hAATtg mouse model. ................................ ..................... 80 4 1 Timeline for the IP multiple injection experiment with 35 SD rats. ...................... 97 4 2 hAAT le vels and hAAT antibody lev els over the time of the IP multiple injection experim ent with 35 SD rats ................................ ................................ .. 98 4 3 Body weight change over the time of the IP multiple injection experiment with 35 SD rats. ................................ ................................ ................................ .......... 99 4 4 All heart function parameters using ECG over the time of the IP multiple injection experiment with 35 SD rats. ................................ ............................... 100 4 5 Final summary of all heart function parameters using ECG of the IP multiple injection experiment with 35 S D rats ................................ ................................ 101 4 6 Measured ECHO data of the IP multiple injection experiment with 35 SD rats 102 4 7 Calculated ECHO data of the IP multiple injection experiment with 35 SD rats 103 4 8 Final summary of all h eart function parameters using ECHO of the IP multiple injection experiment with 35 SD rats ................................ ................................ 104 4 9 Actual organ weight and organ weight/body weight ratios of the IP multiple injection experi ment with 35 SD rats. ................................ ............................... 105

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13 4 10 Representative histology of organs harvested at the endpoint of the IP multiple injection experiment with 35 SD rats. ................................ .................. 106 4 11 Calculated percentage of apoptosis based on TUNEL stained hearts of the IP multiple injection experiment with 35 SD rats ................................ ................... 107 4 12 Timeline for the IV multiple injection experiment with 11 young SD rats. ......... 108 4 13 hAAT levels and hAAT antibody levels over the time of the IV multiple injection experiment with 11 young SD rats ................................ ...................... 109 4 14 Body weight change over the time of the IV multiple injection experiment with 11 young SD rats. ................................ ................................ ............................. 110 4 15 All heart function parameters using E CG over the time of the IV multiple injection experiment with 11 young SD rats ................................ ...................... 111 4 16 Final summary of all heart function parameters using ECG of the IV multiple injection experiment with 1 1 young SD rats ................................ ...................... 112 4 17 Measured ECHO data of the IV multiple injection experiment with 11 young SD rats ................................ ................................ ................................ ............. 113 4 18 Calculated ECHO data of the IV multiple injection experiment with 11 young SD rats ................................ ................................ ................................ ............. 114 4 19 Representative histology of organs harvested at the endpoint of the IV multiple injection experiment with 11 y oung S D rats ................................ ........ 115 4 20 Timeline for the IV multiple injection experiment with 6 old SD rats. ................ 116 4 21 hAAT levels and hAAT anti body levels over the time of the IV multiple injection experiment with 6 old SD rats ................................ ............................. 117 4 22 Body weight change over the time of the IV multiple injection experiment with 6 old SD rats ................................ ................................ ................................ ..... 118 4 23 All heart function parameters using ECG over the time of the IV multiple injection experiment with 6 old SD rats. ................................ ............................ 119 4 24 Measured ECHO data of the IV multiple injection experiment with old SD rats 120 4 25 Calculated ECHO data of the IV multiple injection experiment with 6 old SD rats ................................ ................................ ................................ ................... 121 4 26 Representative histology of organs harvested at the endpoint of the IV multiple injection experiment with 6 old Sprague Dawley rats. ......................... 122

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14 LIST OF ABBREVIATION S %FS L eft ven tricular fractional shortening ALB A lbumin C2C12 M ouse myoblast cell line DMEM DMSO Dimethyl Sulfoxid DOX D oxorubicin ECG E lectrocardiography ECHO E chocardiography EF L eft ventricular ejection fraction ELISA Enzyme Linked Immunosorbent A ssay ESV End systolic volume FBS Fetal Bovine Serum hAAT Human alpha 1 antitrypsin IP I ntraperitoneal IV I ntravenous IVS s I nterventricular septum thickness in systole LVID s L eft ventricular internal di ameter in end systole LVPW s L eft ventricular posterior wall thickness in systole MTT 3 (4,5 di m ethyl t hiazol 2 yl) 2,5 diphenyl 2H t etrazolium bromide P/S Penicillin Streptomycin antibiotic solution PBS Phosphate Buffered Saline SV Stroke volume

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15 Abstract of Dissertation Presented to the Graduate School of the University of Florida in P artial Fulfillme nt of the Requirements for the D egree of Doctor of Philosophy IN VIVO AND IN VITRO PROTECTIVE EFFECT S OF ALPHA 1 ANTITRYPSIN ON D OXORUBICIN INDUCED CYTOTOXICITY By Matthias Fueth December 2011 Chair: Sihong Song Major: Pharmaceutical Sciences The use of the antineoplastic drug d oxorubicin (DOX) is limited by its cumulative dose dependent cardiotoxicity, which may result from in tense cardiac oxidative stress and inflammation, but the mechanism is not yet completely understood. As sho wn in previous studies, human a lpha 1 a ntit rypsin ( h AAT) exhibits anti inflammatory and anti oxidative properties, protect s pancreatic beta cells from cytokine induced apoptosis and reduc es the incidence and severity of collagen induced arthritis. In this study, we investigated the feasibility of u sing h AAT to attenuate d oxorubicin induced cardiotoxicity. For the in vitro studies, cell viability was investigated in both C2C12 cells and primary rat cardiomyocyte cultures and measured by MTT assay utilizing two different treatment strategies. We further performed a protein binding study to investigate h AAT. For the in vivo studies we monitored electrocardiography ( ECG ) in two different mouse models after intraperitoneal injections of d oxorubicin Furthermore three experiments using Sprague Dawley rats were conducted, in which we used echocardiography additionally to monitor heart functio n. Also, in two age different rat model s we administered doxorubicin and h AAT intravenously with a multiple dosing scheme In all animal experiments, body weight s

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16 and cardiac Troponin I levels were measured and the myocardium was pathologically examined with H&E and TUNEL staining The results of in vitro as well as in vivo studies showed that h AAT significantly reduced d oxorubicin induced cytotoxicity and cell death. In vitro investigati on showed that hAAT inhibited cell killing after being exposed to doxorubicin. Doxorubicin also showed no signs of binding to hAAT The protective effects of h AAT were confirme d in vivo with significant prevention of body weight loss, serum troponin I elevation and alterations of QT and QRS interval s in C57BL/6 mice a s well as a significant higher survival in hAAT transgenic mice Despite complications that occurred in our rat model, most of the ECG results in mice were confirmed in this model. T he echocardiog raphy also showed that h AAT prevent ed cardiac damage to some degree We further o bserved a trend of dilated left ventricle s when subjects were not treated with hAAT. Our r esults have provided considerable evidence that AAT may potentially serve as a novel cardio protective agent against d oxorubicin induced cardiac injury.

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17 CHAPTER 1 INTRODUCTION Rationale The estimated cancer prevalence in the United States for the year 200 7 was over 11 million people and it wa s estimated that about 1.6 million new cases of cancer will be diagnosed in 201 1 Also, in the year 20 11 about 571,950 Americans will die of cancer more than 1,500 people a day (SEER Cancer Statistics Review) Cancer i s the second leading cause of death in the US, exceeded only by heart disease. One of every four deaths in the US is from cancer. Since 1990, there have been approximately 5 million cancer deaths. The financial costs of cancer are great for both the person with cancer and for society as a whole. In 2009, the National Institutes of Health estimated the 2008 overall annual costs of cancer were $228.1 billion (American Cancer Society/Cancer Facts & Figures 2009. Atlanta, GA). With these numbers in mind, I thin k there is a greater need than ever for developing more efficient and safer anti cancer treatment Doxorubicin (DOX, Adriamycin ) Anthracyclines primarily d oxorubicin (DOX) are well established and in use worldwide as highly efficacious broad spectrum ant ineoplastic agents for various hemopoietic [2] and solid tumors [3 5] for over ten years The primary factor limiting the clinical use of doxorubicin is its undesirable serious cardiotoxic side effect leading to cardiomyo pathy that leads to congestive heart failure and death. doxorubicin induced acute cardiotoxicity may begin with the initiation of chemotherapy and may resolve, whereas the chronic cardio toxicity develops any time after completion of doxorubicin regimens a 2 body surface area (BSA) [7, 22 24] In a study with 265 pediatric patients, the total cumulative

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18 incidence of doxorubicin induced cardiomyopathy was 7.5 %. Cardiomyopathy manifested in 11 cases already under antineoplastic therapy and in the remaining nine cases after stopping antineoplastic therapy [25] In another retrospective study of 399 patient medical record s, doxorubicin associated cardiomyopathy and congestive heart failure were dose dependent, and the incidence of these complications rose to unacceptably high levels when the cumulative dose of the drug exceeded 550 mg /m 2 BSA Congestive heart failure devel oped in more than 4% of patients who had received a cumulative doxorubicin dose of 500 to 550 mg/m 2 BSA and the incidence rose to more than 18% at a dose of 551 to 600 mg /m 2 BSA and to about 36 percent at a dose of 601 mg/m 2 BSA o r greater [8, 26] Despite extensive clinical utilization, the mechanism of action of anthracyclines remain s a matter of controversy and is mainly considered as follows: intercalation into DNA and interference with DNA unwinding or DNA str and separation and helicase activity, leading to inhibited synthesis of macromolecules; generation of free radicals, leading to DNA damage and/or lipid peroxidation; induction of apoptosis in response to topoisomerase II inhibition [27] Most of these well established theories are reasonable chemotherapy agent by inhibiting tumor cells to further p rogress. As a consequence, in action. Recent studies have revealed that the mechanism underlying doxorubicin induced cardiac injury includes apoptosis of cardiomyocyte s, irreversible damage of DNA and alteration of cardiac energetics due to cardiac oxidative stress, caused by

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19 increased production of reactive oxygen species, with subsequent cardiac inflammation and apoptosis, both of which limit cardiac function [9, 10] Although a variety of approaches to protect the heart against doxorubicin induced cardiotoxicity have been attempted, a treatment to prevent short and long term doxorubicin induced cardiac damage assumedly cause d by oxidative stress, induction of apoptosis and inflammation, remains limited All over the world, researchers have attempted to develop a treatment, showing cardiac protection against doxorubicin induced cardiomyopathy for example by moderate diet rest riction [16] using Dexrazoxane, an iron chelator [17] or treatment with the phytochemical oleuropein [18] as well as other treatm ent strategies and mechanism investigations Even though doxorubicin with its cardiotoxic side effect has been studied for over 50 years, Dexrazoxane (Zinecard ) has been the only Food and Drug Administration (FDA) and European Medicines Agency (EMA) approv ed treatment to prevent long term cardio toxicity caused by anthracyclines. Recently, the FDA and EMA restricted the use of dexrazoxane injection to adult patients, who have already received a certain amount of anthracyclines, in order to reduce incidence of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) especially in children Consequently, it is imperative to find supporting treatments, which may prevent doxorubicin induced cardiac injury. We believe that our research has relevance with r espect to the development of new protective agents to minimize cardiac inflammation and apoptosis. Cardiomyopathy Even though doxorubicin is being used successfully in the treatment of a variety of cancer, its cardio toxic side effect, which can lead to c ardiomyopathy and congestive heart failure, is alarming. Cardiomyopathy is a weakening of the heart muscle or a

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20 change in the heart muscle structural composition. This disease of the heart m uscle is a type of progressive heart disease in which the heart is abnormally enlarged or it makes the heart muscles thicker and more rigid than normal. In rare cases, scar tissue replaces the muscle tissue. As a result, the heart muscle's ability to pump blood is reduced, often causing serious complications such as hear t failure, abnormal heart rhythms, endocarditis ( an inflammation of the heart lining ) and backup of blood into the lungs or rest of the body leading to fluid buildup Usually, cardiomyopathy begins in the heart's lower chambers (the ventricles), but in sev ere cases can affect the upper chambers or atria as well. Among others, the most common types of cardiomyopathy include : Dilated cardiomyopathy, a condition in which the heart becomes weak and large, leading to reduced pump efficiency Hypertrophic cardiom yopathy (HCM) is a condition in which the heart muscle is thickened. This condition makes it harder for blood to leave the heart. HCM is usually passed down through families. I schemic cardiomyopathy is a term used to describe a heart condition, in which th e heart can no longer pump enough blood to the rest of the body due to the buildup of plaque and consequently blocking or narrowing of the small blood vessels that supply blood and oxygen to the heart. Restrictive cardiomyopathy is a group of disorders in which the heart chambers are unable to properly fill with blood because the heart muscle is stiff. Some types of cardiomyopathy run in families, but in many people, however, the cause is unknown. Common causes of cardiomyopathy are:

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21 Alcoholism and cocaine use Chemotherapy drugs, such as doxorubicin Coronary artery disease (ischemic cardiomyopathy) -most common cause End stage kidney disease Genetic defects High blood pressure (hypertension) Infections due to viruses -HIV, Lyme disease, Chagas disease Nutritional deficiencies (such as selenium, thiamine, and calcium) Pregnancy In general, the treatment usually involves medicines, su rgery, other medical procedures and lifestyle changes. Human Alpha 1 Antitrypsin (h AAT) As mentioned previously, many at tempts have been made to maintain damage. Human alpha 1 antitrypsin is a 52 kDa glycoprotein, which inhibits neutrophil elastase and proteinase 3 as well as other proteina ses It is primarily synthesized and expressed in the liver, but also in extrahepatic cells like neutrophils, monocytes, macrophages, alveolar macrophages, intestinal epithelial cells, carcinoma cells and the cornea [29 32] The normal serum level of h AAT is 2 3mg/mL During inflammation, infection or malignant diseases, h AAT levels, as an acute phase reacta nt can rise up to 3 4 fold. In human neutrophils, monocytes and alveolar macrophages, h AAT expression increases in response to inflammatory mediators like IL TNF [33, 34] It has been shown that h AAT is able to completely abolish the acute inflammatory infiltration and connective tissue breakdown [35] h AAT can also inhibit lipopolysaccharide (LPS) stimulated release of TNF release of the anti inflammatory cytokine IL 10 [36] It is further able to protect

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22 significantly against the let hality induced by TNF [37] hAAT can also induce expression of IL1 Ra in human peripheral blood mononuclear cells (PBMC) [38] and reduces ischemia induced apoptosis, inflammation, and acute phase response in kidney [39] There is increasing evidence that ke y activities of h AAT in vitro namely inhibition of endotoxin 10 in human monocyte, are mediated by an elevation of cAMP and activation of cAMP dependent protein kinase A [40] In inflammatory cells, elevation of cellular cAMP either through activation of multiple membrane receptors or inhibition of cAMP catabolism results in inhibition of LPS stimulated cytokine and chem okine release and leukocyte recruitment [41 44] and of T cell activation and proliferation [45] Our previous studies showed that h AAT gene therapy in a NOD mouse model reduced insulitis, decreased level s of insulin auto antibodies, attenuated cellular autoimmunity and prevented type 1 diabetes (an autoimmune disease) [46] Furthermore, neutrophil elastase inhibitors were able to reduce incidence as well as severity of collagen induced arthritis (CIA) in both, rats and mice [47] Our latest studies showed increasing evidence indicating that h AAT treatment with its anti inflammatory and anti oxidative prop erties re duces body weight loss as well as other clinical symptoms in doxorubicin induced cardiotoxicity in mice. Therefore, we propose d that h AAT may be used as a therapeutic drug, altering immune response and protecting tissue from damage in doxorubicin induced cardiomyopathy. We performed a series of in vitro and in vivo experiments with objectives to confirm what seems to be reasonable in the ory.

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23 Objectives Etiologies of cardiomyopathy are diverse, yet, one of the most common is chemotherapy, such as anthracycl ine induced cardio myopathy [1] Anthracyclines, primarily Doxorubicin (DOX) are well established as highly efficacious antineoplastic agents for various hemopoietic [2] and soli d tumors [3 5] However, the cumulative dose dependent cardio toxicity of these agents [4, 6, 7] which has been recognized for more than 20 years [8] continues to limit their therapeutic potential. Recent studies have revealed that the mechanism underlying doxorubicin induced cardiac injury includes apoptosis of cardiomyocytes due to cardiac oxidative stress and cardia c inflammation [9, 10] Human a lpha 1 antitrypsin (hAAT), is a multi functional protein with anti inflammatory and anti oxidative properties. We have already shown that treatment of hAAT prevents type 1 diabetes d evelopment by preventing islet cells from cytokine induced apoptosis in animal models [11] and showed the protective effect of hAAT protein and gene therapy on the development of arthritis in collagen induced arthritis (CIA) and pristane induced arthritis (PIA) mouse mo del [12] Increasing evidence indicate d that due to its anti inflammatory and anti oxidative properties, hAAT may protect cardiac tissue damage. But, the effect of human alpha 1 antitrypsin on doxorubicin induced cardiotox icity has not been tested, yet. The goal of this study was to investigate the feasibility of using the protective effect of hAAT protein therapy on the development of cardiotoxicity in doxorubicin induced cardiomyopathy animal model. We hypothesize d that anti inflammatory and anti oxida tive hAAT can be used as an efficient tool to prevent doxorubicin induced cardiomyopathy.

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24 The three objectives of this study we re designed to provide a comprehensive assessment that hAAT can be used as a new and promising approach to prevent doxorubicin in duced cardiac damage. Objective 1 : E valuat ion O f The Protective Effect Of H uman Alpha 1 A ntitrypsin (h AAT) In V itro The first objective was designed to provide an overview of human a lpha 1 a ntitryp protective properties on d using in vitro systems In addition, these experiments were necessary to develop basic concepts for the experimental conditions used for rodent studies. Objective 1 a : Determination o f d oxorubicin d ose a nd e xposure t ime i n a m ouse m yoblast c ell l ine (C2C12 cells). Before investigating more complex in vitro study designs, we need ed to determine optimal experimental conditions at which doxorubicin starts to show a decrea se in viable cell number. We use d a mouse myoblast cell line (C2C12) and different concen tration of doxorubicin (0g/ml to 200g/ml) as well as va rying exposure times (5, 60, 120 minutes and 16 hours ) to test dose and time dependent effects. Cell viability w as evaluated three days after doxorubicin exposure by MTT assay. The condition that res ulted in 50 60% killing w as used in the following experiments Objective 1 b : Determination of d oxorubicin's b inding e ffect t o h AAT i n v itro Considering the protein binding potential of Doxorubicin [13 15] we fur ther investigate d its binding potential to human alpha 1 antitrypsin. The necessity of this experiment was to proof that hAAT has potential as a protective agent because of its anti apoptotic and anti inflammatory properties, rather than just binding to do xorubicin and thus diminishing not only its toxic side effects, but also its effective anti neoplastic properties. U sing the experimental conditions from our previous experiment we

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25 incubated doxorubicin with different amounts of hAAT (0, 0.5, 1 and 2 mg/m L). Cell viability (reflecting the killing activity of doxorubicin ) was evaluated as described in Objective 1a In addition, we determine d free doxorubicin concentrations based on the separation of free drug from bound drug after incuba ting doxorubicin wit h varying hAAT concentrations and subsequent filtration Objective 1 c : Ev aluation o f t he p rotective e ffect o f h AAT i n C2C12 c ells a n d c ardiomyocytes. As a consequence of these preceding experiments, w e used two different treatment strategies (Preven tion a nd Pretreatment ) and two different hAAT concentrations to finally determine whether or not hAAT has a protective effect on doxorubicin induced cell death. To simulate the pathophysiological conditions to a greater extent we also use d rat primary cardiomyoc ytes. Objective 2 : E valuation O f The Therapeutic Effect O f H uman Alpha 1 A ntitrypsin ( h AAT ) T herapy O n D oxorubicin Induced Cardiomyopathy In Mouse M odels. Based on the observation we achieved from previous studies and our in vitro studies t he aim of the second objective wa s to demonstrate the feasibility of using the protective effect of hAAT protein therapy on the development of cardiotoxicity in doxorubicin induc ed cardiomyopathy mouse model s Objective 2a: Evaluation of hAAT therapy o n doxorubicin indu ced cardiomyopathy using C57BL/6 mice To test this hypothesis and to further confirm the da ta of previous studies, we perform ed an experiment us ing twenty C57BL/6 mice. We use d one high dose intraperitoneal injection of 15mg/kg doxorubicin and two additio nal low dose intraperitoneal injection s of 5mg/kg doxorubicin to induce cardiac damage. Physiological

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26 examination s of the mice were performed using ECG and daily weighing. Blood samples were drawn before and after the doxorubicin injection. Objective 2b: E xamination of the therapeutic potential of h AAT o n doxorubicin induced cardiomyopathy using NOD hAATtg mice. Our group has previously developed a hAAT transgenic mouse line on Non Obese Diabetic (NOD) background. These mice express high levels of hAAT in t he circulation. We used these mice (NOD hAATtg) to test the protective effect of hAAT on doxorubicin induced cardiomyopathy. Age matched normal NOD mice function ed as a control and we induce cardiac damage with two high dose intraperitoneal injection s of 1 5mg/kg doxorubicin Physical and physiological examinations of the animals were performed in the same way as mentioned under objective 2a Objective 3: Examination Of The Therapeutic Potential O f H uman Alpha 1 A ntitrypsin ( h AAT ) T herapy On Doxorubicin Indu ced C ardi omyopathy In R ats. Several other groups have been working with rat models, showing cardiac protection against doxorubicin induced cardiomyopathy by moderate diet restriction [16] using Dexrazoxane, an iro n chelator [17] or treatment with the phytochemical oleuropein [18] as well as other treatment strategies and mechanism investigations We have made the experience that rat s seem to be a more beneficial animal model, because they showed less variation, more consistency, easier hand ling and because rats seem to simulate most of the clinical and hemodynamic changes. The functional refractoriness of doxorubicin induced cardiomy opathy and heart failure observed in humans has been observed in rats [19 21] Supported by our previous observations and the implementing of echocardiography to monitor the heart function more detailed we tried t o accomplish the most promising therapy approach in this distincitve animal model.

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27 Objective 3a: Examination of the therapeutic potential of hAAT therapy o n doxorubicin induced cardiomyopathy in Sprague Dawley rats using an i ntraperitoneal administration r oute From our experience with this administration route in our previous mouse models, w e retained to induce d cardiomyopathy by multiple low dose intraperitoneal doxorubicin injection s W hen rats reach ed an average body weight of 0.25kg which correlates to approximately 8 weeks of age, we treat ed one group with scheduled hAAT intraperitoneal injection s before and after doxorubicin administrations Body weight and h eart function w ere measured weekly using electrocardiography and e chocardiography. Blood s ampl es were drawn weekly and tissues w ere harvested immediately after doxorubicin injections were completed as well as at the end of the experiment These were subjected to pathological examination including haematoxylin and eosin (H&E) and TUNEL staining Obj ective 3b: Examination of the therapeutic potential of hAAT therapy o n doxorubicin induced cardiomyopathy in young Sprague Dawley rats using an i ntravenous administration route To further simulate the treatment practiced in humans, w e induce d cardiomyopath y by multiple low dose intravenous doxorubicin injection s This experiment was in principle designed as the previous one, except we t reat ed one group with scheduled intravenous injection s of hAAT before and after doxorubicin administrations, wh en rats were 8 weeks of age and tissues w ere harvested only at the end of the experiment Objective 3c: Examination of the therapeutic potential of hAAT therapy o n doxorubicin induced cardiomyopathy in old Sprague Dawley rats using an i ntravenous administration route We also investigated the possibility of age related differences in our treatment and consequently initiated this experiment when animals reached an average body weight of

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28 450g which correlates to approximately 18 weeks of age. The study design was identi cal to the design previously described in objective 3b and therefore these experiments were performed simultaneously.

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29 CHAPTER 2 EVALUATION OF THE PR OTECTIVE EFFECT OF H UMAN AL PHA 1 ANTITRYPSIN THERAPY IN VITR O Rationale The aim of the first objective was designed to provide an overview of alpha 1 using in vitro systems and to develop basic concepts for the experimental conditions used for rodent studies. Du e to shortage of availability of cardiomyocyte cell line s we primarily used a mouse myoblast cell line (C2C12) This cell line was harvested from a thigh muscle of C3H mice and differentiates rapidly in culture These cells are capable of forming contracti le myotubes, producing character istic muscle proteins and are useful tool s to investigate their behavior when treated with doxorubicin. We are aware of the structural and characteristical differences between these progenitor cells and fully developed and differentiated cardiomyocyte s. An d even though our main focus is heart muscle cells, doxorubicin is harmful to other cells and organs as well and gave us a satisfying first impression of how cytotoxic this drug is in general In our initial in vitro experiments we therefore used these C2C 12 cells and exposed these to different concentration of doxorubicin as well as varying exposure times to test dose and time dependent effects Secondly, we conducted a protein binding experiment to eliminate the possibility that hAAT diminishes not only i ts toxic side effects, but also its effective anti neoplastic properties by binding to doxorubicin T o simulate to a greater extent the pathophysiological conditions we were fortunate to receive a few cardiomyocytes, which were isolated from neonatal (<1 week of age ) Sprague Dawley rats to investigate if hAAT has a protective effect on

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30 doxorubicin induced cytotoxicity. As a result of the observations from these in vitro experiments, we were able to design the more complex in vivo studies. Materials And Me thods For a complete list of lab materials and equipment which was used for the following in vitro experiments please refer to T able 2 1. Cell Preparation M ouse myoblast cells (C2C12), originally cultured fro m the thigh muscle of C3H mice were cultured on a 100mm cell culture dish in 1X Medium ( 1X DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin Streptomycin antibiotic solution (P/S) at 37C and 5% CO 2 After reaching 80% confluence, approximately 1.25x10 5 C2C 12 cells/well were seeded on a 12 well cell culture cluster. Shortly before reaching complete confluence medium was changed to DMEM supplemented with 2% FBS and 1% P/S, to induce differentiation into myotubes and the cells were kept under these condition s before experiment was initiated Evaluation Of Cell Viability Three days after initiation of doxorubicin exposure, t he difference in cell number was estimated by a colorimetric assay for measuring cell viability called MTT assay Only viable cells are ca pable of converting yellow 3 (4,5 Dimethyl 2 thiazolyl) 2,5 diphenyl 2H tetrazolium bromide (MTT) to a water insoluble blue purple colored formazan derivative (1 (4,5 Dimethylthiazol 2 yl) 3,5 diphenyl formazan). According to ls were incubated w ith 50L MTT (5mg/mL ) at 37C and 5% CO 2 f or 5 hours, solubilized in 600 L dimethyl sulfoxid (DMSO), transferred to a 96

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31 well plate and finally measured spectrophotometrically at 550nm using a 96 w ell m icroplate r eader Data A nalysis For the cellular experiment each treatment was simultaneously performed as a triplicate and the optical density reading at 550nm was averaged and depictured as relative units with Microsoft Excel 2007. The relative units of the control group (no treatment) wa s set at 100% and compared with the other treatments to determine the threshold, which was defined at a 50 60% killing. The si gnificance level was set at 0.05. In the acellular assay, the optical density reading of only 1X DMEM was set to 0% and the unfilt ered 50g/m L doxorubicin in 1X DMEM without any hAAT to 100% T he filtered solutions were described proportionally Experimental Designs Determination Of Doxorubicin Dose And Exposure Time For the determination of the experimental conditions, the medium wa s removed and cells were washed once with PBS and immediately exposed to different concentrations of doxorubicin in DMEM ( 0g/mL to 200g/mL ) for 5, 60, 120 minutes and 16 hours to test dose and time dependent effects (Table 2 2 ) Then, cells were washed w ith PBS and kept in fresh DMEM supplemented with 2% FBS and 1% P/S at 37C and 5% CO 2 For an overall timeline of th i s experiment, please refer to F igure 2 1. Acellular Method Validation Doxorubicin in increasing concentrations ranging from 0g/mL to 200g /mL in either PBS or 1X DMEM supplemented with 2% FBS and 1% P/S were prepared and measu red spectrophotometrically at 49 0nm using a 96 Well Microplate Reader to establish a relative units versus concentration standard curve.

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32 Acellular Evaluation Of Doxorub For the determination of the protein binding potential of doxorubicin under acellular conditions, we incubated different concentrations of hAAT (0.5, 1.0 and 2mg/mL) with 50 g/m L doxorubicin in 1X DMEM supplemented with 2 % FB S and 1% P / S antibiotic solution for 15 minutes at room temperature. We then filtered the solutions using Ultracel 30K centrifugal filters for 45 minutes at 5000RPM as recommended by the manufacturer. A solution with 50g/mL doxorubicin in 1X DMEM suppleme nted with 2 % FBS and 1% P / S antibiotic solution but no hAAT functioned as a control. After filtration, a fraction of these solutions (100 L) were transferred to a 96 well plate and finally measu red spectrophotometrically at 49 0nm using a 96 Well Microplate Reader o h AAT The majority of the filtrates was used on C2C12 cells to test cytotoxicity of doxorubicin after incubation with hAAT and subsequent centrifugal filtration. In addition, we exposed the sa me batch of C2C12 with different doxorubicin concentrations (0, 25, 50 and 100 g/ml) using identical conditions as earlier determined in objective 1a. The latter experiment was performed to show cell viability of identical passaged and differentiated C2C1 2 cells, when exposed to untreated doxorubicin. T he medium was removed and cells were washed once with PBS and immediately exposed to the filtered, unfiltered and untreated doxorubicin solution, for 60 minutes and 16 hours. Finally cells were washed with PBS and kept in fresh DMEM supplemented with 2% FBS and 1% P/S at 37C and 5% CO 2 For an overall timeline of this ex periment, please also refer to F igure 2 1.

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33 Evaluation Of T wo Therapy Strategies To Prevent Doxorubicin Induced Cytotoxicity Using the cond itions initially determined in objective 1a and d e pending on the therapy strategy hAAT i n two different concentrations (0. 5 and 1.0mg/mL ) was added to the existing medium either for 3 days prior (Pretreatment), or for 3 days prior plus 3 days after (Preve ntion) doxorubicin exposure (Figure 2 7). Albumin and heat inactivated hAAT in identical concentrations functioned as controls (Table 2 2) and differences in volume were corrected with appropriate volumes of PBS. P rior to doxorubicin exposure, the medium w as removed and cells were washed once with PBS and immediately exposed to 50 g/mL doxorubicin in DMEM for 60 minutes and 16 hours to induce cell death. Then, cells were washed with PBS and kept in fresh DMEM supplemented with 2% FBS and 1% P/S at 37C and 5% CO 2 and depending on the therapy strategy, spiked with hAAT. For an overall timeline of th is experiment, please refer to F igure 2 7. Results And Discussion Determination Of Doxorubicin Dose And Exposure Time In A Mouse Myoblast Cell L ine (C2C12 C ells). As shown in Figure 2 3 A, a short exposure time of doxorubicin (5 minutes) did not kill cells efficiently, provid ing a n in sufficient margin for future improvement by hAAT An increase in exposure time to 60 minutes (Fig ure 2 3 B) and 16 hours (Fig ure 2 3 D ) resulted in a significant cell killing of 73.3% 8.2 and 63.5% 4.7, respectively, when using 50g/ml doxorubicin leaving us with a more adequate margin to further investigate whether or not hAAT has a protective eff ect on doxorubicin induced cytotoxic ity After being exposed to various doxorubicin concentrations for 60 minutes, m icroscopic pictures of the C2C12 cell s were taken with a 4 0 fold magnification (Figure 2 2 ). These images clearly show a rapid reduc tion in cell number with increased

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34 doxorubic in concentration already 5 hours after being exposed to doxorubicin (Figure 2 2 A), which was even more notable on day 3, shortly before MTT assay was performed (Figure 2 2 B) At a exposure time of 120 minutes, we observed a to severe cell reduction of 81 .94% 2.4 (Figure 2 3 C) leaving no room for improvement or even masking a possible protective effect of hAAT by to extreme conditions. For these reasons, we used 50g/mL doxorubicin for either 60 minutes or 16 hours as the condition of our choice for the following more com plex in vitro study designs Determination Of Doxorubicin's Binding Effect To Human Alpha 1 Antitrypsin (hAAT) In V itro As the name doxorubicin implies, the French word ruby, rubis, meaning the color red, describes already the intense r ed colored drug. With 1X DMEM being also red, due to the colored ph indicator, we first tested the utilization of detecting doxorubicin in 1X DMEM at 490nm, at which doxorubicin has its absorption maximum. These r esults were compared to identical doxorubic in concentration s prepared in colorless PBS. We observed no differences in the absorption profile for our concentration range whe ther we used 1X DMEM or PBS as a solvent (Figure 2 4) This result indicated that doxorubicin in 1X DMEM can be detected accura tely. human alpha 1 antitrypsin, by incubating different amounts of hAAT with a constant concentration of doxorubicin. Using centrifugal filter devices with a cutoff of 30K nom inal molecular weight, we filtered all molecules with a higher molecular weight, including the 52kDa hAAT and doxorubicin if it bound to hAAT. A small amount of these filtrates was subjected to the spectrophotometrical measurement at 490nm. The concentrati on of the filtrate from the solution without hAAT resulted in 36.71% recovery compared to the

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35 unfiltered solution without hAAT. When incubated with 0.5, 1.0 and 2.0mg/ml hAAT we observed similar recoveries of 35.71%, 40.52% and 41.70%, respectively (Figure 2 5). Th ese initial results show ed that doxorubicin d id not bind to hAAT or only b ound it to a minimal extent. We further performed a cellular experiment in the same way as done previously, where we treated C2C12 cells with the filtrates and unfiltered d oxorubicin for 60 minutes (Figure 2 6 A, B) or 16 hours (Figure 2 6 C, D) We observed that incubation of doxorubicin with hAAT before the filtration did not affect its cytotoxicity (Figure 2 6) The s e results were consistent with the observations from our acellular experiment and indicated that doxorubicin does not bind to hAAT and that a possible protective effect of hAAT does not derive from doxorubicin binding to hAAT. Evaluation Of The Protective Effect Of Human Alpha 1 Antitrypsin (hAAT) In C2C12 Cell s A nd C ardiomyocytes. These final in vitro experiments were meant to investigate the protective effect of hAAT on C2C12 cells as well as rat primary Cardiomyocytes and show ed first results of protective properties against doxorubicin induced cytotox icity. We used two therapy strategies (Prevention and Treatment) to determine whether or not h AAT has a protective effect on doxorubicin induced cell death In the p revention experiment with 60 minutes exposure of doxorubicin we observed that doxorubicin induced a significant cell number reduction ( 36.9% 10.0 relative to t he c ontrol (no doxorubicin ), indicat ing that the conditions we chose were appropriate. Human alpha 1 antitrypsin at both concentrations (0.5mg/ml and 1.0 mg/ml) increased cell viability significantly with a cell reduction of only 5.0% 14.3 and 6.9% 11.3, respectively

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3 6 whereas no improvement in cell viability was observed when treated with Albumin or heat inactivated hAAT (Figure 2 8 ). After changing the conditions to 16 hours of doxorub icin exposure, we observed a similar result. hAAT significant ly protected C2C12 cells from doxorubicin induced cell death (Figure 2 9 A) In the pretreat ment experiment, hAAT also increased cell viability significantly (Figure 2 9 B) C2C12 cells incubated with hAAT alone did not alter cell growth (Figure 2 9 ). We have performed a similar experiment using primary car diomyocytes, which were isolated from neonatal (<1 week of age ) Sprague Dawley rats and seeded on 12 well plates. From experience of a collabor ating lab at the University of Florida and due to the fact that these cardiomyocytes are more sensitive a n approximately 100 fold lower dose of doxorubicin (0.58g/ml) but for a longer exposur e time (24 hours) has been used These conditions yield ed to a 67.3% 1 cell reduction between the control group and the doxorubicin only group. No improvement of the cell viability in our p revention group was observed when compared to all other treatments ( DOX alone, Albumin and h eat i nactivated hAAT ) (Figure 2 10 A) It is possible that these conditions might have been too extreme to protect cells

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37 Table 2 1. Complete list of materials and equipment for the in vitro studies Materials Company mouse myoblast cell line (C2C12) Amer ican Type Culture Collection (Manassas, VA, USA) Mediatech, Inc (Manassas, VA, USA) Fetal Bovine Serum (FBS) Mediatech, Inc (Manassas, VA, USA) Penicillin Streptomycin antibiotic solution (P/S) Mediat ech, Inc (Manassas, VA, USA) Doxorubicin hydrochloride Tocris bioscience (Ellisville, MO, USA) 1X Phosphate Buffered Saline Mediatech, Inc. (Manassas, VA, USA) 100mm x 20mm cell culture dish Corning, Inc. (Coring, NY, USA) 12 well cell culture cluster Corning, Inc. (Coring, NY, USA) 96 well Elisa Plate, clear BD Biosciences (Bedford, MA, USA) Disposable serological pipette Corning, Inc. (Coring, NY, USA) 15ml and 50ml centrifuge tubes Corning, Inc. (Coring, NY, USA) Micropipettes Eppendorf AG (Hambu rg, Germany) Pipet Tips USA Scientific, Inc. (Ocala, FL, USA) In vitro toxicology assay kit, MTT based Sigma Aldrich, Co. (St. Louis, MO, USA) Dimethyl Sulfoxid (DMSO) FisherBiotech (Fair Law, NJ, USA) Dynex Technologies MRX Revelation 96 Well Micropla te Reader MTX Lab Systems, Inc. (Vienna, VA, USA) Human alpha 1 antitrypsin Talecris, Inc (Research Triangle Park,NC, USA) Centrifugal Filter Units Millipore Co. (Billierica, MA, USA)

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38 Table 2 2 Determination of d oxorubicin dose and exposure time on C2C12 cells. Cell viability was evaluated on day 3 by MTT assay cell line Treatment DOX [g/ml] for 5 min DOX [g/ml] for 60 min DOX [g/ml] for 120 min DOX [g/ml] for 16hr N C2C12 myoblasts Control --------3 DOX 2.5 2.5 0.5 3 DOX 5 5 1 3 DOX 10 10 12.5 2 3 DOX 25 25 25 5 3 DOX 50 50 50 10 3 DOX 100 100 100 25 3 DOX 200 200 50 3

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39 Figure 2 1. Timeline for doxorubicin dose and exposure time experiment on C2C12 cells

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40 Figure 2 2 Representative images of C2C12 cells, wh ich were exposed to different concentrations of doxorubicin for 60 min. ( A ) 5 hours after doxorubicin exposure; ( B ) on day 3, shortly before MTT assay. After doxorubicin exposure the cells were kept for 3 days in fresh DMEM + 2% FBS at 37C and 5% CO 2 40x Magnification

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41 Figure 2 3 D etermination of the experimental conditions C2C12 cells were exposed to different concentrations of doxorubicin in DMEM for ( A ) 5 min ( B ) 60 min ( C ) 120 min and ( D ) 16 hours to test dose and time dependent effects Cell viab ility is expressed in percent relative to Control using the optical Density reading at 550nm (OD 550nm ) after MTT assay. Dashed line indicates 50% cut off value of C ontrol. Results are expressed as the mean +SD of triplicate assays. ** p

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42 Figure 2 4. Relative units versus concentration standard curve. Increasing amounts of doxorubicin were solved in either 1X DMEM supplemented with 2% F BS and 1% P/S or 1X PBS and measured spectrophotometrically at 490nm. Relative units represent the optical Density reading at 490nm (OD 490nm )

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43 Figure 2 5. Acellular e valuation of W e incubated different conc entrations of hAAT (0.5, 1.0 and 2mg/ml) with 50g/ml doxorubicin in 1X DMEM supplemented with 2% FBS and 1% P/S antibiotic solution. A solution with 50g/ml doxorubicin but no hAAT functioned as a control Solutions were filtered using Ultracel 30K centri fugal filters. Filtrates were transferred to a 96 well plate and measured spectrophotometrically at 490nm doxorubicin recovery is expressed in percent relative to 0 AAT unfil using the optical Density reading a t 490nm (OD 490nm )

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44 Figure 2 6. Cellular e valuation of C2C12 cells were exposed to different concentrations of doxorubicin for ( A ) 60 minutes and ( C ) 16 hours to test dose dependent effects Under identical conditions, the filtered doxorubicin and unfiltered doxorubicin solutions were used on C2C12 cells for ( B ) 60 minutes and ( D ) 16 hours. Finally cells were washed with PBS and kept in fresh DMEM supplemented with 2% FBS and 1% P/S at 37C and 5% CO 2 Cell viability is expressed in percent rel ative to Control using the optical Density reading at 550nm (OD 550nm ) after MTT assay Results are expressed as the mean +SD of triplicate assays.

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45 Table 2 3 Determination of human alpha induced cell death using C2C12 cells. Cell viability was evaluated on day 3 by MTT assay. hi hAAT, heat inactivated human alpha 1 antitrypsin. Therapy Strategy Treatment DOX for 60 min or 16 hr for 3 days before DOX for 3 days after DOX N Prevention Control ------3 DOX 50g/ml ----3 Albumin 50g/ml 0.5 and 1mg/ml 0.5 and 1mg/ml 3 each hi hAAT 50g/ml 0.5 and 1mg/ml 0.5 and 1mg/ml 3 each hAAT 50g/ml 0.5 and 1mg/ml 0.5 and 1mg/ml 3 each Pretreatment Control ------3 DOX 50g /ml ----3 Albumin 50g/ml 0.5 and 1mg/ml --3 each hi hAAT 50g/ml 0.5 and 1mg/ml --3 each hAAT 50g/ml 0.5 and 1mg/ml --3 each

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46 Figure 2 7. Timeline for the three different hAAT treatment strategies. Filled black lines represe nt the duration of hAAT being added to the medium. The black and white filled line represents whether hAAT was added 60 minutes or 16 hours after doxorubicin exposure.

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47 Figure 2 8. Effect of hAAT on doxorubicin induced cytotoxicity in C2C12 cells C2C12 cells were incubated with hAAT before and after (Prevention strategy) 60 minutes doxorubicin (50 g/ml) exposure followed by MTT assay after three days. Cell viability is expressed in percent relative to Control using the optical Density reading at 550nm (OD 550nm ) after MTT assay ( ), negative control (DOX only); Alb, Albumin; iAAT L, heat inactivated hAAT 0.5mg/ml; iAAT H, heat inactivated hAAT 1.0mg/ml; AAT L and AAT H, h AAT 0.5mg /ml and 1.0mg/ml, respectively. Results are expressed as the mean + SD of triplicate assays. ** p value 0.01.

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48 Figure 2 9 Effect o f h AAT on doxorubicin induced cytotoxicity in C2C12 cells. C2C12 cells were incubated with h AAT ( A ) before and after 16 hours doxorubicin (50g/ml) exposure (Prevention strategy); ( B ) only before (Pretreatment) 16 hours doxorubicin (50g/ml) exposure followed by MTT assay on day 3. Cell viability is expressed in percent relative to Control using the optical Density reading at 550nm (OD 550nm ) after MTT assay AAT H, hAAT 1.0mg/ml. Res ults are expressed as the mean +SD of triplicate assays. p value and ** p

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49 Figure 2 10. Effect of hAAT on doxorubicin induced cytotoxicity in rat primary cardiomyocytes. Cardiomyocytes were incubated with hAAT before and after 24 hours DOX ( 0.58 g/ml) exposure (Prevention strategy) followe d by MTT assay on day 3 Results are expressed as the mean + SD results of a triplicate assays ( left) and a duplicate ( right ). Cell viability is expressed in percent relative to Control using the optical Density reading at 550nm (OD 550nm ) after MTT assay ( ), DOX only; Alb l Albumin 0.5mg/ml ; iAAT L, heat inactivated hAAT 0.5mg/ml; AAT l and AAT H, hAAT 0.5mg /ml and 1.0mg/ml, respectively.

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50 CHAPTER 3 EVALUATION OF THE TH ERAPEUTIC EFFECT OF HUMAN ALPHA 1 ANTITRYPSIN (HAAT) THERAPY ON DO XORUBICIN INDUCED CA RDIOMYOPATHY IN MOUS E MODELS Rationale Following our in vitro studies and due to the fact that most of the successful work in our group was experienced in mice, t he second objective was to demonstrate the feasibility of using the protective effect of hAAT protein therapy on the development of cardiotoxicity in doxorubicin induc ed cardiomyopathy mouse model s In order to test the protective effect of hAAT on doxorubicin induced cardiomyopathy in vivo an initial study with nine C57BL/6 mice was performed ea rlier in our lab oratory We conducted a pro phylactic treatment study, in which h uman alpha 1 antitrypsin ( hAAT, n=5) and lactated RL, n=4) was intraperitoneal injected every three days throughout the length of the experiment The injecti on start ed right after mice received a single high dose intraperitoneal injection of doxorubicin (15mg/kg). Ten days before until 14 days after doxorubicin injection, all mice were weig hted daily and received p hysiological examination s twice a week using e lectro c ardiography (ECG). Body w eight change is a common side effect during chemotherapy, both because doxorubicin may reduce appetite and the general toxicity of the drug can lead to tissue wasting We showed that 15 days post doxorubicin injection hAAT a ttenuated body weight loss (93.4% 4.0) compared to the R L group (87.8% 7.4) (Figure 3 1 ). As mentioned above, c ardiomyopathy is a weakening of the heart muscle leading to abnormal cardiac energetics due to cardiac oxidative stress, caused by increased pr oduction of reactive oxygen species, with subsequent cardiac inflammation and

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51 apoptosis These conditions limit cardiac function and are detectable by electrocardiography (ECG). Specifically, a decrease in heart rate and a prolongation of the QT interval a re strong evidences of an insufficient ability of the heart to pump blood into the body Physiological evaluations showed an acute raise in P P interval reflecting the heart rate, in the RL group (0.125s 0.0136) which was significantly prevented by hAAT (0.100s 0.0046) ( Figure 3 2 ). In addition, a significant decrease in QT interval prolongation was observed 4 days (0.041s 0.0010 (hAAT) compared to 0.054s 0.0051 ( RL )) and 8 days (0.043s 0.0038 (hAAT) compared to 0.057s 0.0036 ( RL )) after a single dox orubicin injection with p values of 0.001 and 0.003, respectively ( Figure 3 3 ). Overall, hAAT significantly prevented immediate doxorubicin induced cardiac damage four days after a single IP dose of doxorubicin (Figure 3 4). At the endpoint of this experim ent, even if it seemed like some of the control animals recovered from an initial bad condition, mice receiving hAAT still attenuated the overall worsening condition (Figure 3 5 ). These first promising in vivo results provided evidence that hAAT has great potential in preventing cardiac damage in doxorubicin induc ed cardiomyopathy mouse models, and made us design additional in vivo experiments with greater number of mice and a different mouse model. Materials And Methods For a complete list of lab material s and equipment which was used for the following in vitro experiments please refer to T able 3 1. Electrocardiography (ECG) All Animals were anaesthetized with 3% isoflurane at an oxygen flow rate of 1.0 liter/minute for 3 minutes and kept anaesthetized on a heating pad in a supine position

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52 for the duration of the ECG. The physiological examination was performed twice a week for 15 20 seconds using a 3 Lead ECG with a Dual Bio Amplifier which was interfaced to a PowerLab. Instead of recording the P P interva l to indirectly monitor the heart rate as we did in our previous study, we focused on the m ore precise R R interval method which is the most common measure of heart rate variability. It is used to assess the ventricular rate and it provides useful inform ation about left ventricular dysfunction after MI, heart failure, and hypertrophic cardiomyopathy. Another parameter of interest was a prolonged QT interval, indicating a characteristical risk factor for ventricular tachyarrhythmia by prolonging repolariza tion. This arrhythmia may cease spontaneously or degenerate into ventricular fibrillation. It causes significant hemodynamic compromise and often death. Human Alpha 1 Antitrypsin ELISA In th i s e xperiment, blood samples were drawn from the tail vein one we ek before and several times after doxorubicin administration ( Figure 3 6 ) to determine hAAT levels A sandwich ELISA of b lood samples which were thawed and diluted with ratio s ranging from 1:1000 to 1:4000, was performed a ccording to the manufacturer and measured spectrophotometrically at 450nm. Mouse Cardiac Troponin I In th i s e xperiment, blood samples were drawn from the tail vein before and after doxorubicin administration ( Figure 3 6 ) to evaluate cardiac Troponin I levels (cTnI). Blood samples were th awed and diluted with 1:4 ratio a manual and measured spectrophotometrically at 450nm.

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53 Data A nalysis Fifteen seconds of r ecorded ECG data were analyzed with Lab Chart 7Pro v7.1.2 software (ADInstruments, Colorado Springs, CO, USA) Mouse cardiac troponin I was detected by enzyme linked immunosorbent Assay (ELISA) and measured spectrophotometrically at 450nm with a 96 Well Microplate Reader Each sample was simultaneously performed as a duplicate and the optical density reading at 4 90nm was averaged and depictured as relative units with Microsoft Excel 2007. hAAT was also detected by ELISA and measured spectrophotometrically at 450nm with a 96 Well Microplate Reader Each sample was measured in three different dilutions to ensure rea dings in the linear region of a simultaneously performed standard concentration curve. The optical density reading at 490nm was compared to the standard concentration curve, averaged within the group and finally, the concentrations were depictured with Mic rosoft Excel 2007. Experimental Designs Evaluation Of Human Alpha 1 Antitrypsin (hAAT) Therapy On Doxorubicin Induced Cardiomyopathy U sing C57BL/6 M ice In this experiment, twenty mice were divided randomly into two groups in which 10 mice received intrape ritoneal injections of clinical grade hAAT and the other group the previous study was that the injection of 100 L hAAT (2mg/mouse) (AAT DOX, solution (RL DOX, n=10) in identical volumes every three days, started 7 days prior to doxorubicin injection s

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54 Cardiomyopathy was induced with one initial 100 L high dose intraperitoneal injection of doxorubicin (15mg/kg) on day 14 followed by two 100 l l ow dose intraperitoneal injection of doxorubicin injections (5mg/kg) on day 0 and day 3. All mice were weighted almost daily, the heart function was monitored with electrocardiography (ECG) twice a week and blood samples (> 200L) were drawn from the tail vein before and after doxorubicin injection s and always 24 hours after hAAT injections. These were kept at 80C for later evaluations of cardiac Troponin I levels (cTnI) and hAAT levels At the end of the experiment, all animals were eut hanized by cervica l dislocation. For an overall timeline of this experiment, please refer to Figure 3 6. After doxorubicin injection s if inflammation or tissue damage got too severe IACUC pre approved criteria was utilized to euthanize any animal to avoid any suffering. A nimals Female C57BL/6 mice, 5 6 weeks of age, were purchased from The Jackson Laboratory (Bar Harbor, Maine, 04609 USA) and housed in a pathogen free animal facility at the University of Florida (Gainesville, FL). Animals were acclimatized to standard ACS housing in a 12 h light dark cycle and constant temperature environment for a minimum of three days before being used. The Institutional Animal Care and Use Committees at the University of Florida approved all animal procedures and manipulations Examinati on Of The Therapeutic Potential Of Human Alpha 1 Antitrypsin (hAAT) O n D oxorubicin Induced Cardiomyopathy U sing NOD hAATtg M ice In this experiment Cardiomyopathy was induced with two 100 L high dose i ntraperitoneal injections of doxorubicin (15mg/kg) on day 0 and day 15. Since transgenic animal express high levels of hAAT in to the blood circulation there w as no

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55 need to permanent ly inject these animals with h AAT protein. NOD hAATtg (n=4) and a ge and gender matched normal NOD mice (n=5) were weighted almos t daily, the heart function was monitored with electrocardiography (ECG) twice a week and blood samples (> 200L) were drawn from the tail vein before and after doxorubicin injection and kept at 80C. At the end of the experiment, all animals were euthani zed by cervical dislocation and main ly heart, but also liver and kidney were removed and fixed in formalin and subjected to histological analysis. stained with hematoxylin and eosin (H&E) according to standard method s For an overall timeline of this experiment, please refer to Figure 3 13. After doxorubicin injection s if inflammation or tissue damage got too severe IACUC pre approved criteria was utilized to euthanize any animal to avoid any suffering. Animals Femal e Non Obese Diabetic (NOD) mice were originally purchased from The Jackson Laboratory (Bar Harbor, Maine, USA) and housed in a pathogen free animal facility at the University of Florida (Gainesville, FL, USA). Genetically modifications were done by our lab with the approval of the Institutional Animal Care and Use Committe es at the University of Florida For this experiment, NOD human Alpha 1 Antitrypsin transgenic mice (NOD hAATtg), 5 6 weeks of age, and age and gender matched regular NOD mice (NOD) which functioned as a control, were acclimatized to standard ACS housing in a 12 h light dark cycle and constant temperature environment for a minimum of three days before being used. The Institutional Animal Care and Use Committees at the University of Florida approved all animal procedures and manipulations.

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56 Results A nd Discussion Evaluation Of Human Alpha 1 Antitrypsin (hAAT) Therapy On D oxorubicin Induced Cardiomyopathy U sing C57BL/6 M ice Earlier, we hypothesize d that hAAT will prevent doxorubicin induced cardiomyopathy in vivo To test this hypothesis and to further support the results of our first experiment we performed a similar experi ment using twenty C57BL/6 mice A clinical relevant concentration of hAAT in the blood circulation of our animals is c rucial to prove our theory. We were able to show that after intraperitoneal injections, hAAT concentrations exceeded 1mg/mL for the duration of the experiment (Figure 3 7). Eleven days after the two low dose intraperitoneal injection s of doxorubicin (day 14) a significant prevention in body weight loss was observed (100.5% 4.9 (hAAT) experiment (Fig ure 3 8 ). We further experienced a significant prevention in R R interv al elongation in mice treated with hAAT on day 20 (0.113s 0.0060 (hAAT) compared to 0.139s 0. (0.110s 0.0028 (hAAT) compared to ure 3 9 ). Similar to our previous study, we confi rmed the result of an acute increase in QT i nterval in the control group shortly after the final intraperitoneal injection of doxorubicin whereas the hAAT treated group remained at baseline levels (Fig ure 3 10 ) In addition, a n overall improve ment of the heart function when treated with hAAT was observed in a var iety of other heart function parameters (Figure 3 11 ). Blood samples were drawn before and after doxorubicin injections to also evaluate cardiac Troponin I levels (cTnI). Troponin is a complex of t hree regulatory proteins that is integral to muscle contraction in skeletal and cardiac muscle. Therefore, it is a useful

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57 diagnostic biomarker for various heart disorders, primarily Troponin I, which is a very sensitive and specific indicator for damage to the heart muscle (myocardium). We were not able to detect any cTnI after the initial high dose of doxorubicin indicating that no cardiac damage oc curred at that time point. This led us to the decision to inject two more low doses of doxorubicin, after wh ich we did detect cTnI in the control group with concentration ranging from 0 .95ng/mL up to 2.16n g/mL but no such increase was observed in mice treated with hAAT (Fig ure 3 12 ). Examination O f The Therapeutic Potential Of Human Alpha 1 Antitrypsin (hAAT) O n D oxorubicin Induced Cardiomyopathy U sing NOD h AATtg M ice The intention of this objective was to further demonstrate that hAAT prevent s doxorubicin induced cardiac damage in a different mouse model We therefore performed a n experi ment similar to the one before, but using a transgenic mouse model Our group has previously developed a hAAT transgenic mouse line on Non O bese Diabetic (NOD) background, which is able to express high levels of hAAT in the circulation. Also, Dr. Song et al have shown in the pa st that hAAT prevents type I diabetes in this mouse model. Diabetes is a chronic disease which may ultimately lead to cardiac damage, due to increased superoxide production and several other reasons. Even though this mouse model has a serious disease alrea dy, w e thought the connection between heart disease and diabetes is not that absurd and used this diabetic m ouse model (NOD ) to test the protective effect of hAAT on doxorubicin induced cardiomyopathy. To ensure that all our transgenic animals express hAAT we measured the concentration of each individual animal before initiating the experiment and observed clinical concentrations in the range of 6.2mg/mL to 7.9mg/mL hAAT (Figure 3 14).

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58 F ourteen days after the first high dose intraperitoneal injection of d oxorubicin we observed no difference in any of our parameters of interest This might have indicat ed the chance of non functional and/or degraded doxorubicin and/or the possibility that a single intraperitoneal injection of 15mg/kg doxorubicin, as mention ed in literature many times, is just not suitable for this mouse model. As a result, we boosted the animals with a second high dose of doxorubicin to eventually induce cardiac damage. Shortly after the additional intraperitoneal injection of doxorubicin a n immediate decrease in body weight was observed (day 20: 82.2% 11.5 (NOD hAATtg) compared to 73.3% 3.7 (NOD)) (Fig ure 3 1 5 ). We were able to support the result of previous experiments with a significant increase in R R interval reflecting a decreased h eart rate in the NOD control group (0.307s 0.0594) compared to the NOD hAATtg group (0.168s 0.0379) (Fig ure 3 16 ). In addition a highly significant prevention of the acute QT interval elongation in the NOD hAATtg group (0.024 0.0010) compared to the NOD control group (0.034s 0.0055) was observed (Figure 3 17 ) With the high expression of hAAT in the blood circulation of our transgenic mouse model, we experience an attenuation of the doxorubicin induced cardio malfunction s as seen in a variety of other p arameters (Figure 3 18 ). As a consequence of all these experimental observations the NOD hAATtg mice cohort showed a significant higher survival (26.0days 5.2) compared to the NOD control group (19.8days 0.84) (Figure 3 19 ) The observed baseline levels of cardiac troponin I indicate that the time between the second high dose doxorubicin injection and the end point of the experiment for the NOD mice (3 6 days) was too short to damage the myocardium severely enough to

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59 release detectable amounts of cTnI in the blood circulation F or the same reason, there were no obvious differences in the histology of the organs (Figure 3 20 ). Based on our previous data and with the evidence from this experiment, we can conclude that hAAT does have an overall cardio prot ective effect in this doxorubicin induced cardiomyopathy mouse model.

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60 Table 3 1. Complete list of materials and equipment for the in vivo studies with two different mouse models Materials Company Doxorubicin hydrochloride Tocris bioscience (Ellisville, MO, USA) Human alpha 1 antitrypsin Talecris, Inc (Research Triangle Park, NC, USA) Baxter Health Care (Deerfield, IL, USA) Isoflurane, USP Webster Veterinary (Devens, MA, USA) cc Insulin Syringes BD Consumer Co. (Fran klin Lakes, NJ,USA) Steril needles (20 G1) BD Consumer Co. (Franklin Lakes, NJ, USA) Serum Separator Tubes BD Consumer Co. (Franklin Lakes, NJ, USA) 1X Phosphate Buffered Saline Mediatech, Inc. (Manassas, VA, USA) 96 well Elisa Plate, clear BD Biosci ences (Bedford, MA, USA) Disposable serological pipette Corning, Inc. (Coring, NY, USA) 15ml and 50ml centrifuge tubes Corning, Inc. (Coring, NY, USA) Micropipettes Eppendorf AG (Hamburg, Germany) Pipet Tips USA Scientific, Inc. (Ocala, FL, USA) Balan ce (d=0.1g) Ohaus Corp. (Pine Brook, NJ, USA) Dynex Technologies MRX Revelation 96 Well Microplate Reader MTX Lab Systems, Inc. (Vienna, VA, USA) ECG apparatus 3 Lead ECG with a Dual Bio Amplifier + PowerLab connection (ADInstruments, Colorado Springs, C O, USA) Mouse Cardiac Troponin I ELISA Kit (Life Diagnostics, Inc., West Chester, PA, USA) hAAT ELISA Kit Sigma Aldrich, Co. (St. Louis, MO, USA)

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61 Figure 3 1. Body weight change over the time of the initial experiment with nine C57BL/6 mice hAAT attenuated doxorubicin induced b ody weight loss after a single IP dose of doxorubicin (15mg/kg); 5 of these received cont inuatively h AAT (2mg/mouse) IP injections (hAAT, filled squares) and the other 4 mice ). Full black arrows indicate high dose doxorubicin injection (15mg/kg). R esults are expressed as the group mean SD. p value 0.0 5.

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62 Figure 3 2. P P intervals over the time of the initial experiment with nine C57BL/6 mice hAAT prevented a raise i n R R interval after a single IP dose of doxorubicin (15mg/kg); 5 of these received cont inuatively h AAT (2mg/mouse) IP inje ctions open circles). Heart function was monitored with biweekly ECG examinations with attention to R R interval as an indicator of cardiac damage. Full black arrows indicate high dose doxorubicin injection (15mg/kg). R esults are expressed as the group mean SD. p value 0.0 5.

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63 Figure 3 3 Q T intervals over the time of the initial experiment with nine C57BL/6 mice. hAAT prevented a QT interval prolongation after a single IP dose of doxorubicin (15mg/kg); 5 of these received cont inuatively hAAT (2mg/mouse) IP solution (RL, open circles). Heart function was monitored with biweekly ECG examinations with attention to Q T intervals as an indicator of cardiac damage Full black arrows indicate high dose doxorubicin injection (15mg/kg). Results are expressed as the group mean SD. p value

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64 Figure 3 4 Early time point s ummary of body weight change and all heart function parameters using ECG f or the initial experiment with nine C57BL/6 mice Each parameter evaluated on day 6 was set as the baseline (100%) and the change from the baseline was based on the examination on day 4. In general, hAAT significantly reduced acute doxorubicin induced cardiac damage after a single IP dose of doxorubicin (15mg/kg); 5 of these received cont inuatively hAAT (2mg/mouse) IP injections (hAAT, black bars ) and the other 4 mice white bars ). Heart function was monitored with biweekly ECG examinations Results are expressed as the group mean SD. p value value 0.0 1.

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65 Figure 3 5 End point summary of body weight change and all heart function parameters using ECG f or the initial experiment with nine C57BL/6 mice Each parameter evaluated on day 6 was set as the baseline (100%) and the chang e from the baseline was based on the final examination on day 14. hAAT attenuated doxorubicin induced cardiac damage after a single IP dose of doxorubicin (15mg/kg); 5 of these received cont inuatively hAAT (2mg/mouse) IP injections (hAAT, black bars ) and t white bars ). Heart function was monitored with biweekly ECG examinations Results are e xpressed as the group mean SD.

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66 Figure 3 6 Timeline for the experiment with twenty C57BL/6 mice

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67 Figure 3 7 hAAT levels over the time of the experiment with twenty C57BL/6 mice Ten mice received cont inuatively hAAT (2mg/mouse) intraperitoneal injections every three days. Results a re expressed as the group mean +SD.

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68 Figure 3 8 Body weight change ov er the time of the experiment with twenty C57BL/6 mice hAAT recovered from doxorubicin induced body weight loss after an initial high dose IP injection of doxorubicin (15mg/kg) and two more low dos e IP injections of doxorubicin (5mg/kg) ; 10 mice received cont inuatively hAAT (2mg/mouse) IP injections (filled squares) and the other 10 mice lactated Dotted black arrows indi cate low dose doxorubicin injection s (5mg/kg). Results are expressed as the group mean SD. p value 0.05.

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69 Figure 3 9 Heart function was monitored with ECG examinations with attention to R R intervals over the time of the experiment with twenty C57BL/6 mice hAAT prevented doxorubicin induced R R interval elongation after an initial high dose IP injection of doxorubicin (15mg/kg) and two more low dos e IP injections of doxorubicin (5mg/kg) ; 10 mice received cont inuatively hAAT (2mg/mouse) IP injections (filled squares) and the other 10 mice lactated Dotted black arrows indicate low dose doxorubicin injection s (5mg/kg). Results are expressed as the group mean SD. p value 0.05.

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70 Figure 3 10 Heart function was monitored with ECG examinations with attention to Q T intervals over the time of the experim ent with twenty C57BL/6 mice hAAT attenuated doxorubicin induced Q T interval elongation after an initial high dose IP injection of doxorubicin (15mg/kg) and two more low dos e IP injections of doxorubicin (5mg/kg) ; 10 mice received cont inuatively hAAT (2m g/mouse) IP injections (filled squares) and the other 10 mice lactated Dotted black arrows indicate low dose doxorubicin injection s (5mg/kg). Results are expressed as the group mean SD. p value 0.05.

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71 Figure 3 11 Final summary of body weight change and all h eart function parameters using ECG f or the experiment with twenty C57BL/6 mice Each parameter evaluated on day 2 was set as the baseline (100%) and the change from the baseline was base d on the final examination on day 26. In general, h AAT attenuated doxorubicin induced cardiac damage after an initial high dose IP injection of doxorubicin (15mg/kg) and two more low dos e IP injections of doxorubicin (5mg/kg); 10 mice received continuative ly hAAT (2mg/mouse) IP injections ( black bars white bars ). Results are expressed as the group mean SD.

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72 Figure 3 12 Cardiac Troponin I levels (cTnI levels) of the experiment with twenty C57BL/6 mice. hAAT treated cohort showed no signs of cTnI elevation after an initial high dose IP injection of doxorubicin (15mg/kg) and two more low dos e IP injections of doxorubicin (5mg/kg); 10 mice received continuatively hAAT (2mg/mouse) IP injections (filled squares) and the other 10 mice Results are expressed individually

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73 Figure 3 1 3 Timeline for the experiment with the NOD hAATtg mouse model

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74 Figure 3 14 hAAT levels at the beginning of the experime nt with the NOD hAATtg mouse model. Results a re expressed as the group mean +SD.

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75 Figure 3 1 5 Body weight change over the time of the experiment with the NOD hAATtg mouse model. NOD hAATtg mice attenuated doxorubicin induced body weight loss afte r two high dose intraperitoneal injection s of doxorubicin (15mg/kg); NOD hAATtg, filled squares; NOD, open circles. Full black arrows indicate high dose doxorubicin injection s (15mg/kg). R esults are expressed as the group mean SD. p value 0.0 5.

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76 Figure 3 16 Heart function was monitored with ECG examinations with attention to R R intervals over the time of the experiment with NOD hAATtg mouse model. hAAT prevented doxorubicin induced R R interval elongation after two high dose in traperitoneal injection s of doxorubicin (15mg/kg) ; NOD hAATtg, filled squares; NOD, open circles. Full black arrows indicate high dose doxorubicin injection s (15mg/kg). R esults are expressed as the group mean SD. p value 0.0 5.

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77 Figure 3 1 7 H eart function was monitored with ECG examinations with attention to Q T intervals over the time of the experiment with NOD hAATtg mouse model. hAAT prevented doxorubicin induced Q T interval pro longation after two high dose i ntra p eritoneal injection s of do xorubicin (15mg/kg) ; NOD hAATtg, filled squares; NOD, open circles. Full black arrows indicate high dose doxorubicin injections (15mg/kg). R esults are expressed as the group mean SD. ** p value 0.0 1.

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78 Figure 3 1 8 Final summary of body weight change and all h eart function parameters using ECG f or the experiment with NOD hAATtg mouse model. Each parameter evaluated on day 1 was set as the baseline (100%) and the change from the baseline was based on the final examination on day 20. In general, hAAT endured doxorubicin induced heart failure after two high dose i ntraperitoneal injection s of doxorubicin (15mg/kg) ; NOD hAATtg, black bars; NOD, white bars. R esults are expressed as the group mean SD.

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79 Figure 3 19 Survival curve over the time of the experiment with NOD hAATtg mouse model. NOD hAATtg mice showed a significant higher survival after two high dose i ntra p eritoneal injection s of doxorubicin (15mg/kg) ; NOD hAATtg, filled squares; NOD, open circles. Full black arrows indicate high dose DOX injection (15mg/kg).

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80 Figure 3 2 0 Representative histology of organs harvested at the endpoint of the experiment with NOD hAATtg mouse model. All organs were H&E stained. For each organ: 1x m agnification and 20x magnification; for heart only: additional 40x magnification

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81 CHAP TER 4 EXAMINATION OF THE T HERAPEUTIC POTENTIAL OF HUMAN ALPHA 1 ANTITRYPSIN (HAAT) T HERAPY ON DOXORUBICI N INDUCED CARDIOMYOPATHY IN RA T MODELS Rationale Several other gro ups have been working with rat models, showing cardiac protection against doxorubicin induced cardiomyopathy by various treatment strategi es and mechanism investigations. These Investigations supported our decision to switch to rats, which seem ed to be a m ore beneficial animal model Furthermore, considering the required high standards for precision, the measurement of QT interval is subjective This is because the end of the T wave is not always clearly defined and usually merges gradually with the baselin e. Based on our previous observations, we intend to accomplish the most promising therapy approach in this different animal model and by including echocardiography (ECHO) as an additional method to monitor the heart function more detailed Materials And Me thods For a complete list of lab materials and equipment which was used for the following in vitro experiments please refer to T able 4 1. Animals Sprague Dawley rats were purchased from Charles River Laboratories International, Inc. ( Wilmington, MA 01887 USA ) and housed in a pathogen free animal facility at the University of Florida (Gainesville, FL, USA) Animals were acclimatized to standard ACS housing in a 12 h light dark cycle and constant temperature environment for a minimum of six days before being used. The Institutional Animal Care and Use

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82 Committees (IACUC) at the University of Florida approved all animal procedures and manipulations Echocardiopgraphy (ECHO) Every week cardiac function was evaluated by transthoracic echocardiography using a GE Health Echocardiography Model Vivid7 with a 1 0 Hz transducer to evaluate mainly left ventricle function. Rats were an a esthetized with 2% Isoflurane at an oxygen flow rate of 1.0 liter/minute for 3 minutes and kept anaesthetized on a heating pad in a supin e position for the duration of the ECHO. The chest was shaved, and two dimensional echocardiography was performed. M mode echocardiography of the left ventricle at the papillary muscle level, guided by two dimensional short axis images was recorded L eft v entricle internal diameter at end systole (LVIDs) interventricular septum thickness in systole (IVSs), left ventricular posterior wall thickness in systole (LVPWs) and left ventricular internal diameter in end systole (LVIDs) were measured on the M mode. The left ventricular fractional shortening (% FS), ejection fraction (% EF) and stroke volume (SV) were automatically calculated by the echocardiographic system. D uring the assessment, i mages were obtained from the parasternal short axis and M mode. Elect rocardiography (ECG) All animals were anaesthetized with 2 % isoflurane at an oxygen flow rate of 1.0 liter/minute for 3 minutes and kept anaesthetized on a heating pad in a supine position for the duration of the ECG. The physiological examination was perf ormed once a week for 15 20 seconds using a 3 Lead ECG with a Dual Bio Amplifier which was interfaced to a PowerLab. Heart function parameters of interest, including R R interval, thus heart

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83 rate, Q T and the Q Tc interval, which is the Q T interval correc ted for heart rate, were described earlier under objective 2A. Human Alpha 1 Antitrypsin ELISA A sandwich ELISA of blood samples, which were thawed and diluted with ratios ranging from 1:1000 to 1:4000, was performed according to the manufacturer and measu red spectrophotometrically at 450nm. Human Alpha 1 Antitrypsin Antibody ELISA Since we delivered a human protein to rats, we measured the antibody levels against this foreign protein in the blood serum. We used dilution of the blood samples ranging from 1:100 to 1:400 for our experimental set up. After coding a clear 96 well Elisa Plate with hAAT, we blocked the plate with bovine serum albumin. We then added our diluted blood samples, followed by additional wash steps and subsequent the enzyme linked anti body (goat anti mouse Ig POD). Finally, substrate was added and the plate was measured spectrophotometrically at 450nm Data A nalysis Fifteen seconds of r ecorded ECG data were analyzed with Lab Chart 7Pro v7.1.2 software (ADInstruments, Colorado Springs, C O, USA). All measurements of the ECHO analysis were based on the average of three c onsecutive cardiac cycles. The stroke volume, ejection fraction and fractional shortening were calc ulated according to the formulas : Stroke Volume (SV) = EDV ESV Ejection Fraction (EF) = (SV / EDV) 100% Fractional shortening ( FS ) = [(LVIDd LVIDs)/LVIDd] 100

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84 hAAT was detected by ELISA as described previously. The optical density reading at 490nm was compared to the standard concentration curve, averaged within the group and finally, the concentrations were depictured with Microsoft Excel 2007. hAAT antibody levels were also measured by ELISA. Each sample was simultaneously performed as a duplicate and the optical density reading at 490nm was averaged and depictured as rel ative units with Microsoft Excel 2007. The TUNEL staining was performed at the Pathology Core at the University of Florida. The percentage of apoptosis based on that staining was analyzed with Image Scope version 11.0.2.725 software (Aperio Technologies, Vista, CA, USA) Experimental Designs Examination Of The Therapeutic Potential O f Human Alpha 1 Antitrypsin (h AAT ) Therapy O n doxorubicin Induced C ardiomyopathy In Sprague Dawley Rats U sing An I ntraperitoneal Administration Route. Seven days prior to first doxorubicin injection, w hen rats reach ed a n average body weight of 250g, we divided them randomly into four groups, in which 10 rats received intraperitoneal injections of 1mL clinical grade hAAT (AAT Prevention, 10mg/rat) and the other group s received pho psphate buffered saline (PBS) (PBS, n=9; PBS DOX, n=10; AAT Treatment, n=6) in identical manner and volumes as control s and diffe rent therapy strategy (Table 4 2 ). These injections were repeated every three days for the duration of the entire experiment. In this study, c ardiomyopathy was induced with thirteen 1mL low dose intraperitoneal injections of doxorubicin (1.25mg/kg) starting on day 0 every other day for 24 days to reach a cumulative, total dose of 16.25 mg/kg. PBS group received phopsphate buffere d saline in identical manner and volumes as a control

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85 From seven days after the final doxorubicin injection (day 31) till the end of the experiment, the AAT Treatment group also received 10 mg/rat hAAT every three days instead of PBS. All mice were weigh ted almost daily and the heart function was monitored with electro cardiography (ECG) and e chocardiography (ECHO) once a week Every week, b lood samples (> 750 L) were drawn from the tail vein 24 hours after hAAT injections and kept at 80C for later evalu ations For an early pathological evaluation of the organs, three PBS, four PBS DOX and four AAT Prevention rats were euthanized by cervical dislocation and mainly heart, but also liver, spleen and kidney were removed 24 hours after final doxorubicin injec tion and fixed in formalin and subjected to pathological examination Seven sections of the heart from base to apex, each 50 m apart, were cut and one middle section stained with TUNEL and the rest with hematoxylin and eosin (H&E) according to standard methods At the end of the experiment, all animals were euthanized a nd examined pathologically in the same way mentioned above. For an overall timeline of this experiment, please refer to Figure 4 1. After doxorubicin injection, if inflammation or tissue damage got too severe IACUC pre approved criteria was utilized to eu thanize any animal to avoid any suffering. Examination Of The Therapeutic Potential O f Human Alpha 1 Antitrypsin (h AAT ) Therapy O n doxorubicin Induced C ardiomyopathy In Young Sprague Dawley Rats U sing An I ntravenous Administration Route. Seven days prior t o first doxorubicin injection, w hen rats reach ed a n average body weight of 0.25kg, which correlates to approximately 8 weeks of age, we divided them randomly into two groups, in which 6 rats received intra venous (IV) injections of 1mL clinical grade hAAT ( AATiv Prevention, 10mg/rat) and the other 5 rats received

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86 phopsphate buffered saline (PBS) (PBSiv) in identical manner and volumes as a control (Table 4 3 ). These injections were repeated every three days for the duration of the entire experiment. In cont rast to earlier studies, c ardiomyopathy was induced in all animals with ten 1mL low dose IV injections of doxorubicin (1.25mg/kg) starting on day 0 every other day for 18 days to reach a cumulative, total dose of 12.5 mg/kg. All mice were weighted almost d aily and the heart function was monitored with electro cardiography (ECG) and e chocardiography (ECHO) once a week. Every week, b lood samples (> 750 L) were drawn from the tail vein 24 hours after hAAT injections and kept at 80C for later evaluations For an overall timeline of this experiment, please refer to Figure 4 12. This time only at the end of the experiment, all animals were euthanized by cervical dislocation and mainly heart, but also liver, spleen and kidney were removed and fixed in formalin and subjected to pathological examination as mentioned above. Examination Of The Therapeutic Potential O f Human Alpha 1 Antitrypsin (h AAT ) Therapy O n doxorubicin Induced C ardiomyopathy In Old Sprague Dawley Rats U sing An I ntravenous Administration Route For t he final study, we initiated the experiment, w hen the rats maintained a n average body weight of 0.45kg, which correlates to approximately 18 weeks of age, and divided them randomly into two groups. One of which received intra venous (IV) injections of 1mL c linical grade hAAT seven days after the last doxorubicin injection (AATiv old Treatment, 10mg/rat) and the other group received phopsphate buffered saline (PBS) (PBSiv old) in identical manner and volumes as a control (Table 4 4 ). These injections were rep eated every three days for the remaining time of the experiment.

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87 The study design was identical to the design previously described in objective 3b with young rats and therefore these experiments were performed simultaneously. For an overall timeline of th is experiment, please refer to Figure 4 20. Results A nd Discussion Examination Of The Therapeutic Potential O f Human Alpha 1 Antitrypsin (h AAT ) Therapy O n doxorubicin Induced C ardiomyopathy In Sprague Dawley Rats U sing An I ntraperitoneal Administration Rou te In contrast to our previous studies with different mouse models, w e induce d cardiomyopathy by multiple intraperitoneal doxorubicin injection s. We retained the intraperitoneal administration route, because it delivered promising results in mice. The mult iple low dose regime allowed us to have better control over doxorubicin induced cardiac damage and mimics clinical practice to a greater extend. In addition to ou r previously applied method, ECG and subsequent the known parameters R R P R and Q T interv al as well as the QRS complex, we used Echocardiography to obtain more detailed information. Those hemodynamic data yield ed to more detailed information about the toxic effect of doxorubicin on cardiac function and the potential of h AAT to prevent these ef fects Unlike in mice which showed therapeutic concentrations of hAAT we reached only low levels of hAAT in our rats when also injected intraperitoneally Furthermore, we experienced a high fluctuation in the hAAT concentration over the duration of the e xperiment (Figure 4 2 A ). These observations might be due to a progressive development of antibodies against hAAT, starting approximately one week after the first hAAT intraperitoneal injection (Figure 4 2B) which were also in contrast with our mouse model s whose immune response to the foreign hAAT protein was negligible.

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88 In terms of body weight, all groups showed a normal weight gain in the very beginning of the experiment s standard weight gain curve. In consistency with our previous experiences, doxorubicin receiving rats started to lose some weight, due to increased level of stress and discomfort. With the completion of the doxorubicin injections, all groups started to gain weight again but this time with a difference (Figure 4 3). Compared to our previous experiences, o ne of the differences was a progressive water inclusion in the abdomen cavity, which was easily noticeable when holding the animals. Secondly, besides in the control group (PBS only), it occurred in most of the anim als of all the other groups. The veterinarian at that time diagnosed a serious development of ascites. This condition could have been due to a cardiac damage, decreasing the pump ability of the heart which led to a backup of blood in front of the right atr ium. This increase in portal blood pressure may have be en responsible for forming the pressure gradient and resulting in abdominal ascites. The rats receiving hAAT showed an even stronger progression of this condition, as a possible result of additional os motic water retention after injecting protein molecules into this existing liquid and therefore increasing the osmotic pressure pulling more water into the cavity In spite of this condition, we were able to support the result of previous experiments in o ur mouse models with a significant increase in Q Tc interval on day 49 reflecting an increase in polarization/repolarization time in the PBS+DOX control group (126.33ms 15.4614 ) compared to the AAT+DOX Prev and AAT+DOX Treat group ( 104.46ms 19.0014 and 96.34ms 8.9288, respectively ) (Fig ure 4 4 ). This elevation of Q Tc interval in the PBS+DOX group was persistent till the end point of the experiment,

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89 whereas the hAAT receiving rats remained at baseline level. Overall, t he prevention of cardiac dysfunctio n when subjects were injected with hAAT was reflected in other parameters of the ECG results as well when looking at the baseline change from the final assessment at day 67(Figure 4 5) We were not able to completely confirm these results with our echoca rdiography (Figure 4 6 and Figure 4 7) but did observe an improving trend in rats receiving hAAT when comparing the final assessment with baseline values (Figure 4 8). The absence of detecting a greater improvement in hAAT receiving animal and the control group could have be en due to the earlier mentioned ascites. At the time point of the early tissue harvesting (day 25), all animals receiving doxorubicin had already signs of tissue damage to some degree and we collected several milliliters of the pale yell ow to clear fluid which accumulated in the peritoneal cavity. In addition to this evidence, p athophysiological examination of the organs (early (day 25) and final (day 67) sacrifice) We observed a thick an d inflexible liver caused by cirrhosis, resulting in an increased pressure in the liver blood flow and ultimately in abdominal ascites. As a consequence, we did not observe any alteration in the organ weight versus body weight ratios (Figure 4 9). Images taken of multiple sections of the heart from PBS+DOX rats from base to apex showed signs of a thickening in left ventricle wall, which is common in hypertrophic cardiomyopath y patients (Figure 4 10). The theory that doxorubicin induced cardiac damage is a result of apoptosis, could not be confirmed by TUNEL staining of a middle section of the heart, followed by pixel count analysis (Figure 4 11). We rather believed

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90 that the induced heart failure is a consequence of a general weakening of the heart, due to a lterations in the cardiac energetics than apoptosis of the myocardium. After this first experiment in rats, we concluded that because of the high toxicity of doxorubicin in general, causing damage in multiple tissue s leading to abdominal ascites, and a low concentration of hAAT in the circulation, we were only able to show improvement to a certain degree. Examination Of The Therapeutic Potential O f Human Alpha 1 Antitrypsin (h AAT ) Therapy O n doxorubicin Induced C ardiomyopathy In Young Sprague Dawley Rats U s ing An I ntravenous Administration Route To overcome the complications we ran into, using an intraperitoneal injection route, and to further simulate the treatment practiced in humans, w e induce d cardiomyopathy this time by multiple intravenous (i.v.) doxor ubicin injection s and restricted the total cumulative dose to 12.5mg/kg One reason we were not able to show the therapeutic potential of hAAT in our preceding study with rats was that we did not reach therapeutical relevant concentrations of hAAT in the blood circulation when administered interperitoneally. By injecting hAAT into the tail vain of the rats, we overcame that issue and achieved concentration exceeding 1mg/mL in average (Figure 4 13A). A delayed onset of a rather mild immune response and thus eliminating the foreign protein before accumulating, contributed to these higher levels of hAAT as well (Figure 4 13B). After a normal increase in body weight and, as experienced before, a typical decrease during doxorubicin administration, both groups se emed to recover and gained weight in almost the same manner (Figure 4 14). An argument why this differs from a significant body weight loss seen in our mouse models, could be an extensive

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91 difference in metabolism and a faster recovery due the fact that we did not inject a single high dose of doxorubicin. As in our previous rat model, we had complications at the injection site of doxorubicin. Once more, this illustrates the high potency and toxicity of the drug. Half way through this experiment we observed s evere lesion s at the site of injection most likely due to leaking of the drug into surrounding tissue. The tissue damage and progression of necrosis was too severe and led to an almost complete amputation of the rats by a veterinarian and the eutha nization of some animals on day 38 and day 39 One week after this procedure, all remaining rats seemed to have recovered from surgery and we were allowed to continue with the experiment. Despite this complication, we confirmed the results from previous s tudies in mice and rats by showing prevention of the Q T and Q Tc interval prolongation in rats treated with hAAT At approximately the same time point as in our IP rat model, we observed a significant prolongation in the Q Tc interval in the PBS +DOX cohor t on day 52 (142.6ms 34.9102) compared to the AAT+DOX (107.1 3.4269). This trend sustained almost to the end point of the experiment (Figure 4 15). Even though the rats in the PBS+DOX cohort seemed to recover, hAAT treated rats still showed an overall im provement of cardiac function when comparing the latest ECG assessment with baseline values (Figure 4 16). This protective effect of hAAT continued to show in the results obtained from echocardiography (Figure 4 17 and Figure 4 18). At around the same time point (between day 43 and day 58) we observed a significant prevention of ejection fraction loss and fractional shortening loss in rats treated with hAAT (Figure 4 18).

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92 In contrast to our previous rat IP model, harvested organs looked undamaged to the nak ed eye and also showed no differences in organ weight to b ody weight ratios. The trend in the earlier st udy that showed a thickening of the left ventricle wall was seen in this experiment to some extend as well (Figure 4 19 ). Once again, the apoptosis anal ysis supported the theory that doxorubicin induced cardi ac dysfunction is not a result of apoptosis, but a consequence of a general weakening of the heart, due to alterations in the cardiac energetics Compared to our previous rat model experiment, we achi eved better result s due to a more precise model. Although we experienced severe complications at the site of injection, i ntravenous i njections did not lead to ascites in any of these animals nor to any other internal organ damage but cardiac damage. We w ere also able to maintain therapeutic levels of hAAT using this administration route. Examination Of The Therapeutic Potential O f Human Alpha 1 Antitrypsin (h AAT ) Therapy O n doxorubicin Induced C ardiomyopathy In Old Sprague Dawley Rats U sing An I ntravenous Administration Route This study was designed to investigate the possibility of age related differences in our treatment The study design was identical to the design previously described in objective 3b and therefore these experiments were performed simul taneously. Besides the age difference between these rats and those from the preceding study, we started treat ing the rats with hAAT one week after completion of doxorubicin administration. The animals in this study were initially used for hAAT IV injection practice, but were given several weeks to eliminate any remaining hAAT before participating in this experiment as shown in the i r baseline levels in the beginning (Figure 4 21 A). On the other hand this was the explanation fo r the detection of hAAT antibodi e s right from the start of this study (Figure 4 21 B) As mentioned before we were able to reach

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93 therapeutic concentrations of hAAT by injecting intravenously and observed a steep increase after initiating hAAT injections on day 25. This basically had no e ffect on the antibody development, which further decreased to baseline levels. In both groups, we observed a gradual decrease in body weight, which was greater during doxorubicin injections, and after a short recovery phase, continued till t he end of the s tudy (Figure 4 22 ). As mentioned under objective 3b, these animals were subjected for tail amputation on day 39 as well, but no euthanization and allowed one week of rest before proceeding with the experiment. Possibly due to the age and stage of maturatio n, the first sign of cardiac dysfunction in the PBSiv old+DOX, shown in the ECG results with a significant prolongation in the Q T interval, was detected close to the end point of this study (day 91) (Figure 4 2 3 ). Although we observed a trend of improvem ent in those rats treated with hAAT from the ECG results towards the end of this experiment these were not confirmed by echocardiography (Figure 4 24 and Figure 4 2 5 ) Similar to the young cohort from objective 3b, harvested organs also looked healthy acc ording to their age and thus did not show any differences in organ weight to body weight ratios The trend from earlier st udies that hAAT seemed to prevent a thickening of the left ventricle wall was also not noticed in this experiment, potentially because of natural organ aging (Figure 4 2 6 ). Despite progression of aging the apoptosis analysis showed no more cell degradation than in y ounger subjects

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94 Compared to the younger rats, we came to the conclusion that the matured older rats seemed to be more resi stant to external intervention and recovered faster from the toxic effect of doxorubicin.

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95 Table 4 1. Complete list of materials and equipment for the in vivo studies with three different rat models Materials Company Doxorubicin hydrochloride Tocris bio science (Ellisville, MO, USA) Human alpha 1 antitrypsin Talecris, Inc (Research Triangle Park, NC, USA) Isoflurane, USP Webster Veterinary (Devens, MA, USA) 1ml Tuberculin Syringes BD Consumer Co. (Franklin Lakes, NJ,USA) Steril needles (20 G1) BD Cons umer Co. (Franklin Lakes, NJ, USA) Serum Separator Tubes BD Consumer Co. (Franklin Lakes, NJ, USA) 1X Phosphate Buffered Saline Mediatech, Inc. (Manassas, VA, USA) 96 well Elisa Plate, clear BD Biosciences (Bedford, MA, USA) Disposable serological pipe tte Corning, Inc. (Coring, NY, USA) 15ml and 50ml centrifuge tubes Corning, Inc. (Coring, NY, USA) Micropipettes Eppendorf AG (Hamburg, Germany) Pipet Tips USA Scientific, Inc. (Ocala, FL, USA) Balance (d=0.1g) Ohaus Corp. (Pine Brook, NJ, USA) Bovine serum albumin Sigma Aldrich, Co. (St. Louis, MO, USA) Goat anti mouse Ig POD Sigma Aldrich, Co. (St. Louis, MO, USA) hAAT ELISA Kit Sigma Aldrich, Co. (St. Louis, MO, USA) Dynex Technologies MRX Revelation 96 Well Microplate Reader MTX Lab Systems, Inc (Vienna, VA, USA) Echocardiography apparatus Vivid 7 GE Healthcare (Piscataway, NJ, USA) ECG apparatus 3 Lead ECG with a Dual Bio Amplifier + PowerLab connection (ADInstruments, Colorado Springs, CO, USA)

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96 Table 4 2 Distribution of Sprague Dawley ra ts in IP multiple injection experiment Groups # of rats (N) PBS 6+3 PBS DOX 6+4 AAT Prevention 6+4 AAT Treatment 6 Table 4 3 Distribution of young Sprague Dawley rats in IV multiple injection experiment Groups # of rats (N) PBSiv 5 AATiv Preve ntion 6 Table 4 4 Distribution of old Sprague Dawley rats in IV multiple injection experiment Groups # of rats (N) PBSiv old 3 AATiv old Treatment 3

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97 Figure 4 1. Timeline for the IP multiple injection experiment with 35 Sprague Dawley rats

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98 Figure 4 2. hAAT levels ( A ) and hAAT antibody levels ( B ) over the time of the IP multiple injection experiment with 35 Sprague Dawley rats after multiple low dose IP injection s of doxorubicin (1.25mg/kg). Dashed line represents detection cut off value R e sults are expressed as the group mean +SD

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99 Figure 4 3 Body weight change over the time of the IP multiple injection experiment with 35 Sprague Dawley rats after multiple low dose IP injection s of doxorubicin (1.25mg/kg) Dashed line with filled grey ci rcles represent normal growth rate according to Charles River Laboratories International. Results are expressed as the group mean SD.

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100 Figure 4 4. A ll heart function parameters using ECG over the time of the IP multiple injection experiment with 35 Sprag ue Dawley rats after multiple low dose IP injection s of doxorubicin (1.25mg/kg) Results are expressed as the group mean SD. p between PBS+DOX and AAT+DOX Prev; # p between PBS+DOX and AAT+DOX Treat

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101 Figure 4 5. Final summary of all hear t function parameters using ECG of the IP multiple injection experiment with 35 Sprague Dawley rats after multiple low dose IP injection s of doxorubicin (1.25mg/kg) Each parameter evaluated on day 11 was set as the baseline (100%) and the change from the baseline was based on the final examination on day 67.Results are e xpressed as the group mean SD. # p 5 between PBS+DOX and AAT+DOX Treat

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102 Figure 4 6 Measured ECHO data of the IP multiple injection experiment with 35 Sprague Dawley rats after multiple low dose IP injections of doxorubicin (1.25mg/kg). Results are e xpressed as the group mean SD

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103 Fig ure 4 7 Calculated ECHO data of the IP multiple injection experiment with 35 Sprague Dawley rats after multiple low dose IP injections of doxorubicin (1.25mg/kg). Results are e xpressed as the group mean SD

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104 Figure 4 8 Final summary of all heart funct ion parameters using ECHO of the IP multiple injection experiment with 35 Sprague Dawley rats after multiple low dose IP injections of doxorubicin (1.25mg/kg). Each parameter evaluated on day 11 was set as the baseline (100%) and the change from the basel ine was based on the final examination on day 67.Results are ex pressed as the group mean SD. p between PBS+DOX and AAT+DOX Prev

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105 Figure 4 9 Actual organ weight and organ weight/body weight ratios of the IP multiple injection experiment with 35 Sprague Dawley rats after multiple low dose IP injections of doxorubicin (1.25mg/kg) R esults are expressed as the group mean +SD.

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106 Figure 4 10 Representative histology of organs harvested at the endpoint of the IP multiple injection experiment with 35 Sprague Dawley rats after multiple low dose IP injections of doxorubicin (1.25mg/kg) Sev en slices of the heart from base to apex, each 50 m apart, were H&E stained and the middle slice was TUNEL stained.

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107 Figure 4 11 Calculated percentage of apoptosis based on TUNEL stained hearts of the IP multiple injection experiment with 35 Sprague Daw ley rats after multiple low dose IP injections of doxorubicin (1.25mg/kg) Seven slices of the heart from base to apex, each 50 m apart, were H&E stained and the middle slice was TUNEL stained.

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108 Figure 4 12 Timeline for the IV multiple injection experim ent with 11 young Sprague Dawley rats

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109 Figure 4 13. hAAT levels ( A ) and hAAT antibody levels ( B ) over the time of the IV multiple injection experiment with 11 young Sprague Dawley rats after multiple low dose IV injection s of doxorubici n (1.25mg/kg). Six rats received cont inuatively hAAT ( 10 mg/mouse) IV injections ( AAT iv filled squares) and the other 5 rats PBS ( PBSiv open circles) R esults are expressed as the group mean +SD; represents time point of tail amputation

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110 Figure 4 14. Body weight change over the time of the IV multiple injection experiment with 11 young Sprague Dawley rats after multiple low dose IV injection s of doxorubicin (1.25mg/kg). Six rats received cont inuatively hAAT ( 10 mg/mouse) IV injections ( AAT iv filled sq uares) and the other 5 rats PBS ( PBSiv open circles) R esults are expressed as the group mean +SD; represents time point of tail amputation

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111 Figure 4 15. All heart function parameters using ECG over the time of the IV multiple injection experiment with 11 young Sprague Dawley rats after multiple low dose IV injections of doxorubicin (1.25mg/kg). Six rats received continuatively hAAT (10mg/mouse) IV injections (AATiv, filled squares) and the other 5 rats PBS (PBSiv, open circles). Results are expressed as the group mean +SD; represents time point of tail amputation.* p

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112 Figure 4 16 Fi nal summary of all hear t function parameters using ECG of the IV multiple injection experiment with 11 young Sprague Dawley rats after multiple low dose IV injections of doxorubicin (1.25mg/kg). Eac h parameter evaluated on day 7 was set as the baseline (1 00%) and the change from the baseline was based o n the final examination on day 91 Results are expressed as the group mean SD.

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113 Figure 4 17 Measured ECHO data of the IV multiple injection experiment with 11 young Sprague Dawley rats after multiple lo w dose IV injections of doxorubicin (1.25mg/kg). Results are e xpressed as the group mean SD. represents time point of tail amputation.* p

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114 Figur e 4 18. Calculated ECHO data of the IV multiple injection experiment with 11 young Sprague Da wley rats after multiple low dose IV injections of doxorubicin (1.25mg/kg). Results are e xpressed as the group mean SD. represents time point of tail amputation.* p

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115 Figure 4 19 Representative histology of organs harvested at the endpo int of the IV multiple injection experiment with 11 young Sprague Dawley rats after multiple low dose IV injections of doxorubicin (1.25mg/kg) Seven slices of the heart from base to apex, each 50 m apart, were H&E stained and the middle slice was TUNEL st ained

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116 Figure 4 2 0 Timeline for the IV multiple injection experiment with 6 old Sprague Dawley rats

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117 Figure 4 21 hAAT levels ( A ) and hAAT antibody levels ( B ) over the time of the IV multiple injection experiment with 6 old Sprague Dawley rats after multiple low dose IV injection s of doxorubicin (1.25mg/kg). Three rats received cont inuatively hAAT ( 10 mg/mouse) IV injections ( AAT iv filled squares) and the other 3 rats PBS ( PBSiv open circles) R esults are expressed as the group mea n +SD; represents time point of tail amputation

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118 Figure 4 2 2 Body weight change over the time of the IV multiple injection experiment with 6 old Sprague Dawley rats after multiple low dose IV injection s of doxorubicin (1.25mg/kg). Three rats received cont inu atively hAAT ( 10 mg/mouse) IV injections ( AAT iv filled squares) and the other 3 rats PBS ( PBSiv open circles) R esults are expressed as the group mean +SD; represents time point of tail amputation

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119 Figure 4 2 3 All heart function parameters using ECG o ver the time of the IV multiple injection experiment with 6 old Sprague Dawley rats after multiple low dose IV injections of doxorubicin (1.25mg/kg). Three rats received continuatively hAAT (10mg/mouse) IV injections (AATiv, filled squares) and the other 3 rats PBS (PBSiv, open circles). Results are expressed as the group mean +SD; represents time point of tail amputation.* p ; p value

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120 Figure 4 2 4 Measured ECHO data of the IV multiple injection experiment with 6 old Sprague Dawley rats after multiple low dose IV injections of doxorubicin (1.25mg/kg). Resu lts are e xpressed as the group mean SD. represents time point of tail amputation

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121 Figure 4 2 5 Calculated ECHO data of the IV multiple injection experiment with 6 old Sprague Dawley rats after multiple low dose IV injections of doxorubicin (1.25mg/kg). Results are e xpressed as the group mean SD. represents time point of tail amputation.* p

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122 Figure 4 2 6 Representative histology of organs harvested at the endpoint of the IV multiple injection experiment with 6 old Sprague Dawley rats after multiple low dose IV injections of doxorubicin (1.25mg/kg) Seven slices of the heart from base to apex, each 50 m apart, were H&E stained and the middle slice was TUNEL stained

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123 CHAPTER 5 CONCLUSION The overall objective of this project was to inve stigate the feasibility of using the protective effect of hAAT protein therapy on the development of cardiotoxicity in doxorubicin induced cardiomyopathy animal model. First, the in vitro experiments demonstrated that human a lpha 1 a ntitrypsin showed a pr otective effect on acute doxorubicin induced cell death which does not derive f rom doxorubicin binding to hAAT, and the most effective therapy strategy turned out to be the Prevention strategy This evidence is in consistency with the results from our pre vious study in which we demonstrated th cells against apoptosis [28] All this confirmed our theory and gave reason to believe, that the anti inflammatory and anti oxidative properties of hAAT might play an important role for preventing tissue injuries in animal models. Despite the awareness that C2C12 cells doxorubicin is even against the more robust skeleton muscle cells and how hAAT improved the cell viability. In general, a more accurate and consistent system to test ex vivo is highly desired. Secondly, t is mainly considered to be from its intercalation into DNA and interf erence with DNA unwinding or DNA strand separation and helicase activity, leading to inhibited synthesis notably in the heart, has to be the result of an additional mechani sm, since cardiac tissue is not fast dividing and intercalation would have only a minor to no effect Recent studies have revealed t hat the mechanism underlying doxorubicin induced cardiac

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124 injury includes irreversible damage of DNA and alteration of cardia c energetics due to cardiac oxidative stress, caused by increased production of reactive oxygen species, with subsequent cardiac inflammation and apoptosis [6, 7] These are more likely the reasons for anthracyclin cardio toxic side effects [5] especially since cardiomyocytes lack of catalase to process these excessive reactive oxygen species. Therefore, several in vivo experiments with objectives to confirm what seemed to be reasonable in theory were conducted using different animal models. Two different mouse models and three different strategies in a rat model gave a compre hensive assessment that hAAT could be used as a new and promising treatment to prevent cardiac damage. In both distinct mouse models, C57BL/6 mice and the Non Obese Diabetic human alpha 1 a ntitrypsin transgenic mice (NOD hAATtg mice) t he anti inflammatory and a nti apoptotic effects of human alpha 1 a ntitrypsin are the reason s for an overall successful ly improved cardiac function after doxorubicin administration In these two models, hAAT attenuated body weight loss, prevented cardiac dysfunctions, as demonstrated in a variety of heart function parame ters utilizing electrocardiography, and reduced signs of myocardial damage induced by doxorubicin. Latter was based on c ardiac Troponin I which is a specific diagnostic biomar ker for various heart disorders and was not detected in C57BL/6 mice when treate d with hAAT. Besides the practicability of a transgenic animal, this already diseased mouse model showed a highly significant survival after receiving two high doses of doxorubicin These anti apoptotic and anti inflammatory effects of hAAT have been demon strated recently by our lab showing the protective effect of hAAT protein and gene therapy on the

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125 development of arthritis in collagen induced arthritis (CIA) and pristane induced arthritis (PIA) mouse model [27] Although we achieved promising evidence in these mouse models, the measurement of QT interval is rather subjective considering the required high standards fo r precision This is because the end of the T wave is not always clearly defined and usually merges gradually with the baseline Echocar diography was the answer to the question for more accuracy. This non invasive method delivered more detailed information about the actual stage of the heart condition. But, even with many years of experience, I questioned the accuracy when utilized in mice as some researchers did. That was one reason why we switched from mouse to rat model. Several other groups have been working with rat models, showing cardiac protection against doxorubicin induced cardiomyopathy by moderate diet restriction [16] using Dexrazoxane, an iron chelator [17] or treatment with the phytochemical oleuropein [18] as well as other treatment strategies and me chanism investigations. In all three therapy strategies, intraperitoneal injection, intravenous in young and in old rats, we observed that hAAT improved cardiac function and prevented cardiomyopathy to some degree. The intraperitoneal administration route of doxorubicin seemed not suitable for our purposes, since it caused severe abdominal ascites and internal organ damage I therefore doubt all who used this injection route to induce cardiac damage even with a different dosing regimen. After this first exp eriment in rats, we concluded that we were only able to show minor improvement, because of the high

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126 tox icity of doxorubicin in general causing result changing complications and leading to low concentra tions of hAAT in the circulation The intravenous injec tion route of doxorubicin and hAAT did lead to a cleaner model of heart dysfunction and therapeutic levels of hAAT. But, due to the extremely high toxicity of doxorubicin, complications in form of necrotic tissue at the site of injection occurred to a poin t, at which we were forced to amputate the tail of most of the animals. In spite of this condition, we were able to support the result of previous experiments in mouse models with a significant increase in Q Tc interval and other heart function parameters of the ECG results in hAAT naiv rats The theory that doxorubicin induced cardiac damage is a result of apoptosis could not be confirmed by histological examination. Instead, we observed a trend of thickening in the left ventricle wall in rats not treated with hAAT, We rather believed that the induced heart failure is a consequence of a general weakening of the heart, due to alterations in the cardiac energetics than apoptosis of the myocardium. Compared to the younger rats, we came to the conclusion that the matured older rats seemed to be more resistant to external intervention and recovered faster from the toxic effect of doxorubicin Future studies will have to focus on improvement of the therapeutic effect by optimizing the dose and timing of hAAT del ivery, including a rAAV mediated hAAT gene therapy and a pre implanted catheter for precise intravenous administration of doxorubicin and the protective agent. Even with some major complications a fter all these in vitro and in vivo experiments, we conclud ed that hAAT has potential to protect against cardiac damage caused by oxidative stress leading to inflammation

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127 LIST OF REFERENCES 1. Morrow, W.R., Cardiomyopathy and heart transplantation in children. Curr Opin Cardiol, 2000. 15 (4): p. 216 23. 2. Hitchcock Bryan, S. Gelber R Cassady JR Sallan SE The impact of induction anthracycline on long term failure free survival in childhood acute lymphoblastic leukemia. Med Pediatr Oncol, 1986. 14 (4): p. 211 5. 3. Bonadonna, G., M. Zambetti, and P. Valagussa, Sequen tial or alternating doxorubicin and CMF regimens in breast cancer with more than three positive nodes. Ten year results. JAMA, 1995. 273 (7): p. 542 7. 4. Ettinghausen, S.E., Bonow RO, Palmeri ST, Seipp CA, Steinberg SM, White DE, Rosenberg SA. Prospective study of cardiomyopathy induced by adjuvant doxorubicin therap y in patients with soft tissue sarcomas. Arch Surg, 1986. 121 (12): p. 1445 51. 5. Fisher, B., Redmond C, Wickerham DL, Bowman D, Schipper H, Wolmark N, Sass R, Fisher ER, Jochimsen P, Legault Poisson S, Doxorubicin containing regimens for the treatment of stage II breast cancer: The National Surgical Adjuvant Breast and Bowel Project experience. J Clin On col, 1989. 7 (5): p. 572 82. 6. Praga, C., Beretta G, Vigo PL, Lena z GR, Pollini C, Bonadonna G, et al., Adriamycin cardiotoxicity: a survey of 1273 patients. Cancer Treat Rep, 1979. 63 (5): p. 827 34. 7. Young, R.C., R.F. Ozols, and C.E. Myers, The anthracycline antineoplastic drugs. N Engl J Med, 1981. 305 (3) : p. 139 53. 8. Lefrak, E.A., Pitha J, Rosenheim S, Gottlieb JA. A clinicopathologic analysis of adriamycin cardiotoxicity. Cancer, 1973. 32 (2): p. 302 14. 9. Kim, K.H., G.Y. Oudit, and P.H. Backx, Erythropoietin protects against doxorubicin induced cardiomyopathy via a phosphatidyl inositol 3 kinase dependent pathway. J Pharmacol Exp Ther, 2008. 324 (1): p. 160 9. 10. Muller, I., D. Niethammer, and G. Bruchelt, Anthracycline derived chemotherapeutics in apoptosis and free radical cytotoxicity (Review). Int J Mol Med, 1998. 1 (2): p. 49 1 4. 11. Zhang, B., Lu Y, Campbell Thompson M, Spencer T, Wasserfall C, Atkinson M, S ong S. Alpha1 antitrypsin protects beta cells from apoptosis. Diabetes, 2007. 56 (5): p. 1316 23.

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132 BIOGRAPHICAL SKETCH Matthias Fueth was born in 1977, in Du esseldorf, Germany. He earned a bachelor degree in p harmacy from the Technische Universitt Braunschweig, Germany in 2005 and received his EU Pharmacy license in 2006. He practiced as a pharmacist from 2005 to 2007. After internships in pharmaceutical comp anies (Schwarz Pharma and Henkel KGaA), in the Department of Pharmaceutics at the University of Florid a in 2005 and in a c ommunity pharmacy, he joined the PhD program in the Department of Pharmaceutics, College of Pharmacy of the University of Florida, wor king under the supervision of Dr Sihong Song in August 2007. During that time he gained experience in the following techniques: Tissue cell culture, PCR, Cloning, Plasmid DNA Isolation and Purification, ELISA, designing and performing studies with rodents Electrocardiography, Echocardiography and organ tissue harvesting, tissue staining and imaging. Matthias Fueth received his Doctor of Philosophy degree in Pharmaceutical Sciences in December 2011.