<%BANNER%>

The Application of Polyamine Pharmacophores as Vectors for Intracellular Transport of Therapeutic Agents

Permanent Link: http://ufdc.ufl.edu/UFE0021084/00001

Material Information

Title: The Application of Polyamine Pharmacophores as Vectors for Intracellular Transport of Therapeutic Agents
Physical Description: 1 online resource (197 p.)
Language: english
Creator: Yao, Hua
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: desferrithiocin, intracellular, iron, kidney, l1210, metabolism, polyamine, toxicity, uptake
Medicinal Chemistry -- 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

Abstract: There is no doubt that iron is essential for life. However, this metal also plays critical role in many diseases. Prominent amongst the iron mediated diseases are various transfusional iron overload disorders; iron overload is of most significance. These conditions are usually managed with iron chelators. (S)-4,5-dihydro-2-(3-hydroxy-2-pyridinyl)-4-methyl-4-thiazolecarboxylic acid (desferrithiocin, DFT) is one of the first discovered orally active iron chelators. In spite of the severe nephrotoxicity associated with DFT, the molecule was recognized as a valuable pharmacophore for designing orally effective iron chelators with less toxicity. Previously, a series of structure activity relationship studies were conducted with various DFT analogues. (S)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid (S)-4'-(HO)-DADFT was identified as an orally effective iron chelator with a profoundly improved organ toxicity profile. However, like other iron chelators, DFT-based iron chelators rarely reached high intracellular concentrations. Therefore, a universal tool is needed for intracellular delivery of iron chelators. In this study, polyamines were exploited for delivering therapeutic agents, particularly iron chelators such as L1 and the DFT analogue (S)-4'-(HO)-DADFT. When using a polyamine as a vectoring agent, the charge property of the molecule that is going to be vectored (cargo molecule) is crucial. A neutral or positively charged, but not a negatively charged, cargo fragment should be compatible with the cellular transport of a polyamine conjugate. This concept was consistent with the transport properties of the polyamine conjugates SPM-L1, NSPD-(S)-4'-(HO)-DADFT and NSPD-(S)-4'-(HO)-DADFT-Ethyl Ester. The data revealed that when the cargo molecule was neutrally charged, the conjugate was able to compete with SPD for the cellular transport and accumulate intracellularly to a millimolar level when used at a low micromolar concentration. However, NSPD-(S)-4'-(HO)-DADFT, the polyamine conjugate having a negatively charged cargo fragment, is a poor transport competitor and accumulated at low levels intracellularly. The iron clearing efficiency of NSPD-(S)-4'-(HO)-DADFT and NSPD-(S)-4'-(HO)-DADFT-Ethyl Ester in bile-duct-cannulated rodents indicated that polyamine conjugation significantly improved the iron clearing activity of the parent chelator (S)-4'-(HO)-DADFT. The metabolic studies of these two conjugates in rodents showed that not only the expected stepwise N1-deaminopropylation occurred at the polyamine vector part, surprisingly enough, but also the oxidative deamination took place at the terminal primary nitrogen.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Hua Yao.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Bergeron, Raymond J.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021084:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021084/00001

Material Information

Title: The Application of Polyamine Pharmacophores as Vectors for Intracellular Transport of Therapeutic Agents
Physical Description: 1 online resource (197 p.)
Language: english
Creator: Yao, Hua
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: desferrithiocin, intracellular, iron, kidney, l1210, metabolism, polyamine, toxicity, uptake
Medicinal Chemistry -- 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

Abstract: There is no doubt that iron is essential for life. However, this metal also plays critical role in many diseases. Prominent amongst the iron mediated diseases are various transfusional iron overload disorders; iron overload is of most significance. These conditions are usually managed with iron chelators. (S)-4,5-dihydro-2-(3-hydroxy-2-pyridinyl)-4-methyl-4-thiazolecarboxylic acid (desferrithiocin, DFT) is one of the first discovered orally active iron chelators. In spite of the severe nephrotoxicity associated with DFT, the molecule was recognized as a valuable pharmacophore for designing orally effective iron chelators with less toxicity. Previously, a series of structure activity relationship studies were conducted with various DFT analogues. (S)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid (S)-4'-(HO)-DADFT was identified as an orally effective iron chelator with a profoundly improved organ toxicity profile. However, like other iron chelators, DFT-based iron chelators rarely reached high intracellular concentrations. Therefore, a universal tool is needed for intracellular delivery of iron chelators. In this study, polyamines were exploited for delivering therapeutic agents, particularly iron chelators such as L1 and the DFT analogue (S)-4'-(HO)-DADFT. When using a polyamine as a vectoring agent, the charge property of the molecule that is going to be vectored (cargo molecule) is crucial. A neutral or positively charged, but not a negatively charged, cargo fragment should be compatible with the cellular transport of a polyamine conjugate. This concept was consistent with the transport properties of the polyamine conjugates SPM-L1, NSPD-(S)-4'-(HO)-DADFT and NSPD-(S)-4'-(HO)-DADFT-Ethyl Ester. The data revealed that when the cargo molecule was neutrally charged, the conjugate was able to compete with SPD for the cellular transport and accumulate intracellularly to a millimolar level when used at a low micromolar concentration. However, NSPD-(S)-4'-(HO)-DADFT, the polyamine conjugate having a negatively charged cargo fragment, is a poor transport competitor and accumulated at low levels intracellularly. The iron clearing efficiency of NSPD-(S)-4'-(HO)-DADFT and NSPD-(S)-4'-(HO)-DADFT-Ethyl Ester in bile-duct-cannulated rodents indicated that polyamine conjugation significantly improved the iron clearing activity of the parent chelator (S)-4'-(HO)-DADFT. The metabolic studies of these two conjugates in rodents showed that not only the expected stepwise N1-deaminopropylation occurred at the polyamine vector part, surprisingly enough, but also the oxidative deamination took place at the terminal primary nitrogen.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Hua Yao.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Bergeron, Raymond J.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021084:00001


This item has the following downloads:


Full Text

PAGE 1

THE APPLICATION OF POLYAMINE PHARMACOPHORES AS VECTORS FOR INTRACELLULAR TRANSPORT OF THERAPEUTIC AGENTS By HUA YAO 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 2007 1

PAGE 2

Copyright 2007 by Hua Yao 2

PAGE 3

To my husband, Meiguo Xin; my daughter, Anni Xin; my son Anthony Xin; and my parents, GuoWei Yao and Hui Min Song; for all their love and support 3

PAGE 4

ACKNOWLEDGMENTS I feel I have achieved a great thing in my life at this point. However, this achievement comes with the help and support of many individuals. First and foremost, I would like to express my sincerest appreciation to my mentor, Dr. Raymond J. Bergeron, who gave me this great opportunity to fulfill my dream. I started in his laboratory as a fulltime biological technician twelve years ago. After six years in this position I decided to enter the graduate program in medicinal chemistry and continue my research with Dr. Bergeron. With the help of his ongoing guidance, encouragement, patience, and friendship, I was able to complete my project and became an independent scientist. He has taught me as much about life as about science. His attitude and dedication to science that led to his successes always inspired me. I would also like to extend my sincere appreciation to my supervisory committee members, Dr. Margaret O. James, Dr. Kenneth Sloan, and Dr. Michael J. Katovich, for their valuable time and constructive comments, which improved the overall quality of my dissertation. Furthermore, I would like to acknowledge my friends and colleagues in and out of Dr. Bergerons laboratory, for their invaluable help. Dr. William Weimar, now a brilliant professor at the Lecom Bradenton School of Pharmacy, assisted me as early as my first day in this laboratory. I would also like to express my sincere thanks to Dr. James McManis and Dr. Neelam Bharti for providing most of the organic compounds used in this project; their help on my questions on synthetic organic chemistry is also highly appreciated. My gratitude also goes to Ms. Elizabeth Nelson, for her technical assistance in HPLC analysis. I would also like to thank Ms. Jan Wiegand and Ms. Tanaya Lindstrom, for their helps with the animal studies. My special thanks go to Mrs. Kathy Bergeron and Mrs. Carrie A. Blaustein, for their professional editorial instructions on my dissertation. My appreciation also goes to Mrs. Rosemary Smith, our dear senior secretary, for her sense of humor and kind heart, which have always cheered me and 4

PAGE 5

brought me happiness. I would also like to thank Dr. Richard R. Streiff for his kind suggestions throughout my dissertation study. I acknowledge all members of the Electron Microscopy Core Facility of the College of Medicine at the University of Florida, especially Dr. Jill Verlander Reed, Ms. Wencui Zhen, and Mrs. Sharon Matthews. I would also like to thank Mr. Neal Benson of the Flow Cytometry Core Laboratory of College of Medicine at the University of Florida. I will always owe a great debt to my parents, Dr. Guo Wei Yao and Mrs. Hui Min Song, who have always supported and cared for me with their selfless love. Finally, I would like to give the deepest gratitude to Mei Guo Xin, my dear husband, whose firm, unconditional love and endless support were always behind me. I give special thanks to my lovely daughter, Anni Xin, who has always stayed beside me and has grown up with me. My gratitude also goes to my beloved son, Anthony Xin, who was born during my final stage of graduate study, a truly precious gift of God. Every doubt in life disappears with his pure eyes and brighting smile. 5

PAGE 6

TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES .........................................................................................................................10 LIST OF FIGURES .......................................................................................................................12 LIST OF ABBREVIATIONS ........................................................................................................15 ABSTRACT ...................................................................................................................................21 CHAPTER 1 INTRODUCTION..................................................................................................................23 1.1 Overview of Polyamines..............................................................................................23 1.1.1 Structures and Physiological Functions in Eukaryotes......................................23 1.1.1.1 Chemical Structures and Physicochemical Properties..............................23 1.1.1.2 Interaction with DNA/RNA......................................................................24 1.1.1.3 Involvement in Hypusine Synthesis..........................................................26 1.1.1.4 Role in Protein Synthesis and Function....................................................27 1.1.1.5 Interaction with NMDA Receptors and Other Ion Channel Receptors.....28 1.1.1.6 Involvement in Signal Transduction, Cell Growth and Apoptosis...........31 1.1.2 Polyamine Cellular Transport............................................................................35 1.1.2.1 Transport in Escherichia coli....................................................................35 1.1.2.2 Transport in Saccharomyces cerevisiae....................................................36 1.1.2.3 Transport in Mammalian Cells..................................................................36 1.1.3 Polyamine Biosynthesis in Mammalian Cells...................................................38 1.2 Iron and Iron Chelators................................................................................................43 1.2.1 Iron in Biological Systems.................................................................................43 1.2.2 Iron Dynamics in Microorganisms....................................................................45 1.2.2.1 Siderophores..............................................................................................45 1.2.2.2 Iron-Chelator Complex Formation and the Chelate Effect.......................46 1.2.2.3 Iron Uptake, Transport and Utilization in Microbes.................................49 1.2.3 Iron Uptake, Storage, and Homeostasis in Mammalian Cells...........................51 1.2.3.1 Transferrin and Transferrin Receptor........................................................51 1.2.3.2 Cellular Uptake of Iron by the Transferrin/Transferrin Receptor System........................................................................................................52 1.2.3.3 Cellular Iron Storage.................................................................................53 1.2.3.4 Cellular Iron Homeostasis Controlled by the IRE/IRP System................54 1.2.4 Iron Absorption, Distribution, and Utilization in Humans................................55 1.2.5 Iron Mediated Diseases......................................................................................56 1.2.6 Iron Chelators as Therapeutics...........................................................................57 1.3 Research Objectives.....................................................................................................58 6

PAGE 7

2 DESFERRITHIOCIN-BASED IRON CHELATORS...........................................................72 2.1 Structure-Activity Relationships among DFT Analogues...........................................72 2.2 Specific Aims...............................................................................................................74 2.3 Materials and Methods.................................................................................................74 2.3.1 Cell Culture........................................................................................................74 2.3.1.1 Culture of L1210 Cells..............................................................................74 2.3.1.2 Culture of Human Renal Proximal Tubule Epithelial Cells......................75 2.3.2 Determination of Antiproliferative Activity (IC 50 )............................................75 2.3.2.1 IC 50 Measurements in L1210 Cells...........................................................75 2.3.2.2 IC 50 Measurements in HPTC Cells...........................................................75 2.3.3 Measurement of Lipophilicity (Log P app )..........................................................76 2.3.4 Stoichiometry Determination by Jobs Plot.......................................................76 2.3.5 Light Microscopy...............................................................................................77 2.3.5.1 Dosing of Animals....................................................................................77 2.3.5.2 Vesicular Perfusion of Kidneys.................................................................77 2.3.5.3 Tissue Microdissection and Embedding...................................................77 2.3.5.4 Light Microscopy Examination of Kidney Thick Sections.......................78 2.3.6 Fluorescent Microscopy.....................................................................................78 2.3.7 Lysosome Stability Assessment by Flow Cytometry........................................78 2.3.8 Synthesis............................................................................................................78 2.4 Results and Discussion................................................................................................79 2.4.1 Lipophilicity, Antiproliferative Activity, and Animal Toxicity........................79 2.4.1.1 Relationship among Lipophilicity, IC 50 in L1210 Cells, and Animal Toxicity......................................................................................................79 2.4.1.2 IC 50 in Human Renal Proximal Tubule Epithelial Cells...........................81 2.4.2 Stoichiometry of the Complex Formed between (S)-4'-(HO)-DADFT-PE and Fe(III)..........................................................................................................82 2.4.3 Animal Toxicity of (S)-4'-(HO)-DADFT and (S)-4'-(HO)-DADFT-PE Examed by Light Microscopy............................................................................82 2.4.4 Observation Lysosome Changes in HPTC cells by Fluorescent Microscopy...83 2.4.5 Lysosome Stability Assessment by Flow Cytometry........................................85 3 POLYAMINES AS VECTORS.............................................................................................97 3.1 Review of the Polyamine Analogues...........................................................................97 3.1.1 Structure Activity Relationships of Polyamine Analogues...............................97 3.1.2 The Metabolism of Polyamine Analogues.......................................................100 3.2 The History of Using Polyamines and Polyamine Analogues as Vectors.................101 3.3 The Model Conjugate Molecules...............................................................................102 3.3.1 The Role of Charge in the Choice of Cargo Molecules...................................102 3.3.2 Design Concept................................................................................................103 3.3.3 Materials and Methods.....................................................................................104 3.3.3.1 Stoichiometry Determination by Jobs Plot............................................105 3.3.3.2 Kinetics of Cellular Transport.................................................................105 3.3.3.3 Antiproliferative Activity Determination................................................106 3.3.3.4 HPLC Analysis of Cellular Polyamine Pools.........................................106 7

PAGE 8

3.3.3.5 HPLC Analysis of Cellular Drug Concentration.....................................107 3.3.3.6 Ornithine Decarboxylase (ODC) Assay..................................................108 3.3.3.7 S-Adenosylmethionine Decarboxylase (AdoMetDC) Assay..................109 3.3.3.8 Spermine/Spermidine N 1 -Acetyltransferase (SSAT) Assay....................109 3.3.4 Results and Discussion....................................................................................110 3.3.4.1 Stoichiometry of the Complex formed between SPM-L1 and Fe(III)....110 3.3.4.2 The Effect of Conjugation on Cellular Transport, Intracellular Uptake, and Polyamine Pools................................................................................110 3.3.4.3 Impact on Antiproliferative Activity.......................................................112 3.3.4.4 Impact on Polyamine Metabolic Enzymes..............................................112 3.4 Polyamine Vectored DFT Analogues........................................................................113 3.4.1 Design Concept................................................................................................113 3.4.1.1 Intracellular Accumulation of DFT Analogues.......................................113 3.4.1.2 Objectives................................................................................................114 3.4.1.3 Specific Aims..........................................................................................114 3.4.2 Materials and Methods.....................................................................................115 3.4.2.1 General Methods.....................................................................................115 3.4.2.2 Syntheses.................................................................................................115 3.4.2.3 Stoichiometry Determination by Jobs Plot............................................119 3.4.2.4 Cell Culture.............................................................................................120 3.4.2.5 HPLC Analysis of the Tissue and Cellular Concentration of (S)-4'-(HO)-DADFT and NSPD-(S)-4'-(HO)-DADFT-EE................................120 3.4.2.6 Mass Spectra Analysis of the Rat Liver Concentration of the Diacid Metabolites of NSPD-(S)-4'-(HO)-DADFT-EE......................................121 3.4.2.7 Iron Clearing Efficiency Measurement...................................................122 3.4.3 Results and Discussion....................................................................................122 3.4.3.1 Stoichiometry of the Complex Formed between NSPD-(S)-4'-(HO)-DADFT and Fe(III)..................................................................................122 3.4.3.2 Effect of Conjugation at 4'-hydroxyl of (S)-4'-(HO)-DADFT on Cellular Transport and Uptake of the Molecule......................................122 3.4.3.3 The Effect of Conjugation on Antiproliferative Activity........................123 3.4.3.4 Effect on Polyamine Metabolic Enzymes...............................................124 3.4.3.5 Metabolism of the Polyamine Moiety of the Conjugate NSPD-(S)-4'-(HO)-DADFT-EE in Rat Liver................................................................125 3.4.3.6 Effect of Polyamine Conjugation at C-4' Hydroxyl on the Iron-Clearing Efficiency of (S)-4'-(HO)-DADFT............................................126 3.5 Cellular Localization of Polyamine Conjugates........................................................128 3.5.1 Background......................................................................................................128 3.5.2 Specific Aims...................................................................................................128 3.5.3 Materials and Methods.....................................................................................128 3.5.3.1 General Methods.....................................................................................128 3.5.3.2 Cell Culture.............................................................................................129 3.5.3.3 Fluorescence Spectra of NBD-GABA and NBD-GABA-NSPD............129 3.5.3.4 Fluorescence Microscopy........................................................................129 3.5.4 Results and Discussion....................................................................................130 8

PAGE 9

3.5.4.1 Cellular Transport and Antiproliferative Properties of NBD-GABA and NBD-GABA-NSPD..........................................................................130 3.5.4.2 Spectrometry of Fluorescent Polyamine NBD-GABA-NSPD................130 3.5.4.3 Cellular Localization of NBD-GABA-NSPD.........................................130 3.6 Polyamine Vectored Anthracene-9-Carboxylic Acid................................................131 3.6.1 Background......................................................................................................131 3.6.2 Specific Aims...................................................................................................132 3.6.3 Materials and Methods.....................................................................................132 3.6.3.1 General....................................................................................................132 3.6.3.2 Light Microscopy Examination of the Cell Morphology........................132 3.6.4 Results and Discussion....................................................................................133 3.6.4.1 Antiproliferative Activities of Anthracene-9-carboxylic Acid and NSPD-Anthorate......................................................................................133 3.6.4.2 Effect of NSPD-Anthroate on Cell Morphology.....................................133 3.7 (S)-4'-(HO)-DADFT-NSPD Ester..............................................................................134 3.7.1 Specific Aims...................................................................................................134 3.7.2 Synthesis..........................................................................................................135 3.7.3 Material and Methods......................................................................................135 3.7.4 Results and Discussion....................................................................................135 3.7.4.1 Effect on Cell Proliferation.....................................................................135 3.7.4.2 Cellular Transport, Intracellular Uptake of NSPD-OH and the Impact on Polyamine Pools..................................................................................136 3.7.4.3 Cellular Transport, Intracellular Uptake of (S)-4'-(HO)-DADFT-NSPD ester and the Impact on Polyamine Pools.....................................136 4 CONCLUSION.....................................................................................................................166 LIST OF REFERENCES.............................................................................................................168 BIOGRAPHICAL SKETCH.......................................................................................................197 9

PAGE 10

LIST OF TABLES Table page 2-1 Effect of structural modifications of DFT on iron clearing efficiency and organ toxicity in primates and rodents.........................................................................................93 2-2 UV wavelengths used for measuring Log P app of DFT analogous....................................94 2-3 Lipophilicity (Log P app ), antiproliferative activity (IC 50 ), and animal tolerability of DFT analogues...................................................................................................................95 2-4 Antiproliferative activity (IC 50 ) of DFT analogues in normal human kidney proximal tubule cells.........................................................................................................................96 3-1 Cellular transport, accumulation of polyamine analogues and effect on polyamine pools and IC 50 ..................................................................................................................154 3-2 Effect of polyamine analogues on polyamine metabolic enzymes..................................155 3-3 Cellular transport, uptake, and the impact on polyamine pools of the SPM-terephthalic acid conjugate (NTS) and the SPM-terephthalic monomethyl ester conjugate (NTS-ME).......................................................................................................156 3-4 Cellular transport, uptake of L1 and SPM-L1, and the impacts on polyamine pools and IC 50 ............................................................................................................................157 3-5 Effect of L1 and SPM-L1 on ODC, AdoMetDC, and SSAT in L1210 cells...................158 3-6 Cellular uptake of representative DFT analogues............................................................158 3-7 Cellular transport, uptake of NSPD-(S)-4'-(HO)-DADFT and NSPD-(S)-4'-(HO)-DADFT-EE......................................................................................................................159 3-8 Antiproliferative activities of NSPD-(S)-4'-(HO)-DADFT, NSPD-(S)-4'-(HO)-DADFT-EE, and their cargo molecules...........................................................................159 3-9 Antiproliferative properties of DFT analogues and their polyamine conjugates on JURKAT cells..................................................................................................................160 3-10 Effect of (S)-4'-(CH 3 O)-DADFT and (S)-4'-(CH 3 O)-DADFT-EE and their polyamine conjugates on ODC, AdoMetDC, and SSAT in L1210 cells...........................................160 3-11 Iron clearing efficiency of (S)-4'-(HO)-DADFT and its polyamine conjugates in rats...161 3-12 Transport properties and antiproliferative activities of NBD-GABA and its polyamine conjugate NBD-GABA-NSPD......................................................................161 10

PAGE 11

3-13 Fluorescence excitation and emission wavelenghts of NBD, NBD-GABA and its polyamine conjugate NBD-GABA-NSPD......................................................................162 3-17 Cellular transport, uptake of (S)-4'-(HO)-DADFT and (S)-4'-(HO)-DADFT-NSPD ester, and the impact on polyamine pools........................................................................164 3-18 Stability of (S)-4'-(HO)-DADFT-NSPD ester in cell culture medium............................165 11

PAGE 12

LIST OF FIGURES Figure page 1-1 Structures of the common natural polyamines...................................................................59 1-2 Biosynthesis of hypusine and the maturation of eIF5A.....................................................60 1-3 Illustration of a NMDAR subunit......................................................................................61 1-4 The polyamine biosynthetic network.................................................................................62 1-5 Mechanism of the ODC-catalyzed decarboxylation of L-ornithine..................................63 1-6 The representative synthetic inhibitors of polyamine metabolism....................................63 1-7 The mechanism of the AdoMetDC-catalyzed decarboxylation of adenosylmethionine...64 1-8 The mechanism of the SSAT assay...................................................................................64 1-9 Bovine serum amine oxidase catalyzed oxidative deamination of polyamines.................65 1-10 Chemical structures of representative catecholamide iron chelators.................................66 1-11 Chemical structures of representative hydroxamate iron chelators...................................67 1-12 Chemical structures of miscellaneous siderophores..........................................................68 1-13 Chemical structures of 2,3-dihydroxy-N,N-dimethylbenzamide (DHBA)........................68 1-14 Iron transport in mammalian cells.....................................................................................69 1-15 Regulation of cellular iron homeostasis by the IRP/IRE system.......................................70 1-16 Daily human body iron storage, distribution, and utilization............................................71 1-17 The structures of some representative iron chelators under clinical investigations...........71 2-1 Structural modifications carried on DFT...........................................................................86 2-2 Illustration of the dissecting positions in rat kidney..........................................................87 2-3 Scheme for the synthesis of (S)-4'-(HO)-DADFT-PE.......................................................87 2-4 The relationship between IC 50 and Log P app of 4'-substituted DFT analogues..................88 2-5 Jobs plot of (S)-4'-(HO)-DADFT-PE...............................................................................88 12

PAGE 13

2-6 Light microscopy of the kidney proximal tubules in rats treated with (S)-4'-(HO)-DADFT-PE or (S)-4'-(HO)-DADFT..................................................................................89 2-7 Cytotoxicity of DFT analogues in HPTC cells evaluated by acridine orange fluorescent staining............................................................................................................91 2-8 Lysosome stability of DFT analogue treated HPTC cells assessed by flow cytometry....92 3-1 Metabolic transformation of DENSPM and DEHSPM in animal models.......................139 3-2 Nitroimidazole-polyamine (1 and 2) and chlorambucil-polyamine (3) conjugates.........140 3-3 A model of the polyamine transport apparatus complex.................................................140 3-4 Scheme for the synthesis of NTS and NTS-ME..............................................................141 3-5 Scheme for the synthesis of SPM-L1...............................................................................141 3-6 Structural illustration of the (SPM-L1) 3 Fe complex........................................................142 3-7 Jobs plots of L1 and its polyamine conjugate SPM-L1..................................................142 3-8 The two linkage sites for the polyamine conjugation in (S)-4'-(HO)-DADFT................142 3-10 Scheme for the synthesis of NSPD-(S)-4'-(HO)-DADFT-EE and NSPD-(S)-4'-(HO)-DADFT............................................................................................................................143 3-11 Jobs plot of NSPD-(S)-4'-(HO)-DADFT........................................................................144 3-12 Schematic illustration of the metabolism of the polyamine moiety of the NSPD-(S)-4'-(HO)-DADFT-EE in rodents.......................................................................................145 3-13 Hepatic metabolism of the two NSPD conjugates of (S)-4'-(HO)-DADFT in rodents...146 3-14 ESI mass spectrometry and MS/MS identification of metabolite 12 of NSPD-(S)-4'-(HO)-DADFT-EE............................................................................................................147 3-15 Biliary ferrokinetics of (S)-4'-(HO)-DADFT and its polyamine conjugates NSPD-(S)-4'-(HO)-DADFT and NSPD-(S)-4'-(HO)-DADFT-EE in rodents.............................148 3-16 Structure of polyamine conjugated fluorescent probes....................................................148 3-17 Fluorescence excitation (in blue) and emission (in red) spectra of NBD-GABA and NBD-GABA-NSPD.........................................................................................................149 3-18 Fluorescence microscopy of NBD-GABA-NSPD accumulation in SK-HEP-1 cells.....150 3-19 Scheme for the synthesis of the conjugate NSPD-ANTH...............................................151 13

PAGE 14

3-20 Light microscopy of L1210 cells treated with Anthroic acid or NSPD-anthroate..........152 3-21 Scheme for the synthesis of (S)-4'-(HO)-DADFT-NSPD ester.......................................153 14

PAGE 15

LIST OF ABBREVIATIONS (S)-3',4'-(CH 3 O) 2 -DADFT 4,5-dihydro-2-(2-hydroxy-3,4-dimethoxyphenyl)-4-methylthiazole-4(S)-carboxylic acid (S)-3',4'-(CH 3 O) 2 -DADMDFT 4,5-dihydro-2-(2-hydroxy-3,4-dimethoxyphenyl)-4-thiazole-4(S)-carboxylic acid (S)-4'-(CH 3 O)-DADFT (S)-4,5-dihydro-2-(2-hydroxy-4-methoxyphenyl)-4-methyl-4-thiazolecarboxylic acid (S)-4'-(HO)-DADFT (S)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid (S)-4'-(HO)-DADFT-NSPD ester norspermidine 4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methylthiazole-4(S)-carboxylate (S)-4'-(HO)-DADFT-PE (S)-4,5-dihydro-2-[2-hydroxy-4-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylic acid (S)-4'-(HO)-DADMDFT (S)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-thiazole carboxylic acid (S)-5'-(CH 3 O)-DADFT (S)-4,5-dihydro-2-(2-hydroxy-5-methoxyphenyl)-4-methyl-4-thiazolecarboxylic acid (S)-5'-(CH 3 O)-DADMDFT 4,5-dihydro-2-(2-hydroxy-5-methoxyphenyl)-4thiazole-4(S)-carboxylic acid (S)-DADFT (S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-methyl-4thiazolecarboxylic acid (S)-DADMDFT (S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-thiazolecarboxylic acid (S)-DMDFT (S)-4,5-dihydro-2-(3-hydroxy-2-pyridinyl)-4thiazolecarboxylic acid 6-ADESPM 6-amino-N 1 ,N 12 -diethylspermine AA atomic absorption ABC ATP-binding cassette AcSPM acetylated spermine 15

PAGE 16

ADH aldehyde dehydrogenase AdoMetDC (SAMDC) S-adenosylmethionine decarboxylase AMPARs -amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptors AO 3,6-dimethylaminoacridine; acridine orange ASA N-hydroxysuccinimidyl-4-azidosalicylic acid ATD amino-terminal domain CDKs cyclin-dependent kinases CHO Chinese hamster ovary cells CID collision induced dissociation CNS central nervous system CPENSPM N 1 -ethyl-N 11 -[(cycloheptyl)methyl]-4,8-diazaundecane CuAOs copper-dependent amine oxidases DAO diamine oxidase dcAdoMet decarboxylated adenosylmethionine DEHSPM N 1 ,N 14 -diethylhomospermine DENSPD N 1 ,N 7 -diethylnorspermidine DENSPM N 1 ,N 11 -diethylnorspermine DESPD N 1 ,N 8 -diethylspermidine DESPM N 1 ,N 12 -diethylspermine DFMO DL-difluoromethylornithine DFO desferrioxamine DFT 4,5-dihydro-2-(3-hydroxy-2-pyridinyl)-4-methyl thiazole-4(S)-carboxylic acid DFT (S)-2-(3-hydroxypyridin-2-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylic acid, desferrithiocin 16

PAGE 17

DHBA 2,3-dihydroxy-N,N-dimethylbenzamide DIEA N,N-diisopropylethylamine DIPAD diisopropyl azodicarboxylate DLM1 tumor stroma and activated macrophage protein DMF N,N-dimethylformamide DMT1 divalent metal transporter 1 DTT dithiothreitol eIF5A eukaryotic initiation factor 5A ESI electrospray ionization mass spectrometry EtOAc ethyl acetate FDESPM N 1 ,N 12 -bis(2,2,2-trifluoroethyl)spermine GABA -aminobutyric acid Hb hemoglobin Hepes 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid HGF hepatocytes growth factor HPTCs normal human renal proximal tubule epithelial cells HSPD homospermidine HSPM N,N'-bis(4-aminobutyl)-1,4-butanediamine, homospermine IBD inflammatory bowel disease i-PrI 2-iodopropane IREs iron responsive elements IRPs iron regulatory proteins KARs kainic acid receptors L1, Deferiprone, Ferriprox 3-hydroxy-1,2-dimethylpyridin-4(1H)-one L1210 murine lymphocytic leukemia cells 17

PAGE 18

LIP labile iron pool LTP long-term potentiation MANT N-methylanthranylic acid MAO monoamine oxidase MAPK mitogen-activated protein kinase MDL 72527 N 1 ,N 4 -bis(2,3-butadienyl)-1,4-butanediamine MGBG methylglyoxal bisguanylhydrazone MK-801 (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine Mops 3-morpholinopropanesulfonic acid N 1 -AcSPD N 1 -acetylspermidine N 1 -AcSPM N 1 -acetylspermine N 4 -BzDESPD N-(3-(ethylamino)propyl)-N-(4-(methylamino)butyl)benzamide N 4 -BzDESPM N-(3-(ethylamino)propyl)-N-(4-(3-(ethylamino)propylamino)butyl)benzamide NAD + nicotinamide adenine dinucleotide NB human neuroblastoma cells NEt 3 triethylamine NMDARs N-methyl-D-aspartate Receptors NSEs non-specific esterases NSPD N 1 -(3-aminopropyl)propane-1,3-diamine NSPD-(S)-4'-(HO)-DADFT (S)-4,5-dihydro-2-[2-hydroxy-4-(12-amino-5,9-diazadodecyloxy)phenyl]-4-methyl-4-thiazole carboxylic Acid NSPD-(S)-4'-(HO)-DADFT-EtE ethyl (S)-4,5-Dihydro-2-[2-hydroxy-4-(12-amino5,9-diazadodecyloxy)phenyl]-4-methyl-4-thiazole carboxylate 18

PAGE 19

NSPD-ANTH 4-(3-(3-aminopropylamino)propylamino)butyl anthracene-9-carboxylate; NSPD-anthroate NSPD-OH N 1 -(3-(hydroxyamino)propyl)propane-1,3-diamine NSPM N,N'-bis(3-aminopropyl)1,3-propanediamine, norspermine NTBI non-transferrin-bound-iron NTS N 1 -(4-carboxy)benzoylspermine; N 1 -terephthaloylspermine NTS-ME N 1 -(4-Carbomethoxy)benzoylspermine; spermine-monomethylterephthalate conjugate O.D. optical density ODC ornithine decarboxylase PAO N 1 -acetyl-spermine/spermidine oxidase PASMCs rat pulmonary artery smooth muscle cells PCD programmed cell death PIH pyridoxal isonicotinoyl hydrazone PIP(4,4,4) N,N'-bis(4-piperidinylmethyl)-l,4-diaminobutane PIP(5,4,5) N,N'-bis-[2-(4-piperidinyl)ethyl]-l,4-diaminobutane PLP pyridoxal 5'-phosphate PPh 3 triphenylphosphine PUT 1,4-butanediamine; putrescine PVP polyvinylpyrrolidone Rb retinoblastoma protein RE reticuloendothelial RTKs receptor tyrosine kinases SAO serum amine oxidase 19

PAGE 20

SAR structure-activity-relationship Ser serine SMO spermine oxidase SODs superoxide dismutases SPD N-(3-aminopropyl)-1,4-butanediamine; spermidine SPM N,N'-bis(3-aminopropyl)-1,4-butanediamine; spermine SPM-L1 1-(12-amino-4,9-diazadodecyl)-2-methyl-3-hydroxy-4(1H)-pyridinone SPM-LA spermine-lipoic acid conjugate SSAT spermidine/spermine N 1 -acetyltransferase TAAB TAAB 812 resin Tf transferrin TfR transferrin receptor TGase protein-glutamine -glutamyl transferase THF tetrahydrofuran Thr threonine TNF tumor necrosis factor TRIS 2-amino-2-hydroxymethyl-1,3-propanediol TsCl p-toluenesulfonyl chloride Tyr tyrosine UTRs untranslated regions 20

PAGE 21

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE APPLICATION OF POLYAMINE PHARMACOPHORES AS VECTORS FOR INTRACELLULAR TRANSPORT OF THERAPEUTIC AGENTS By Hua Yao August 2007 Chair: Raymond J. Bergeron Major: Pharmaceutical Sciences There is no doubt that iron is essential for life. However, this metal also plays critical role in many diseases. Prominent amongst the iron mediated diseases are various transfusional iron overload disorders; iron overload is of most significance. These conditions are usually managed with iron chelators. (S)-4,5-dihydro-2-(3-hydroxy-2-pyridinyl)-4-methyl-4-thiazolecarboxylic acid (desferrithiocin, DFT) is one of the first discovered orally active iron chelators. In spite of the severe nephrotoxicity associated with DFT, the molecule was recognized as a valuable pharmacophore for designing orally effective iron chelators with less toxicity. Previously, a series of structure activity relationship studies were conducted with various DFT analogues. (S)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid [(S)-4'-(HO)-DADFT] was identified as an orally effective iron chelator with a profoundly improved organ toxicity profile. However, like other iron chelators, DFT-based iron chelators rarely reached high intracellular concentrations. Therefore, a universal tool is needed for intracellular delivery of iron chelators. In this study, polyamines were exploited for delivering therapeutic agents, particularly iron chelators such as L1 and the DFT analogue (S)-4'-(HO)-DADFT. When using a polyamine as a vectoring agent, the charge property of the molecule that is going to be vectored (cargo 21

PAGE 22

molecule) is crucial. A neutral or positively charged, but not a negatively charged, cargo fragment should be compatible with the cellular transport of a polyamine conjugate. This concept was consistent with the transport properties of the polyamine conjugates SPM-L1, NSPD-(S)-4'-(HO)-DADFT and NSPD-(S)-4'-(HO)-DADFT-Ethyl Ester. The data revealed that when the cargo molecule was neutrally charged, the conjugate was able to compete with SPD for the cellular transport and accumulate intracellularly to a millimolar level when used at a low micromolar concentration. However, NSPD-(S)-4'-(HO)-DADFT, the polyamine conjugate having a negatively charged cargo fragment, is a poor transport competitor and accumulated at low levels intracellularly. The iron clearing efficiency of NSPD-(S)-4'-(HO)-DADFT and NSPD-(S)-4'-(HO)-DADFT-Ethyl Ester in bile-duct-cannulated rodents indicated that polyamine conjugation significantly improved the iron clearing activity of the parent chelator (S)-4'-(HO)-DADFT. The metabolic studies of these two conjugates in rodents showed that not only the expected stepwise N 1 -deaminopropylation occurred at the polyamine vector part, surprisingly enough, but also the oxidative deamination took place at the terminal primary nitrogen. 22

PAGE 23

CHAPTER 1 INTRODUCTION 1.1 Overview of Polyamines As the name implies, polyamines are organic molecules usually containing at least two primary or secondary amino nitrogen atoms. The naturally occurring polyamines exist in virtually every life form, from bacteria to human beings. Since the first discovery of spermine phosphate crystal by Dutch biologist van Leeuwenhoek Anthonii (1632-1723), 1 polyamines have held the attention of researchers in a number of different fields, from basic science to medicine. 1.1.1 Structures and Physiological Functions in Eukaryotes 1.1.1.1 Chemical Structures and Physicochemical Properties From a chemical structural perspective, polyamines are simple, aliphatic, either linear or branched amines. Although there are a number of different polyamines, the most relevant ones to the current study are 1,4-butanediamine (putrescine, PUT), N-(3-aminopropyl)-1,4-butanediamine (spermidine, SPD), N,N'-bis(3-aminopropyl)-1,4-butanediamine (spermine, SPM), and 1,5-pentanediamine (cadaverine Figure 1-1). They are also the most common polyamines formed intracellularly in eukaryotes. The amino groups contained in polyamines render the molecules basic; the pK a values for natural polyamines usually range from 8 to 11. 2, 3 The protonation of the polyamine nitrogens takes place in a stepwise fashion. For example, as demonstrated by 13 C-NMR titration, 2 in the stage of monoprotonation of SPD (i.e., when 1 equivalent of acid was added), 32% of both the primary aminopropyl nitrogen and the secondary nitrogen atoms were protonated, while 43% of the primary aminobutyl nitrogen atom was protonated. In the diprotonation stage, 67% of the primary aminopropyl nitrogen atom, 42% of the secondary nitrogen atom, and 86% of the primary aminobutyl nitrogen atom were protonated. In the triprotonation stage, these nitrogen 23

PAGE 24

atoms were 94%, 95%, and 92% protonated. Moreover, at pH 7.4, the protonation fraction was 92%, 86% and 100% for the aminopropyl nitrogen, the secondary nitrogen, and the aminobutyl nitrogen atom respectively. Therefore, at the physiological pH, SPD exists predominantly as a trication. The extent of protonation of a polyamine is dependent on the length of the methylene backbone between two neighbouring nitrogens. 4, 5 The observation from three tetraamine series, N,N'-bis(3-aminopropyl)-1,3-propanediamine (norspermine, NSPM), SPM, and N,N'-bis(4-aminobutyl)-1,4-butanediamine (homospermine, HSPM), demonstrated that the relative abundance of tetracation at pH 7.4 increased with the number of methylene groups present in the molecule. As longer methylene backbones separating the consecutive cationic nitrogens can minimize the electrostatic repulsion, 2 the actual fractions of the tetracationic species at pH 7.4 in NSPM, SPM, and HSPM were 54%, 76%, and 85% respectively. 5 Moreover, the substituent groups on nitrogen atoms also can play important roles in determining the charge status of polyamine analogues. For example, bis-ethylation at the terminal primary nitrogen of these analogues always increased the fraction of tetracation by about 10%. 5 However, substitution of the two ethyl groups in N 1 ,N 12 -diethylspermine (DESPM) by two trifluoroethyl groups completely suppressed the tetracation species; the trifluoroethyl compound N 1 ,N 12 -bis(2,2,2-trifluoroethyl)spermine (FDESPM) actually exists as a dication instead of a tetracation (the fraction value was 99%) because of the strong electron withdrawing ability of the -trifluoroethyl groups leading a reduction in pK a of the terminal nitrogens. 4 1.1.1.2 Interaction with DNA/RNA Because of their polycationic nature, it is not surprising that polyamines can bind to anionic macromolecules such as DNA, RNA, proteins and phospholipids. The binding is largely 24

PAGE 25

electrostatic interactions occurring between the protonated positively charged polyamine nitrogens and anionic phosphate or carboxyl groups of these cellular constituents. Although hydrophobic interactions cannot be excluded, they represent only a minimum component in the free energy (G) of binding. 6 As demonstrated by X-ray crystallography, SPM spans the major groove of G-C base pairs in DNA. 7, 8 Most DNA-protein interactions are in the major groove, which suggests some important roles in DNA replication and gene transcription that may be played by the polyamine-DNA interaction. DNA conformation is changed upon the binding of polyamines; 9 the binding causes a transition of DNA from the right-handed B-form to the left-handed Z-form. 10, 11 The extent of the transition is dependent on the number of net positive charges on the polyamine molecule under the physiological conditions. For example, SPM is more effective than SPD in the induction of the B-Z transition; however, PUT and acetylated SPD are not effective inducers. 10 The functional implications of these transitions are very interesting. In Z-DNA, there is no major groove present, but only one region is similar to the B-DNA minor groove. The major groove-forming base pairs are usually buried in the center of the helix in B-DNA but produce a bulging surface in Z-DNA. 12 Even though the major groove in B-DNA is already wide and easily accessible to proteins, the exposure of these base pairs may facilitate more specific protein bindings. Therefore, although less common than B-DNA in nature, Z-DNA can have important biological functions. Its appearance relies on physiological activities at the time. This is strongly manifested by the expression of the human oncoprotogene C-MYC. 13 Z-DNA is formed within three regions proximate to the promoters of C-MYC when it is expressed; more interestingly, the Z-conformation is transformed back to the normal B-form when the transcription is turned off. 14 25

PAGE 26

Certain Z-DNA-binding proteins, e.g., ADAR1, a double-stranded RNA adenosine deaminase, have been identified either by crystallography 15 or computer programs searching similar sequence motifs such as DLM1 (tumor stroma and activated macrophage protein) and E3L (a variola virus protein). 12 The strong association between polyamines and DNA was thought to stabilize the structure of DNA, as demonstrated in vitro by the observation that DNA-SPD complexes were resistant to DNase I 16 and the fact that polyamines, especially SPD and SPM increased the melting temperatures (T m ) of DNA duplex and triplex. This association was detected in nucleated erythrocytes 17 and in some other cells such as replicating intestinal cells. 18 Recent evidence suggests that the nuclear-deposited polyamines actually can affect the nuclear remodeling via histone modification. 19 At 4 mM, PUT and SPD repressed the transglutaminase (protein-glutamine -glutamyltransferase, EC 2.3.2.13, TGase) catalyzed histone polymerization; PUT was about twice as active as SPD, while SPM had no effect. The differential effects among these polyamines on histone modification were probably due to their different carbon chain lengths. 19 While polyamines play a number of different roles via charge mediated interactions with both DNAs and RNAs, they also serve as indispensable fragments in some cellular macromolecules. 1.1.1.3 Involvement in Hypusine Synthesis The unusual amino acid hypusine [N -(4-amino-2-hydroxybutyl)lysine] provides a good example of polyamines as critical fragments in a regulatory system. Hypusine was initially discovered in bovine brain 20 and was later found to exist widely in eukaryote and archaebacteria. 21, 22 Eukaryotic initiation factor 5A (eIF5A, formerly as eIF4D) 21, 23 is the only hypusine-containing protein (18 kDa) discovered in nature to date; the amino acid sequence surrounding hypusine is highly conserved in this protein. 24, 25 Hypusine is essential for 26

PAGE 27

mammalian cell proliferation; rapidly dividing cells such as CHO have high rate of hypusine synthesis, 26 which is low in the senescent IMR-90 human diploid fibroblasts. 27 Hypusine is formed shortly after translation of the eIF5A mRNA. SPD, but not SPM, is required for the biosynthesis of this unusual amino acid. 28 SPD serves as a 4-aminobutyl donor to the lysine residue on the eIF5A precursor in the formation of deoxyhypusine, the first step of the hypusine biosynthesis (Figure 1-2). 29 The enzyme deoxyhypusine synthase (EC 2.5.1.46) is responsible for catalyzing this nicotinamide adenine dinucleotide (NAD + ) dependent step. 30 Hypusine is formed subsequently by hydroxylation on the side chain of deoxyhypusine via the deoxyhypusine hydroxylase (EC 1.14.99.29). 31 1.1.1.4 Role in Protein Synthesis and Function While SPD is an indispensable participant in eIF5A synthesis, it is not surprising that polyamines are also involved in many steps of other gene transcriptions and protein syntheses. For example, multi-fold increases of protein synthesis were observed in both mammalian and bacterial systems as a result of the polyamine stimulated amino acid incorporation. 32, 33 Polyamines could also modulate the synthesis of occludin, a tight-junction specific integral membrane protein. 34 Depletion of intracellular polyamines in the differentiated IEC-6 cells by the irreversible ornithine decarboxylase (ODC) inhibitor-difluoromethylornithine (DFMO) significantly decreased the occludin protein synthesis, while exogenous SPD and PUT reversed the inhibition effect of polyamine depletion. Not only can polyamines stimulate or suppress protein synthesis, but they can also directly or indirectly regulate functions and activities of many different proteins, from ion channel receptors (see section 1.1.1.5) to enzymes such as kinases and phosphatases that are involved in signaling pathways (see section 1.1.1.6). 27

PAGE 28

1.1.1.5 Interaction with NMDA Receptors and Other Ion Channel Receptors Early understanding about the interaction between polyamines and ion channels was obtained from the studies on argiopine, a polyamine-containing spider venom. 35 It was discovered that this substance was actually a blocker for the glutamate-activated ion channels, which mediate synaptic transmissions in vertebrates. 36 There are three subtypes in the ionotropic glutamate receptor (iGluRs) superfamily, namely -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), kainic acid receptors (KARs), and N-methyl-D-aspartate receptors (NMDARs). 36 NMDARs are located throughout many regions of the brain including the hippocampus. On a molecular structural basis, the native NMDARs are hetero-oligomers that are usually composed of four or five subunits. Different combinations of NR1 and NR2A-D or NR3A-B subunits at different stoichiometries can produce specific receptors. 37, 38 As shown in Figure 1-3, each subunit of the receptor protein has four major domains. Two of them, namely the amino-terminal domain (ATD) and the amino-binding domain, are located at the large extracellular site. The ATD functions as a regulatory domain, which mediates subunit assembly and provides binding sites for receptor modulators such as Zn 2+ H + and polyamines. The amino-binding domain is also called the ligand-binding domain; it provides the clamshell-shaped ligand binding sites for glycine on NR1 and glutamate on NR2. 39 Like other receptors, the NMDAR proteins are transmembraneous, and the membrane spanning region is formed by three protein threads (M1, M3, and M4) plus a re-entrant hairpin P-loop (M2) that forms the channel pore. The fourth domain is formed by the intracellular carboxy-terminus which is responsible for the alternative splicing and target protein interactions. 40, 41 28

PAGE 29

On the depolarized plasma membrane, NMDARs are activated by the excitatory neurotransmitter L-glutamate and its co-agonist glycine. The entry of Na + and Ca 2+ to the postsynaptic neuron through the activated receptor ion channels enables the receptors to mediate multiple CNS functions. For example, the NMDARs play important roles in long-term potentiation (LTP), which is often associated with the learning and memory functions of CNS. 42 However, over stimulation of the NMDARs may lead to epilepsy or excitotoxicity induced neurodegeneration. 43 Therefore, under normal physiological conditions, both the onset and duration of the receptor stimulation are tightly controlled. While extracellular Mg 2+ acts as an open-channel blocker of NMDARs in a voltage-dependent manner, the endogenous polyamines SPD and SPM exhibit dual modulating effects. The effects of polyamines on NMDARs were recognized by an earlier observation that SPD enhanced the binding of (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine (dizocilpine, or MK-801), an open-channel blocker for NMDARs, to the rat brain cortical membranes by decreasing its K d for the receptor. 44 In later studies the mode of action of these polyamines on MK-801 binding exhibited a biphasic characteristic, i.e., they are antagonists at high micromolar concentrations, but agonists at low micromolar concentrations. 5, 45 Furthermore, the antagonistic potency of SPM and its synthetic analogues on MK-801 binding was shown to be positively correlated with two major factors: the length of methylene backbones and the geometry of terminal alkyl groups. 5 It was suggested that both the charge-screening 46 and the direct channel block played important roles in antagonistic effects of polyamines. 47 In addition to polyamines, Zn 2+ and H + are also endogenous ligands that can modulate NMDARs. 48, 49 All these mediating molecules exert their actions at distinctive sites on the receptor protein. Earlier studies suggested that there should be specific polyamine recognition 29

PAGE 30

sites on NMDAR because both the PUT and cadaverine antagonized the stimulating effects brought by SPD or SPM on MK-801 binding to NMDAR; 45 these sites are different from those for Mg 2+ and Zn 2+ 50 As for SPM, there are three separate binding sites on the NMDAR, 47 more specifically and most likely on the NR1 subunit. 51 Two of these binding sites are located at the extracellular domain of the receptor; they are the sites for SPM modulation of the glutamate and glycine binding, respectively. The third site resides in the channel pore, and the linear polyamines can reach this site from both sides of the membrane and induce the voltage-dependent block of the channel. 39 Some other SPM-mediated effects on the NMDARs may occur. Different from the glycine-independent mechanism, the glycine-dependent stimulation results from the SPM-induced increase of receptor affinity for glycine. SPM may also decrease the receptor affinity for glutamate and regulate the proton-sensitivity of NMDARs. 49 Under normal physiological conditions, the receptors are tonically blocked by extracellular protons. The pH-sensitive element on the receptor has been identified as a lysine residue at the position 211 of the NR1 subunit exon 5, which encodes a surface loop located near the polyamine binding site and is proposed as a constituent modulator for the receptor. 49 Therefore, it is clear that the modulations of NMDARs by extracellular SPM are multitudinal. The transmembrane nature of the channel pore also allows channel modulation by intracellular polyamines. From the electrophysiological studies on the rat hippocampus cultures, it was demonstrated that the intracellular source of SPM regulates the NMDARs by decreasing the open probability of the channel without affecting MK-801 binding affinity. 51 Not only do polyamines modulate NMDARs, but they also play regulatory roles on the other two iGluRs, AMPARs and KARs, together called non-NMDARs. 52 All the three iGluRs 30

PAGE 31

are topologically similar 53 but are functionally different because of their unique subunit combinations. 36 SPM can mediate the inward rectification of the Ca 2+ -permeable subtype receptors of the non-NMDAR family. 47 The study of tetraamines with various methylene group spacings suggested that the hydrophobic interactions between polyamines and glutamate receptors provide the major binding energy. 52 Through the interactions with NMDARs and a few other ion channels, polyamines may affect many signaling events in the mammalian brain based on the fact that Ca 2+ is an important second messenger molecule in the signal transduction process. 1.1.1.6 Involvement in Signal Transduction, Cell Growth and Apoptosis Mammalian cells can respond to various stimulations induced by external/internal substances (signaling molecules, or ligands) such as neurotransmitters, growth factors, and hormones. Receptor proteins, which are divided into three major families G-protein coupled receptors, enzyme-linked receptors, and ionotropic receptors, can detect these stimuli (see section 1.1.1.5) through the receptor-ligand interactions. For ligands such as growth factors and neurotransmitters, signals are transferred from the outside to the inside of a cell through the binding of these ligands to their detectors, the corresponding membrane-spanning receptors on the cell surface. These ligand-receptor interactions may induce conformational changes in the cytosolic domain of the receptor molecule. 54 Consequently, the intrinsic kinase activity of the receptor changes at the intracellular domain. Kinases are a class of enzymes that phosphorylate target proteins on their hydroxyl-containing amino acids, e.g., serine (Ser), threonine (Thr), and tyrosine (Tyr) residues. Phosphorylation of the receptor molecule will affect its association with cytosolic mediators that relay signals to effector enzymes such as kinases and metabolic enzymes. The series cascade of events eventually affects various cellular biological processes, 31

PAGE 32

such as gene expression, cell proliferation, cell migration, cell survival/apoptosis, and metabolism. The cascade induced by growth factors (mitogens), the so-called mitogen-activated protein kinase (MAPK) pathways, 55 will lead to cell proliferation, differentiation, and stress response associated events. These pathways invariably act through the enzyme-linked receptors that include receptor tyrosine kinases (RTKs) and receptor serine/threonine kinases (RS/TKs). The Ras/Raf/MEK/ERK (also known as Ras/MAPK) cascade is among one of the best-defined MAPK mediated signaling pathways. 56 Polyamines have long been recognized as versatile regulators in both normal and neoplastic cell proliferation and transformation. 57 Accordingly, it is expected that interconnections should exist between polyamines and the MAPK signaling pathways. A positive loop was found to connect MAPK, ODC and polyamines. 56 Activation of the MAPK pathway in L1210-DR cells, the DFMO-resistant mouse leukemia cells that over-produce ODC and thus have higher PUT and SPD amounts, 58, 59 up-regulated ODC gene expression and led to an increase of polyamine levels. More interestingly, the enhanced polyamine levels in turn led an increase of MAPKs activity. Another study using hypoxic rat pulmonary artery smooth muscle cells (PASMCs) demonstrated the unique dependence of the p38 MAP kinase phosphorylation on PUT uptake. 60 The ODC gene was identified as one of the target genes of the proto-oncogene c-myc, 61-63 which is expressed virtually in all proliferating normal cells. Overexpression of the ODC gene not only promoted malignant transformation of many cell types but also increased tumor cell invasion. 64-66 Accordingly, the ODC gene is also considered a proto-oncogene. 56, 67 The most 32

PAGE 33

important function of proto-oncogenes is to participate in growth signal transduction via regulation of the cell cycle, 68 i.e., the phases of the growth kinetics of cells. Generally, upon receiving signals from growth factors, eukaryotic cells enter the first gap phase (G 1 ), in which cells grow along with the cellular organelle duplications, while the RNA and protein synthesis are initiated. The G 1 phase is quite long because in this period that cells are checked for DNA errors, which are to be repaired if there are any. Sequentially, the cells progress into the S phase, during which DNA replicates and chromosomes duplicate; the cells will have 4-fold of haploid DNA at the end of this phase. Next the cells are to be checked again in the second gap phase (G 2 ). If DNA replication is completed, the cells enter the mitosis (M) phase and go through the last examination the spindle attachment check-up. Finally, every cell that has passed the checkpoint produces a daughter cell via a process called cytokinesis All the cells including those daughter cells may start this whole cycling process again. Cell cycle progression is controlled by various cyclins and their protein kinase partner cyclin-dependent kinases (CDKs). A cyclin forms a complex with its corresponding CDK and activates this CDKs protein kinase function. The phosphorylation activities of the cyclin-CDK complexes provide the driving force for the cell cycle. Basically, all mammalian cells have five different types of cyclins, A, B, D, E, and F; each type of cyclin binds to specific CDKs. 69 For example, cyclin A binds to and activates unphosphorylated CDK2, and this cyclin A-CDK2 complex regulates the S phase entry and G 1 /S transition. 70 It has been proven that polyamines participate in the regulation of cyclin A. The interaction of polyamines with cyclin A provides the active site on the protein for the binding of the substrate protein of cyclin A as well as ATP. 71 This in turn affects the S phase progression because the size of polyamine pools can influence the elongation step of DNA replication during the S phase. 72 33

PAGE 34

In addition to ODC, other regulatory genes of the polyamine metabolic pathway such as AdoMetDC and SSAT are also expressed during the cell cycle. 73 Both ODC and AdoMetDC are the rate-limiting regulators for polyamine biosynthesis. For example, in Chinese hamster ovary (CHO) cells, the mRNA amounts of both the enzymes doubled along with their enzyme activities at the end of the S phase. 72 The changes of polyamine regulatory enzyme amounts during cell cycle progression were also observed in other cells such as normal human dermal fibroblasts. 67 Moreover, intracellular polyamine contents double during each cycle, but in different cell cycle phases for each individual polyamine. In CHO cells, PUT and SPM contents double in different phases, the late S phase through the S/G 2 transition and G 1 through S phase respectively, but the SPD level continues increasing during the entire cycle. 74 While polyamine levels fluctuate during cell cycle progression, manipulation of cellular polyamine levels by specific inhibitors of the polyamine metabolic pathway or by synthetic polyamine analogues inevitably disrupts the normal cell cycle. For example, the ODC-specific inhibitor DFMO or the AdoMetDC-specific inhibitor SAM486A 75 arrested the human neuroblastoma (NB) cell growth in G 1 phase. 76 This cell cycle block was due to the inhibition of the phosphorylation on retinoblastoma protein (Rb), a G 1 /S transition regulator. By the same mechanism, treatment of MALME-3M melanoma cells with the polyamine analogue N 1 ,N 11 -diethylnorspermine (DENSPM) depleted intracellular polyamine levels by potent inhibition of both ODC and AdoMetDC activities and super-induction of SSAT activity, 77 thus arresting the cell cycle in the G 1 phase and G 2 /M transition. 78 More importantly, an enhanced expression of the wild-type tumor suppressor p53 gene was observed ahead of the Rb phosphorylation inhibition; this indicated the critical role played by the p53 gene. The same treatment on SK-MEL-28, human melanoma cells similar to MALME-3M but containing mutated p53, did not 34

PAGE 35

cause cell cycle block; rather, these cells left the cell cycle and turned to another pathway, apoptosis. 78 Apoptosis, or programmed cell death (PCD), 78-80 is a highly organized regulatory process that occurs in all normal cells to maintain cellular homeostasis. It plays pivotal roles in embryogenesis, cell proliferation, differentiation and tissue turn over, etc. The exact mechanisms underlying polyamine-induced apoptosis remain to be clarified. Nevertheless, it is usually caused by disrupted intracellular polyamine homeostasis. Since apoptosis is a genetically relevant event, exactly how the polyamines respond to this process depends on the cell types and growth stages. 81, 82 To illustrate the existing confusion regarding the role of polyamine in apoptosis, cellular polyamine levels do not necessarily correlate with the activity of ODC (see section 1.1.3), either increased or decreased polyamine pools were observed during apoptosis. For example, paralleled with upregulated ODC activity, the polyamine pools are increased in both the tumor necrosis factor (TNF) induced normal human fibroblasts apoptosis 83 and the hepatocytes growth factor (HGF) induced HepG2 cells apoptosis. 84 In contrast, although ODC activity is enhanced, the polyamine pools are diminished in the apoptotic cells, such as TNF-induced human cervical carcinoma cells, 85 rodent T-cell hybridoma cells expressing human TNF receptors R55 or R75, 86 and human promyeloma leukemic HL-60 cells. 87 1.1.2 Polyamine Cellular Transport 1.1.2.1 Transport in Escherichia coli Polyamines can be taken up into cells by using the polyamine transport apparatus (polyamine transporters). The transport is saturable, energy dependent, and additionally, may or may not be Na + -dependent. 88 Both prokaryotic and eukaryotic cells possess specific transport systems for PUT, SPD, and SPM. Three types of polyamine transporters have been identified in 35

PAGE 36

Escherichia coli to date. 89 The first two types are the PUT-specific PotF/G/H/I system and the SPD-specific PotA/B/C/D system. PotF/G/H/I system is only responsible for the PUT uptake process. 89 Both systems belong to the ATP-binding cassette (ABC) transporter superfamily. All ABC transport proteins use the energy provided by binding ATP to drive the transport of various molecules across virtually every type of cellular membrane. 90 The PotA through PotI are functional component proteins for these transport systems. More specifically, PotD and PotF represent periplasma substrate binding proteins, PotB, PotC, PotH and PotI are channel forming transmembrane proteins, and PotA and PotG are membrane associated ATPase. The third type of E.coli polyamine transporters contains the transmembrane protein PotE, which is active for both the uptake and excretion of PUT from the cells. 1.1.2.2 Transport in Saccharomyces cerevisiae Similarly to prokaryotes, unicellular eukaryotic yeast cells have multiple polyamine transport proteins. There are four families of polyamine transport proteins, TPO1 through TPO4, which were discovered in Saccharomyces cerevisiae plasma and vacuolar membranes. 91, 92 However, they are responsible for the excretion rather than the uptake of polyamines. 92 While TPO2 and TPO3 are preferentially used by SPM, TPO1 and TPO4 are used by all the three native polyamines. 1.1.2.3 Transport in Mammalian Cells The fact that multiple polyamine transporters exist in both bacteria and yeast underscores that the exquisite regulation of intracellular polyamine contents is of crucial importance for cell survival. In mammalian cells, the molecular structures of these transport proteins are currently unknown; nonetheless, many of their important properties have been discovered. The specificity of the transporter for a particular native polyamine is mostly cell type dependent. For example, the B-lymphocytes have two different types of transporters: the low affinity type is for both PUT 36

PAGE 37

and SPD transport and the high affinity type is for SPD transport only. 81 However, several other cells, such as the cultured cerebellar astrocytes, the murine leukemic L1210 cells, and the bovine lymphocytes, have only a single high affinity transport system for all three native polyamines. 93-95 Two overlapping transporter systems have been found to coexist in the pulmonary artery smooth muscle cells: one is PUT specific while the other is used by all three natural polyamines. 60 The integral polyamine transport system is composed of two distinct processes, which are responsible for either the uptake or the excretion of polyamines. 88, 96 In response to the diminution of intracellular polyamine pools, cells use the transport apparatus to uptake polyamines from the environment, whereas excretion of polyamines may occur when they are accumulated. 96 Moreover, the transport system is tightly regulated by an endogenous protein, antizyme. Antizyme was initially identified as a polyamine-inducible ODC suppressor, 97 which binds and targets ODC for degradation via the non-lysosomal, ubiquitin-independent pathway by the 26S proteasome. 98 In ODC-overproducing mouse breast cancer cells that accumulate polyamines abnormally, the polyamine uptake was inhibited by antizyme, whereas the polyamine excretion was stimulated. 99 Therefore, the intracellular polyamine levels can be finely adjusted by antizyme via controlling of both the uptake and excretion for optimal cell growth and survival. 100 Significantly, the polyamine transporters have surprisingly broad structural tolerance; 101 even compounds structurally very different from linear polyamines can be taken up by cells via the polyamine transport system. This is well demonstrated by the ability of the herbicide N,N'-dimethyl-4',4'-bipyridylium (paraquat) to compete with PUT for transport in rat lung slices. 102 MGBG, an anticancer agent mentioned previously (Section 1.1.3), can also accumulate in cells 37

PAGE 38

by sharing the polyamine transport system. 103, 104 Additionally, a large variety of other polyamine derivatives and analogues can enter cells by using the polyamine transporters. This unique property of the polyamine transporters renders them an important target for cancer chemotherapeutics 105-107 and as potential conduit for polyamine vectoring of various cargo molecules. Clearly, the polyamine transport apparatus plays a critical role in cellular polyamine homeostasis. However, transporters themselves are not sufficient to perform the entire task; they have to act together with polyamine metabolic processes as an integral polyamine regulating system. 1.1.3 Polyamine Biosynthesis in Mammalian Cells Polyamine metabolism consists of both an anabolic and a catabolic component. The sequential biosynthesis of polyamines in mammalian cells (Figure 1-4) starts with the formation of PUT via the decarboxylation of the amino acid L-ornithine, one of the biodegradation products of arginine from the urea cycle. This step requires ODC, the first polyamine biosynthesis rate limiting enzyme with a short half-life (10 60 min). 108 Eukaryotic ODC is a pyridoxal 5'-phosphate (PLP) dependent enzyme 109 and requires the disulfide reducing agent dithiothreitol (DTT) for maximum enzyme activity in vitro. 110, 111 Like all other PLP-dependent enzymes, ODC binds PLP to form an ODC-PLP intermediate via a Schiff base with its active site lysine residue (Lys-69) 112 (Figure 1-5). Nucleophilic attack of the Schiff base carbon by the -amino nitrogen of L-ornithine produces a geminal diamine intermediate. Upon releasing ODC from the intermediate, an ornithine aldimine is formed. The loss of CO 2 from the C -carboxylate of the ornithine fragment produces a quinonoid intermediate. Protonation of the quinonoid by an 38

PAGE 39

unknown active site amino acid 113 generates a putrescine aldimine, which regenerates PLP and produces putrescine simultaneously upon condensation with water. ODC has long been targeted for polyamine biosynthesis inhibition. DL-Difluoromethylornithine (DFMO), 114 an ornithine analogue (Figure 1-6), was designed to target ODC as an anticancer agent. 115 It is an irreversible enzyme inhibitor, i.e., it binds to ODC and acts as the substrate for the enzyme; the enzyme is inhibited because the product remains at the active site rather than being released. 116, 117 Unfortunately, it failed to deplete cellular SPM; therefore, the arrest of growth of cancer cells by DFMO is only a cytostatic effect. The next higher polyamine SPD is formed by the donation of an aminopropyl fragment by the decarboxylated adenosylmethionine (dcAdoMet) to one of the terminal amino nitrogens of PUT by spermidine synthase (EC 2.5.1.16). 118 Addition of another aminopropyl fragment to the N 8 -end of the SPD molecule yields the tetraamine SPM by spermine synthase (EC 2.5.1.22). 119 The enzyme activities of both the SPD and SPM synthases are subjected to product-inhibition, i.e. they are inhibited in a feedback mechanism by SPD or SPM. DcAdoMet, the common aminopropyl donor in both steps above, is derived from the amino acid methionine. Adenosylation by adenosylmethionine synthetase (also called methionine adenosyltransferase or AdoMet synthetase) (EC 2.5.1.6) with the consumption of ATP generates AdoMet, which is subsequently decarboxylated by S-adenosylmethionine decarboxylase (AdoMetDC, or SAMDC) (EC 4.1.1.50) to give dcAdoMet. Similar to ODC, AdoMetDC is also a short-lived enzyme. However, unlike ODC which uses PLP as the cofactor for the catalysis of decarboxylation reaction, AdoMetDC uses pyruvoyl prosthetic group to participate in a similar reaction. 120 This pyruvoyl-dependent decarboxylation reaction proceeds according to the mechanism shown in Figure 1-7. First, nucleophilic attack of 39

PAGE 40

the pyruvoyl carbonyl by the -amino nitrogen of S-adenosylmethionine (S-AdoMet) generates a Shiff base intermediate. Subsequent loss of a CO 2 from the carboxylate of the imine forms a decarboxylated intermediate, which is protonated and attacked by H 2 O to produce the decarboxylated S-AdoMet and regenerate AdoMetDC enzyme. The decarboxylated S-AdoMet serves as an important aminopropyl donor in the biosynthesis of polyamines SPD and SPM (Figure 1-4). As expected, AdoMetDC can be a potential target for inhibition of SPD and SPM formation. In fact, the chemotherapeutic agents methylglyoxal bisguanylhydrazone (MGBG) 121 (Figure 1-6) and its analogue SAM486A 122 are the two most potent AdoMetDC inhibitors. While the intracellular polyamines are formed mainly by means of biosynthesis, the backconversion of SPM to SPD and SPD to PUT is of equal importance in self-regulation of polyamine pools in mammalian cells. The catabolism of SPM and SPD begins with the enzyme acetyl-CoA:Spermidine/Spermine N 1 -acetyltransferase (SSAT) (EC 2.3.1.57), a flavin adenine dinucleotide (FAD) dependent enzyme 123 that catalyzes the transfer of an acetyl group from acetyl Coenzyme A (Acetyl-CoA) to the N 1 -aminopropyl nitrogen of SPM and SPD to produce the acetylated SPM (N 1 -AcSPM) and the acetylated SPD (N 1 -AcSPD) respectively (Figure 1-8). Like the two key regulatory enzymes ODC and AdoMetDC in the biosynthetic pathway, SSAT also has a very short half-life (20 40 min). 124, 125 Interestingly, SPD was found to be a better substrate for SSAT than SPM 126 Because of its unsymmetrical structure, SPD has another acetylation product, N 8 -AcSPD. However, instead of SSAT, a histone acetylase acetyl-CoA:Spermidine N 8 -acetyltransferase (SPD N 8 acetylase) was suggested to be responsible for acetylation of the aminobutyl moiety of SPD to produce N 8 -AcSPD. 127, 128 40

PAGE 41

While N 8 -AcSPD is deacetylated or excreted, N 1 -AcSPD and N 1 -AcSPM are oxidized by N 1 -acetyl-spermine/spermidine oxidase (PAO) (EC 1.5.3.11) to generate PUT and SPD respectively, along with 3-acetamidopropanal and H 2 O 2 as byproducts. 129, 130 PAO is constitutive and has a half-life of 48 h. 131 The enzyme activity of PAO is highest among all the polyamine metabolism enzymes in rat tissues 132 and is abundant in virtually all tissues of vertebrates. 128 Furthermore, cultured tumor cells in the late logarithmic phase had higher PAO activity than cells in the exponential phase. For example, PAO activity was enhanced in the aged cells suggesting facilitation of excretion of unwanted polyamines. 133 Degradation of polyamines by PAO produces 3-acetamidopropanal and H 2 O 2 as the byproducts; this process was proposed as a major cytotoxic mechanism for the polyamine analogue N 1 -ethyl-N 11 -[(cycloheptyl)methyl]-4,8,-diazaundecane (CPENSPM) against non-small cell lung carcinoma cells. 80 Likewise, the polyamine oxidation by amine oxidases is also implicated in brain damage 131, 134 and cytotoxicity in human fibroblasts 135 and human colon cancer cells. 136 In addition to PAO, several other amine-oxidizing enzymes, namely spermine oxidase (SMO), diamine oxidase (DAO), monoamine oxidase (MAO), and serum amine oxidase (SAO), are also pertinent to oxidative polyamine catabolism. Both PAO and MAO require the cofactor FAD to cleave all primary, secondary, and tertiary amino groups, but they have different cellular localizations: cytoplasm and peroxisomes for PAO 137 but outer mitochondrial membrane for MAO. 138 Functionally, MAO is primarily involved in the catabolism of those biogenic monoamines, such as neurotransmitters dopamine and serotonin; 139 whereas PAO is responsible for polyamine oxidation as ascribed previously. Although SPM is not the preferred substrate for PAO, it can be specifically oxidized by SMO to SPD. Moreover, SMO and PAO produce different byproducts, 3-aminopropanal and 3-acetamidopropanal respectively. 140 Based on the 41

PAGE 42

fact that the over accumulation of SPM to millimolar concentration imposes direct toxicity in cells such as BHK kidney cells 141 and cerebellar granule neuron cells, 142 multiple catabolic routes existing for SPM may lead to efficient detoxification of this compound. Many inhibitors of PAO have been discovered during the past several decades. N 1 ,N 4 -bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) (Figure 1-6) is among the most potent and selective ones. 134, 143 It dramatically decreased polyamine levels 143 while leading to the accumulation of N 1 -AcSPD and N 1 -AcSPM. 144 Unlike PAO and MAO, both DAO and SAO are copper-dependent oxidases (CuAOs) that require 2,4,5-trihydroxylphenylalanine (TOPA) quinone as the cofactor. 139 Putrescine and spermidine can be oxidized by DAO to 4-aminobutanal and putrescine and 3-aminopropanal respectively. However, while spermine is a good substrate for DAO in rodent tissues, 145 it cannot be oxidized by the enzyme in the in vitro system. 128 This discrepancy might be due to the product (12-Amino-4,9-diaza-dodecanal) inhibition effect occurring to the in vitro system which lacks aldehyde-metabolizing enzymes, such as aldehyde dehydrogenase (ADH). 131 One of the significant aspects of DAO is the deamination of PUT to -aminobutyric acid (GABA). Although the function of PUT-derived GABA in mammalian cells or tissues outside the CNS needs to be fully elucidated, it was observed that the PUT-derived GABA exists in high levels in the intestines, tumors, 128 and stomach of fasting rats. 146 While functionally similar to DAO, SAO is usually less important because of its low activity in normal human and rodent plasma. 128 However, its activity is increased during pregnancy to protect against spontaneous abortion. 147 In contrast with humans and rodents, SAO is present in high amounts in ruminant sera such as fetal bovine serum (BSAO), which is responsible for the polyamine induced cytotoxicity in cell culture systems, 148 because BSAO 42

PAGE 43

catalyzes the oxidative deamination of polyamines, generating hydrogen peroxide and ammonia (Figure 1-9). For example, SPM can be oxidized by BSAO to a dialdehyde N,N'-bis(3-propionaldehyde)-1,4-diaminobutane plus hydrogen peroxide and ammonia, 149 or to SPD and 3-aminopropanal, which is unstable and can spontaneously form acrolein. 150 However, SPD can be oxidized by BSAO to N'-(4-aminobutyl)-aminopropionaldehyde plus hydrogen peroxide and ammonia. 149 It is for this particular reason that aminoguanidine, a specific SAO inhibitor, is always included at micromolar concentrations in cell culture media. 151 1.2 Iron and Iron Chelators 1.2.1 Iron in Biological Systems Iron is the fourth most abundant element and the second most abundant metal in the earths crust. Many important redox proteins require iron as a cofactor. There are three well known families of such proteins. The first, haemoproteins, consists oxygen carrier proteins (hemoglobins and myoglobins), oxygen activating proteins (cytochrome oxidase, peroxidases, catalases, and cytochrome P450s), and electron transport proteins (cytochrome a, b and c). In these haemoproteins, iron binds to the four nitrogens on the pyrrole rings of porphyrin. The second, iron-sulfur proteins in which iron is bound to the sulfur atom on cysteine to form iron-sulfur clusters, which are involved in electron transport and redox functions. The last, superoxide dismutases (SODs), are mononuclear non-heme enzymes with iron(III) located at the active sites. 152 Although iron deficiency is of great concern to human health, 153 it is also known that excess iron is involved in numerous diseases due to the unique chemical properties of the metal. As a transition metal, iron is highly redox active. Although iron has many oxidation states from -2 to +6, Fe(II) and Fe(III) are of principal interest in this study. Clearly, iron can act as both a reducing agent and an oxidizing agent. The two oxidation states of iron can interconvert in the 43

PAGE 44

presence of molecular oxygen and produce superoxide anion (O 2 ) (Equation 1-1). However, the superoxide anion can further transmute into hydrogen peroxide (H 2 O 2 ) and oxygen either spontaneously or via the catalysis by superoxide dismutases (SODs) (Equation 1-2). Hydrogen peroxide is a very strong and unstable oxidizing agent that can decompose spontaneously into water and oxygen in air. Intracellularly, this decomposition is catalyzed by some housekeeping enzymes such as glutathione peroxidase (Equation 1-3) and catalase (Equation 1-4). However, if intracellular hydrogen peroxide is present at unmanageable concentrations by these enzymes, or produced at cellular sites where these enzymes are unavailable, it will impose great danger to cells because it can pass through membranes easily. If it encounters Fe(II), hydrogen peroxide can oxidize Fe(II) to Fe(III) by the Fenton reaction (Equation 1-5), generating a hydroxyl radical (HO ) and a hydroxide ion. The hydroxyl radical is a very reactive free radical species 154 that can lead to oxidative stresses at cellular sites where it forms. The toxic effects of this radical are well documented. It can initiate lipid peroxidation chain reactions 155, 156 by abstracting a H atom at polyunsaturated fatty acid side chains and generate lipid hydroperoxide (ROOH), which can undergo the Fenton type reaction in the presence of Fe(II) and generate alkoxy radicals (RO ). Therefore, the lipid rich cell membrane is a major target for this hydroxyl radical induced damage. DNA is another sensitive target of this radical. The DNA single strand break usually results from the oxidation of the sugar moiety by hydroxyl radical. 157, 158 Additionally, this radical can also oxidize an amino acid residue of a protein at the site where Fe(II) binds; this modification may subsequently lead to proteolysis. 159 The reduction of Fe(III) back to Fe(II) occurs in the presence of biological reducing agents such as ascorbic acid, 160 superoxide anion (O 2 ), glutathione etc., thus this free radical generating cycle cannot continue if there is either a free radical scavenger or iron chelator present. 44

PAGE 45

Fe(II)+O2Fe(III)+O2.(1-1) 2O2.-+2H+H2O2+O2 SOD (1-2) 2-Glutathione-SH(Glutathione-S)2-H2O22H2O (1-3) 2H2O22H2O+O2Catalase (1-4) Fe(II) + H 2 O 2 Fe(III) + HO + OH (1-5) Ascorbic acid, Superoxide anion Compared with Fe(II) which is readily soluble in water, Fe(III) usually exists as ferric hydroxide [Fe(OH) 3 ] and is extremely insoluble (the solubility product K sp = 2 x 10 -39 ), limiting its concentration to about 10 -18 M at the physiological pH. 152, 161 Because of the ease of oxidation of Fe(II) to Fe(III) and the abundance of O 2 ferrous iron is of limited availability, and the insoluble ferric iron has to be complexed and rendered soluble for use by organisms. 1.2.2 Iron Dynamics in Microorganisms 1.2.2.1 Siderophores All microorganisms require iron for survival. To overcome the iron accessibility problem, they synthesize and secrete relatively low molecular weight, highly iron(III)-specific chelating agents, siderophores (Greek for iron carrier), to assimilate iron from extracellular resources. 162 These chelators form soluble complexes with iron(III), so that microorganisms can use the iron available from their surroundings. Many siderophores have been isolated from microorganisms up to date; these compounds are classified into two major groups, the 2,3-dihydroxybenzoyl group containing catecholamides (Figure 1-10) and the N-hydroxy amide containing hydroxamates (Figure 1-11). 45

PAGE 46

Interestingly, although these catecholamides and hydroxamates are structurally very different, they often share a common feature, polyamine backbones. For example, while catecholamides L-agrobactin and L-parabactin have SPD backbone, L-vibriobactin and L-vulnibactin have NSPD backbone. Hydroxamates desferrioxamine and bisucaberin have cadaverine backbone, whereas rhodotorulic acid has a PUT backbone. The hydroxamates are usually synthesized by microorganisms that exist under relatively high iron concentrations, while the catecholamides are synthesized when iron is present in low concentrations. 163 Generally, because having the two neighboring phenolic hydroxyls, catecholamide chelators bind iron much more tightly than hydroxamate chelators. 164, 165 In addition to catecholamides and hydroxamates, there are some other iron chelating compounds produced by microbes such as rhizoferrin, pyochelin, and desferrithiocin (DFT) (Figure 1-12). 1.2.2.2 Iron-Chelator Complex Formation and the Chelate Effect Fe(III) generally forms an octahedral hexacoordinate complex with an iron chelator (ligand, L). There are two kinds of equilibrium constants used for describing the complex forming process: one is K (formation constant, Equation 1-7 to 1-12), which stands for the stepwise equilibrium constant; the other is (Equation 1-13 to 1-19), which is the overall equilibrium constant. 163 Fe(III)LL-Fe(III) 1[L-Fe(III)] = [Fe(III)][L]K (1-7) 2L-Fe(III)LL-Fe(III) 22[L-Fe(III)] = [L-Fe(III)][L]K (1-8) 46

PAGE 47

23L-Fe(III)LL-Fe(III) 332[L-Fe(III)] = [L-Fe(III)][L]K (1-9) 34L-Fe(III)LL-Fe(III) 443[L-Fe(III)] = [L-Fe(III)][L]K (1-10) 45L-Fe(III)LL-Fe(III) 554[L-Fe(III)] = [L-Fe(III)][L]K (1-11) 56L-Fe(III)LL-Fe(III) 665[L-Fe(III)] = [L-Fe(III)][L]K (1-12) Fe(III)LL-Fe(III) 1[L-Fe(III)] = [Fe(III)][L] (1-13) 2Fe(III)2LL-Fe(III) 222[L-Fe(III)] = [Fe(III)][L] (1-14) 3Fe(III)3LL-Fe(III) 333[L-Fe(III)] = [Fe(III)][L] (1-15) 4Fe(III)4LL-Fe(III) 444[L-Fe(III)] = [Fe(III)][L] (1-16) 5Fe(III)5LL-Fe(III) 555[L-Fe(III)] = [Fe(III)][L] (1-17) 6Fe(III)6LL-Fe(III) 666[L-Fe(III)] = [Fe(III)][L] (1-18) The relationship between the stepwise and the overall equilibrium constants can be related as following general equation (Equation 1-19): n123456KKKKKK......K n (1-19) When comparing the complex formation process between a multidentate chelator and a metal ion with the corresponding process involving a comparable monodentate ligand, it is always found that the overall formation constant of the former is greater; that is, multidentate 47

PAGE 48

ligands always form more thermodynamically stable metal complexes than analogous ligands with less coordination donors, a well known phenomenon called the chelate effect. This effect can be explained by comparing the Fe(III)-complex formation constant of enterobactin with that of 2,3-dihydroxy-N,N-dimethylbenzamide (DHBA, figure 1-13), an analogous bidentate ligand of enterobactin. Enterobactin is hexadentate and can form a very tight octahedral complex with Fe(III) with a formation constant of 10 52 However, the for DHBA is only 10 40.24 which is almost 12 log-units lower than that of enterobactin. 166 Recall that G = RT ln K, and G = H -TS. The changes of enthalpy (H) for the complex formation steps usually are very close for forming a metal complex with either a multidentate or a chelator with less donors. 167 Therefore, the chelate effect observed is considered essentially an entropy effect; that is, increase of disorder in the system upon complex formation. For example, one molecule of enterobactin binds one atom of Fe(III) with displacement of six molecules of H 2 O surrounding Fe(III) in solution, hence net five molecules are exchanged in the system. However, three molecules of DHBA are needed to bind one molecule of Fe(III); while there are still six molecules of H 2 O displaced, only a net of three molecules are exchanged in the system. Therefore, upon binding Fe(III), the hexadentate enterobactin leads to a higher increase of the entropy than the bidentate DHBA does. The formation constants of various chelators are often tend to be difficult to compare when they are measured under different conditions, for example, pH, as can be seen in following calculation of the stability constant (Equation 1-20) of a fully protonated hexadentate iron chelator: -3+6Fe(III) + HL FeL + 6H 48

PAGE 49

-3+6ML6[FeL][H] [Fe(III)][HL]K (1-20) For example, the stability constant is 10 -0.97 for the fully protonated enterobactin (H 6 ent) but 10 52 for the fully deprotonated enterobactin (ent 6). 168 Therefore, it is more helpful to use free Fe(III) concentration in solution at a given pH, total Fe(III), and total chelator concentration. 166 Conventionally, it is measured at pH 7.4 with 10 M of ligand and 1 M of Fe(III) present. The free Fe(III) concentration is represented by pM value (pM = -log[Fe(III)]); thus, a stronger iron chelator has a higher pM value. As mentioned in the last section, catecholamides bind Fe(III) much more tightly than hydroxamates do. The formation constants for catecholamides can be as high as 10 52 M -1 for enterobactin, which corresponds to a pM value of 35.5. 166 However, the formation constants for hydroxamates are often many log units lower; for example, it is 10 31 M -1 and corresponds to a pM value of 25.9 for desferrioxamine B. 163 1.2.2.3 Iron Uptake, Transport and Utilization in Microbes As introduced in section 1.2.2.1, siderophores are synthesized by microbes and then released into the extracellular environment as a means of iron acquisition. Once formed, the siderophore-Fe(III) complex is recognized and transported by specific membrane receptor and transport systems. All Gram-negative bacteria have an outer membrane on which those specific receptors for siderophore-iron complex reside. For example, E. coli use the outer membrane receptor protein FhuA to facilitate the transport of ferric-chromes across the outer membrane, then use the ATP-binding cassette (ABC) transporters to ferry these complexes across the cytoplasmic membrane. 169, 170 However, in Gram-positive bacteria such as Listeria monocytogenes and Streptococcus pyogenes that lack an outer membrane but have a permeable cell wall, the iron can usually reach the cytoplasmic membrane directly, where the periplasmic ABC transporters can carry iron into the cytoplasm. 171, 172 49

PAGE 50

While there are numerous ferric iron transport systems which exist in microbes, 152 they are subdivided into two classes: the high affinity system and the low affinity system. These two systems may coexist in a single microbial species and lead to a biphasic kinetic profile for the ferric-siderophore complex transport. This is well demonstrated in the parabactin-mediated iron transport in Paracoccus denitrificans, a Gram-negative bacterium. 163, 173 The high affinity transport system operated at L-parabactin concentration below 1 M with a apparent K m of 0.24 0.06 M; whereas the low affinity system operated at L-parabactin concentration between 1 and 10 M, and has a apparent K m of 3.9 1.2 M. Then how can microbes use iron since siderophores bind Fe(III) so tightly? Basically, two mechanisms have been proposed: a ferrisiderophore reductase mechanism and a ligand-exchange mechanism. Ferrisiderophore reductases have been purified and characterized in species such as Pseudomonas aeruginosa 174 and Saccharomyces cerevisiae. 175 It was assumed that once the ferric-siderophore complex was taken up by microorganisms, Fe(III) was reduced to Fe(II) by the enzyme and released thereafter. Alternative to the ferrisiderophore reductase mechanism, the ligand-exchange mechanism was found to exist in Aeromonas hydrophila, Escherichia coli and many other microorganisms. 176 This mechanism proposed that once the ferric-siderophore complex encountered the membrane bound receptor, Fe(III) would bind to the ferric-free siderophore that initially already bound to the receptor; this Fe(III)-exchange induced a conformational change to the receptor protein so that the newly formed Fe(III)-siderophore complex could enter the cytoplasm and is released there. The receptor protein then returned to its original conformation, but with the siderophore originally complexed to Fe(III). Additionally, the ferric-siderophore transport systems of microorganisms are highly stereospecific; this stereospecificity requirement is part of the recognition process for the ferric50

PAGE 51

siderophore receptors and was classically exemplified by the uptake of ferric-enterobactin complex in E. coli. 177 While the outer membrane receptor recognized the right-handed () complex formed between Fe(III) and the synthetic L-enterobactin, it rejected the left-handed () complex formed between Fe(III) and D-enterobactin. Another interesting observation is that the racemic parabactin (D,L-parabactin) inhibited E. coli growth at an initial period but failed to do so later. 178 This is because at the early growth stage the deprivation of iron by exogenous parabactin induced the siderophore biosynthetic system in E. coli. Later on, the microbe produced its own siderophores such as enterobactin in concentration high enough to compete with D,L-parabactin. The ability of some pathogenic microbes to sequester iron from transferrin, 179, 180 lactoferrin, 181 and heme 182, 183 of the host to sustain their own growth has been known for decades. Thus, chelators intended to be used as therapeutics should be tested against possible pathogens and must not be able to support microbial growth. 1.2.3 Iron Uptake, Storage, and Homeostasis in Mammalian Cells 1.2.3.1 Transferrin and Transferrin Receptor In contrast to bacteria, mammals use the transferrin/transferrin receptor system to transport iron. The human transferrin (hTf) (M.W. ~ 80 kD) is a serum glycoprotein that structurally resembles human lactoferrin. Each hTf has two homologous lobes termed Cand N-lobe. Each lobe contains a series of -helices and a -sheet domains which interact with each other to form a cleft for iron binding. 184 Four residues are responsible for the binding of Fe(III) in both the Cand N-lobe binding sites: one aspartate, two tyrosines, and one histidine. 185, 186 Additionally, a carbonate molecule plays a synergistic role in the hTf binding of Fe(III). 187 Both the Cand N-lobe can bind one ferric ions with very high affinity (K d = 10 -23 M), yet reversibly. 184, 188 51

PAGE 52

The human transferrin receptor (hTfR or hTfR1) 189 is a 90-kDa homodimeric membrane associated glycoprotein that serves as the guard of the iron uptake system. The two subunits are connected by two cysteine disulfide bonds and each subunit is characterized by three distinct domains. 190 The N-terminal cytoplasmic domain containing residues 1 67 is involved in Fe(III) 2 -Tf-TfR complex endocytosis. The single-pass transmembrane domain of the residues 68 88 is for docking the receptor protein to the cell membrane. The large C-terminal extracellular domain comprised of the residues 89 760 contains the Tf-recognition sites; the amino acids 646 648 (Arg-Gly-Asp) are suggested to be critical in Tf binding by a mutagenesis study. 191 1.2.3.2 Cellular Uptake of Iron by the Transferrin/Transferrin Receptor System As illustrated in Figure 1-14, uptake of iron starts with the binding of one diferric-Tf to each of the two homologous TfR arms (Steps 1 and 2). Next, the complex enters into a clathrin-coated pit by invagination of the cell membrane and is internalized to an endocytotic vesicle, which matures into an endosome soon after (Step 3). Iron is then liberated from the Tf-TfR complex in endosome and the free Fe(II) is exported out of the endosome by the divalent metal transporter 1 (DMT1); this step also requires the endosomal ATP-driven proton pump (H-ATPase) (Step 4). + Transferrin remains bound to its receptor at the acidic pH of endosome and lysosome even after iron is released (step 5). 184, 192 Once getting out to the extracellular space (pH = 7.4), apoTf is dissociated from TfR (step 6). The freed apoTf continues to bind more Fe(III). One Tf molecule may go through one to two hundred such iron cycles before its degradation. 193 The mechanism of Fe(III)/Fe(II) reduction occurring in step 4 has long been debated. Previously it was thought that Fe(III) was reduced to Fe(II) by the acidic environment in the endosome (pH ~ 5.5). 194-196 However, just the pH shift from neutral to around 5 is not sufficient for the Fe(III)/Fe(II) reduction. 193 It is likely that the protonation of the synergistic carbonate ion 52

PAGE 53

is the first step of the iron releasing, which occurs before the Tf changes from the closed to open conformation. 197 The open conformation may provide access to a ferrireductase Steap3, which reduces Fe(III) to Fe(II). 198 The much weaker affinity of Fe(II) for Tf (K d = 10 -9 M) 199 than that of Fe(III) further promotes the release of iron from Tf. The transferrin receptor is also suggested to facilitate the iron release in the endosome. 185 Significantly, one of the most recent studies 193 demonstrated that at pH 5.6, the binding of TfR to the iron-borne Tf significantly raised the reduction potential of Fe(III) to Fe(II) to 300 mV, which otherwise was 500 mV at pH 7.4 and was too low for the reduction to occur physiologically. 1.2.3.3 Cellular Iron Storage Ferritin is the major cellular storage site for excess iron. It is a 50-kD cytosolic protein consisting of 24 polypeptide chains, which are a collection of 22-kDa H-chains and 20-kDa L-chains. The apo-ferritin is a hollow sphere with a 12-nm outer shell and a 8-nm inner shell. 200 Each ferritin molecule can hold up to 4,500 iron atoms in its inner shell. 201 The iron storage process starts with the entrance of Fe(II) into the ferritin shell, via the funnel-shaped hydrophilic three-fold channels on the protein. 202 Fe(II) is then oxidized to Fe(III) by ferroxidase located at the H-chain in the presence of O 2 and the resulting Fe(III) moves to the L-chain inside the protein shell. Iron is stored in ferritin in the form of mineralized hydrous ferric oxide that resembles ferrihydrite (5Fe 2 O 3 9H 2 O). 203 The mineral crystals grow by a process involving oxidation of Fe(II) to Fe(III), nucleation and mineralization. 200 In addition to ferritin, there is another iron storage protein called hemosiderin. Iron saturated ferritin is easily aggregated and its protein shell is degraded by lysosomal enzymes to form the end-product called hemosiderin; 204, 205 it was found that the predominant component of hemosiderin is the denatured form of the ferritin H-chain in the liver of iron overloaded patients. 206 Hemosiderin is in high amount particularly in iron-overloaded conditions. 207, 208 The 53

PAGE 54

iron to protein ratio is higher in hemosiderin than in ferritin. Hemosiderin iron may exist in different forms depending on different sources, such as ferrihydrite (5Fe 2 O 3 .9H 2 O) in normal human spleen, amorphous Fe (III) oxide in primary haemochromatosis patients, 209 and goethite (-FeOOH) in patients receiving regular transfusion therapy. 210 Therefore, the iron bioavailability from hemosiderin may vary, e.g., it will be low with the one having higher amount of goethite iron than those having the ferrihydrite as the major iron form. 210 1.2.3.4 Cellular Iron Homeostasis Controlled by the IRE/IRP System The iron released from transferrin in endosome is transported to the cytosol (Section 1.2.3.2) and enters a transit pool 211 called the labile iron pool (LIP) 212, 213 or the chelatable iron. 214 However, the LIP was also found to exist in other cellular organelles such as mitochondria, lysosome, and nucleus. 215 The LIP contains both Fe(II) and Fe(III) that are weakly bound to various cellular low molecular weight chelators such as citrates, phosphates, carboxylates, phospholipids, and polypeptides. 216, 217 Therefore, although LIP represents only three to five percent of the total cellular iron, 218 it is able to cause oxidative stress in cells when out of control. 219 Therefore, mammalian cells have developed a mechanism to keep iron in this pool at equilibrium. The size of the LIP is tightly controlled by the post-transcriptional regulation of the TfR and the ferritin expression 220 via a distinctive IRE/IRP mechanism. Iron responsive elements (IREs) are highly conserved stem-loop structural motifs in the untranslated regions (UTRs) of TfR1 or ferritin (both the Hand L-chains). Ferritin mRNA has only one IRE motif at its 5'-UTR for the protein translation. However, TfR1 possesses five IREs at its 3'-UTR for the mRNA stability regulation. 221 IREs confer binding sites, the stem-loop motifs, for iron regulatory proteins (IRPs). Iron regulatory proteins are cytosolic RNA-binding proteins, which have two 54

PAGE 55

forms, IRP1 and IRP2. IRP1 is homologous to the mitochondrial aconitase, a [4Fe-S] containing enzyme, and exists in cells with either high or low iron levels. However, IRP2 lacks aconitase activity and is available only in iron depleted cells. 222 As depicted in Figure 1-15, when the cellular iron pool runs low, IRP1 lacks a [4Fe-S] cluster and aconitase activity; both the IRP1 and the IRP2 bind to IREs with high affinity. 223 This results in the downregulation of ferritin protein synthesis by repressed translation of ferritin mRNA (upper left panel). Meanwhile, the binding of IRPs to IREs stabilizes the TfR1 mRNA (lower left panel). 220 On the other hand, when cellular iron is in surplus, IRP1 has a [4Fe-S] cluster and switches to the aconitase activity; the IRP1 is inactivated for the IRE-binding, 222, 224 and the IRP2 is degraded by the ubiquitin-endonuclease pathway (lower right panel). 225 However, the elevated iron pool results an active ferritin mRNA translation (upper right panel). 222, 224 Although both IRP1 or IRP2, contributes more to the cellular iron homeostasis, 226 they play different roles. For example, inactivation of IRP2 induced iron overload in mice, 227 while IRP1 disrupted animals were functionally normal. 228 1.2.4 Iron Absorption, Distribution, and Utilization in Humans In humans, the body iron concentration is very well maintained mainly through the tightly regulated iron absorption in the small intestine. A normal individual acquires 8 18 mg of iron from diets containing both heme iron and non-heme iron. 229 The dietary non-heme iron is absorbed by enterocytes in the duodenum, where ferric iron is reduced to ferrous iron by the ferric reductase Dcytb, and taken up via the apical membrane of enterocytes by DMT1 (Nramp2). 230 Next, the ferrous iron is transported out of the enterocyte by the ferroportin Ireg1 at the basolateral membrane. Before it enters the body circulation, Fe(II) is oxidized to Fe(III) by 55

PAGE 56

the membrane bound ferroxidase hephaestin, a homologous protein of the plasma copper-containing ferroxidase ceruloplasmin. 231, 232 Compared with non-heme iron, the dietary heme iron has higher bioavailability; 233, 234 about two thirds of the body iron is derived from heme iron in non-vegetarians. 235 The transport mechanism of heme-iron is quite different from that of non-heme iron. 236 First, hemoglobin is enzymatically digested in the intestinal lumen and the heme enters enterocyte in the intact metalloporphyrin form and is degraded by mucosal heme oxygenase. 237 Finally, the iron released from heme enters circulation as inorganic iron. Because human body lacks an efficient iron excretion pathway under normal physiological status, 238, 239 in spite of a total iron storage of 2.3 3.8 g, the iron absorption and excretion is only 1 2 mg daily (Figure 1-16). 240 Iron(III) in circulation is bound to Tf and transported to various tissues for use and storage. As illustrated in Figure 1-16, 20 30% of the total body iron is stored in ferritin or hemosiderin in the liver and heart, around 10% is stored in bone marrow, and another 10% is stored in myoglobin (Mb), cytochromes, and iron-containing enzymes. 163 The serum plasma iron only represents about 0.1% of the total body iron. Most of the iron (60 70%) is incorporated into the hemoglobin (Hb) during erythropoiesis. At the end of their lifetime (~ 120 days), erythrocytes are degraded by macrophages of the reticuloendothelial (RE) system and the Kupffer cells of the liver by phagocytosis. Iron is eventually released from Hb heme and bound to Tf or ferritin. 241 1.2.5 Iron Mediated Diseases Iron-related diseases usually arise as the result of an imbalance between iron absorption and utilization. Although iron-deficiency associated anemia is a very common public health issue, iron-overload disorders are also of great concern; 242 approximately 1 6% of Americans are affected by these diseases. 240 Among the many different types of the iron overload, the 56

PAGE 57

primary iron overload (hemochromatosis) and the secondary iron overload are the two principle types. The most intensively studied type-1 hemochromatosis is a result of the mutation in HFE gene 243, 244 which encodes a transmembrane glycoprotein HFE that modulate iron uptake. Type-1 hemochromatosis is characterized by the increased intestinal iron absorption that eventually leads to iron overloading in both the RE system and parenchymal tissues. 245 Secondary iron overload is typically caused by chronic blood transfusion therapy that is required for diseases such as hereditary -thalassemia major, sickle cell anemia, and myelodysplasia. 163, 246 Every unit of transfused blood can load about 200 mg of heme iron in an adult body. 245 Because there is no physiological mechanism for iron excretion in human, the loaded iron is eventually accumulated in the body. In the plasma, high Tf saturation results non-transferrin-bound-iron (NTBI) in circulation. 247 NTBI is redox-active and can thus cause cellular damage to organelles such as mitochondria and lysosomes. Organs such as liver, heart, and pancreas are the major sites where the iron accumulates. Many complications may develop as iron-overload progresses, such as cirrhosis 248 with cancer, cardiac fibrosis with heart failure and diabetes mellitus. 249 Additionally, many forms of infections caused by bacteria, fungi, yeast, and HIV-1 are aggravated due to the impairment of host immune defense by excessive iron. 250-253 Furthermore, localized iron-overload is also found in neuron-degeneration associated with Alzheimers (AD) and Parkinsons diseases (PD). 254 1.2.6 Iron Chelators as Therapeutics Hereditary hemochromatosis can be simply handled with phlebotomy. 255 However, the secondary iron overload can only be managed with iron chelators to promote the excretion of the excessively accumulated iron. 160 Deferoxamine B mesylate (Desferal, DFO) is the first approved iron chelator for clinical use in 1960s. Although DFO does work in patients, it is orally 57

PAGE 58

ineffective due to its hydrophilicity and high molecular weight. Therefore, DFO has a poor GI absorption; only five to ten percent of subcutaneously administered DFO can bind iron. 256 In addition, the plasma half-life of DFO is extremely short, about 0.3 h; 257, 258 the patient compliance is unsatisfactory due to its burdensome dosing regimen: subcutaneous infusion at the rate of 12 24 hours per day, 5 6 days per week. 259 Over the past several decades, many research groups have focused on searching for orally active iron chelators that can substitute for DFO. Some of the representative orally active iron chelators are shown in Figure 1-17, e.g., Ferriprox (the older names are L1, CP20 and Deferiprone, 1), 260 ICL670A (Deferasirox, 2), 261, 262 GT56-252 (Deferitrin, 3). 263-265 However, each of these synthetic iron chelators have problems regarding specificity and toxicity. 256, 266-268 Nonetheless, better understanding has been gained regarding the structural requirements in the development of effective, yet safe, iron chelators. In addition to managing iron overload, iron chelators have been tested in cancer therapy, 269-271 Wilsons disease, 272, 273 alcohol liver disease, 256, 274 ischemia/reperfusion, 275, 276 malaria, 277-279 and neurodegenerative diseases. 280 1.3 Research Objectives Based on the wide therapeutic potentials of iron chelators (Section 1.2.6) and the potential of using polyamine as vectors, the current study is aimed at expanding the concept and feasibility of applying polyamines and their analogues to: (1) vector iron chelators to facilitate their cellular uptake, and further to enhance their antineoplastic activity, (2) increase the iron clearance efficiency in experimental animals, and (3) vector other therapeutic agents to improve their efficacy. 58

PAGE 59

H2N NH2 H2N HN H2N NH2 NH2 NH HN NH2 H2N 1243 Figure 1-1. Structures of the common natural polyamines (1) putrescine (PUT), (2) spermidine (SPD), (3) spermine (SPM), and (4) cadaverine. 59

PAGE 60

HN O O NH2 NH2 HN H2N Lysine residue ineIF5A precursorSpermidineHN O O NH H2N Deoxyhypusine ineIF5A intermediate+ Deoxyhypusine SynthaseHN O O NH H2N OH Deoxyhypusine Hydroxylase12Hypusine in active eIF5AH2N N NH2 NAD+NADH + H+ NH2 H2N Figure 1-2. Biosynthesis of hypusine and the maturation of eIF5A Step 1, spermidine serves as a 4-aminobutyl donor to the lysine residue on the eIF5A precursor in the formation of deoxyhypusine intermediate, which is catalyzed by the enzyme deoxyhypusine synthase; Step 2, hydroxylation on the side chain of deoxyhypusine by deoxyhypusine hydroxylase generate hypusine. 60

PAGE 61

Figure 1-3. Illustration of a NMDAR subunit Four major domains are shown here: the amino-terminal domain (ATD) and the amino-binding domain (S1 and S2) at the extracellular site; the transmembrane domain (M1, M3, M4 and P-loop); the carboxy-terminus (CTD) at the intracellular domain. 61

PAGE 62

H2N NHNH2H2NNH2H2NNH2PutrescineNHNH2SpermidineSpermineNHHNNHHNHNHNNH2HN OHO+NH2ArginineOHOOrnithineH2NH2NNH2ArginaseSPD synthaseSPM synthaseN1-AcSPMH2NHNOOHONHNH2N1-AcSPDNHOHOSNH2CO2HMethionineNNNNNH2OOHOHHHHHSH2NCO2H+ATPPPi, PiS-AdoMetNNNNNH2OOHOHHHHHSH2N+dcAdoMetAdoMet synthetaseAdoMetDCH2NNH2OureaAcetyl-CoAAcetyl-CoACO23-AcetamidopropanalCO2O+ H2O2+ H2O2ODCSSATPAOSSATPAO3-AcetamidopropanalSMOHO H2O2 +3-AminopropanalNH2HO4-AminobutanalNH2HOOGABAADHADHOHOON-Acetyl--alanineADHDAO Figure 1-4. The polyamine biosynthetic network ODC, ornithine decarboxylase; S-AdoMet, S-adenosylmethionine; dcAdoMet, decarboxylated Sadenosylmethionine; AdoMetDC, S-adenosylmethionine decarboxylase; SMO, spermine oxidase; DAO, diamine oxidase; PAO, N 1 -acetyl-spermine/spermidine oxidase; ADH, aldehyde dehydrogenase; N 1 -AcSPM, N 1 -acetylspermine; N 1 -AcSPD, N 1 -acetylspermidine. 62

PAGE 63

H3N NH3 NH C O H O CH3 O P PLPdipolarion ODCLys-69 NH C N H O CH3 O P H ODC ornithinealdimineODC-PLP(Schiffbaseintermediate)H3N O H2N O H3N O HN O C NH O CH3 O P H3N H NH CH NH O CH3 O P Quinonoidintermediate ODCNH2 H geminaldiamineintermediateH3N O N O CH NH O CH3 O P (CH2)4 (CH2)4 CO2 Putrescine ODC(CH2)4 NH2 OrnithineH+ H2OH2O H3N H NH CH NH O CH3 O P H putrescinealdimine Figure 1-5. Mechanism of the ODC-catalyzed decarboxylation of L-ornithine First, ODC binds PLP to form an Schiff base intermediate with the residue Lys-69. Nucleophilic attack by L-ornithine produces a geminal diamine intermediate. An ornithine aldimine is formed upon releasing ODC from the intermediate. The loss of CO 2 from the ornithine fragment produces a quinonoid intermediate. Protonation of the quinonoid generates a putrescine aldimine, which regenerates PLP and produces putrescine simultaneously upon the water attack. H2NOHH2NOCF2HDFMOH2NHNNNNHNH2NHCH3NHMGBGH2CCNHHNCCH2MDL 72527 Figure 1-6. The representative synthetic inhibitors of polyamine metabolism DFMO, DL-difluoromethylornithine; MGBG, methylglyoxal bisguanylhydrazone; MDL 72527, N 1 ,N 4 -bis(2,3-butadienyl)-1,4-butanediamine. 63

PAGE 64

RS NH2 CH3 H3C O O AdoMetDC +RS CH3 H3C N O AdoMetDC O OH CO2RS CH3 H3C N OAdoMetDC RS CH3 H3C NH H+ OAdoMetDC RS NH3 CH3 H3C O O AdoMetDC +S-adenosylmethionineAdoMetDC enzyme withpyruvoyl prothetic groupShiff base intermediateDecarboxylated S-adenosylmethionineH2O O OH H2O Figure 1-7. The mechanism of the AdoMetDC-catalyzed decarboxylation of adenosylmethionine Nucleophilic attack of the pyruvoyl carbonyl by the -amino nitrogen of S-adenosylmethionine (S-AdoMet) generates a Shiff base intermediate. Subsequent loss of a CO 2 from this imine intermediate forms a decarboxylated intermediate, which is protonated and attacked by H 2 O to produce the decarboxylated S-AdoMet and regenerate AdoMetDC enzyme. H2N NH HN NH2 H2N NH HN HN CH3 O SSATAcCoAH2N NH NH2 NH NH NH2 SSATAcCoA H3C O Figure 1-8. The mechanism of the SSAT assay AcCoA, acetyl Coenzyme A. 64

PAGE 65

H2N NH HN NH2 H2N NH NH2 H NH2 O H2O2H NH HN H O H2O2NH3 BSAO++++BSAOH2N NH NH2 H2O2H NH NH2 NH3++BSAOO (1)(2)SpermineSpermidineSpermidineO N,N'-Bis(3-propionaldehyde)-1,4-diaminobutaneN'-(4-aminobutyl)-aminopropionaldehyde3-Aminopropanal acroleinH O +NH3 Figure 1-9. Bovine serum amine oxidase catalyzed oxidative deamination of polyamines Scheme for (1) SPM and (2) SPD. 65

PAGE 66

HN N NH O O NO OH OH HO HO OH O OH HN N NH O O NO OH OH HO HO OH O HN N NH O NO OH OH OH O OH O ON OH OH HN N NH O NO OH OH OH O O ON OH O O HN O O O O NH NH O OH HO OH OH O OH HO O 12345 Figure 1-10. Chemical structures of representative catecholamide iron chelators (1) L-agrobactin (Agrobacterium tumefaciens), (2) L-parabactin (Paracoccus denitrificans), (3) L-vibriobactin (Vibrio cholerae), and (4) L-vulnibactin (Vibrio vulnificus), (5) enterobactin (Escherichia coli). Polyamine backbones are shown in bold bonds, the microorganism that produces each compound is shown in parentheses. 66

PAGE 67

HN N O N H2N O OH NH N CH3 O O O OH OH HN N O N H2N O OH NH N O O O OH OH OH O N NH HN N O O O O HO OH NHHN N N CH3 H3C OH OH O O O O 1234 Figure 1-11. Chemical structures of representative hydroxamate iron chelators (1) desferrioxamine B (Streptomyces pilosus), (2) desferrioxamine G (Hafnia alvei), (3) bisucaberin (Vibrio salmonicida), and (4) rhodotorulic acid (Rhodotorula pilimanae). Polyamine backbones are shown in bold bonds, the microorganism that produces each compound is shown in parentheses. 67

PAGE 68

HO2C HN NH CO2H O O HO OH HO2C CO2H OH SN N H CO2H CH3 H N OH SN CO2H CH3 123 Figure 1-12. Chemical structures of miscellaneous siderophores (1) rhizoferrin (Rhizopus arrhizus), (2) pyochelin (Pseudomonas aeruginosa), and (3) desferrithiocin (Streptomyces antibioticus). Polyamine backbones are shown in bold bonds, the microorganism that produces each compound is shown in parentheses. N O OH HO CH3 CH3 Figure 1-13. Chemical structures of 2,3-dihydroxy-N,N-dimethylbenzamide (DHBA) an analogous monodentate ligand of enterobactin. 68

PAGE 69

Figure 1-14. Iron transport in mammalian cells Steps 1-2, binding of one diferric-Tf to each of the two homologous TfR arms; Step 3, the Tf-TfR complex enters into a clathrin-coated pit by invagination of the cell membrane; Step 4, Fe(III) is liberated from the Tf-TfR complex in endosome, and the free Fe(II) is exported out of the endosome by the divalent metal transporter 1 (DMT1); Step 5, iron is released in lysosome, and transferrin remains bound to its receptor there; Step 6, apoTf is dissociated from TfR when the complex is in the extracellular space. 69

PAGE 70

Figure 1-15. Regulation of cellular iron homeostasis by the IRP/IRE system The size of the intracellular iron pool posttranscriptionally regulates the binding of IRPs to IREs vice versa. Iron responsive elements (IREs) are conserved hair-pin structural motif that located at the untranslated regions of both TfR1 and ferritin Hand L-chains. Iron regulatory proteins (IRPs) are cytosolic RNA-binding proteins that have two forms: IRP1 and IRP2. While Ferritin mRNA has only one IRE motif at its 5'-UTR for the protein translation, TfR1 possesses five IREs at its 3'-UTR for the mRNA stability regulation. Under low iron conditions, IRPs bind to IREs with high affinity, resulting inefficient ferritin mRNA translation but stabilize the TfR1 mRNA. However, when the intracellular iron pool is increased, IRP1 is switched to the aconitase function and inactivated toward the binding to IRE. IRP2 is degraded via the ubiquitin mediated protease pathway. On the other hand, the ferritin mRNA translation is activated to respond the high cellular iron concentrations. 70

PAGE 71

Iron Intake8 18 mg duodenum absorption1 2 mgPlasma3 4 mg Bone Marrow300 mgErythropoiesisas Hb, 1800 mg Macrophage600 mg Muscle (as Mb), 300 mg Liver and HeartFerritin & Hemosiderin, 1000 mg Losses1 2 mg Figure 1-16. Daily human body iron storage, distribution, and utilization. N O OH CH3 CH3 NNN HO CO2H HO OH SN CO2HCH3HO 123 Figure 1-17. The structures of some representative iron chelators under clinical investigations (1) L1 (Deferiprone), (2) ICL670A, and (3) GT56-252. 71

PAGE 72

CHAPTER 2 DESFERRITHIOCIN-BASED IRON CHELATORS 2.1 Structure-Activity Relationships among DFT Analogues One of the first discovered orally active iron chelators, (S)-4,5-dihydro-2-(3-hydroxy-2-pyridinyl)-4-methyl-4-thiazolecarboxylic acid (desferrithiocin, DFT), 281 is a tridentate siderophore that has three donor groups: a phenolic oxygen, a thiazoline nitrogen, and a carboxyl. 282 DFT forms a stable 2:1 Fe(III) complex with a formation constant of 4 x 10 29 M -1 283 Although it worked well in both the bile duct-cannulated rodent model 284 and the iron-overloaded C. apella primate model 281 for clearing iron, it exhibited severe nephrotoxicity. 285 Nevertheless, DFT was recognized as a valuable pharmacophore for designing orally effective iron chelators with less toxicity. 282, 286, 287 A series of SARs were carried out with various DFT analogues in past years. 288-292 Preliminary studies were focused on determining the basic structural requirements that are compatible with the iron clearing activity of DFT. Three simple manipulations were conducted initially (Figure 2-1): (1) removal of the aromatic nitrogen to generate (S)-DADFT [(S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-methyl-4-thiazolecarboxylic acid, desazadesferrithiocin], (2) deletion of the thiazoline methyl group to give (S)-DMDFT [(S)-4,5-dihydro-2-(3-hydroxy-2-pyridinyl)-4-thiazolecarboxylic acid, desmethyldesferrithiocin], and (3) simultaneous elimination of both the aromatic nitrogen and the thiazoline methyl to produce (S)-DADMDFT [(S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-thiazolecarboxylic acid, desazadesmethyldesferrithiocin]. It was found that the thiazoline methyl and the pyridine nitrogen were not the prerequisites for the iron clearing properties of DFT. 288 In the iron-overloaded C.apella primates, (S)-DMDFT had a much lower iron clearing efficiency (ICE, see Table 2-1 for the definition) of 4.8 2.7% than the parent chelator DFT did, 16.1 8.5%. 72

PAGE 73

However, both (S)-DADFT and (S)-DADMDFT had a slightly lower ICE, 13.1 4.0% and 12.4 7.6%, respectively (Table 2-1). 163 Additional structural modifications were made to the already simplified framework, (S)-DADMDFT. The changes include alterations of the distance between the chelating centers, modifications of the thiazoline ring, benz-fusions, and changes of the configuration at the C-4 chiral center (Figure 2-1). All of these structural alterations led to the dramatic decrease or loss of the iron clearing activity. (S)-DADMDFT was identified as the simplest framework that can retain the iron clearing activity of DFT. As mentioned earlier, DFT was severely nephrotoxic. Unfortunately, although both (S)-DADFT and (S)-DADMDFT had comparable ICE and were not nephrotoxic, they showed severe organ toxicity in the GI track in rodents. The other derivative (S)-DMDFT satisfied the toxicity requirements, but had a low ICE (Table 2-1). Therefore, further modifications were performed on (S)-DADFT and (S)-DADMDFT to solve the toxicity problem. With these modifications which include alteration of the lipophilicity (log P app ) and/or redox potential, the pharmacological properties of these compounds such as organ distribution, disposition, and toxicity profile might change accordingly. The changes in log P app and/or redox potential were made through adding aromatic ring substituents and/or adding or removing the thiazoline methyl group. The aromatic ring substituents can be either electron-donating or electron-withdrawing. By addition of an electron-donating group such as in the hydroxylated derivatives (S)-4'-(HO)-DADFT [(S)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-methyl-4thiazolecarboxylic acid] and (S)-4'-(HO)-DADMDFT [(S)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-thiazolecarboxylic acid], the organ toxicities were dramatically reduced as compared with their corresponding parent drugs, (S)73

PAGE 74

DADFT and (S)-DADMDFT respectively. In the iron-overloaded primates, although the ICE of (S)-4'-(HO)-DADMDFT (5.3 1.7%) was several fold lower than its parent compound (S)-DADMDFT, (S)-4'-(HO)-DADFT had an ICE the same as its parent compound (S)-DADFT (Table 2-1). It is very interesting to discover that the organ toxicity was lower in those ligands with lower lipophilicity, as seen in the pair (S)-DADMDFT and (S)-4'-(HO)-DADMDFT, which had log P app values of -0.93 and -1.33 respectively, 160 and also in the pair (S)-DADFT and (S)-4'-(HO)-DADFT, which had log P app values of -0.53 and -1.05 respectively. 290 2.2 Specific Aims The DFT analogues of interest will be (1) evaluated for their antiproliferative activity in L1210 or human proximal tubule epithelial cells to determine if this could be of value in predicting their animal toxicity; (2) investigated for the correlation between cellular or animal toxicity and the lipophilicity; (3) studied for the nephrotoxicity at the light microscopic level. 2.3 Materials and Methods 2.3.1 Cell Culture 2.3.1.1 Culture of L1210 Cells Murine lymphocytic leukemia cells (L1210 cells) were grown in tissue culture flasks containing RPMI-1640 medium which was supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS), 14 mM Hepes, 7 mM Mops buffer, 1 mM aminoguanidine, and 1 mM of additional L-glutamine. Cells were routinely kept in the logarithmic phase by subculturing and incubating in a 37 C water-jacketed 5% CO 2 and 95% humidified incubator (Forma Scientific Inc.). 74

PAGE 75

2.3.1.2 Culture of Human Renal Proximal Tubule Epithelial Cells Normal human renal proximal tubule epithelial cells (HPTC) (Cambrex BioScience Walkersville Inc. East Rutherford, NJ) were grown in tissue culture flasks containing renal epithelial medium REGM TM BulletKit (Cambrex BioScience Walkersville Inc. East Rutherford, NJ) supplemented with 5%(v/v) FBS. Cells were subcultured when they reached 80% confluency and incubated in a 37 C water-jacketed 5% CO 2 and 95% humidified incubator. 2.3.2 Determination of Antiproliferative Activity (IC 50 ) 2.3.2.1 IC 50 Measurements in L1210 Cells The growth inhibitory properties (IC 50 ) of iron chelators were determined as described elsewhere. 293 Briefly, L1210 cells in logarithmic growth (0.5 x 10 5 /mL) were seeded in T-25 tissue culture flasks. Cells were incubated in media containing compound of interest at graduated concentrations of 100 M, 30 M, 10 M, 3 M, 1 M, 0.3 M, 0.1 M and 0.03 M under conditions described above for the desired time course. IC 50 is defined as the concentration of compound resulting in fifty percent of growth arrest as compared with non-treated cells, and it is calculated by the following formula: [] []% 100 [] []TreatedcellinitialcellControlGrowthControlcellinitialcell (2-1) 2.3.2.2 IC 50 Measurements in HPTC Cells HPTC cells (1.5 x 10 3 cells per cm 2 ) between passage 3 to 7 were seeded in T-25 tissue culture flasks. Cells were incubated in media containing compound of interest at graduated concentrations of 100 M, 30 M, 10 M, 3 M, 1 M, 0.3 M, 0.1 M and 0.03 M under conditions described in Section 2.2.1.1 for the desired time course. An overnight period was allowed for cells to attach to the flask surface before introducing compound solutions. IC 50 is defined and calculated in Section 2.2.1.1. 75

PAGE 76

2.3.3 Measurement of Lipophilicity (Log P app ) The octanol-H 2 O partition data were expressed as distribution coefficients uncorrected for partial ionization of the acids and were all measured at pH 7.4 (50 mM TRIS buffer) using UV spectrometry. Both the aqueous (Tris buffer) and organic (1-octanol) solvents were saturated before experiments. The measurements were done by using a shake flask direct measurement. 294 Quadruplicate samples were vigorously agitated with HPLC grade 1-octanol overnight on a lab rotator (Barnstead International, Dubuque, IA). The two layers were allowed to settle at least for 2 h before separation. The measurements were made at 24 C using a Shimadzu model 2501PC UV spectrometer (Columbia, MD), the optical density (O.D.) was measured at two different wavelengths for each compound (Table 2-2). 2.3.4 Stoichiometry Determination by Jobs Plot The stoichiometry of the complex formed between the conjugate (S)-4'-(HO)-DADFT-polyether [(S)-4'-(HO)-DADFT-PE] and Fe(III) was determined spectrophotometrically. 293 DFT has been known to form a 2:1 ligand-Fe(III) complex 283 and was used as a positive control for this assay. Solutions containing different ligand:Fe(III) ratios were prepared by mixing appropriate volumes of 0.9 mM (S)-4'-(HO)-DADFT-PE in 100 mM TRIS-Cl (pH 7.4), and 0.5 mM Fe(III) nitrilotriacetate (NTA) in 100 mM TRIS-Cl (pH 7.4), so that [(S)-4'-(HO)-DADFT-PE] + [Fe(III)] = 1.0 mM. The 0.5 mM Fe(III)-NTA solution was prepared immediately prior to use by dilution of a 50 mM Fe-(III)-NTA stock solution with TRIS buffer. The Fe(III)-NTA stock solution was prepared by mixing equal volumes of 100 mM ferric ammonium sulfate [NH 4 Fe(SO 4 ) 2 ] and 200 mM Na 3 NTA. The iron content was verified by atomic absorption (AA) spectroscopy. 76

PAGE 77

2.3.5 Light Microscopy 2.3.5.1 Dosing of Animals Male Sprague Dawley rats (200~300 g) were administered po. with 474 mol/kg of either (S)-4'-(HO)-DADFT-PE or (S)-4'-(HO)-DADFT twice daily for 7 days. Total of twelve rats were used for this study, four controls, four treated with (S)-4'-(HO)-DADFT-PE and the remaining four were treated with (S)-4'-(HO)-DADFT. 2.3.5.2 Vesicular Perfusion of Kidneys One day post drug, the rats were anesthetized with sodium ketamine/xylyzine intraperitoneally. Midline incision was made in the abdomen and the abdominal aorta was isolated, cleansed of extraneous tissue and cannulated with a 1.5 inch, 18-gauge needle. The vessels of rat were rinsed with Tyrodes buffer containing 3% PVP (pH 7.35, 282 mOsm). Both kidneys were then perfusion fixed with 1% glutaraldehyde in Tyrodes buffer containing 3% PVP (pH 7.36, 347 mOsm) and cut off and immersed in a glass vial containing fresh fixative at room temperature. The kidneys were rinsed with Tyrodes buffer (pH 7.34, 290 mOsm) twice after 4 h. 2.3.5.3 Tissue Microdissection and Embedding One of the two kidneys was dissected. Tissue samples (1-mm 3 ) were cut from kidney outer cortex (O.C.), inner cortex (I.C.) and outer stripe (O.S.) according to the following diagram (Figure 2-2). These tissue samples were then placed in sodium cacodylate, followed by the treatment with osmic acid in sodium cacodylate at 4 C under dark. After two rinses with cacodylate buffer, the samples were dehydrated in graded concentrations of ethyl alcohol and infiltrated with propylene oxide overnight. Finally, they were embedded in TAAB with DMP-30 and then polymerized for 2 3 days. 77

PAGE 78

2.3.5.4 Light Microscopy Examination of Kidney Thick Sections Thick sections (500 nm) were cut from TAAB embedded tissue, mounted onto glass slides and stained with Toludine Blue, the whole tissue slices were observed under the Zeiss Axioskop microscope. 2.3.6 Fluorescent Microscopy HPTC cells were seeded in a two-well collagen I coated glass slides and cultured for one day, followed by treatment with 100 M iron chelators of interested for another day. After discard the old media and rinsed with PBS, the cells were stained with acridine orange AO in culture medium (5 g/mL) for 15 minutes at 37 C, then washed three times with PBS. Cells were observed under a Zeiss Axioplan2 fluorescence microscope equipped with four fluorescence filters and a RGB color Spot digital camera. All the pictures were taken under the same exposure conditions. 2.3.7 Lysosome Stability Assessment by Flow Cytometry HPTCs were seeded in T-25 tissue culture flasks and cultured for one day to allow attachment, followed by exposure to 100 M iron chelators for another day. After rinsing with PBS, the cells were stained with AO in culture medium (5 g/mL) at 37 C. After 30 min, the cell layers were washed three times with PBS, collected in 1 mL ice-cold PBS by scraping with a rubber policeman and transferred into 12 x 75 mm polystyrene tubes. Lysosome integrity was assessed by using a FACSort flow cytometer (BD Biosciences) in the FL3 channel and the histogram data were analyzed by the CellQuest software. 2.3.8 Synthesis The conjugate (S)-4,5-dihydro-2-[2-hydroxy-4-[2-[(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylic acid [(S)-4'-(HO)-DADFT-PE, 4] was synthesized starting from the 78

PAGE 79

esterification of (S)-4'-(HO)-DADFT (1) with 2-iodopropane (i-PrI) and N,N-Diisopropylethylamine (DIEA) in N,N-Dimethylformamide (DMF) to form isopropyl 2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylate [(S)-4'-(HO)-DADFT-iPr ester, 2] (Figure 2-3). This ester was then alkylated with tri(ethylene glycol) monomethyl ether in the presence of diisopropyl azodicarboxylate (DIPAD) and triphenylphosphine (PPh 3 ) in tetrahydrofuran (THF) to provide (S)-4,5-dihydro-2-[2-hydroxy-4-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylate (3). Hydrolysis of the conjugate ester isopropyl 3 with 50% NaOH in methanol followed by acidification with 1 N HCl afforded the product 4. The detailed synthetic method was described elsewhere. 263 2.4 Results and Discussion 2.4.1 Lipophilicity, Antiproliferative Activity, and Animal Toxicity 2.4.1.1 Relationship among Lipophilicity, IC 50 in L1210 Cells, and Animal Toxicity Previous studies have determined the simplest platform of DFT, (S)-DADMDFT, had severe GI-toxicity, although it had comparable iron clearing activity with DFT. Two 4'-hydroxylated derivatives, (S)-4'-HO-DADFT and (S)-4'-HO-DADMDFT, were found to have substantially reduced GI-toxicity in rats. 290, 295 However, while (S)-4'-HO-DADMDFT had no sign of organ toxicity and about 60% reduced ICE in primates, (S)-4'-HO-DADFT presented with mild dose-limiting toxicity in the kidneys, particularly in the proximal tubule epithelium; nevertheless, it retained the ICE of its parent compound (S)-DADFT. 290 In this study, all the compounds of interest were tested for their antiproliferative activity in L1210 cells to determine if this could be of value in predicting their animal toxicity. As indicated in Table 2-3, each case of removing the thiazoline methyl resulted in decreasing of the lipophilicity and increasing of the IC 50 value of the corresponding compound. For example, (S)-DADFT was more lipophilic than its demethylated derivative (S)-DADMDFT; the log P app value 79

PAGE 80

of these two analogues was -0.53 and -0.93 respectively. Accordingly, the IC 50 of (S)-DADFT was about 3-fold lower than that of (S)-DADMDFT. Similarly, with a log P app value of -1.05, (S)-4'-(HO)-DADFT was more lipophilic than (S)-4'-(HO)-DADMDFT, whose log P app was -1.33. The removal of the thiazoline methyl group in this case diminished the antiproliferative activity, the IC 50 increased from 16 M for (S)-4'-(HO)-DADFT to over 100 M for (S)-4'-(HO)-DADMDFT (Table 2-3). In the 4'-methoxylated pair, again, this correlation is apparent. Decreasing of the partition coefficient by demethylation resulted in almost 3-fold loss of the antiproliferative activity; the IC 50 value increased from 6 M for (S)-4'-(CH 3 O)-DADFT to 15 M for (S)-4'-(CH 3 O)-DADMDFT. Clearly, the thiazoline methyl group is a major determinant of the lipophilicity for the DFT analogues; decreased lipophilicity caused by this demethylation will inevitably reduce the antiproliferative activity of these compounds. Previously it was found that the animal toxicity of a iron chelator was inversely related to its partition coefficient (log P app ). 160 The current results demonstrated a very similar correlation between antiproliferative activity (IC 50 ) and partition coefficient of the C-4' DFT derivatives (Figure 2-4); an inverse correlation between these two properties was evident. More importantly, as shown in Table 2-3, within each family of the C-4' derivatives of (S)-DADFT or (S)-DADMDFT, the compound with both the highest IC 50 and the log P app values was well tolerated in animals. For example, all the rats treated with (S)-4'-(HO)-DADFT at a dose of 384 mol/kg/d for 10 days survived, but those rats treated with either (S)-DADFT or (S)-4'-(CH 3 O)-DADFT at the same dose were all dead by either day 5 or day 6 respectively. Similarly, all the rats given (S)-4'-(HO)-DADMDFT at a dose of 384 mol/kg/d for 10 days survived, but those treated with (S)-DADMDFT were all dead by day 5. 80

PAGE 81

Although (S)-4'-(HO)-DADFT was well tolerated in rats when given at 384 mol/kg/d, it still presented with mild nephrotoxicity in a dose-limiting manner. 290 Therefore, structural modifications were made to (S)-4'-(HO)-DADFT to resolve this toxicity problem yet retain the iron clearing activity. One of these new derivatives is the less lipophilic molecule, (S)-4,5-dihydro-2-[2-hydroxy-4-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylic acid [(S)-4'-(HO)-DADFT-PE]. (S)-4'-(HO)-DADFT-PE was found to have an increased ICE in both the rodent and primate models. When administered po in rats at a dose of 300 mol/kg, the compound had an ICE of 5.5 1.9%, while its parent drug (S)-4'-(HO)-DADFT had an ICE of only 1.1 0.8% when used at the same conditions. Moreover, the ICE of this polyether compound was 25.4 7.4% in primates, again, significantly higher than that of (S)-4'-(HO)-DADFT, 16.8 7.2%. 263 With a log P app value of -1.10, (S)-4'-(HO)-DADFT-PE inhibited 50% of L1210 cells growth at 18 M, which was 3-fold higher than that of (S)-4'-(HO)-DADFT, 6 M (Table 2-3). The animal tolerability study was in accord with the IC 50 evaluation; all the rats treated with (S)-4'-(HO)-DADFT-PE under the same condition as for (S)-4'-(HO)-DADFT survived 263 (Table 2-3). We elected to take a closer look at the origin of the reduced toxicity of the polyether compound (S)-4'-(HO)-DADFT-PE. 2.4.1.2 IC 50 in Human Renal Proximal Tubule Epithelial Cells Animal histopathological study showed that the tissue damages caused by (S)-4'-(HO)-DADFT were mostly in the proximal tubule epithelia. 160 Therefore, it was decided to assess the antiproliferative activity of the DFT-based iron chelators in the normal human proximal tubule epithelial cells (HPTCs) to compare their toxicity. The cells were exposed to the drug of interest for 96 h and their cell counts were compared with the untreated control cells (Table 2-4). 81

PAGE 82

As shown in Table 2-4, among the tested drugs, DFT had the highest activity; it inhibited 50% of the HPTC cells growth at 3 M. (S)-DADFT was the next active one, its 96 h IC 50 (10 M) was 3-fold higher than that of DFT. The demethylation further decreased the activity; the IC 50 of (S)-DADMDFT increases to 18 M. In keeping with the finding that the hydroxylation at C-4' substantially reduced the animal toxicity of DFT analogues, both derivatives, (S)-4'-(HO)-DADFT and (S)-4'-(HO)-DADMDFT, had much higher 96 h IC 50 in HPTC cells than their corresponding parent compounds, (S)-DADFT and (S)-DADMDFT. While (S)-4'-(HO)-DADFT increased the IC 50 to 35 M, with an IC 50 more than 100 M, (S)-4'-(HO)-DADMDFT was virtually inactive in arresting HPTC cell growth at 96 h. Interestingly, the data obtained from HPTC cells are in agreement with those from L1210 cells and also correlate well with the animal tolerability study (Table 2-3). 2.4.2 Stoichiometry of the Complex Formed between (S)-4'-(HO)-DADFT-PE and Fe(III) The stoichiometry of the complex formed between (S)-4'-(HO)-DADFT-PE and Fe(III) demonstrated a 2:1 ligand-Fe(III) complex (Figure 2-5), which was the same as that of the cargo molecule (S)-4'-(HO)-DADFT. 296 2.4.3 Animal Toxicity of (S)-4'-(HO)-DADFT and (S)-4'-(HO)-DADFT-PE Examed by Light Microscopy Although the origin of the dose-limiting nephrotoxicity of (S)-4'-(HO)-DADFT was unclear, the main target was in the proximal tubules. In this study, the newly developed compound (S)-4'-(HO)-DADFT-PE, which was shown to be well tolerated in rats and had a low antiproliferative activity in the cell culture model (Section 2.4.1.1), was compared with its parent drug, (S)-4'-(HO)-DADFT. Based on the preliminary dose-range finding study, it was decided that a dose of 237 mol/ kg/dose (474 mol/kg/d) of each drug to be administered in the non-iron-overloaded rats for 7 days. 82

PAGE 83

The above two drugs were given po. by gavage to the rodents twice daily for 7 days. One day postdrug the kidneys were perfusion-fixed and one kidney from each rat was dissected. Tissue samples of 1 mm 3 were cut from the kidney cortexes and embedded in TAAB resin. Thick sections (500 nm) cut from the embedded tissue were observed under light microscope. It was found that the proximal and distal tubules of kidneys from the control animals showed normal tubular architecture (Figure 2-6, top). The proximal tubules of kidneys from the polyether drug treated rodents (Figure 2-6, bottom) were indistinguishable from those of the control animals; the distal tubules present with occasional vacuolization but were otherwise normal. However, animals treated with (S)-4'-(HO)-DADFT (Figure 2-6, middle) showed regional, moderate to severe vacuolization in the proximal tubules, a loss of the brush border, and tubular extrusions toward the lumen; the distal tubules showed moderate to severe vacuolization. 2.4.4 Observation Lysosome Changes in HPTC cells by Fluorescent Microscopy Lysosomes are cellular organelles that contain large amount of acid hydrolases; they are responsible for the digestion of cellular macromolecules via the autophagocytotic pathway. 297, 298 The degradation of iron-containing metalloproteins and ferritin in lysosome can release the redox-active iron in this organelle, and thus makes it very sensitive to the iron induced oxidative stress. 299 Under oxidative stresses, lysosome membrane are ruptured and the hydrolytic enzymes are released into the cytosol; cells eventually undergo apoptosis as the result. 300 Doulias et al. demonstrated that DFO was taken up by the cells via the endocytotic pathway and delivered into the lysosomes. 301 Although the mechanism by which the other iron chelators, such as DFT and its analogues, are taken up by cells is currently unknown, it is plausible that they share the same pathway as DFO. One of the objectives of the current study is to investigate the mechanism of the nephrotoxicity induced by DFT-based iron chelators at the subcellular level. It is currently 83

PAGE 84

speculated that these compounds cause nephrotoxicity via the lysosomal pathway. To prove this hypothesis, the HPTC cells treated with several representative DFT analogues were observed under microscope after staining with acridine orange. Acridine orange (3,6-dimethylaminoacridine, AO) is a lysosomotropic metachromatic fluorescent agent; it exists as a dimmer when trapped inside of lysosomes and shows intense red fluorescence when irradiated with blue light, but shows green fluorescence in other cellular structures such as the cytosol. Therefore, AO has been widely used as an excellent non-enzymatic lysosome marker. The ability of lysosomes to concentrate AO depends on the lysosomal membrane integrity, thus lysosome breaching will lead cells to lose red fluorescence. Iron chelators (100 M) treated or untreated (Control) HPTC cells were stained with AO, and the digital fluorescence pictures were taken (Figure 2-7). After one day exposure, HPTC cells treated with DFT had many apoptotic nuclei, more importantly, they have substantially reduced AO-induced red fluorescence (Figure 2-7, right upper panel) as compared with the control cells (Figure 2-7, left upper panel). Although the (S)-DADFT (Figure 2-7, left lower panel) treated cells also lost some red fluorescence, the level of the reduction is much lower. However, both the DFT and (S)-DADFT treated cells presented with giant vacuoles surrounding the nucleolus (Figure 2-7, yellow arrows). The identity of the vacuoles is currently unclear. Among the three drugs, (S)-DADMDFT (Figure 2-7, right lower panel) caused the least damage to the HPTC cells: the loss of red fluorescence was minimal, and the vacuoles were absent. These results are quite consistent with the previous data of antiproliferative activity determination (Section 2.3.1.2) 84

PAGE 85

2.4.5 Lysosome Stability Assessment by Flow Cytometry To further confirm the above findings quantitatively, HPTC cells treated with 100 M iron chelators DFT, (S)-DADFT, and (S)-DADMDFT were subjected to the flow cytometry study along with untreated controls by observing the changes of the AO-induced red fluorescence intensity in the FL3 channel. Those cells with red fluorescence were gated, and their percentages are indicated above the bar (M1) in each panel of Figure 2-8. When compared with the control cells, DFT treated cells led to a reduction of 16% in the red fluorescence. The reduction caused by (S)-DADFT was less than that of DFT, 8.7%. However, the (S)-DADMDFT treated HPTC cells did not show any lysosome damage, their red fluorescence amount was same as that of control cells (Figure 2-8). The above results are well in keeping with the previous microscopic observations (Section 2.3.4) and also correlate well with the cell proliferation inhibition studies. These data together suggest the nephrotoxicity induced by the DFT-based iron chelators is due to the lysosome damage in the proximal tubule epithelial cells. 85

PAGE 86

N OH SN CH3 OOH DFT OH SN CH3 OOH N OH SN H OOH (S)-DMDFT(S)-DADFT N OH SN H OOH (S)-DADMDFT N -CH3-CH3 OH SN H OOH AlterationsofDistancebetweenChelatingCenters OH ON H OOH ThiazolingRingModifications OH SN OOH (R)-DADMDFT H ChangesinconfigurationatC-4 OH SN H OOH Benz-fusionAdditionofSubstituentstotheAromaticRing OH SN H OOH (S)-4'-(HO)-DADMDFTHO AdditionofSubstituentstotheAromaticRing OH SN CH3 OOH (S)-4'-(HO)-DADFTHO (1)(2)(3)(3)(4)(5)(6)(7)(8)(9) Figure 2-1. Structural modifications carried on DFT which led to discovery of (a) the fundamental framework compatible with iron clearing and (b) system can reduce toxicity significantly while maintain iron clearing efficiency (ICE). 86

PAGE 87

Figure 2-2. Illustration of the dissecting positions in rat kidney O.C., outer cortex; I.C., inner cortex, and O.S., outer stripe. OH SN CH3 OOH OH SN CH3 OO OH SN CH3 OO HO O HO O O O a12 OH SN CH3 OOH O O O O bc 34 Figure 2-3. Scheme for the synthesis of (S)-4'-(HO)-DADFT-PE Reagents: (a) i-PrI, DIEA, DMF, R.T.,14 d; (b) CH 3 [O(CH 2 ) 2 ] 3 OH, DIPAD, PPh 3 THF, 5 C, 1d; (c) 50% NaOH, CH 3 OH, 1 N HCl, R.T., 1d. 87

PAGE 88

Figure 2-4. The relationship between IC and Log P of 4'-substituted DFT analogues 50 app Figure 2-5. Jobs plot of (S)-4'-(HO)-DADFT-PE The theoretical mole fraction maximum for a 2:1 ligand/Fe complex of 0.667; a linear intercept maximum of 0.674 was observed, indicating a 2:1 complex formed between (S)-4'-(HO)-DADFT-PE and Fe(III). 88

PAGE 89

Figure 2-6. Light microscopy of the kidney proximal tubules in rats treated with (S)-4'-(HO)-DADFT-PE or (S)-4'-(HO)-DADFT Rats were administered po with either (S)-4'-(HO)-DADFT-PE or (S)-4'-(HO)-DADFT twice daily for 7 d at a dose of 237 mol/kg. Thick sections (500 nm) were cut from the TAAB embedded kidney proximal tubule samples, mounted onto glass slides and stained with Toludine Blue. The whole tissue slices were observed under the Zeiss Axioskop microscope. The proximal tubules from rats treated with (S)-4'-(HO)-DADFT-PE are almost indistinguishable from those of the control animals. However, the proximal tubules from rats treated with (S)-4'-(HO)-DADFT display moderate to severe damage (tubules marked with an asterisk). Magnification = 400x. 89

PAGE 90

Figure 2-6 Continued 90

PAGE 91

Figure 2-7. Cytotoxicity of DFT analogues in HPTC cells evaluated by acridine orange fluorescent staining 91

PAGE 92

Figure 2-8. Lysosome stability of DFT analogue treated HPTC cells assessed by flow cytometry Cells with red fluorescence (AO-induced) were gated, and their percentages are indicated above the bars (M1) in each panel. 92

PAGE 93

Table 2-1. Effect of structural modifications of DFT on iron clearing efficiency and organ toxicity in primates and rodents Structural Modifications of Desferrithiocin Iron Clearing Efficiency% (ICE)* (rat) Iron Clearing Efficiency% (ICE)* (primate) Organ Toxicity (rat) DFT 5.5 3.2 16.1 8.5 Severe nephrotoxicity (S)-DADFT 2.7 0.5 13.1 4.0 (300 mol/kg) Severe GI toxicity (S)-DMDFT 2.4 0.6 4.8 2.7 Well-tolerated, all histopathologies normal (S)-DADMDFT 1.4 0.6 12.4 7.6 (300 mol/kg) Severe GI toxicity (S)-4'-(HO)-DADMDFT 2.9 2.8 4.2 1.4 Well tolerated (S)-4'-(HO)-DADFT 1.0 0.4 13.4 5.8 Mild nephrotoxicity The structural modifications are illustrated in Figure 2-1. Iron clearing efficiency% (ICE) is defined as the percentage ratio of the chelator-induced iron clearance over the theoretical iron clearance (TCL, i.e. total iron-binding capacity of iron chelator administered). All the ligands were given at 150 mol/kg po in both animal models unless noted in parentheses. Iron chelator toxicities were histopathologically evaluated in rat organs. 93

PAGE 94

Table 2-2. UV wavelengths used for measuring Log P app of DFT analogous Compound Wavelength (nm) max max (S)-DADFT 250 314 (S)-4'-(HO)-DADFT 270 302 (S)-4'-(CH 3 O)-DADFT 270 300 (S)-4'-(HO)-DADFT-PE 271 370 (S)-4'-(HO)-DADMDFT 270 302 (S)-4'-(CH 3 O)-DADMDFT 270 300 94

PAGE 95

Table 2-3. Lipophilicity (Log P app ), antiproliferative activity (IC 50 ), and animal tolerability of DFT analogues Compound Log P app a IC 50 (M) b 48 h/96 h Tolerability c NOHSNCO2HCH3 DFT -1.77 d 3.5/2.0 OHSNCO2HCH3 (S)-DADFT -0.53 6/6 All rats dead by day 5. OHSNCO2HCH3HO (S)-4'-(HO)-DADFT -1.05 e 16/17 All rats survived. OHSNCO2HCH3H3CO (S)-4'-(CH 3 O)-DADFT -0.70 6/5 All rats dead by day 6. OHSNCO2HCH3OOOO (S)-4'-(HO)-DADFT-PE -1.10 18/16 All rats survived. N OH SN CO2H H (S)-DMDFT n.a. 10/12 Not determined OHSNCO2HH (S)-DADMDFT -0.93 d 18/18 All rats dead by day 5. OHSNCO2HHHO (S)-4'-(HO)-DADMDFT -1.33 d > 100/> 100 All rats survived. OHSNCO2HHH3CO (S)-4'-(CH 3 O)-DADMDFT -0.89 15/13 Not determined a Data are expressed as the log of the fraction in the octanol layer (log P app ). Measurements were done in TRIS buffer, pH 7.4, using a shake flask direct method. b IC 50 values were estimated from growth curves for L1210 cells grown in the presence of nine different concentrations of tested compounds spanning four logarithmic units from 0, 0.03, 0.1, 0.3, 1.0, 3, 10, 30, and 100 M. IC 50 data are presented as the mean of at least two experiments with variation from the mean typically 10-25%. c All compounds were given to the rats po at a dose of 384 mole/kg/d up to 10 days. 263 d Reproduced from published work. 160 e Reproduced from published work. 290 95

PAGE 96

Table 2-4. Antiproliferative activity (IC 50 ) of DFT analogues in normal human kidney proximal tubule cells Compound structure Abbreviation 96 h IC 50 ( M) NOHSNCO2HCH3 DFT 3 OHSNCO2HCH3 (S)-DADFT 10 OHSNCO2HCH3HO (S)-4'-(HO)-DADFT 35 OHSNCO2HH (S)-DADMDFT 18 OHSNCO2HHHO (S)-4'-(HO)-DADMDFT > 100 OHSNCO2HCH3OOOO (S)-4'-(HO)-DADFT-PE IC 50 values were estimated from growth curves for HPTC cells grown in the presence of nine different concentrations of tested compounds spanning four logarithmic units: 0, 0.03, 0.1, 0.3, 1.0, 3, 10, 30, and 100 M. IC 50 data are presented as the mean of at least two experiments with variation from the mean typically 10-25%. 96

PAGE 97

CHAPTER 3 POLYAMINES AS VECTORS 3.1 Review of the Polyamine Analogues 3.1.1 Structure Activity Relationships of Polyamine Analogues Early SAR studies indicated that small structural alterations in polyamine analogues often led to significant changes in their biological activities, including the transport affinity for the polyamine transport apparatus. 5, 302-304 Because the polyamine transporter provides biological counteranions for the cationic amino nitrogens of polyamines, the charge disposition of a polyamine analogue plays a critical role in determining its transport property. 305 For example, the tetraamine N 1 ,N 12 -bis(2,2,2-trifluoroethyl)spermine (FDESPM) retains very similar steric features to its tetracationic parent molecule, N 1 ,N 12 -diethylspermine (DESPM), but it behaves as a dication at physiological pH. 5 Consequently, FDESPM competes very poorly with SPD for polyamine transport (K i = 285 M, Table 3-1), whereas DESPM is an excellent transport competitor (K i = 1.6 M). A poor uptake ratio (the ratio of the intracellular compound concentration over the treatment concentration) of less than 0.2 makes FDESPM ineffective at depleting polyamine pools, and thus not active against L1210 cells (IC 50 > 100 M). On the other hand, DESPM accumulates intracellularly to a concentration of 1780 M when cells are exposed to the analogue at 30 M for 48 h. It completely depletes PUT and SPD, and significantly reduces SPM to 22% of the control. Therefore, DESPM is very active against L1210 cells at both 48 h (IC 50 = 30 M) and 96 h (IC 50 = 0.18 M). The importance of the charge disposition of a polyamine analogue on its biological activities is also demonstrated with the studies of two piperidine analogues of DESPM, N,N'-Bis(4-piperidinylmethyl)-l,4-diaminobutane [PIP(4,4,4)] and N,N'-Bis-[2-(4-piperidinyl)ethyl]-l,4-diaminobutane [PIP(5,4,5)]. In PIP(4,4,4), the distances between the two neighbouring 97

PAGE 98

nitrogens and the two terminal nitrogens are very close to those of DESPM; 305 thus, the charge disposition of PIP(4,4,4) at physiological pH is also similar to DESPM. PIP(4,4,4) competes very well with SPD (K i = 4.9 M) for cellular transport, and the uptake ratio of PIP(4,4,4) is more than 20-fold higher than that of DESPM (1259 vs. 59). However, PIP(4,4,4) is only as effective as DESPM at depleting PUT and SPD; it reduces PUT, SPD, and SPM to 0%, 5%, and 50% of controls respectively (Table 3-1). Moreover, although PIP(4,4,4) is 15-fold more active (IC 50 = 2 M) than DESPM (IC 50 = 30 M) against L1210 at 48 h, it is as active (IC 50 = 0.1 M) as DESPM at 96 h (Table 3-1). PIP(5,4,5), another piperidine analogue of DESPM, behaves quite differently from PIP(4,4,4). As compared with DESPM, both the distances between the two neighbouring nitrogens and the two terminal nitrogens of PIP(5,4,5) are much longer. 305 Consequently, PIP(5,4,5) has a decreased affinity for the polyamine transporter (K i = 18.1 M) and a much lower uptake ratio of 15 than PIP(4,4,4) or DESPM. Not surprisingly, although PUT is depleted to 0% in the PIP(5,4,5) treated cells, SPD and SPM remain 27% and 89% respectively (Table 3-1). Although PIP(5,4,5) is not active against L1210 cells at 48 h, it is almost as active as both DESPM and PIP(4,4,4) at 96 h (IC 50 = 0.3 M). Early studies indicated that other determinants can also affect the intracellular transport of polyamine analogues, for example, the number of nitrogens in the molecule, the length of the methylene spacing, and the size of the terminal alkyl substituents. Generally, tetraamines such as DESPM are better transport competitors than the corresponding triamine analogues, e.g., N 1 ,N 8 -diethylspermidine (DESPD). With a K i value (19 M) over 10-fold higher than that of DESPM (1.6 M), DESPD has a lower uptake ratio (46) than DESPM (59) (Table 3-1). Although DESPD 98

PAGE 99

can similarly deplete both PUT and SPD to 0% and 5% of control as compared with DESPM, it only reduces SPM to 74%, which is much higher than 22% in the cells treated with DESPM. The effect of methylene spacing on polyamine transport is exemplified by a group of (bis)ethylated tetraamines having methylene backbone of (3,3,3), (3,4,3), and (4,4,4), i.e., N 1 ,N 11 -diethylnorspermine (DENSPM), DESPM, and N 1 ,N 14 -diethylhomospermine (DEHSPM). The analogues with longer methylene spacing, DESPM and DEHSPM, have low K i of 1.6 and 1.4 M respectively, while DENSPM, with the shortest methylene spacing, has a K i value of 17 M. The uptake ratio is even more sensitive to methylene spacing of polyamine analogues. Analogues with longer methylene spacing have higher uptake ratios. For example, DENSPM, DESPM, and DEHSPM have uptake ratios of 24, 59, and 294, respectively. DEHSPM is not as active as the two analogues with shorter methylene spacing at depleting polyamine pools: it reduces PUT and SPD to 49% and 40% of control respectively, but does not have any effect on SPM. Both DENSPM and DESPM deplete PUT and SPD to 0 6% of control and reduce SPM to less than 30% of control (Table 3-1). 302, 303 These two analogues have similar antiproliferative activities at 48 h (IC 50 : 20 30 M), and 96 h (IC 50 : 0.18 2 M). However, DEHSPM is much more active than the other two analogues; its 48 and 96 h IC 50 values are 0.2 M and 0.07 M, respectively. Altering the terminal substituents also has a significant impact on the transport property of polyamine analogues. This is best seen in a family of homospermine analogues, DMHSPM, DEHSPM, DIPHSPM, and DTBHSPM. 302 Changing the terminal alkyl groups from methyl to tert-butyl leads to the gradual decrease of the affinity for the polyamine transporter; the K i values of DMHSPM, DEHSPM, DIPHSPM, and DTBHSPM are 0.97 M, 1.4 M, 8.1 M, and 56 M, respectively (Table 3-1). The ability of these compounds to deplete intracellular polyamine pools is consistent with their affinities for the polyamine transporter. DMHSPM, DEHSPM, and 99

PAGE 100

DIPHSPM deplete PUT to 0%, while DTBHSPM reduces PUT to 83%. DMHSPM and DEHSPM also deplete SPD to 0%, while DIPHSPM and DTBHSPM reduce SPD to 17% and 85% respectively. All four analogues reduce SPM level to 30%, 61%, 83%, and 94% respectively. The antiproliferative activity of these compounds roughly correlates with the sizes of the substituents. The three analogues with relatively small alkyl groups, DMHSPM, DEHSPM, and DIPHSPM inhibit L1210 cell growth at nanomolar concentrations at 96 h, i.e., 0.32 M, 0.07 M and 0.06 M, while DTBHSPM, the analogue with the bulky group arrests cell growth at micromolar concentration (3 M) at 96 h. 3.1.2 The Metabolism of Polyamine Analogues Polyamine analogues are basically metabolized in two major steps, dealkylation and oxidation. Terminally alkylated polyamine analogues have to be N-dealkylated before any further metabolic transformation can occur. The dealkylated analogue is then metabolized by the same enzyme system, i.e., the SSAT/PAO, as for the natural polyamines (Figure 1-3). This metabolic pathway for polyamine analogues was demonstrated with DENSPM in mouse, dog, 306 and primates 307 DENSPM was shown to be metabolized by the route illustrated in Figure 3-1. Basically, the metabolism involved N-deethylation and stepwise loss of the aminopropyl fragments catalyzed by the SSAT/PAO system. 307 However, because DEHSPM only contains 4-aminobutyl fragments, the compound simply experienced the terminal N-deethylation steps (Figure 3-1). 308 The final metabolite HSPM was accumulated to a significant amount (84%) only 1-day post-drug and persisted for several more days in the dog, leading to a chronic toxicity in the animal. 100

PAGE 101

Therefore, it is of great importance to consider the metabolic behavior of a particular polyamine analogue whenever the intention is to utilize it as a vectoring agent (see later in Section 3.4.3.5). It has been demonstrated that tetraamines are typically more effective than their corresponding triamines at repressing the polyamine metabolic enzymes ODC and AdoMetDC. 303 For example, while the bisethylated tetraamines, e.g. DESPM, DEHSPM, and DENSPM suppress ODC activity to 3-10% of controls, and AdoMetDC activity to 28-42% of controls in L1210 cells, the corresponding bisethylated triamines, e.g. DESPD, DEHSPD, and DENSPD only suppress ODC activity to 30-80% of controls and AdoMetDC activity to 45-90% of controls (Table 3-2). 3.2 The History of Using Polyamines and Polyamine Analogues as Vectors Natural polyamines were exploited for delivering antibiotic nitroimidazole compounds) 309 and the alkylating agent chlorambucil (compound 3, Figure 3-2). 310 into tumor cells in early 90s. In the case of nitroimidazole-polyamine conjugates, those with SPD fragment (compound 1 and 2, Figure 3-2) had higher affinities for the polyamine transport apparatus (K i = 0.6 1.5 M) than the conjugates containing other polyamine fragments; however, the cytotoxicity was not well correlated with the transport affinity for the most conjugates tested. The animal studies of spermidine-chlorambucil demonstrated tissue accumulation and enhanced toxicity of the conjugated compound over the parent drug chlorambucil. 311 The use of polyamines as vectors for targeted delivery of DNA-intercalating agents was extensively studied by Phanstiel et al. 312-314 This research focused on conjugating various DNA-crosslinking compounds such as acridine, anthracene, naphthalene, and pyrene to polyamines and polyamine analogues. The antineoplastic activity of these conjugates was dependent on 101

PAGE 102

many factors such as the structure of the polyamine vectors and the linkage between the polyamines and the compounds being conjugated. For example, an acridine-polyamine conjugate with a N-alkyl linkage was more active than one with an amide linkage against tumor cells. 315 This result is in keeping with earlier findings from a series of N 4 and N 1 ,N 8 -SPD derivatives. 105 Polyamines have also been used as vectors for amino acids. 316 Among the 21 polyamine-amino acid conjugates synthesized, ornithine-SPM, lysine-SPM, and D-tryptophan-SPM were identified as the most active agents that against MDA-MB-231 human breast cancer cells; their IC 50 values were 3.0 M, 5.0 M, and 2.5 M respectively. 316 With this information in hand, there is reason to believe that polyamines and their analogues can serve as vectors for variety of molecules. The established charge and steric boundary conditions set by the polyamine transporter, together with the history of using polyamines and their analogues as vectors provide the foundation for the current research project. 3.3 The Model Conjugate Molecules 3.3.1 The Role of Charge in the Choice of Cargo Molecules As introduced in Section 3.1.1.2, the critical property of the polyamine transport apparatus is the charge recognition of the polyamines and their analogues. Therefore, when using a polyamine as a vectoring agent, the charge property of the molecule that is going to be vectored (cargo molecule) is crucial. The polyamine transporter provides biological counteranions for the cationic polyamine nitrogens (Figure 3-3). Thus, a neutral or positively charged cargo fragment should be compatible with the cellular transport of a polyamine conjugate. However, based on simple electrostatics, a negatively charged cargo fragment is not likely to be compatible with the polyamine transporter. 296 102

PAGE 103

3.3.2 Design Concept In addition to managing iron overload, a few of the iron chelators, such as parabactin 269 DFO 317 pyridoxal isonicotinoyl hydrazone (PIH) analogues 318 L1 319, 320 and DFT, 321, 322 have been tested and proven to be effective at inhibiting cancer cell growth in vitro. These chelators work by keeping apo-ribonucleotide reductase (apo-RR) from accessing iron, thus preventing assembly of the active enzyme. 269 RR is a rate limiting enzyme for DNA synthesis 323 and iron is required for the enzymes activity. Iron depletion by chelators will make RR inactive and limit the cancer cell growth. 324, 325 However, while the iron sources are limited in cell culture media, there is a constant and abundant supply of Tf-bound iron in the tumor of whole animals; this can significantly weaken the effectiveness of iron chelators at arresting tumor cells in vivo. For example, DFO was shown to be active at arresting cancer cell growth in culture; 322, 326, 327 however, it was not effective at neuroblastoma xenografts. 328 To assess the importance of the charge on the cargo molecule, two analogues having different charge characters, terephthalic acid and 4-(methoxycarbonyl)benzoic acid (monomethylterephthalate), were conjugated with SPM to give 4-(3-(4-(3-aminopropylamino)butylamino)propylcarbamoyl)benzoic acid (N 1 -terephthaloylspermine, NTS) and methyl 4-(3-(4-(3-aminopropylamino)butylamino)propylcarbamoyl)benzoate (SPM-monomethylterephthalate, NTS-ME) (Figure 3-4). These compounds were chosen because of their similarity in size and sweep volume, and also because of their different charges. 296 Therefore, it is expected that the neutrally charged NTS-ME should be transported successfully, but the negatively charged NTS should not. These two compounds were synthesized as shown in Figure 3-4. 296 Briefly, acylation of N 1 ,N 4 ,N 9 -Tris(tertbutoxycarbonyl) spermine (1) with the 1,1'-carbonyldiimidazole (CDI)-activated monomethyl terephthalate (2) produced the intermediate 3. Deprotection of 3 with HCl 103

PAGE 104

generated one of the final products, triamine amide methyl ester NTS-ME (6). A second final product was generated by hydrolyzing the methyl ester of 3 with NaOH to give the Boc-protected intermediate 4, which was then cleaved with trifluoroacetic acid (TFA) to afford the compound N 1 -terephthaloylspermine (NTS, 5). The current study focuses on achieving higher intracellular level of iron chelators by using polyamines as the vectors. The feasibility of the concept was first tested with a simple bidentate ligand, 1,2-dimethyl-3-hydroxypyridin-4-one (L1); this was based on the fact that L1 itself does not achieve high intracellular concentrations (1 M). 293 The first model compound was thus designed as the polyamine-hydroxypyridinone conjugate, 1-(12-amino-4,9-diazadodecyl)-2-methyl-3-hydroxy-4(1H)-pyridinone (SPM-L1). L1 is a neutral molecule; therefore, its polyamine conjugate SPM-L1 should be transported in cells successfully. This conjugate molecule was synthesized as shown in Figure 3-5. 293 Briefly, 3-Benzyloxy-2-methyl-4-pyrone (1) and spermine hydrochloride in NaOH were reacted in aqueous ethanol to produce the intermediate 2. The final product SPM-L1 (3) was generated after the hydrogenolysis of the benzyl protecting group of 2 with 10% Pd-C in aqueous ethanol. These three polyamine conjugates were evaluated in L1210 cells for their (1) ability to compete with radiolabeled spermidine for the polyamine transport apparatus (K i ), (2) cellular uptake, and (3) impact on polyamine pools. Additionally, SPM-L1 and its cargo molecule L1 were also evaluated in L1210 cells for their antiproliferative activities (IC 50 ) and effects on the polyamine enzymes ODC, AdoMetDC, and SSAT. 3.3.3 Materials and Methods All the chemicals used in this study were purchased from Sigma (Sigma-Aldrich Co. St. Louis, MO) otherwise noted. Cell growth media and sera were purchased from Invitrogen 104

PAGE 105

(Invitrogen Co. Carlsbad, CA). Cell lines were obtained from ATCC (American Type Culture Collection, Manassas, VA) unless noted. Tissue culture flasks were purchased from Corning Co. (Corning, NY) or BD Biosciences Co. (Rockville, MD). [ 3 H]SPD (specific activity 27.6 Ci/mmole), [114 C]-L-Ornithine (specific activity 47.4 mCi/mmole), [Acetyl-114 C]-CoenzymeA, specific activity 50-60 mCi/mmol), and scintillation cocktail Biofluor TM were purchased from New England Nuclear (PerkinElmer Life and Analytical Sciences, Inc., Wellesley, MA). S-Adenosyl-L-[carboxyl14 C]-methionine (specific activity > 55 mCi/mmole) was purchased from Amersham (Amersham Biosciences, Piscataway, NJ). 3.3.3.1 Stoichiometry Determination by Jobs Plot The stoichiometry of the complex formed between the conjugate SPM-L1 and ferric ion was determined spectrophotometrically ( max = 459 nm). 293 L1 ( max = 455 nm) has been known to form a 3:1 ligand-Fe(III) complex 329 and was used as a positive control for this assay. Solutions containing different ligand:Fe(III) ratios were prepared by mixing appropriate volumes of 0.5 mM SPM-L1 in 100 mM TRIS-Cl (pH 7.4), and 0.5 mM Fe(III) nitrilotriacetate (NTA) in 100 mM TRIS-Cl (pH 7.4), so that [SPM-L1] + [Fe(III)] = 1.0 mM. The 0.5 mM Fe(III)-NTA solution was prepared immediately prior to use by dilution of a 50 mM Fe-(III)-NTA stock solution with TRIS buffer. The Fe(III)-NTA stock solution was prepared by mixing equal volumes of 100 mM ferric ammonium sulfate [NH 4 Fe(SO 4 ) 2 ] and 200 mM Na 3 NTA. The iron content was verified by atomic absorption (AA) spectroscopy. 3.3.3.2 Kinetics of Cellular Transport The ability of polyamine analogues, iron chelators, and polyamine-iron chelator conjugates to compete with SPD for uptake into cells (K i ) by the polyamine transport apparatus was determined as described elsewhere 293 Briefly, approximately 3 ~ 5 X 10 8 cells in exponential 105

PAGE 106

growth were cooled on ice and centrifuged at 1200 rpm, 4 C for 10 min. The cell pellet was washed once with cold DPBS and centrifuged again. The pellet was then resuspended in 13 mL cold normal growth medium. After warming up to 37 C, a 500 L-aliquot of the cell suspension was added at the rate of 5-sec intervals to each pre-warmed 15 mL-centrifuge tube containing assay mixture (500 L of growth medium with 1 M, 2 M, 4 M, 6 M, 8 M, 10 M of unlabeled SPD, and 10 L of [ 3 H]SPD). The cells in the assay mixtures were then incubated in a 37 C shaking water bath. After 20 min, the tubes were chilled in ice slurry for 5 min and centrifuged. The pellets were washed at least twice with 5 mL ice-cold PRMI-1640 containing 1 mM SPD, and were lysed with 0.5 mL of 0.1% Triton X-100 thereafter. The lysate was transferred to a scintillation vial containing 10 mL scintillation cocktail Biofluor TM The CPM was counted with a liquid scintillation counting machine (Beckman model # LS6500) at the rate of 1 min per sample. The data were analyzed by fitting to Lineweaver-Burk plots. K i was calculated using the formula: [1iappmDrugKKK ] (3-1) 3.3.3.3 Antiproliferative Activity Determination The method for determination of the antiproliferative activity (IC 50 ) is the same as described in Section 2.2.1.1. 3.3.3.4 HPLC Analysis of Cellular Polyamine Pools Cells in logarithmic growth (~ 5 x 10 5 /mL) were treated with the compound of interest at a concentration close or equal to its 48 h IC 50 value. Cells were centrifuged and washed with ice-cold PBS three times. Perchloric acid (0.6 N) was added to the pellet at the final cell concentration of 1 x 10 7 /mL. The samples were freeze-fractured by alternating immersions in 106

PAGE 107

liquid nitrogen and hot tap water three times. PUT, SPD and SPM levels in the perchloric acid-extracts were determined by the dansylchloride derivatization method on HPLC as described elsewhere. 330 3.3.3.5 HPLC Analysis of Cellular Drug Concentration Cellular Concentration of L1 and SPM-L1 L1210 cells in logarithmic growth (~ 5 x 10 5 /mL) were treated with 50 M of L1 or 0.2 M of SPM-L1. After 48 h, cells were collected and handled by the same method as the polyamine pools analysis. Samples containing SPM-L1 were initially dansylated. All samples were cleaned with a C18 SPE column and the resulting solution was placed on a C18 (5 m) HPLC column in a concentration in the range of 500 4500 pmol/100 mL. The two mobile phases used for analytical separation were: mobile phase A, 20% buffer/80% CH 3 OH; mobile phase B, 98% buffer/2% CH 3 CN. The buffer consisted of potassium phosphate (10 mM) and EDTA (2 mM), pH 2.9. Under these conditions, SPM-L1 was eluted at 47.1 min. L1 was measured by HPLC with UV detection 331 using the same mobile phases as for SPM-L1. Cellular Concentration of NTS and NTS-ME L1210 cells in logarithmic growth (~ 5 x 10 5 /mL) were treated with 100 M of NTS or NTS-ME. After 48 h, cells were collected and handled by the same method as the polyamine pools analysis. The supernatant of each sample was injected directly onto the column or diluted with mobile phase A (see below). Analytical separation was performed on a Waters Symmetry C18 column (5 m) with a guard column using a Rainin HPLC system. The buffer employed was sodium octanesulfonate (2.5 mM) in potassium phosphate (25 mM), pH 3.0. Mobile phase A consisted of 5% CH 3 CN, 95% buffer; mobile phase B consisted of 60% CH 3 CN, 40% buffer. Postcolumn derivatization used a boric acid buffer system (H 3 BO 3 3.1% w/v; KOH, 2.6% w/v; 107

PAGE 108

2-mercaptoethanol, 0.6% v/v) that contained o-phthaldialdehyde (10 mL of a 4% w/v solution in CH 3 OH per L of buffer). A Shimadzu fluorescence detector (ex, 340 nm; em, 445 nm) was used. 3.3.3.6 Ornithine Decarboxylase (ODC) Assay The ODC assay was designed to determine the suppressive effect of polyamine derivatives on ODC expression post-transcriptionally. L1210 cells in log-phase (~ 5 x 10 5 /mL) were treated with the drug of interest at 1 M for 4 hours, and then harvested by centrifugation. The pellet was washed twice with 30 mL ice-cold DPBS and resuspended in an ice-cold buffer (25 mM Tris-HCl, 100 M EDTA, 1.25 mM DTT and 405 M pyridoxal 5'-phosphate, pH 7.2) to obtain a suspension containing 4 x 10 7 cells per mL. This suspension was homogenized for 15 sec with an ultrasonic processor. The homogenates were further centrifuged at 20,000 r.p.m. for 5 min in a 4 C cold room. A 200 L-aliquot of the supernatant was added to the bottom of a 10 mL side-armed assay flask (Kimble/Kontes Co., Vineland, NJ); 200 L 1 M benzethonium hydroxide was then added to the plastic center well (Kimble/Kontes Co., Vineland, NJ) containing a piece of Whatman 3mm filter paper. The substrate (50 L), i.e., the mixture of L-ornithine and [114 C]-L-ornithine (final L-orinithine concentration was 2.25 mM), was then added to the homogenate, the flask was kept sealed and incubated in a shaking water bath at 37 C for 30 min. The reaction was stopped by chilling the flask on ice. After injection of 0.5 mL 1 M sulfuric acid into the flask, the assay mixtures were further incubated for 15 min in the water bath. At the end of the assay, the plastic center wells containing the filter paper were transferred into the corresponding scintillation vials with 15 mL of Biofluor. The radioactivities were counted for 2 min by a liquid scintillation counter described in Section 2.3. The results were expressed as the percentage of the radioactive counts of the treated cells over that of the untreated control cells. DEHSPM (1 M) treated cells served as the positive control for this assay. 108

PAGE 109

3.3.3.7 S-Adenosylmethionine Decarboxylase (AdoMetDC) Assay The AdoMetDC assay was designed to determine the suppressive effect of polyamine derivatives on AdoMet expression post-transcriptionally. Cells in log-phase were treated with the drug of interest at 1 M for 6 h and then processed as described in Section 2.4. Aliquot of 200 L of the supernatant was added to the bottom of a 10 mL side-armed assay flask; 200 L of 1 M Benzethonium Hydroxide was then added to the filter paper in the center well. The substrate (50 L), i.e., the mixture of 2.5 mM L-Adomet and 0.11 mM S-adenosyl-L-[carboxyl14 C]-methionine in the homogenization buffer containing 15 mM PUT, was added to the homogenate. The reaction was carried out as described in section 2.4. The results were expressed as the percentage of the radioactive counts of the treated cells over that of the untreated control cells. DEHSPM (1 M) treated cells served as the positive control in this assay. 3.3.3.8 Spermine/Spermidine N 1 -Acetyltransferase (SSAT) Assay This assay was designed to measure the SSAT activity in mammalian cells treated with polyamine derivatives. Briefly, after exposure with the compound of interest at 10 M for 48 h, cells were centrifuged and washed twice with 20 mL ice-cold DPBS. The pellets were resuspended in ice-cold homogenization buffer (5.0 mM HEPES and 1.0 mM DTT in ddH 2 O, pH 7.6) to obtain 2.5 x 10 7 cells per mL and then disrupted for 15 sec with an ultrasonic processor. The homogenates were centrifuged at 10,000 g for at least 15 min and the supernatant was centrifuged again at 12,000 g for 2-4 h in a cold room. The final supernatant (50 L), i.e., the enzyme homogenate, was added to a slim-type 400 L microcentrifuge tube containing 50 L of substrate solution (150 mM HEPES containing 6 mM SPD and 0.5 Ci [Acetyl-114 C]-CoEnzymeA, pH 7.6). This mixture was incubated in a 37 C water bath for 15 min. The reaction was stopped by chilling the tube in ice-water slurry. Hydroxylamine (20 L of 1.0 M) 109

PAGE 110

was added to each tube, and then heated in a boiling water bath for 3 min. The reaction mixture was centrifuged at 12,000 g and 50 L of the supernatant was applied to a piece of Whatman P81 cellulose phosphate paper (pre-treated with 1 N HCl) placed in a plastic vial. The paper was allowed to stand in a vial containing distilled water for 30 min, then rinsed with deionized water. After air-drying, the paper was transferred to a scintillation vial containing 15 mL of Biofluor TM and counted for 2 min. The results were expressed as the percentage of the radioactive counts of the treated cells over that of the untreated control cells. In this assay, 2 M DENSPM treated cells served as the positive control. 3.3.4 Results and Discussion 3.3.4.1 Stoichiometry of the Complex formed between SPM-L1 and Fe(III) Although the bidentate iron chelator L1 forms a 3:1 octahedral ligand-metal complex with ferric iron, 332 the stoichiometry of the chelator-Fe(III) complex formed between the polyamine conjugated chelator SPM-L1 is not known. Based on the HSAB theory, 333, 334 it is also possible for the hard acid Fe(III) to have primary amine nitrogen as a ligand donor. Therefore, the stoichiometry of the complex formed between SPM-L1 and Fe(III) was measured. The result showed that both the conjugate and its parent ligand L1 form 3:1 ligand-Fe(III) complexes (Figure 3-6 and Jobs plot, Figure 3-7 A and B). Therefore, the conjugation of L1 with SPM does not affect the stoichiometry of the Fe(III) complex. 3.3.4.2 The Effect of Conjugation on Cellular Transport, Intracellular Uptake, and Polyamine Pools SPM-monomethyl terephthalate (NTS-ME) was a potent transport competitor with an K i value of 3.1 M (Table 3-3). However, its corresponding free acid SPM-terephthalic acid (NTS), with a K i value of 27 M, was not as good a competitor. It is important to point out that the terephthalate fragment is negatively charged at physiological pH, the condition of the 110

PAGE 111

experiment. When L1210 cells were treated with 100 M of NTS for 48 h, the intracellular amount of the conjugate was below the detection limit (70 M) and the compound uptake ratio was lower than 0.7 (Table 3-3). However, in 100 M of NTS-ME treated cells at 48 h, the intracellular concentration of conjugate was as high as 434 22 M and the uptake ratio (4.3) was 6-fold higher than that of NTS. These results were in agreement with their affinities for the polyamine transporter, and strongly supported the importance of the charge of the cargo molecule in determining the transport property of a polyamine conjugate. Neither NTS-ME nor NTS was effective at depleting native polyamine pools; the PUT levels remained 42 5% and 53 8% of control value in NTS-ME and NTS treated L1210 cells respectively (Table 3-3). While NTS barely affected SPD and SPM levels, NTS-ME raised SPD to 109 19% and SPM to 122 16% of controls respectively. Although L1 is not a competitor for SPD uptake by the polyamine transporter in L1210 cells, its polyamine conjugate SPM-L1, with a low K i value (3.7 M), is an excellent one (Table 3-4). Therefore, SPM-L1 was taken up by cells efficiently; after a 24 h treatment at 0.2 M, the intracellular concentration of SPM-L1 in L1210 cells reached to 203 18 M, which was 1000-fold more than the treatment dose (uptake ratio = 1015). The native polyamines also reached slightly higher levels in the conjugate treated cells than those in the controls; the levels of PUT, SPD, and SPM were 118 8%, 125 1%, and 121 2% of controls, respectively (Table 3-4). AT 48 h, the conjugate increased the intracellular SPM-L1 concentration to 388 36 M. This does not seem to be very high as compared with the intracellular concentration of polyamine analogues, which are usually in the millimolar range (Table 3-1). However, considering the low treatment concentration of 0.2 M, the actual uptake ratio (1940) of SPM-L1 was very high. It is very interesting that unlike the 24 h treatment, the 48 h treatment decreased the PUT and SPD 111

PAGE 112

levels to 33 1% and 64 5% of control respectively (Table 3-4); however, the SPM level was further increased to 139 9% of control. Not surprisingly, when cells were treated with 50 M L1 at 48 h, the intracellular L1 concentration was only about 1 M. Like in the SPM-L1 treated cells at 24 h, PUT, SPD and SPM were not depleted in L1 treated cells; their levels were 86 10%, 124 4%, and 146 5% of controls, respectively. 3.3.4.3 Impact on Antiproliferative Activity L1 was active against L1210 cells only at high concentrations; its IC 50 was about 50 M at both 48 and 96 h (Table 3-4). The data were consistent with results (70 110 M) obtained from rat and human hepatocellular carcinoma cell lines. 321 This could be due to the low uptake ratio of L1 (~0.02), as demonstrated in L1210 cells (Table 3-4). On the other hand, at both 48 and 96 h, SPM-L1 was highly active against L1210 cells with IC 50 of 0.18 M, which was almost 300-fold lower than that of L1. These results are in keeping with the high uptake ratio of SPM-L1 (1940) in L1210 cells (Table 3-4). 3.3.4.4 Impact on Polyamine Metabolic Enzymes The conjugate SPM-L1 was more effective than L1 at regulating polyamine metabolic enzymes ODC, AdoMetDC, and SSAT. Treatment of L1210 cells with 1 M of SPM-L1 at 4 h significantly reduced ODC activity to 26 6%, whereas its cargo molecule L1 did not affect ODC activity (88 32%). In the 6-h treated cells, AdoMetDC activity was increased to 121 5% with L1 (1 M) but decreased to 72 11% with SPM-L1 (1 M). Moreover, in the 48-h treated cells, SPM-L1 (10 M) stimulated SSAT activity to 205 45%, but L1 (10 M) decreased SSAT activity to 72 7% (Table 3-5). 112

PAGE 113

3.4 Polyamine Vectored DFT Analogues 3.4.1 Design Concept 3.4.1.1 Intracellular Accumulation of DFT Analogues Desferrithiocin (DFT) is a well known orally effective siderophore with potent iron clearing activity; however, as explained in Chapter 2, the severe nephrotoxicity 285 impedes its clinical usefulness. Structural alterations of this molecule generated several modified DFT analogues including (S)-4'-(HO)-DADFT, which had a profoundly improved organ toxicity profile and a comparable iron clearing efficiency (ICE) in primates but a decreased ICE in rodents. 290 The finding of the inverse relationship between toxicity and lipophilicity (Chapter 2) among those DFT analogues led to the generation of the C-4' methoxylated derivatives including (S)-4'-(CH 3 O)-DADFT, which had a better access to target organs (heart, liver, and pancreas) that are most affected by iron-overload than its less lipophilic parent (S)-4'-(HO)-DADFT. C-4' methoxylation significantly improved the lipophilicity and ICE of DFT analogues, as seen in (S)-4'-(CH 3 O)-DADFT and (S)-4'-(CH 3 O)-DADMDFT; 160 however, it did not change the compounds toxicity profile (Table 2-3). The recent discovery of the polyether derivative (S)-4'-(HO)-DADFT-PE solved the nephrotoxicity problem associated with (S)-4'-(HO)-DADFT. Additionally, the polyether compound had improved lipophilicity and ICE (Section 2.3.1.1). 263 Like other iron chelators such as L1, DFT-based iron chelators rarely reached high intracellular concentrations (Table 3-6). For example, in L1210 cells treated with (S)-4'-(HO)-DADMDFT for 48 h, the compound only achieved an intracellular concentration of 31 M, which was more than three-fold lower than the treatment concentration (100 M, Table 3-6). Although the intracellular concentration of (S)-4'-(HO)-DADFT (130 M) was higher than the treatment dose (100 M), the uptake ratio was still quite low (1.30). Despite the higher 113

PAGE 114

lipophilicities as compared to their corresponding parent chelators (Chapter 2), both (S)-4'-(CH 3 O)-DADMDFT and (S)-4'-(CH 3 O)-DADFT achieved only low intracellular concentrations. In L1210 cells treated with (S)-4'-(CH 3 O)-DADMDFT (15 M) or (S)-4'-(CH 3 O)-DADFT (10 M) for 48 h, the compounds intracellular concentration was only 20 M (an uptake ratio of 1.30) or below the detection limit (Table 3-6). 3.4.1.2 Objectives In spite of the high iron clearing efficiency and low animal toxicity of the polyether derivative of (S)-4'-(HO)-DADFT (Chapter 2), a more universal tool is still needed for delivering iron chelators intracellularly. Based on the successful use of polyamines as vectoring agents for the bidentate iron chelator L1 (Section 3.3.4), the current study explores the utility of polyamines as vectors to enhance the intracellular delivery of DFT-based iron chelators, and to improve the ICE of these chelators as well. 3.4.1.3 Specific Aims The current study aims to (1) evaluate polyamine conjugates NSPD-(S)-4'-(HO)-DADFT and NSPD-(S)-4'-(HO)-DADFT-EE and their cargo molecules (S)-4'-(HO)-DADFT and (S)-4'-(HO)-DADFT-EE in L1210 cells for their ability to compete with the radiolabeled SPD for the polyamine transport apparatus (K i ); (2) study their effects on intracellular uptake and cell proliferation (IC 50 ); (3) investigate their suppressive effects on polyamine metabolic enzymes ODC, AdoMetDC, and SSAT; (4) obtain the metabolic profiles of the conjugates NSPD-(S)-4'-(HO)-DADFT-EE and NSPD-(S)-4'-(HO)-DADFT; and (5) compare their iron clearing efficiency with the parent compound (S)-4'-(HO)-DADFT in the non-iron-overloaded bile-duct-cannulated rat model. 296 114

PAGE 115

3.4.2 Materials and Methods 3.4.2.1 General Methods The materials and methods for K i IC 50 ODC, AdoMetDC, and SSAT measurements were described in Section 3.4. HPLC analysis of cellular polyamine pools was described in Section 3.3.2.3. 3.4.2.2 Syntheses All the reagents used for syntheses were purchased from Aldrich Chemical Co. (Milwaukee, WI). All solvents used were Fisher Optima-grade. In reactions involving chelators, distilled solvents and glassware were presoaked in 3 N HCl for 15 min. Silica gel 70-230 from Fisher Scientific was utilized for column chromatography, and silica gel 32-63 from Selecto Scientific, Inc.(Suwanee, GA) was used for flash column chromatography. Optical rotations were run at 589 nm (sodium D line) utilizing a Perkin-Elmer 341 polarimeter with c as g of compound/100 mL of solution. NMR spectra were obtained at 400 MHz ( 1 H) or 100 MHz ( 13 C) on a Varian Mercury-400BB. Chemical shifts () for 1 H spectra are given in parts per million (ppm) downfield from tetramethylsilane for organic solvents (CDCl 3 not indicated) or sodium 3-(trimethylsilyl)-propionate-2,2,3,3-d 4 for D 2 O. Chemical shifts () for 13 C spectra are given in ppm referenced to 1,4-dioxane ( 67.19) in D 2 O or to the residual solvent resonance in CDCl 3 ( 77.16) or CD 3 OD ( 49.00). Coupling constants (J) are in hertz. The base peaks are reported for the ESI-FTICR mass spectra. (S)-4'-(HO)-DADFT was chosen as the model cargo molecule for polyamine conjugation. There were two potential sites for conjugation in this molecule: the 4'-hydroxyl and the thiazoline carboxyl (sites I and II in Figure 3-8). If site I was chosen for affixing the polyamine vectors, the cargo fragment would carry a negative charge on the thiazoline carboxyl, one would 115

PAGE 116

anticipate an unsuccessful cellular transport of the conjugated molecule. As demonstrated previously in the terephthalic acid system, this problem could be circumvented by esterification of the carboxyl group. Initially, two conjugated molecules of (S)-4'-(HO)-DADFT were designed: (S)-4,5-dihydro-2-[2-hydroxy-4-(12-amino-5,9-diazadodecyloxy)phenyl]-4-methyl-4-thiazolecarboxylic acid [NSPD-(S)-4'-(HO)-DADFT] and its ethyl ester analogue, ethyl (S)-4,5-dihydro-2-[2-hydroxy-4-(12-amino-5,9-diazadodecyloxy)phenyl]-4-methyl-4-thiazolecarboxylate [NSPD-(S)-4'-(HO)-DADFT-EE]. To allow the coupling reaction to occur at the C-4' hydroxyl (site I) of (S)-4'-(HO)-DADFT, the triamine NSPD was chemically modified at one of the two terminal amines to generate the Boc-protected NSPD-OTs (5, Figure 3-9). Synthesis of N 1 -(4-hydroxybutyl)-N 1 ,N 4 ,N 7 -tris(tert-butoxycarbonyl)norspermidine (4) The triamine NSPD (1) was initially transformed to the amino alcohol N 1 -(3-(hydroxyamino)propyl)propane-1,3-diamine (3, NSPD-OH) by adding 4-chloro-1-butanol 2 (11.03 g, 102 mmol) dropwise in a solution of 1 in 1-butanol (250 mL) in the presence of KI (1.66 g, 12.01 mmol) and K 2 CO 3 (7.03 g, 4.23 mmol). After refluxed under N 2 for 24 h, the reaction mixture was slowly cooled to room temperature and the solid was filtered and discarded. The filtrate was concentrated under high vacuum to yield the crude product 3 which was used directly for the following reaction without further purification. Next, a solution of di-tert-butyl dicarbonate (131.41 g, 600 mmol) in THF (100 mL) was added dropwise to a solution of 3 (20.3 g, 100 mmol) in 50% THF (300 mL) and the mixture was stirred overnight at room temperature. Following solvent removal by rotary evaporation, the residue was dissolved in EtOAc (100 mL) and dH 2 O (100 mL). The aqueous layer was extracted twice with EtOAc (100 mL). The pooled extractant was washed sequentially with 0.5 M citric acid, dH 2 O, and saturated NaCl and dried 116

PAGE 117

over Na 2 SO 4 After solvent was removed under high vacuum, the product was purified by gravity column chromatography, eluting with 50:50:2 EtoAc/hexanes/CH 3 OH, provided 4 (40%) as a colorless oil: 1 H NMR 1.44-1.46 (m, 27 H), 1.5-1.8 (m, 8 H), 3.02-3.34 (m, 10 H), 3.67 (t, 2 H, J = 5.9), 4.78 and 5.27 (2 br s, 1 H); 13 C NMR 25.20, 27.84, 28.57, 28.60, 28.61, 29.82, 37.72, 44.10, 45.01, 47.01, 62.57, 79.57, 155.75, 156.16, 174.84; HRMS m/z calcd for C 25 H 50 N 3 O 7 504.3648 (M+ H), found 504.3640. Synthesis of N 1 ,N 4 ,N 7 -Tris(tert-butoxycarbonyl)-N 1 -[4-(tosyloxy)butyl]norspermidine (5) p-Toluenesulfonyl chloride (2.15 g, 11.28 mmol) in dry CH 2 Cl 2 (15 mL) was added to a solution of 4 (3.79 g, 7.52 mmol) in dry CH 2 Cl 2 (15 mL) followed by triethylamine (2.14 mL, 15.36 mmol) at 0 C. The reaction was stirred overnight at room temperature and the mixture was concentrated by rotavap. The residue was dissolved in EtOAc (200 mL), which was washed with 125-mL portions of 8% NaHCO 3 0.5 M citric acid, dH 2 O, and saturated NaCl. Solvent removal and flash chromatography, eluting with 5% CH 3 OH/CH 2 Cl 2 afforded 4.22 g (85%) of 5 as a light yellow oil: 1 H NMR 1.40-1.85 (m, 35 H), 2.45 (s, 3 H), 3.04-3.30 (m, 10 H), 4.03 (t, 2 H, J = 6.0), 5.28 (s, 1 H), 7.35 (d, 2 H, J = 8.1), 7.78 (d, 2 H, J = 8.4); 13 C NMR 21.28, 21.59, 22.86, 23.12, 25.81, 26.20, 26.82, 30.90, 31.18, 34.41, 37.00, 44.35, 53.53, 54.18, 80.19, 125.72, 125.83, 127.82, 128.84, 129.83, 133.06, 140.15, 141.98, 149.30. With the Boc-protected NSPD-OTs in hand, the polyamine conjugate NSPD-(S)-4'-(HO)-DADFT and its ethyl ester NSPD-(S)-4'-(HO)-DADFT-EE were synthesized (Figure 3-10). 296 Synthesis of Ethyl (S)-4,5-Dihydro-2-[2-hydroxy-4-[12-(tert-butoxycarbonylamino)-5,9-bis(tert-butoxycarbonyl)-5,9-diazadodecyloxy]phenyl]-4-methyl-4-thiazolecarboxylate (7) A mixture of 5 (4.22 g, 6.41 mmol), 6 (1.87 g, 6.65 mmol) 296 and freshly prepared 0.20 M sodium ethoxide in CH 3 CH 2 OH (15 mL, 3.0 mmol) was stirred at 60-70 C for overnight. After 117

PAGE 118

the white solid was filtered, the filtrate was concentrated by rotary evaporation. The residue was dissolved in CHCl 3 (100 mL) and was washed with H 2 O and saturated NaCl. Solvent removal and column chromatography on silica gel, eluting with 5% EtOAc/CH 2 Cl 2 gave 2.02 g (41%) of 7 as a pale yellow oil: [] 25 +17.0 (c 1.00, CHCl 3 ); 1 H NMR 1.30 (t, 3 H, J = 7.2), 1.4-1.8 (s + m, 35 H), 3.02-3.33 (m + d, 11 H, J = 11.4), 3.83 (d, 1 H, J = 11.1), 3.98 (t, 2 H, J = 5.8), 4.24 (2 q, 2 H, J = 7.2), 6.41 (dd, 1 H, J = 8.6, 2.4), 6.46 (d, 1 H, J = 2.4), 7.28 (d, 1 H, J = 6.6); 13 C NMR 14.18, 24.57, 26.42, 26.50, 28.54, 28.56, 28.58, 29.81, 37.51, 39.92, 43.67, 44.97, 46.79, 61.91, 67.78, 79.52, 79.74, 83.21, 101.39, 107.24, 109.81, 131.77, 155.58, 156.10, 161.33, 163.29, 170.87, 172.91, 182.00. Synthesis of Ethyl (S)-4,5-Dihydro-2-[2-hydroxy-4-(12-amino-5,9-diazadodecyloxy)phenyl]-4-methyl-4-thiazolecarboxylate Trihydrochloride (8) Compound 7 (0.020 g, 0.026 mmol) was dissolved in EtOH (1 mL). Concentrated HCl (0.25 mL) was added to above solution dropwise with ice cooling. The reaction was warmed to room temperature and was stirred 16 h. Concentration under high vacuum gave 0.014 g (93%) of 8 as a white solid: [] 26 +67.7 (c 0.62, H 2 O); 1 H NMR (D 2 O) 1.29 (t, 3 H, J = 7.2), 1.81 (s, 3 H), 1.83-1.96 (m, 4 H), 2.02-2.20 (m, 4 H), 3.04-3.25 (m, 10 H), 3.62 (d, 1 H, J = 12.0), 4.01 (d, 1 H, J = 12.3), 4.17 (t, 3 H, J = 5.8), 4.30 (q, 2 H, J = 7.2), 6.59 (d, 1 H, J = 2.4), 6.67 (dd, 1 H, J = 9.0, 2.4), 7.65 (d, 1 H, J = 9.0); 13 C NMR (D 2 O) 13.79, 23.05, 23.30, 24.34, 25.96, 29.27, 37.14, 39.27, 45.00, 45.26, 45.33, 48.13, 64.57, 68.76, 76.83, 102.00, 107.11, 109.81, 134.44, 159.47, 161.46, 167.21, 172.88; HRMS m/z calcd for C 23 H 39 N 4 O 4 S 467.2692 (M + H, free amine), found 467.2685. Synthesis of (S)-4,5-Dihydro-2-[2-hydroxy-4-[12-(tert-butoxycarbonylamino)-5,9-bis(tert-butoxycarbonyl)-5,9-diazadodecyloxy]phenyl]-4-methyl-4-thiazolecarboxylic Acid (9) 118

PAGE 119

Treatment of 7 (1.55 g, 2.02 mmol) with 1 M methanolic NaOH (40 mL, 40 mmol) at room temperature for 6 h, acidification with 1 N HCl to pH 1.0, and solvent removal in vacuo gave a pink solid. Extraction with EtoAc (2 x 100 mL), washed with dH 2 O (100 mL) and saturated NaCl (100 mL), then dried over Na 2 SO 4 followed by concentration under high vacuum gave a brownish oil 9 of 1.45 g (97%). [] 26 +8.8 (c 1.02, CH3OH); 1 H NMR 1.38-1.84 (s + m, 35 H), 3.02-3.32 (m + d, 11 H, J = 11.4), 3.87 (d, 1 H, J = 11.4), 3.98 (t, 2 H, J = 5.6), 6.41 (d, 1 H, J = 8.7), 6.48 (s, 1 H), 7.28 (d, 1 H, J = 6.0); 13 C NMR 24.64, 26.46, 28.57, 28.59, 28.62, 29.00, 37.34, 39.91, 45.03, 46.86, 67.79, 79.76, 79.91, 83.05, 101.48, 107.37, 109.71, 131.88, 155.74, 161.39, 162.23, 163.40, 174.85, 186.55; HRMS m/z calcd for C 36 H 59 N 4 O 10 S 739.3952 (M + H), found 739.3898. Synthesis of (S)-4,5-Dihydro-2-[2-hydroxy-4-(12-amino-5,9-diazadodecyloxy)phenyl]-4-methyl-4-thiazolecarboxylic Acid Trihydrochloride (10) Compound 9 (0.096 g, 0.13 mmol) was dissolved in dry CH 2 Cl 2 (5 mL) Concentrated HCl (1 mL) was added to above solution dropwise with ice bath cooling. The reaction was warmed to room temperature and was stirred 16 h. Concentration under high vacuum gave 0.068 g (96%) of 10 as a white solid: [] 26 +50.8 (c 0.295, H 2 O); 1 H NMR (D 2 O); 13 C NMR (D 2 O) 23.04, 23.30, 24.35, 25.94, 37.15, 39.18, 44.92, 45.00, 45.27, 45.34, 48.13, 68.86, 75.64, 101.95, 106.23, 110.22, 134.96, 161.39, 168.10, 174.69, 182.87; HRMS m/z calcd for C 21 H 34 N 4 O 4 S 439.2379 (M + H, free amine), found 439.2374. 3.4.2.3 Stoichiometry Determination by Jobs Plot The stoichiometry of the complex formed between the conjugate NSPD-(S)-4'-(HO)-DADFT and Fe(III) was determined spectrophotometrically. 293 DFT has been known to form a 2:1 ligand-Fe(III) complex 283 and was used as a positive control for this assay. Solutions 119

PAGE 120

containing different ligand:Fe(III) ratios were prepared by mixing appropriate volumes of 0.5 mM NSPD-(S)-4'-(HO)-DADFT in 100 mM TRIS-Cl (pH 7.4), and 0.5 mM Fe(III) nitrilotriacetate (NTA) in 100 mM TRIS-Cl (pH 7.4), so that [NSPD-(S)-4'-(HO)-DADFT] + [Fe(III)] = 1.0 mM. The 0.5 mM Fe(III)-NTA solution was prepared immediately prior to use by dilution of a 50 mM Fe-(III)-NTA stock solution with TRIS buffer. The Fe(III)-NTA stock solution was prepared by mixing equal volumes of 100 mM ferric ammonium sulfate [NH 4 Fe(SO 4 ) 2 ] and 200 mM Na 3 NTA. The iron content was verified by atomic absorption (AA) spectroscopy. 3.4.2.4 Cell Culture Human T lymphocytic leukemia cells (Jurkat cells) were cultured by the same conditions for L1210 cells as described in Section 2.2.1.1. Because this cell line originated from humans unlike L1210, which was from mice, it was chosen to study the species-dependent antiproliferative effect of DFT analogues. 3.4.2.5 HPLC Analysis of the Tissue and Cellular Concentration of (S)-4'-(HO)-DADFT and NSPD-(S)-4'-(HO)-DADFT-EE Male Sprague-Dawley rats (250-350 g) were administered the compounds that have been dissolved in ddH 2 O subcutaneously. At 2, 4, 6, 8, and 12 h of treatment with NSPD-(S)-4'-(HO)-DADFT-EE or NSPD-(S)-4'-(HO)-DADFT, the rodents were sacrificed and the livers were removed. The liver samples were homogenized in 0.6 N HClO 4 at a ratio of 1:2 (w/v). The equal volume of iron-free H 2 O as HClO 4 was then used to rinse the homogenizer probe; this rinsing solution was combined with the homogenized sample and mixed well. This homogenate was centrifuged and the supernatant (200 L) was injected onto a HPLC column. For analysis of cellular uptake of compounds, L1210 cells in logarithmic growth (~ 5 x 10 5 /mL) were treated with 100 M of (S)-4'-(HO)-DADFT or NSPD-(S)-4'-(HO)-DADFT-EE. 120

PAGE 121

After 4 h and 12 h, cells were collected and washed with PBS at 4 C. Pellet was resuspended in mobile phase A (see below) to achieve a concentration of 1 x 10 7 cells/mL. The suspension was centrifuged and the supernatant (200 L) was injected onto a HPLC column. Analytical separation was performed on a reversed-phase Supelco Discovery RP Amide C16 column (250mm x 4.6 mm, 5 m) on a Rainin HPLC system, and the UV detector was set at 300 nm. The buffer employed was sodium octanesulfonate (2.5 mM) in potassium phosphate (25 mM), pH 3.0. Mobile phase A consisted of 5% CH 3 CN, 95% buffer; mobile phase B consisted of 60% CH 3 CN, 40% buffer. The mobile phases were pumped at a flow rate of 1.5 mL/ min. The solvent gradient program employed an initial 10-min isocratic portion with 5% mobile phase B (95% A), followed by a linear gradient increase to 80% mobile phase B at 35 min, a 5-min ramp to 100% B held for 10 min, and ramping back to 5% mobile phase B for 8 min. This method had a detection limit of 0.2 M as a direct injection. The concentrations were calculated from the peak area fitted to calibration curves by non-weighted least squares linear regression with Rainin Dynamax HPLC Method Manager software. 3.4.2.6 Mass Spectra Analysis of the Rat Liver Concentration of the Diacid Metabolites of NSPD-(S)-4'-(HO)-DADFT-EE A SupelClean 1-mL C-18 cartridge (Supelco, Bellefonte, PA) was used for solid-phase extraction (SPE) of the supernatant obtained after centrifugation of the rat liver homogenate from 2 h post-dosing with NSPD-(S)-4'-(HO)-DADFT-EE. The cartridge packing was pre-wetted with CH 3 CN (2 mL) and washed with 3% (v/v) aqueous acetic acid solution (2 mL) before loading the sample. After washing with 1% (v/v) aqueous acetic acid solution (2 mL), the sample was eluted with 300 L of a mixture of H 2 O, CH 3 CN, and glacial acetic acid at a ratio of 74/25/1 (v/v/v). Electrospray ionization (ESI) mass spectra were acquired on a quadrupole ion trap instrument (LCQ, ThermoFinnigan, San Jose, CA) operated with the manufacturers Xcalibur 1.3 software. 121

PAGE 122

Full-scan product ion spectra (MS/MS) were recorded at 1.0-u precursor ion isolation width, and the activation amplitude was adjusted to 1.75 V (35% of the maximum value) to obtain collision induced dissociation (CID). Accurate mass measurements were performed on an Applied Biosystems (Foster City, CA) QSTAR XL hybrid quadrupole/time-of-flight instrument in the full-scan acquisition mode. 3.4.2.7 Iron Clearing Efficiency Measurement The iron clearing efficiency of iron chelators and polyamine iron chelator conjugates were measured according to the published method. 296 3.4.3 Results and Discussion 3.4.3.1 Stoichiometry of the Complex Formed between NSPD-(S)-4'-(HO)-DADFT and Fe(III) As was observed in SPM-L1, polyamine conjugation did not change the stoichiometry of the ligand-Fe(III) complex. The stoichiometry of the complex formed between the conjugate NSPD-(S)-4'-(HO)-DADFT and Fe(III) demonstrated a 2:1 ligand-Fe(III) complex (Figure 3-11), which was the same as that of the cargo molecule (S)-4'-(HO)-DADFT. 296 3.4.3.2 Effect of Conjugation at 4'-hydroxyl of (S)-4'-(HO)-DADFT on Cellular Transport and Uptake of the Molecule The cellular transport and intracellular uptake properties of the two conjugates NSPD-(S)-4'-(HO)-DADFT and NSPD-(S)-4'-(HO)-DADFT-EE were evaluated in L1210 cells. As shown in Table 3-7, the conjugate acid NSPD-(S)-4'-(HO)-DADFT did not efficiently compete with [ 3 H]-SPD for uptake (K i = 73 M), whereas the corresponding ester NSPD-(S)-4'-(HO)-DADFT-EE was a good transport competitor (K i = 5.7 M). When L1210 cells were treated with 100 M of the acid for 4 h, only 71 23 M of the compound was detected intracellularly. An extended treatment of 12 h did not markedly increase the intracellular concentration of the acid (75 24 M). However, in the 100 M ester treated cells at 4 h, NSPD-(S)-4'-(HO)-DADFT-EE 122

PAGE 123

concentrated to 948 107 M, and its hydrolysis product NSPD-(S)-4'-(HO)-DADFT reached 2984 286 M. Furthermore, the ethyl ester conjugate reached to an even higher concentration of 1503 194 M in the 12-h treated cells; additionally, its hydrolysis product NSPD-(S)-4'-(HO)-DADFT also reached 3100 403 M. These observations are in keeping with the idea that using an ethyl to mask the carboxyl of NSPD-(S)-4'-(HO)-DADFT can significantly improve the intracellular uptake of this compound, and the ethyl ester can be efficiently hydrolyzed by cellular esterases. These results are also consistent with those obtained from NTS and NTS-ME (Section 3.3.4.2) and further underscore the importance of the charge carried by the cargo molecule on the intracellular delivery of polyamine conjugates. 3.4.3.3 The Effect of Conjugation on Antiproliferative Activity IC 50 values of iron chelators usually remain constant in spite of treatment time (Chapter 2, Table 2-2). For example, IC 50 value of (S)-DADFT in L1210 cells is 6 M at both 48 h and 96 h. The IC 50 value of the polyamine vectored iron chelator SPM-L1 also remained unchanged during treatment (Section 3.3.4.2). This phenomenon was again observed in the two polyamine conjugates of (S)-4'-(HO)-DADFT at the 48h and 96 h treatments (Table 3-8). The IC 50 was 1.5 M for the conjugate ester NSPD-(S)-4'-(HO)-DADFT-EE and was 40 M for the conjugate acid NSPD-(S)-4'-(HO)-DADFT at both 48 h and 96 h. While the conjugated ester was more active than the conjugated acid at inhibiting L1210 cell growth, the non-conjugated ester (S)-4'-(HO)-DADFT-EE also had a much lower IC 50 (9.0 M and 6.0 M at 48 and 96 h respectively) than that of its acid counterpart (S)-4'-(HO)-DADFT (16 M and 17 M at 48 and 96 h, respectively). The polyamine conjugated NSPD-(S)-4'-(HO)-DADFT-EE but not the corresponding acid NSPD-(S)-4'-(HO)-DADFT outperformed the non-conjugated compound (S)-4'-(HO)-DADFT-EE at inhibiting L1210 cell growth. 123

PAGE 124

To further demonstrate the advantage of polyamine vectoring, several DFT analogues and the above two NSPD conjugates of (S)-4'-(HO)-DADFT were tested for their antiproliferative activity in another lymphocytic leukemia cell line, Jurkat Clone E6-1 (Table 3-9). Because of its longer doubling time as compared with L1210 cells (26 h versus 11 h), Jurkat cells were exposed to the compounds of interest for 96 h and 192 h to obtain data comparable to L1210 cells. Interestingly, except for (S)-4'-(CH 3 O)-DADFT, which had a lower IC 50 at 192 h (1.5 M) than at 96 h (5.3 M) in Jurkat cells and was more active than in L1210 cells at the longer treatment time (Table 3-9), the other three tested non-polyamine-vectored DFT analogues had similar potency against both Jurkat and L1210 cells. In contrast, both conjugates NSPD-(S)-4'-(HO)-DADFT and NSPD-(S)-4'-(HO)-DADFT-EE were more active in Jurkat cells than in L1210 cells. The 96 h and 192 h IC 50 values of both NSPD-(S)-4'-(HO)-DADFT and NSPD-(S)-4'-(HO)-DADFT-EE in Jurkat cells were lower than in 48 h and 96 h treated L1210 cells. Finally, the conjugate ester was more active than its acid analogue against both the human and rodent cells; again, this underscores the importance of charge of the cargo molecule in determining the biological activity of the conjugate. 3.4.3.4 Effect on Polyamine Metabolic Enzymes Although NSPD itself reduced ODC to 11% of control, 303 NSPD conjugation to either (S)-4'-(HO)-DADFT or (S)-4'-(HO)-DADFT-EE did not have a significant impact on ODC activity. Both NSPD-(S)-4'-(HO)-DADFT and its parent compound (S)-4'-(HO)-DADFT diminished ODC activity to 76 5% and 71 2% respectively. The esters NSPD-(S)-4'-(HO)-DADFT-EE and (S)-4'-(HO)-DADFT-EE reduced ODC activity to 59 3% and 61 8% respectively (Table 3-10). In the case of AdoMetDC, the two conjugates decreased the enzyme activity to about 74%, but the two cargo compounds did not affect this enzyme. None of the four compounds had 124

PAGE 125

any impact on SSAT activity; the enzyme activity remained around 100% of control in each case. 3.4.3.5 Metabolism of the Polyamine Moiety of the Conjugate NSPD-(S)-4'-(HO)-DADFT-EE in Rat Liver The metabolism of both the conjugate ester and acid, NSPD-(S)-4'-(HO)-DADFT-EE and NSPD-(S)-4'-(HO)-DADFT were investigated in non-iron-overloaded, bile-duct-cannulated rats. 296 These rats were given either the compound sc at a dose of 300 M/kg and sacrificed after 2, 4, 6, 8, and 12 h. The livers were then removed and prepared for HPLC and ESI-MS analysis. 296 Basically, the metabolic profile of these two compounds resembled that of typical polyamine analogues as exemplified by DENSPM (Figure 3-1), i.e., the stepwise deaminopropylation by the SSAT/PAO system (Figure 3-12, right part). Both the diamine metabolite 16 and the metabolite monoamine 18 were detected in rat livers by HPLC using synthetic compounds 16 and 18 296 as the standards. When rats were given either the conjugate ester 8 or the conjugate acid 10 for 4 h, the diamine metabolite 16 reached a peak liver concentration of 168 M and 128 M respectively (Figure 3-13). The metabolite 16 remained at concentrations higher than 130 M 8 h post-drug in the conjugate treated rats, whereas it decreased to 100 M 6 h post-drug in the conjugate acid treated rats. However, the monoamine metabolite 18 never reached 50 M in both the conjugates treated rats. When the conjugate ester 8 was administered, the parent drug disappeared as early as 2 h post-drug in the rat liver, while compound 10 was detected as one of the metabolites at a concentration of 85 M and it sustained at this concentration until 6 h post-drug (Figure 3-13 panel 2). Thus, it appeared that the ester had been degraded to its corresponding acid by the non-specific serum esterases in the rodent tissue, although the exact esterase is yet to be identified. This result agreed with that obtained from the earlier studies in L1210 cells (Table 3-7). 125

PAGE 126

Surprisingly, there were major difference between the metabolism of the simple linear polyamine DENSPM and the two conjugates, the principal metabolite in rats treated with either conjugate was found to be the dicarboxylic acid 12 (Figure 3-13), which was detected by ESI-MS (Figure 3-14). This metabolite achieved the peak liver concentration above 400 M at 2 h in each case (Figure 3-13). It remained at this level for 6 h in rats treated with the conjugate ester 8, but dropped to 328 M only 2 h post-drug in rats treated with the conjugate acid 10. Although the metabolic route of generating 12 is yet to be identified, the PAO/DAO system is believed to be involved; 296 this proposed route is depicted by the dotted arrows in Figure 3-12. The conjugate acid 10 was oxidatively deaminated by PAO/DAO at its primary amine, generating the hypothesized aldehyde intermediate 11, which was subsequently oxidized to the acid metabolite 12 by the enzyme aldehyde dehydrogenase (ADH). 335 A similar pathway was also proposed for the formation of the diacid 14, which was generated from the diamine metabolite 16. The total ligand (SUM, metabolites 12, 10, 16, and 18) achieved a concentration over 600 M 2 h post-drug and was the highest (697 M) after 4 h in the rats given the conjugate ester 8 (Figure 3-13 lower panel); this high liver concentration was sustained until 6 h post-drug and dropped to 400 M and 217 M after 8 h and 12 h. Although the total ligand reached peak concentration (682 M) as early as 2 h post-drug in the rats treated with the conjugate acid 10, it decreased immediately after 4 h to 553 M and further to around 280 M between 6 h and 8 h and finally to 134 M at 12 h post-drug. Both the intact conjugate 10 and the parent drug (S)-4'-(HO)-DADFT never reached a concentration higher than 150 M (Figure 3-13 upper panel). 3.4.3.6 Effect of Polyamine Conjugation at C-4' Hydroxyl on the Iron-Clearing Efficiency of (S)-4'-(HO)-DADFT The iron excretion promoted by the two polyamine conjugated iron chelators NSPD-(S)-4'-(HO)-DADFT and NSPD-(S)-4'-(HO)-DADFT-EE, as well as the parent iron chelator (S)-4'126

PAGE 127

(HO)-DADFT, was measured in non-iron-overloaded, bile-duct-cannulated rats. All the animals received the drugs at a dose of 300 M/kg sc, the bile and urine samples were collected after 24 h (for both polyamine conjugates) or 48 h (for the parent chelator) and iron concentrations were measured by flame atomic absorption (AA) spectroscopy. 284 Recall that iron clearing efficiency (ICE) was calculated by dividing the net iron excretion by the total iron binding capacity of a iron chelator and expressed as a percentage (Chapter 2). 282 As shown in Table 3-11, while the parent chelator (S)-4'-(HO)-DADFT had an ICE of 1.1 0.6%, its conjugate acid had a nine-fold higher ICE, 9.2 2.6%. Although the corresponding ethyl ester conjugate NSPD-(S)-4'-(HO)-DADFT-EE did not survive the cleavage by non-specific serum esterases and existed largely as the free acid shortly after administered (Section 3.4.3.5), its ICE (13.6 3.3%) was still higher than that of NSPD-(S)-4'-(HO)-DADFT (9.2 2.6%). The advantage of the bile-duct cannulated rat model is the ease of collecting bile samples at desired time points, this makes the monitoring the kinetics of iron excretion convenient. As demonstrated in Table 3-11, the modes of excretion of iron in all three compounds treated animals were essentially same. However, the kinetics of iron excretion associated with these compounds was significantly different (Figure 3-15). The iron excreted in bile reached the peak at 4 h and dropped back to the baseline at 25 h in (S)-4'-(HO)-DADFT treated rodents. The peak of iron excretion in both NSPD-(S)-4'-(HO)-DADFT and NSPD-(S)-4'-(HO)-DADFT-EE treated rodents occurred at 6 h. The iron excretion did not fall to the baseline until 50 h post-drug in the case of the conjugate acid NSPD-(S)-4'-(HO)-DADFT. However, a more protracted iron excretion was observed in the conjugate ester NSPD-(S)-4'-(HO)-DADFT-EE treated animals; the iron excretion did not return to the baseline even after 72 h. These observations were in keeping with the metabolism of NSPD-(S)-4'-(HO)-DADFT and NSPD-(S)-4'-(HO)-DADFT-EE 127

PAGE 128

(Section 3.4.3.5), i.e., the liver concentration of NSPD-(S)-4'-(HO)-DADFT was higher in the conjugate ester treated animals than in the conjugate acid treated ones after 2 h administration of these drugs. 3.5 Cellular Localization of Polyamine Conjugates 3.5.1 Background Although the information about molecular structure of the mammalian polyamine transporter is scarce, the biochemical properties of the protein have been well studied (Chapter 1, Section 1.1.2.3). One important feature of the transporter is its broad structural tolerance 101, 303 allowing the development of several fluorescent probes, e.g., N 4 -NBD-SPD, 336 Monofluorescein isothiocyanate-spermine,(FL-SPM) 337 N 4 -SPD-MANT, 338 and N 4 -SPD-C 2 -BODIPY 339 (Figure 3-16), for monitoring the cellular fate of polyamine analogues. These polyamine fluorophore adducts had comparable affinities with their parent polyamines for the polyamine transporter, and their subcellular distribution was exclusively cytoplasmic. More specifically, these fluorescent polyamines accumulated to granular cellular structure resembling endocytotic vesicles or lysosomes. 338 3.5.2 Specific Aims The current study aims to (1) to evaluate the conjugate NBD-GABA-NSPD as well as the corresponding cargo molecule NBD-GABA in L1210 cells for their abilities to compete with [ 3 H]SPD for the polyamine transport apparatus (K i ), and their effects on intracellular uptake and cell proliferation (IC 50 ); and (2) to observe the cellular compartmentalization of NBD-GABA-NSPD by fluorescence microscopy. 3.5.3 Materials and Methods 3.5.3.1 General Methods The materials and methods for K i and IC 50 measurements were described in Section 2.2.1. 128

PAGE 129

3.5.3.2 Cell Culture L1210 cells were cultured as described in Section 2.2.1. Human liver ascites adenocarcinoma cells (SK-Hep-1) were purchased from ATCC. Cells were grown in tissue culture flasks containing DMEM medium which was supplemented with 5% (v/v) FBS, 15 mM Hepes-Mops, and 1 mM aminoguanidine. Cells at 80% confluency were subcultured and incubated in a 37 C water-jacketed 5% CO 2 and 95% humidified incubator. 3.5.3.3 Fluorescence Spectra of NBD-GABA and NBD-GABA-NSPD The fluorescence excitation and emission spectra of NBD-GABA and NBD-GABA-NSPD were measured with 0.1 mM solutions in ddH 2 O at 25 C in quartz cuvettes of 1 cm path-length and slit width of 2 nm by a PerkinElmer LS55 luminescence spectrometer (PerkinElmer Analytical Instruments, Shelton, CT). The spectra were recorded within the wavelength of 200 600 nm with FL WinLab software. 3.5.3.4 Fluorescence Microscopy SK-HEP-1, a human liver adenocarcinoma cell line that is endothelial origin, was chosen for this study because the cells have large lysosomes. Cells (5 X 10 5 ) were seeded in a 35-mm culture dish containing one piece of tissue culture microscopic disc and cultured at 37 C overnight. The spent medium was displaced with 2.0 mL of fresh medium with or without NH 4 Cl (10 mM) and incubated for 1 h, then followed by treatment with 100 M of either NBD-GABA or NBD-GABA-NSPD for 30 min with or without the continued presence of NH 4 Cl. Finally, acridine orange (5 g/mL) was added to all culture dishes and the cells were further incubated at 37 C for 15 min. After rinsing with fresh PBS three times, each disc was mounted onto a microscopic slide and topped with a cover slip, then observed under a Zeiss Axioplan2 fluorescence microscope. 129

PAGE 130

3.5.4 Results and Discussion 3.5.4.1 Cellular Transport and Antiproliferative Properties of NBD-GABA and NBD-GABA-NSPD It has been previously demonstrated (Section 3.3.4.1 and 3.4.3.1) that polyamine conjugated compounds are good competitors for polyamine transport, even when their corresponding cargo molecules are not. Here again, NBD-GABA-NSPD had a good affinity (K i = 4.2 M) for the polyamine transporter (Table 3-16). Although NBD-GABA was not very active (IC 50 > 100 M) against L1210 cells, its polyamine conjugate NBD-GABA-NSPD inhibited 50% of cell growth at 50 M at 48 h and 18M at 96 h (Table 3-12). 3.5.4.2 Spectrometry of Fluorescent Polyamine NBD-GABA-NSPD The fluorescence excitation and emission wavelengths of NBD-GABA-NSPD (Figure 3-17 B) and its parent compound NBD-GABA (Figure 3-17 A) were measured with their 0.1 mM solutions in ddH 2 O and compared with the wavelengths of NBD (Table 3-13). Although the excitation wavelengths of NBD-GABA-NSPD (400 nm) and NBD-GABA (430 nm) were different from that of NBD (465 nm), all three compounds had emission wavelengths around 545 nm. 3.5.4.3 Cellular Localization of NBD-GABA-NSPD As previously demonstrated, fluorescent polyamine probes usually accumulate to granular cellular structure resembling endocytotic vesicles or lysosomes (Section 3.5.1). Therefore, it was assumed that the fluorescent polyamine conjugate NBD-GABA-NSPD would accumulate in the same cellular compartments. To confirm this hypothesis, some the cells were pre-treated with NH 4 Cl (10 mM) for 1 h. Non-drug-treated control cells ( NH 4 Cl) were stained with the lysosome/endosome fluorescent dye LysoTrackerRed. As seen in Figure 3-18, the intensity of red fluorescence in the NH 4 Cl pre-treated control cells (A) was much weaker than in the regular 130

PAGE 131

control cells (B); this indicates that the 1-h treatment with NH 4 Cl (10 mM) was sufficient to raise the lysosomal pH in SK-HEP-1 cells and thus could prevent the accumulation of lysosomotropic molecules. In NBD-GABA treated cells, the intensity of green fluorescence was apparently the same regardless of pre-treatment with (Figure 3-18, C) or without NH 4 Cl (Figure 3-18, D). However, the lysosomal pH influenced the uptake of NBD-GABA-NSPD significantly. The intensity of green fluorescence was very weak in cells that were pre-treated with NH 4 Cl and diffusive in the cytoplasm (Figure 3-18, E), whereas it was much stronger in cells without NH 4 Cl pre-treatment (Figure 3-18, F). More importantly, the most intensive green fluorescence in these cells (Figure 3-18, F) was granular in appearance and had a reticular perinuclear pattern, which resembled what has been seen in the control cells without the NH 4 Cl pre-treatment (Figure 3-18, B). Therefore, it can be concluded that most of the fluorescent polyamine conjugate NBD-GABA-NSPD accumulated in lysosomes (or endosomes) after uptake intracellularly. However, the Golgi apparatus cannot be excluded because of its tight association with lysosome/endosome in the endocytotic pathway. 340-342 Indeed, the Golgi, especially the trans-Golgi-network (TGN), was indicated as a minor accumulation site for the fluorescent polyamine N 4 -SPD-MANT because of the proposed expression of the putative polyamine transporter protein or the relatively low organelle pH of ~5.9 in this compartment. 343 3.6 Polyamine Vectored Anthracene-9-Carboxylic Acid 3.6.1 Background Chloride channel (Cl channel) is a large family of pore-forming proteins that reside in the plasma or organelle membrane of many cells 344 including lymphocytes 345, 346 These proteins function as regulators for cell/organelle volume, ion homeostasis, transepithelial transport, and electrical excitability. 347 Anthracene-9-carboxylic acid (9-AC) is a known Cl channel blocker 348 131

PAGE 132

and has been demonstrated to have a cardioprotective effect on myocardial ischemia in experimental animals 349 or in single Guinea pig myocyte 350 Interestingly, 9-AC also has been shown to have an inhibitory effect (IC 50 : 100 500 M) on human leukemic cell proliferation. 351 However, the high IC 50 value suggests this compound is not very potent, we thus attempted to use polyamines to vector 9-AC in order to achieve higher potency against malignant leukemic cells. The polyamine conjugate NSPD-anthroate (NSPD-ANTH) was thus designed and synthesized according to the scheme shown in Figure 3-19. First, anthracene-9-carboxylic acid (1) was chlorinated with thionyl chloride (SOCl 2 ) in dichloromethane to afford the chlorinated intermediate 2, which was then reacted with the Boc-protected triaminoalcohol 3 in the presence of the base triethylamine (TEA) to generate the ester 4. Deprotection of the Boc groups in 4 with HCl in CH 2 Cl 2 produced the final product 5 as a trihydrochloride salt (NSPD-anthroate, NSPD-ANTH). 3.6.2 Specific Aims The specific aims of the current study are to assess (1) the antiproliferative activities, (2) transport competition ability, and (3) morphological impact of 9-AC and NSPD-ANTH in L1210 cells. 3.6.3 Materials and Methods 3.6.3.1 General The materials and methods for the K i and IC 50 measurements were described in Section 3.4. L1210 cells were cultured as described in Section 2.2.1. 3.6.3.2 Light Microscopy Examination of the Cell Morphology L1210 cells in logarithmic growth were incubated with NSPD-ANTH (100 M) or 9-AC (100 M) in growth medium at 37 C for 20 min. A thin film of the cells on a microscope slide was fixed in absolute methanol for 30 sec. The slides were immersed in a modified Wright132

PAGE 133

Giemsa stain solution (0.4% w/v in methanol, pH 6.8) (Sigma co., St. Louis, MO) for 10 min and flushed with PBS and then dH 2 O. The slides were allowed to air-dry and examined with 40x objective under a Zeiss Axioplan2 microscope. 3.6.4 Results and Discussion 3.6.4.1 Antiproliferative Activities of Anthracene-9-carboxylic Acid and NSPD-Anthorate It was demonstrated previously (Section 3.3.4.1 and 3.4.3.1) that polyamine conjugated compounds were good competitors for the SPD transport, even though their corresponding cargo molecules were not. Here again, NSPD-ANTH had a good affinity for the polyamine transporter (K i = 6.7 M), whereas 9-AC was non-competitive (Table 3-14). Anthracene-9-carboxylic acid (9-AC) was not particularly active against L1210 cell growth (IC 50 > 100 M); this is in agreement with the results obtained from two other lymphocytic cell lines, HL-60 and Jurkat, in which 9-AC suppressed cell growth at IC 50 between 100 500 M. 351 However, the polyamine vectored 9-AC, NSPD-anthroate, had an IC 50 value of 5 M at both 48 and 96 h (Table 3-12). Similarly, NBD-GABA was not very active (IC 50 > 100 M), whereas NBD-GABA-NSPD inhibited 50% of cell growth at 50 M and 18M at 48 h and 96 h respectively. 3.6.4.2 Effect of NSPD-Anthroate on Cell Morphology As seen in Table 3-14, the polyamine conjugate of 9-AC, NSPD-ANTH, was very active against the mouse lymphocytic leukemic cells L1210 at 48 h (IC 50 = 5 M). Actually, this conjugate also had a fast onset in L1210 cells, which could be observed under a light microscope (Figure 3-20). L1210 cells were stained with Wright-Giemsa stain after a brief (20 min) exposure to either 9-AC or NSPD-ANTH. The majority of 9-AC treated cells (Figure 3-20, B) appeared shrunken as compared with the untreated control cells (Figure 3-20, A). Loss of cell volume has 133

PAGE 134

been observed to occur in almost all apoptotic cells, 352 and one of the most important functions of the Cl channel is to regulate cell volume. 353 Blocking of the Cl channel may prevent the efflux of both K + and Cl ions and thus was suggested to have protective effects in cortical neuron cells against apoptotic cell death. 354 However, a Cl channel may interact with other signaling pathways and thus a given Cl channel blocker may play different roles in cell proliferation and apoptosis. 355 This may explain the growth inhibition effect observed with 9-AC in both the current study and earlier study. 351 However, the morphological appearance of those NSPD-ANTH treated L1210 cells was significantly different from both the control cells and the 9-AC treated cells. After 20 min, most of the NSPD-ANTH treated cells were heavily lysed (light purple in color, Figure 3-20 C), while those unlysed cells were darker and their nuclei were rounder. This dramatic impact of NSPD-ANTH on cell morphology is in keeping with its marked antiproliferative activity. Therefore, polyamine conjugation of 9-AC remarkably increased the potency of this compound against the malignant leukemic cells. 3.7 (S)-4'-(HO)-DADFT-NSPD Ester 3.7.1 Specific Aims As demonstrated previously in both cultured cells (Section 3.4.3.2) and animal models (3.4.3.5), the polyamine conjugated iron chelator NSPD-(S)-4'-(HO)-DADFT-EE metabolized to its corresponding acid NSPD-(S)-4'-(HO)-DADFT in high intracellular and liver concentrations; however, (S)-4'-(HO)-DADFT was never detected as one of the metabolic products, although the monoamine conjugate 18 (Figure 3-12) was. This indicates that Site I in (S)-4'-(HO)-DADFT molecule is not ideal for a polyamine vector if delivery of the parent DFT chelators is the objective. A polyamine vector that can deliver the parent chelator into cells or tissues where it is released would be of interest. 134

PAGE 135

The specific aim here is to construct a labile (S)-4'-(HO)-DADFT polyamine conjugate linking via an ester at site II (Figure 3-8) of (S)-4'-(HO)-DADFT molecule. This polyamine conjugate (S)-4'-(HO)-DADFT-NSPD ester will be then evaluated in L1210 cells for its (1) effects on intracellular uptake and cell proliferation (IC 50 ), (2) ability to compete with [ 3 H]SPD for the polyamine transport apparatus (K i ), and (3) intracellular uptake and effect on polyamine pools. 3.7.2 Synthesis The synthesis of the (S)-4'-(HO)-DADFT-NSPD ester began with the tris(Boc) protected polyamine reagent NSPD-OTs (1, Figure 3-21), whose synthesis was described earlier (Figure 3-9). This molecule was converted to the iodid 2 with NaI in acetone. Coupling of 2 to (S)-4'-(HO)-DADFT (3) in the presence of DIEA in DMF generated the tris(Boc) protected ester 4. Removing of the Boc groups in 4 with TFA in CH 2 Cl 2 generated the final product, (S)-4'-(HO)-DADFT-NSPD ester (5). 3.7.3 Material and Methods The materials and methods for the measurement of K i and IC 50 and HPLC analysis were the same as in Section 3.4.2.1. 3.7.4 Results and Discussion 3.7.4.1 Effect on Cell Proliferation As discussed in Section 3.4.1, when designing a polyamine-iron chelator conjugate, any additional cytotoxicity from the polyamine vector is undesirable. Therefore, the antiproliferative activity of NSPD-OH, the polyamine fragment of both the acid and ester conjugates of (S)-4'-(HO)-DADFT, was evaluated to ascertain its practicability as a vector for iron chelators. The antiproliferative activity of NSPD-OH was very low; its 48-h and 96-h IC 50 values were 350 M and 25 M, much higher than that of NSPD at 1.3 M and 0.8 M, respectively (Table 3-15). 135

PAGE 136

Interestingly, (S)-4'-(HO)-DADFT-NSPD ester had a higher IC 50 (25 M) than its cargo molecule (S)-4'-(HO)-DADFT (IC 50 = 16 M, Table 2-2) at 48 h; nevertheless, both compounds had the basically same 96-h IC 50 values of 15 17 M (Table 3-15). 3.7.4.2 Cellular Transport, Intracellular Uptake of NSPD-OH and the Impact on Polyamine Pools NSPD-OH had a decreased affinity for the polyamine transporter (K i = 42.8 M) as compared with NSPD (K i = 7.2 M) (Table 3-16). This is in agreement with the finding that hydroxylation of a polyamine on its carbon chain leads to an increase of the K i value. For example, both the bis-hydroxylated polyamines (HO) 2 -DEHSPM and (HO) 2 -DENSPM, either in the (R,R)or (S,S)-configuration, had higher K i values than their corresponding parent molecules DEHSPM and DENSPM. 356 Although both NSPD and NSPD-OH accumulated intracellularly to the same extent, 2138 204 M and 2516 37 M respectively, the uptake ratio of NSPD (475) was much higher than that of NSPD-OH (25.2). At 48 h, both NSPD (4.5 M) and NSPD-OH (100 M) effectively depleted PUT to 0%, NSPD depleted SPD to 12 5% while NSPD-OH depleted SPD to 30 1%. However, NSPD was less effective than NSPD-OH at depleting SPM, which remained 83 8% of control in the NSPD treated cells, but 51 1% in the NSPD-OH treated cells. 3.7.4.3 Cellular Transport, Intracellular Uptake of (S)-4'-(HO)-DADFT-NSPD ester and the Impact on Polyamine Pools Unlike NSPD-(S)-4'-(HO)-DADFT-EE, which had a high affinity (K i = 5.7 M, Table 3-7) for the polyamine transporter in L1210 cells, (S)-4'-(HO)-DADFT-NSPD ester (K i = 34.6 M, Table 3-7) did not compete with SPD for transport as well. Not surprisingly, the chelator (S)-4'-(HO)-DADFT itself was not a competitive inhibitor of polyamine transport. 136

PAGE 137

When L1210 cells were treated with 100 M (S)-4'-(HO)-DADFT-NSPD ester (Figure 3-23, 5) for 2 h, the compound was hardly detectable intracellularly, and the metabolite (S)-4'-(HO)-DADFT reached a concentration of 42 4 M, whereas NSPD-OH accumulated to 136 7 M (Table 3-17). However, in the cells treated with 100 M (S)-4'-(HO)-DADFT for 2 h, 58 5 M of the parent drug could be detected intracellularly. Neither the conjugate nor the cargo depleted the cellular polyamine pools. The conjugate (S)-4'-(HO)-DADFT-NSPD ester raised PUT, SPD, and SPM to 115 2%, 113 13%, and 114 30% respectively, while the cargo (S)-4'-(HO)-DADFT increased PUT to 127 1%, SPD to 120 15%, and SPM to 131 42%. In the cells treated with (S)-4'-(HO)-DADFT-NSPD ester (100 M) at 4 h, the NSPD-OH concentration increased to 280 19 M, no conjugate molecule could be detected (Table 3-17), and the concentration of (S)-4'-(HO)-DADFT decreased to 37 5 M. However, in the cells treated with (S)-4'-(HO)-DADFT (100 M) at 4 h, the compound concentration increased slightly to 65 1 M. Similar to the 2-h treatment, polyamine pools remained close to the control levels at 4 h in both the conjugated and non-conjugated chelator treated cells. In conjugate treated cells, PUT, SPD, and SPM levels were 106 5%, 108 1%, and 113 % respectively; similarly, in (S)-4'-(HO)-DADFT treated cells, the polyamine levels were 137 3%, 109 2%, and 109 % respectively. However, a prolonged exposure of 48 h with the conjugate at 25 M had profound effect on polyamine pools. PUT, SPD, and SPM levels were reduced to 7 0%, 41 4%, and 76 9% respectively. Moreover, NSPD-OH accumulated to a concentration of 1000 128 M. On the other hand, in cells treated with 20 M (S)-4'-(HO)-DADFT at 48 h, the native polyamines remained at the same levels of controls. 137

PAGE 138

The unexpected low intracellular accumulation of both the conjugate [(S)-4'-(HO)-DADFT-NSPD ester] and cargo [(S)-4'-(HO)-DADFT] molecules regardless of treatment time (Table 3-17) made the chemical stability of (S)-4'-(HO)-DADFT-NSPD ester questionable. Therefore, the conjugate was tested and compared with (S)-4'-(HO)-DADFT at different conditions: in supernatant that cells treated with drugs and in cell culture medium formulated with or without FBS. As shown in Table 3-18, it is not surprising that the concentration of (S)-4'-(HO)-DADFT in supernatant was virtually constant regardless of the treatment time. However, the concentration of (S)-4'-(HO)-DADFT-NSPD ester in supernatant decreased with longer treatment time, from 113 M at 2 h to 92 M at 4 h and only 3 M at 48 h. On the other hand, the ratio of (S)-4'-(HO)-DADFT in (S)-4'-(HO)-DADFT-NSPD ester treated samples increased with longer treatment time, from 12% at 2 h to 20% at 4 h and 97% at 48 h. To study if non-specific serum esterases in the serum (FBS) used by the culture medium played a role in the degradation of (S)-4'-(HO)-DADFT-NSPD ester, in the absence of cells, the compound was incubated in the medium with or without FBS for 2 h, 4 h, and 48 h. Apparently, (S)-4'-(HO)-DADFT-NSPD ester showed very similar properties in both media; the concentration of (S)-4'-(HO)-DADFT-NSPD ester decreased while the concentration of its degradation product (S)-4'-(HO)-DADFT increased with the time. At 2 h, about 20% of (S)-4'-(HO)-DADFT-NSPD ester was degraded to (S)-4'-(HO)-DADFT; at 4 h, this number increased to about 40%. At 48 h, most of the conjugate (97%) was degraded (Table 3-18). Therefore, it seems that the chemical instability of (S)-4'-(HO)-DADFT-NSPD ester is structure related; conjugation of a polyamine vector at the carboxyl of (S)-4'-(HO)-DADFT via an ester bond may not be practical. 138

PAGE 139

A. HN HN HN HN HN HN HN NH2 HN HN NH2 H2N HN HN NH2 H2N HN NH2 HN NH2 H2N NH2 DENSPMMENSPMNSPMMENSPDMEDAPNSPDDAP B. HN NH HN NH HN NH HN NH2 H2N NH HN NH2 DEHSPMMEHSPMHSPM Figure 3-1. Metabolic transformation of DENSPM and DEHSPM in animal models MENSPM, monoethylnorspermine; NSPM, norspermine; MENSPD, monoethylnorspermidine; NSPD, norspermidine; MEDAP, monoethyldiaminopropane; DAP, 1,3-diaminopropane; MEHSPM, monoethylhomospermine; HSPM, homospermine. 139

PAGE 140

N NH HN HN N NN OH OH NN NO2 NO2 H2N N NH2 N OH N O2N 123 NH N Cl Cl O H2N N HN NH2 Figure 3-2. Nitroimidazole-polyamine (1 and 2) and chlorambucil-polyamine (3) conjugates R C Figure 3-3. A model of the polyamine transport apparatus complex R = H or alkyl; C = cargo molecule to be vectored by polyamines. Basically, the cargo molecule can be neutral or positively or negatively charged. 140

PAGE 141

HN N N NH2 Boc Boc Boc O CH3 HO O O +HN N N NH Boc Boc Boc O O CH3 O a HN N N NH Boc Boc Boc O OH O H2N HN NH NH O O CH3 O bcH2N HN NH NH O OH O d651234 Figure 3-4. Scheme for the synthesis of NTS and NTS-ME. Reagents: (a) CDI, CH 2 Cl 2 ;(b) HCl, CH 3 OH; (c) 1 N NaOH, CH 3 OH, then 1 N HCl; (d) TFA, CH 2 Cl 2 O O CH3 OBn H2N NH HN N CH3 OBn O NH2 NH HN NH2 +aH2N NH HN N CH3 OH O b 123 Figure 3-5. Scheme for the synthesis of SPM-L1 Reagents: (a) 2 N NaOH, EtOH, then HCl (b) H 2 /Pd-C/EtOH. 141

PAGE 142

N HO O H3C N HO O H3C HN N OH O CH3 Fe HN HN NH2 HN NH2 NH NH NH2 Figure 3-6. Structural illustration of the (SPM-L1) 3 Fe complex. Figure 3-7. Jobs plots of L1 and its polyamine conjugate SPM-L1 The theoretical mole fraction maximum for a 3:1 ligand/Fe complex of 0.75 was observed in both instances. OH SN CH3 OOH HO 1'2'3'4'5'6'site Isite II Figure 3-8. The two linkage sites for the polyamine conjugation in (S)-4'-(HO)-DADFT I, 4'-hydroxyl, and II, thiazoline carboxyl. 142

PAGE 143

H2N NH NH2 Cl OH H2N NH NH OH +123a BocNH N Boc N OTs Boc 5b BocNH N Boc N OH Boc 4c Figure 3-9. Scheme for the synthesis of NSPD-OH and the Boc-protected NSPD-OTs. Reagents: (a) 1-butanol, KI, K 2 CO 3 ; (b) (Boc) 2 O, 50% THF; (c) TsCl, NEt 3 CH 2 Cl 2 OH SN CH3 OOC2H5 O N N NH BocNH N Boc N OTs Boc OH SN CH3 OOC2H5 HO Boc Boc Boc OH SN CH3 OOH O N N NH Boc Boc Boc OH SN CH3 OOC2H5 O NH NH H2N OH SN CH3 OOH O NH NH H2N abcd NSPD-(S)-4'-(HO)-DADFT-EtENSPD-(S)-4'-(HO)-DADFT5678910+3HCl 3HCl Figure 3-10. Scheme for the synthesis of NSPD-(S)-4'-(HO)-DADFT-EE and NSPD-(S)-4'-(HO)-DADFT Reagents: (a) Na, CH 3 CH 2 OH, 60-70 C; (b) EtOH, concentrated HCl, 0 C, then R.T.; (c) 1N NaOH, CH 3 OH, then 1 N HCl; (d) concentrated HCl, dry CH 2 Cl 2 0 C, then R.T. 143

PAGE 144

Figure 3-11. Jobs plot of NSPD-(S)-4'-(HO)-DADFT The theoretical mole fraction maximum for a 2:1 ligand/Fe complex of 0.667; a linear intercept maximum of 0.675 was observed, indicating a 2:1 complex formed between NSPD-(S)-4'-(HO)-DADFT and Fe(III). 144

PAGE 145

OH SN CH3 OOC2H5 O NH NH H2N OH SN CH3 OOH O NH NH H2N OH SN CH3 OOH O NH NH HO OH SN CH3 OOH O NH H2N O OH SN CH3 OOH O NH H OH SN CH3 OOH O H2N O 8esterase10 OH SN CH3 OOH O NH NH H PAO/DAOO ADHSSAT OH SN CH3 OOH O NH NH NH 1511 H3C O 12 PAO OH SN CH3 OOH O NH NH H3C O SSAT PAO OH SN CH3 OOH O NH HO O ADHPAO/DAO1413161718 Figure 3-12. Schematic illustration of the metabolism of the polyamine moiety of the NSPD-(S)-4'-(HO)-DADFT-EE in rodents Two distinct metabolic routes were found after the conjugate ester was degraded to the conjugate acid by the non-specific serum esterase: (1) the oxidative deamination pathway (left part of the scheme, the dotted arrows indicate the pathway is hypothetic) through PAO/DAO and aldehyde dehydrogenase (ADH); (2) The typical SSAT/PAO pathway (right part of the scheme). 145

PAGE 146

Figure 3-13. Hepatic metabolism of the two NSPD conjugates of (S)-4'-(HO)-DADFT in rodents Both the compounds were given to rats sc at 300 mol/kg. (1) Concentration of NSPD-(S)-4'-(HO)-DADFT and its metabolites in rat liver. The parent chelator (S)-4'-(HO)-DADFT was administered under the same conditions and its liver concentration is shown here for comparison. (2) Concentration of NSPD-(S)-4'-(HO)-DADFT-EE and its metabolites in rat liver. Total (Parent + metabolites); Diacid metabolite 12; Diamine metabolite 16; Administered compound /metabolite 10; Monoamine metabolite 18; Administered compound (S)-4'-(HO)-DADFT. 146

PAGE 147

Figure 3-14. ESI mass spectrometry and MS/MS identification of metabolite 12 of NSPD-(S)-4'-(HO)-DADFT-EE (8). (a) Full-scan MS by ESI of the rat liver extract after 2 h administration of the conjugate ester 8. Accurate mass of the molecular ion of the metabolite 12 is given in italics and only matches to C 21 H 31 N 3 O 6 S + H + (calculated 454.2012, = 3.1 ppm) within 10 ppm accuracy, when the following search limits in elemental composition are set: C 24, H 40, N 4, O 6 and S 1; (b) Product-ion spectrum (CID) from m/z 454. 147

PAGE 148

Figure 3-15. Biliary ferrokinetics of (S)-4'-(HO)-DADFT and its polyamine conjugates NSPD-(S)-4'-(HO)-DADFT and NSPD-(S)-4'-(HO)-DADFT-EE in rodents Each compound was given to rats at 300 M/kg sc. Legend: closed triangle, (S)-4'-(HO)-DADFT; opened circle, NSPD-(S)-4'-(HO)-DADFT; closed square, NSPD-(S)-4'-(HO)-DADFT-EE. H2N N NH2 NON NO2 HN HN NH NH2 O O HO OH O HN S NHOHN H2N N NH2 N NB F F HN NH S NH O NH2 H2N O 1234 Figure 3-16. Structure of polyamine conjugated fluorescent probes 1, N 4 -NBD-SPD; 2, Monofluorescein isothiocyanate-spermine (FL-SPM); 3, N 4 -SPD-MANT; 4, N 4 -SPD-C 2 -BODIPY. 148

PAGE 149

A. B. Figure 3-17. Fluorescence excitation (in blue) and emission (in red) spectra of NBD-GABA and NBD-GABA-NSPD A) NBD-GABA (0.1 mM in ddH 2 O); B) NBD-GABA-NSPD (0.1 mM in ddH 2 O). 149

PAGE 150

w/ NH 4 Cl w/o NH 4 Cl A. B. C. D. E. F. Figure 3-18. Fluorescence microscopy of NBD-GABA-NSPD accumulation in SK-HEP-1 cells Cells were pre-treated with (A, C, and E) or without (B, D, and F) NH 4 Cl (10 mM) for 1 h, and exposed to the corresponding compounds for 30 min). A and B represent control cells; C and D represent NBD-GABA (100 M) treated cells; E and F represent NBD-GABA-NSPD (100 M) treated cells. Magnification: 60x (A and B), 40x (C-F). 150

PAGE 151

CO2H COCl BocNH N Boc N OH Boc BocHN N Boc N Boc O O NH2 HN HN O O abc12345 Figure 3-19. Scheme for the synthesis of the conjugate NSPD-ANTH Reagents: (a) SOCl 2 CH 2 Cl 2 ; (b) CH 2 Cl 2 TEA; (c) CH 2 Cl 2 HCl. 151

PAGE 152

A. B. C. Figure 3-20. Light microscopy of L1210 cells treated with Anthroic acid or NSPD-anthroate Cells were stained with Wright-Giemsa stain after each drug treatment at 37 C. A, normal L1210 cells; B, cells treated with Anthroic acid (100 M) for 20 min; B, cells treated with NSPD-anthroate (100 M) for 20 min. 152

PAGE 153

BocNH N Boc N OTs Boc HN N N I Boc Boc Boc OH SN CH3 OOH HO HN N N Boc Boc Boc OH SN CH3 O HO O NH2 HN HN OH SN CH3 O HO O 12345abc Figure 3-21. Scheme for the synthesis of (S)-4'-(HO)-DADFT-NSPD ester Reagents: (a) NaI, acetone; (b) DIEA, DMF; (c) TFA, CH 2 Cl 2 153

PAGE 154

Table 3-1. Cellular transport, accumulation of polyamine analogues and effect on polyamine pools and IC 5 0 Treatment Conc. Intracellular Conc. c Polyamine Pools e Compound a K i (M) b (M) (pmol/10 6 cells) Uptake ratio d PUT SPD SPM IC 50 (M) f 48 / 96 h NHNHHNHN DESPM (3,4,3) 1.6 30 1780 20 59 0 0 22 30 / 0.18 NH HN HN CF3 NH F3C FDESPM (3,4,3) 285 100 < 20 < 0.2 103 103 108 >100 / >100 NH HN NH HN PIP (4,4,4) 4.9 2.0 2518 91 1259 0 5 50 2.0 / 0.1 NH HN N N PYR(4,4,4) > 500 100 87 95 99 >100 / 80 NHNHHN DESPD (3,4) 19 100 4610 46 0 5 74 ~100 / 0.7 NHNHNH DENSPD (3,3) 250 100 3670 36 0 17 74 >100 / >100 NH HN NH DEHSPD (4,4) 19 25 4610 184 0 6 114 22 / 0.35 NHNHNHNH DENSPM (3,3,3) 17 100 2440 24 0 6 30 20 / 2 NHHNNHHN DEHSPM (4,4,4) 1.4 10 2940 294 0 0 61 0.2 / 0.07 NH HN NH HN DMHSPM (4,4,4) 0.97 100 1490 14.9 0 0 30 >100 / 0.32 NH HN NH HN DIPHSPM (4,4,4) 8.1 20 0 17 83 20 / 0.06 NH HN NH HN DTBHSPM (4,4,4) 56 100 83 85 94 >100 / 3 a The number in the parentheses next to each abbreviation indicates the methylene spacing lengths between the neighbouring nitrogens of the molecule. b K i determinations were made by following analogue competition for uptake against [ 3 H]SPD by PA transporter. All polyamine analogues exhibited simple substrate-competitive inhibition of [ 3 H]SPD transport. c Cellular drug amount is expressed as pmol/10 6 cells. Untreated L1210 cells (10 6 ) correspond to about 1 L volume; therefore, the concentration can be estimated as M. 154

PAGE 155

d The uptake ratio is defined as the ratio of the intracellular analogue concentration over the treatment concentration. e Putrescine (PUT), spermidine (SPD), and spermine (SPM) levels after 48 h of treatment are given as percentages of polyamine found in untreated controls. Typical control values in pmol/10 6 L1210 cells are PUT = 260 59, SPD = 3354 361, and SPM = 658 119. f IC 50 values were estimated from growth curves for L1210 cells grown in the presence of nine different concentrations of tested compounds spanning four logarithmic units: 0, 0.03, 0.1, 0.3, 1.0, 3, 10, 30, and 100 M. IC 50 data are presented as the mean of at least two experiments with variation from the mean typically 10-25%. Table 3-2. Effect of polyamine analogues on polyamine metabolic enzyme s Compound ODC a AdoMetDC a SSAT a DESPD 30 68 1380 DEHSPD 47 90 640 DENSPD 80 45 780 DESPM 3 28 460 DEHSPM 7 41 140 DENSPM 10 42 1500 a Enzyme activity is expressed as percent of untreated control for ODC (1 M at 4 h), AdoMetDC (1 M at 6 h), and SSAT (10 M at 48 h for all of the above analogues except DENSPM, which is 2 M). Each experiment included a positive control which had a known, reproducible impact on enzyme activities (mean S.D.): 1 M DEHSPM reduced ODC to 7 3% of untreated control; 1 M DEHSPM reduced AdoMetDC to 41 6% of untreated control; and 2 MDENSPM increased SSAT levels to 1471 120% of untreated control. Data shown in the table represent the mean of two to three triplicate experiments and have variances consistent with those suggested by the positive control data presented above. b,c Reproduced data. 302, 305 155

PAGE 156

Table 3-3. Cellular transport, uptake, and the impact on polyamine pools of the SPM-terephthalic acid conjugate (NTS) and the SPM-terephthalic monomethyl ester conjugate (NTS-ME). Treatment Conc. Intracellular Conc. b Uptake ratio c Polyamine Pools d Compound K i (M) a (M) (pmol/10 6 cells) PUT SPD SPM HOOCO2H Terephthalic acid N.C. 100 < 50 < 0.5 105 12 119 6 110 11 H2NNHHNHNOCO2H NTS 27 100 < 70 < 0.7 53 8 91 4 93 6 HOOCO2CH3 Monomethyl Terephthalate N.C. 100 < 70 < 0.7 119 4 117 5 101 6 H2NNHHNHNOCO2CH3 NTS-ME 3.1 100 434 22 4.3 42 5 109 19 122 16 a K i determinations were made by following compound competition for uptake against [ 3 H]SPD by the PA transporter. NTS and NTS-ME exhibited simple substrate-competitive inhibition of [ 3 H]SPD transport. N.C., non-competitive. b L1210 cells were treated with either NTS or NTS-ME at 100 M for 48 h. Cellular drug amount is expressed as pmol/10 6 cells. Untreated L1210 cells (10 6 ) correspond to about 1 L volume; therefore, the concentration can be estimated as M. c The uptake ratio is defined as the ratio of the intracellular analogue concentration over the treatment concentration. d Putrescine (PUT), spermidine (SPD), and spermine (SPM) levels after 48 h of treatment are given as percent polyamine found in untreated controls. Typical control values in pmol/10 6 L1210 cells are PUT = 260 59, SPD = 3354 361, and SPM = 658 119. 156

PAGE 157

Table 3-4. Cellular transport, uptake of L1 and SPM-L1, and the impacts on polyamine pools and IC 50 Intracellular Conc. b Polyamine Pools d IC 50 (M) e Compound K i (M) a Treatment (pmol/10 6 cells) Uptake ratio c PUT SPD SPM 48 / 96 h NOOHCH3CH3 L1 N.C. 50 M, 48 h ~ 1 ~ 0.02 86 10 124 4 146 5 46 / 55 NOHOCH3NHHNN2 H SPM-L1 3.7 0.2M, 24 h 203 18 1015 118 8 125 1 121 2 0.18 / 0.18 SPM-L1 0.2 M, 48 h 388 36 1940 33 1 64 5 139 9 a K i determinations were made by following compound competition for uptake against [ 3 H]SPD by the PA transporter. SPM-L1 exhibited simple substrate-competitive inhibition of [ 3 H]SPD transport. N.C.: noncompetitive. b L1210 cells were treated with either 50 M of L1 for 48 h, or 0.2 M of SPM-L1 for 48 h. Cellular drug amount is expressed as pmol/10 6 cells. Untreated L1210 cells (10 6 ) correspond to about 1 L volume; therefore, the concentration can be estimated as M. c The uptake ratio is defined as the ratio of the intracellular analogue concentration over the treatment concentration. d Putrescine (PUT), spermidine (SPD), and spermine (SPM) levels after 48 h of treatment are given as percent polyamine found in untreated controls. Typical control values in pmol/10 6 L1210 cells are PUT = 260 59, SPD = 3354 361, and SPM = 658 119. e IC 50 values were estimated from growth curves for L1210 cells grown in the presence of nine different concentrations of tested compounds spanning four logarithmic units: 0, 0.03, 0.1, 0.3, 1.0, 3, 10, 30, and 100 M. IC 50 data are presented as the mean of at least two experiments with variation from the mean typically 10-25%. 157

PAGE 158

Table 3-5. Effect of L1 and SPM-L1 on ODC, AdoMetDC, and SSAT in L1210 cells Compound ODC a AdoMetDC a SSAT a L1 88 32 121 5 72 7 SPM-L1 26 6 72 11 205 45 a Enzyme activity is expressed as percent of untreated control for ODC (1 M at 4 h), AdoMetDC (1 M at 6 h), and SSAT (10 M at 48 h for all of the above analogues except DENSPM, which is 2 M). Each experiment included a positive control which had a known, reproducible impact on enzyme activities (mean S.D.): 1 M DEHSPM reduced ODC to 7 3% of untreated control; 1 M DEHSPM reduced AdoMetDC to 41 6% of untreated control; and 2 MDENSPM increased SSAT levels to 1471 120% of untreated control. Data shown in the table represent the mean of two to three triplicate experiments and have variances consistent with those suggested by the positive control data presented above. Table 3-6. Cellular uptake of representative DFT analogues Intracellular Conc. a Uptake ratio b Compound Treatment (pmol/10 6 cells) OHSNCO2HHHO (S)-4'-(HO)-DADMDFT 100 M, 48 h 31 5 0.31 OHSNCO2HHH3CO (S)-4'-(CH 3 O)-DADMDFT 15 M, 48 h 20 5 1.30 OHSNCO2HCH3HO (S)-4'-(HO)-DADFT 100 M, 12 h 130 21 1.30 OHSNCO2HCH3H3CO (S)-4'-(CH 3 O)-DADFT 10 M, 48 h u.d. a Cellular drug amount is expressed as pmol/10 6 cells. Untreated L1210 cells (10 6 ) correspond to about 1 L volume; therefore, the concentration can be estimated as M. u.d., under the detection limit. b The uptake ratio is defined as the ratio of the intracellular analogue concentration over the treatment concentration. 158

PAGE 159

Table 3-7. Cellular transport, uptake of NSPD-(S)-4'-(HO)-DADFT and NSPD-(S)-4'-(HO)-DADFT-EE a L1210 cells were treated with either compounds at 100 M for the time period indicated. Cellular drug amount is expressed as pmol/10 6 cells. Untreated L1210 cells (10 6 ) correspond to about 1 L volume; therefore, the concentration can be estimated as M. Intracellular Concentration (M) a Treatment Concentration 100 (M) NSPD-(S)-4'-(HO)-DADFT NSPD-(S)-4'-(HO)-DADFT-EE K i (M) b NSPD-(S)-4'-(HO)-DADFT 4 h 71 23 73 NSPD-(S)-4'-(HO)-DADFT-EE 4 h 2984 286 948 107 5.7 NSPD-(S)-4'-(HO)-DADFT 12 h 75 24 NSPD-(S)-4'-(HO)-DADFT-EE 12 h 3100 403 1503 194 b K i determinations were made by following compound competition for uptake against [ 3 H]SPD by the PA transporter. NSPD-(S)-4'-(HO)-DADFT and NSPD-(S)-4'-(HO)-DADFT-EE exhibited simple substrate-competitive inhibition of [ 3 H]SPD transport. Table 3-8. Antiproliferative activities of NSPD-(S)-4'-(HO)-DADFT, NSPD-(S)-4'-(HO)-DADFT-EE, and their cargo molecules Compound IC 50 (M) a 48 h 96 h OHSNCO2HCH3HO (S)-4'-(HO)-DADFT 16 17 OHSNCO2HCH3ONHNHH2N NSPD-(S)-4'-(HO)-DADFT 40 40 OH SN CO2C2H5CH3HO (S)-4'-(HO)-DADFT-EtE 9.0 6.0 OHSNCO2C2H5CH3ONHNHH2N NSPD-(S)-4'-(HO)-DADFT-EtE 1.5 1.5 a IC 50 values were estimated from growth curves for L1210 cells grown in the presence of nine different concentrations of tested compounds spanning four logarithmic units: 0, 0.03, 0.1, 0.3, 1.0, 3, 10, 30, and 100 M. IC 50 data are presented as the mean of at least two experiments with variation from the mean typically 10-25%. 159

PAGE 160

Table 3-9. Antiproliferative properties of DFT analogues and their polyamine conjugates on JURKAT cells Compound IC 50 (M) a 96 h 192 h DFT 5.4 (3.5) b 5.1 (2.0) DADFT 8.5 (6.0) 7.0 (6.0) DADMDFT 17 (18) 17 (18) (S)-4'-(CH 3 O)-DADFT 5.3 (6) 1.5 (5) NSPD-(S)-4'-(HO)-DADFT 23 (40) 11 (40) NSPD-(S)-4'-(HO)-DADFT-EE 0.5 (1.5) 0.4 (1.5) a IC 50 values were estimated from growth curves for JURKAT cells grown in the presence of nine different concentrations of tested compounds spanning four logarithmic units: 0, 0.03, 0.1, 0.3, 1.0, 3, 10, 30, and 100 M. IC 50 data are presented as the mean of at least two experiments with variation from the mean typically 10-25%. b IC 50 values of the same compound on L1210 cells are included in parentheses for comparisons. Table 3-10. Effect of (S)-4'-(CH 3 O)-DADFT and (S)-4'-(CH 3 O)-DADFT-EE and their polyamine conjugates on ODC, AdoMetDC, and SSAT in L1210 cells a Enzyme activity is expressed as percent of untreated control for ODC (1 M at 4 h), AdoMetDC (1 M at 6 h), and SSAT (10 M at 48 h for all of the above analogues except DENSPM, which is 2 M). Each experiment included a positive control which had a known, reproducible impact on enzyme activities (mean S.D.): 1 M DEHSPM reduced ODC to 7 3% of untreated control; 1 M DEHSPM reduced AdoMetDC to 41 6 % of untreated control; and 2 MDENSPM increased SSAT levels to 1471 120% of untreated control. Data shown in the Compound ODC a AdoMetDC a SSAT a (S)-4'-(CH 3 O)-DADFT 71 2 113 4 109 2 NSPD-(S)-4'-(HO)-DADFT 76 5 73 9 n.d. (S)-4'-(CH 3 O)-DADFT-EE 61 8 99 3 103 3 NSPD-(S)-4'-(HO)-DADFT-EE 59 3 74 2 101 6 160

PAGE 161

table represent the mean of two to three triplicate experiments and have variances consistent with those suggested by the positive control data presented above. Table 3-11. Iron clearing efficiency of (S)-4'-(HO)-DADFT and its polyamine conjugates in rats Iron Excretion b Compound Iron Clearing Efficiency % a bile urine (S)-4'-(HO)-DADFT 1.1 0.6 93 7 NSPD-(S)-4'-(HO)-DADFT 9.2 2.6 97 3 NSPD-(S)-4'-(HO)-DADFT-EE 13.6 3.3 99 1 a Both the polyamine conjugates were given to non-iron-overloaded, bile-duct-cannulated rats at 300 M/kg sc. The net iron excretion after 48 h (for both the conjugates) or 24 h [for the parent iron chelator (S)-4'-(HO)-DADFT] was calculated by subtracting the iron excretion of the control animals from the iron excretion of the treated animals. Iron clearing efficiency % is defined as the percentage ratio of net iron excretion/total iron binding capacity of the chelator administered. b Iron excretion in either bile or urine samples was expressed as percentage of the total iron excretion. Table 3-12. Transport properties and antiproliferative activities of NBD-GABA and its polyamine conjugate NBD-GABA-NSPD Compound K i ( M) a IC 50 ( M) b 48 h 96 h HONHONONNO2 NBD-GABA N.C. > 100 > 100 H2NNHNHONHONONNO2 NBD-GABA-NSPD 4.2 50 18 a K i determinations were made by following compound competition for uptake against [ 3 H]SPD by the PA transporter. NBD-GABA-NSPD exhibited simple substrate-competitive inhibition of [ 3 H]SPD transport. N.C., non-competitive. b IC 50 values were estimated from growth curves of L1210 cells grown in the presence of nine different concentrations of tested compounds spanning four logarithmic units: 0, 0.03, 0.1, 0.3, 1.0, 3, 10, 30, and 100 M. IC 50 data are presented as the mean of at least two experiments with variation from the mean typically 10-25%. 161

PAGE 162

Table 3-13. Fluorescence excitation and emission wavelenghts of NBD, NBD-GABA and its polyamine conjugate NBD-GABA-NSPD Compound Fluorescence Wavelength (nm) excitation emission NBD a 465 545 NBD-GABA 430 540 NBD-GABA-NSPD 400 550 a Source: Molecular Probes (Invitrogen Co., Carlsbad, CA). Table 3-14. Transport properties and antiproliferative activities of 9-AC and its polyamine conjugate NSPD-ANTH. Compound K i ( M) a IC 50 ( M) b 48 h 96 h CO2H 9-AC N.C. > 100 > 100 H2NHNHNOCO NSPD-ANTH 6.7 5 5 a K i determinations were made by following compound competition for uptake against [ 3 H]SPD by the PA transporter. NSPD-ANTH exhibited simple substrate-competitive inhibition of [ 3 H]SPD transport. N.C., non-competitive. b IC 50 values were estimated from growth curves of corresponding cell lines grown in the presence of nine different concentrations of tested compounds spanning four logarithmic units: 0, 0.03, 0.1, 0.3, 1.0, 3, 10, 30, and 100 M. IC 50 data are presented as the mean of at least two experiments with variation from the mean typically 10-25%. 162

PAGE 163

Table 3-15. Antiproliferative activities of the conjugate (S)-4'-(HO)-DADFT-NSPD ester and the polyamine fragment NSPD-OH Compound IC 50 ( M) a 48 h 96 h H2N NH NH2 NSPD 1.3 0.8 H2N NH NH OH NSPD-OH 350 25 OHSNCH3HOOOHNHNN2 H (S)-4'-(HO)-DADFT-NSPD ester 25 15 a IC 50 values were estimated from growth curves for L1210 cells grown in the presence of nine different concentrations of tested compounds spanning four logarithmic units: 0, 0.03, 0.1, 0.3, 1.0, 3, 10, 30, and 100 M. IC 50 data are presented as the mean of at least two experiments with variation from the mean typically 10-25%. Table 3-16. Cellular transport, uptake of NSPD and NSPD-OH, and impact on polyamine pools Intracellular Conc. b Polyamine Pools d Compound K i (M) a Treatment (M) (pmol/10 6 cells) Uptake ratio c PUT SPD SPM NSPD 7.2 4.5 2138 204 475 0 12 5 83 8 NSPD-OH 42.8 100 2516 37 25.2 0 30 1 51 3 a K i determinations were made by following compound competition for uptake against [ 3 H]SPD by the PA transporter. NSPD and NSPD-OH exhibited simple substrate-competitive inhibition of [ 3 H]SPD transport. b L1210 cells were treated with each compounds for 48 h at the concentration indicated. Cellular drug amount is expressed as pmol/10 6 cells. Untreated L1210 cells (10 6 ) correspond to about 1 L volume; therefore, the concentration can be estimated as M. 163

PAGE 164

c The uptake ratio is defined as the ratio of the intracellular analogue concentration over the treatment concentration. Table 3-17. Cellular transport, uptake of (S)-4'-(HO)-DADFT and (S)-4'-(HO)-DADFT-NSPD ester, and the impact on polyamine pools a L1210 cells were treated with compounds for 48 h at the concentration indicated. Cellular drug amount is expressed as pmol/10 6 cells. Untreated L1210 cells (10 6 ) correspond to about 1 L volume; therefore, the concentration can be estimated as M. Intracellular Conc. ( M) a Polyamine Pools c Treatment NSPD-OH Cargo Conjugate K i (M) b PUT SPD SPM (S)-4'-(HO)-DADFT-NSPD ester 100 M, 2 h 136 37 42 4 trace 34.6 115 2 113 13 114 30 (S)-4'-(HO)-DADFT 100 M, 2 h 58 5 0.0 N.C. 127 1 120 15 131 42 (S)-4'-(HO)-DADFT-NSPD ester 100 M, 4 h 280 19 37 5 0.0 106 5 108 1 113 3 (S)-4'-(HO)-DADFT 100 M, 4 h 65 1 0.0 137 3 109 2 109 5 (S)-4'-(HO)-DADFT-NSPD ester 100 M, 48 h 41 2 0.0 (S)-4'-(HO)-DADFT-NSPD ester 25 M, 48 h 1000 128 7 0 41 4 76 9 (S)-4'-(HO)-DADFT 100 M, 48 h 33 2 0.0 (S)-4'-(HO)-DADFT 20 M, 48 h 101 3 100 2 95 5 b K i determinations were made by following compound competition for uptake against [ 3 H]SPD by the PA transporter. (S)-4'-(HO)-DADFT-NSPD ester exhibited simple substrate-competitive inhibition of [ 3 H]SPD transport. N.C., non-competitive. 164

PAGE 165

c Putrescine (PUT), spermidine (SPD), and spermine (SPM) levels after 48 h of treatment are given as percent polyamine found in untreated controls. Typical control values in pmol/10 6 L1210 cells are PUT = 260 59, SPD = 3354 361, and SPM = 658 119. Table 3-18. Stability of (S)-4'-(HO)-DADFT-NSPD ester in cell culture medium Concentration ( M) b Ratio c Treatment Sample a Cargo Conjugate [Cargo]/{[Cargo] + [Conjugate]} Supernatant 16 1 113 3 12% medium + FBS, w/o cells 21 2 99 5 18% (S)-4'-(HO)-DADFT-NSPD ester 100 M, 2 h medium FBS, w/o cells 18 1 101 4 15% Supernatant 82 2 medium + FBS, w/o cells (S)-4'-(HO)-DADFT 100 M, 2 h medium FBS, w/o cells Supernatant 23 1 92 3 20% medium + FBS, w/o cells 42 1 60 1 41% (S)-4'-(HO)-DADFT-NSPD ester 100 M, 4 h medium FBS, w/o cells 37 4 57 1 39% Supernatant 82 3 medium + FBS, w/o cells (S)-4'-(HO)-DADFT 100 M, 4 h medium FBS, w/o cells Supernatant 93 5 3 1 97% medium + FBS, w/o cells 88 1 2 0 98% (S)-4'-(HO)-DADFT-NSPD ester 100 M, 48 h medium FBS, w/o cells Supernatant 88 2 medium + FBS, w/o cells (S)-4'-(HO)-DADFT 100 M, 48 h medium FBS, w/o cells a L1210 cells were treated with either compounds for 48 h at the concentration indicated, samples were collected from three different sources: from the supernatant after centrifugation of cultured cells; from the fetal bovine serum (FBS) formulated culture medium but without cells; from the basal medium without FBS and cells. b Concentrations were determined by HPLC and expressed in M. c The ratio is calculated by dividing the concentration of (S)-4'-(HO)-DADFT by the total concentration of (S)-4'-(HO)-DADFT and (S)-4'-(HO)-DADFT-NSPD ester present in each sample. Experiments were done in triplicates. 165

PAGE 166

CHAPTER 4 CONCLUSION Traditionally, polyamine analogues are used as antineoplastics; other than that, they are also attractive means for drug delivery. This study is focused on exploiting polyamine analogues as vectors for iron chelators such as L1 and DFT analogues and other molecules. The results obtained from the model compounds NTS, NTS-ME and SPM-L1 demonstrated the critical importance of the charge of a cargo molecule in determining the transport property of a polyamine conjugate. That is, a neutral or positively charged cargo fragment is compatible with the cellular transport of a polyamine conjugate, but a negatively charged cargo fragment is not. For example, while L1 hardly accumulated in L1210 cells, the polyamine conjugate SPM-L1 accumulated to a concentration 1940-fold of the treatment concentration. Moreover, SPM-L1 was about 300-fold more active than L1 at inhibiting L1210 cell growth. Similarly, the polyamine conjugate NSPD-(S)-4'-(HO)-DADFT-EtE was successfully taken up by L1210 cells; the drug and its hydrolyzed metabolite NSPD-(S)-4'-(HO)-DADFT accumulated to a concentration of over 400-fold of the treatment concentration only after 4 h. Both the polyamine conjugate acid NSPD-(S)-4'-(HO)-DADFT and ester NSPD-(S)-4'-(HO)-DADFT-EtE had much higher ICE in rodents; meanwhile, the ethyl ester conjugate had a more protracted iron excretion in the animals. The metabolic profile of these two conjugates followed the typical polyamine analogue metabolism, the stepwise deaminopropylation by the SSAT/PAO system. However, the major metabolite present in animals treated by either the conjugates were the oxidative deaminated product, which is a significant finding and should be considered in the future designing of polyamine conjugates. In current study, polyamine analogue NSPD was also tested for the feasibility of vectoring a Cl channel blocker anthracene-9-carboxylic acid (9-AC). The conjugate NSPD-ANTH gained 166

PAGE 167

great affinity for the polyamine transporter in L1210 cells and had significantly elevated cytotoxicity against L1210 cells which was demonstrated by the antiproliferative study and the light microscopic observation. 167

PAGE 168

LIST OF REFERENCES 1. Leeuwenhoek, A. V., Observationes D. Anthonii Leeuwenhoek De Natis E Semine Genitali Animalculis. Philos. Trans. R. Soc. 1678, 12, 1040-1043. 2. Kimberly, M. M., and Goldstein, J. H., Determination of pK a Values and Total Proton Distribution Pattern of Spermidine by Carbon-13 Nuclear Magnetic Resonance Tirations. Anal. Chem. 1981, 53, 789-793. 3. Aikens, D.; Bunce, S.; Onasch, F.; Parker, R., 3rd; Hurwitz, C.; Clemans, S., The Interactions between Nucleic Acids and Polyamines. II. Protonation Constants and 13 C-NMR Chemical Shift Assignments of Spermidine, Spermine, and Homologs. Biophys Chem 1983, 17, (1), 67-74. 4. Bergeron, R. J.; Weimar, W. R.; Wu, Q.; Austin, J. K., Jr.; Mcmanis, J. S., Impact of Polyamine Analogues on the NMDA Receptor. J Med Chem 1995, 38, (3), 425-428. 5. Bergeron, R. J.; Weimar, W. R.; Wu, Q.; Feng, Y.; Mcmanis, J. S., Polyamine Analogue Regulation of NMDA MK-801 Binding: A Structure-Activity Study. J Med Chem 1996, 39, (26), 5257-5266. 6. Drew, H. R.; Dickerson, R. E., Structure of a B-DNA Dodecamer. III. Geometry of Hydration. J Mol Biol 1981, 151, (3), 535-556. 7. Williams, L. D.; Frederick, C. A.; Ughetto, G.; Rich, A., Ternary Interactions of Spermine with DNA: 4'-Epiadriamycin and Other DNA: Anthracycline Complexes. Nucleic Acids Res 1990, 18, (18), 5533-5541. 8. Raspaud, E.; Durand, D.; Livolant, F., Interhelical Spacing in Liquid Crystalline Spermine and Spermidine-DNA Precipitates. Biophys J 2005, 88, (1), 392-403. 9. Basu, H. S.; Feuerstein, B. G.; Deen, D. F.; Lubich, W. P.; Bergeron, R. J.; Samejima, K.; Marton, L. J., Correlation between the Effects of Polyamine Analogues on DNA Conformation and Cell Growth. Cancer Res 1989, 49, (20), 5591-5597. 10. Thomas, T. J.; Gunnia, U. B.; Thomas, T., Polyamine-Induced B-DNA to Z-DNA Conformational Transition of a Plasmid DNA with (Dg-Dc)N Insert. J Biol Chem 1991, 266, (10), 6137-6141. 11. Wang, A. J.; Quigley, G. J.; Kolpak, F. J.; Van Der Marel, G.; Van Boom, J. H.; Rich, A., Left-Handed Double Helical DNA: Variations in the Backbone Conformation. Science 1981, 211, (4478), 171-176. 168

PAGE 169

12. Rich, A.; Zhang, S., Timeline: Z-DNA: The Long Road to Biological Function. Nat Rev Genet 2003, 4, (7), 566-572. 13. Wolfl, S.; Wittig, B.; Rich, A., Identification of Transcriptionally Induced Z-DNA Segments in the Human C-Myc Gene. Biochim Biophys Acta 1995, 1264, (3), 294-302. 14. Wittig, B.; Wolfl, S.; Dorbic, T.; Vahrson, W.; Rich, A., Transcription of Human C-Myc in Permeabilized Nuclei Is Associated with Formation of Z-DNA in Three Discrete Regions of the Gene. Embo J 1992, 11, (12), 4653-4663. 15. Schwartz, T.; Rould, M. A.; Lowenhaupt, K.; Herbert, A.; Rich, A., Crystal Structure of the Zalpha Domain of the Human Editing Enzyme Adar1 Bound to Left-Handed Z-DNA. Science 1999, 284, (5421), 1841-1845. 16. Baeza, I.; Gariglio, P.; Rangel, L. M.; Chavez, P.; Cervantes, L.; Arguello, C.; Wong, C.; Montanez, C., Electron Microscopy and Biochemical Properties of Polyamine-Compacted DNA. Biochemistry 1987, 26, (20), 6387-6392. 17. Antony, T.; Thomas, T.; Shirahata, A.; Sigal, L. H.; Thomas, T. J., Selectivity of Spermine Homologs on Triplex DNA Stabilization. Antisense Nucleic Acid Drug Dev 1999, 9, (2), 221-231. 18. D'agostino, L.; Di Luccia, A., Polyamines Interact with DNA as Molecular Aggregates. Eur J Biochem 2002, 269, (17), 4317-4325. 19. Sato, N.; Ohtake, Y.; Kato, H.; Abe, S.; Kohno, H.; Ohkubo, Y., Effects of Polyamines on Histone Polymerization. J Protein Chem 2003, 22, (3), 303-307. 20. Shiba, T.; Mizote, H.; Kaneko, T.; Nakajima, T.; Kakimoto, Y., Hypusine, a New Amino Acid Occurring in Bovine Brain. Isolation and Structural Determination. Biochim Biophys Acta 1971, 244, (3), 523-531. 21. Park, M. H.; Wolff, E. C.; Folk, J. E., Hypusine: Its Post-Translational Formation in Eukaryotic Initiation Factor 5a and Its Potential Role in Cellular Regulation. Biofactors 1993, 4, (2), 95-104. 22. Bartig, D.; Klink, F., Determination of the Unusual Amino Acid Hypusine at the Lower Picomole Level by Derivatization with 4-Dimethylaminoazobenzene-4'-Sulphonyl Chloride and Reversed-Phase High-Performance or Medium-Pressure Liquid Chromatography. J Chromatogr 1992, 606, (1), 43-48. 23. Park, M. H., The Essential Role of Hypusine in Eukaryotic Translation Initiation Factor 4d (eIF-4D). Purification of eIF-4D and Its Precursors and Comparison of Their Activities. J Biol Chem 1989, 264, (31), 18531-18535. 169

PAGE 170

24. Xu, A.; Jao, D. L.; Chen, K. Y., Identification of mRNA That Binds to Eukaryotic Initiation Factor 5a by Affinity Co-Purification and Differential Display. Biochem J 2004, 384, (Pt 3), 585-590. 25. Cooper, H. L.; Park, M. H.; Folk, J. E.; Safer, B.; Braverman, R., Identification of the Hypusine-Containing Protein Hy + as Translation Initiation Factor eIF-4D. Proc Natl Acad Sci U S A 1983, 80, (7), 1854-1857. 26. Torrelio, B. M.; Paz, M. A.; Gallop, P. M., Cellular Proliferation and Hypusine Synthesis. Exp Cell Res 1984, 154, (2), 454-463. 27. Chen, Z. P.; Chen, K. Y., Dramatic Attenuation of Hypusine Formation on Eukaryotic Initiation Factor 5a During Senescence of IMR-90 Human Diploid Fibroblasts. J Cell Physiol 1997, 170, (3), 248-254. 28. Chattopadhyay, M. K.; Tabor, C. W.; Tabor, H., Spermidine but Not Spermine Is Essential for Hypusine Biosynthesis and Growth in Saccharomyces Cerevisiae: Spermine Is Converted to Spermidine in vivo by the FMS1-Amine Oxidase. Proc Natl Acad Sci U S A 2003, 100, (24), 13869-13874. 29. Murphey, R. J.; Gerner, E. W., Hypusine Formation in Protein by a Two-Step Process in Cell Lysates. J Biol Chem 1987, 262, (31), 15033-15036. 30. Chen, K. Y.; Liu, A. Y., Biochemistry and Function of Hypusine Formation on Eukaryotic Initiation Factor 5a. Biol Signals 1997, 6, (3), 105-109. 31. Murphey, R. J.; Tome, M. E.; Gerner, E. W., Hypusine Biosynthesis in Protein and Its Biological Consequences. Adv Exp Med Biol 1988, 250, 449-458. 32. Atkins, J. F.; Lewis, J. B.; Anderson, C. W.; Gesteland, R. F., Enhanced Differential Synthesis of Proteins in a Mammalian Cell-Free System by Addition of Polyamines. J Biol Chem 1975, 250, (14), 5688-5695. 33. Praisler, R.; Mannlein, E.; Aschhoff, H. J.; Mach, M.; Kersten, W., Polyamine Requirement for Microbial Protein Synthesis: Structural Specificity in Cell-Free Systems of Escherichia Coli. Hoppe Seylers Z Physiol Chem 1984, 365, (9), 1155-1162. 34. Guo, X.; Rao, J. N.; Liu, L.; Zou, T.; Keledjian, K. M.; Boneva, D.; Marasa, B. S.; Wang, J. Y., Polyamines Are Necessary for Synthesis and Stability of Occludin Protein in Intestinal Epithelial Cells. Am J Physiol Gastrointest Liver Physiol 2005, 288, (6), G1159-1169. 35. Grishin, E. V.; Volkova, T. M.; Arsen'ev, A. S.; Reshetova, O. S.; Onoprienko, V. V., [Structural-Functional Characteristics of Argiopine--the Ion Channel Blockers from the Spider Argiope Lobata Venom]. Bioorg Khim 1986, 12, (8), 1121-1124. 170

PAGE 171

36. Mayer, M. L., Glutamate Receptor Ion Channels. Curr Opin Neurobiol 2005, 15, (3), 282-288. 37. Kashiwagi, K.; Pahk, A. J.; Masuko, T.; Igarashi, K.; Williams, K., Block and Modulation of N-Methyl-D-Aspartate Receptors by Polyamines and Protons: Role of Amino Acid Residues in the Transmembrane and Pore-Forming Regions of NR1 and NR2 Subunits. Mol Pharmacol 1997, 52, (4), 701-713. 38. Cull-Candy, S.; Brickley, S.; Farrant, M., NMDA Receptor Subunits: Diversity, Development and Disease. Curr Opin Neurobiol 2001, 11, (3), 327-335. 39. Herin, G. A.; Aizenman, E., Amino Terminal Domain Regulation of NMDA Receptor Function. Eur J Pharmacol 2004, 500, (1-3), 101-111. 40. Furukawa, H.; Gouaux, E., Mechanisms of Activation, Inhibition and Specificity: Crystal Structures of the NMDA Receptor NR1 Ligand-Binding Core. Embo J 2003, 22, (12), 2873-2885. 41. Huggins, D. J.; Grant, G. H., The Function of the Amino Terminal Domain in NMDA Receptor Modulation. J Mol Graph Model 2005, 23, (4), 381-388. 42. Nicoll, R. A.; Malenka, R. C., Expression Mechanisms Underlying NMDA Receptor-Dependent Long-Term Potentiation. Ann N Y Acad Sci 1999, 868, 515-525. 43. Hartmann, J.; Ransmayr, G.; Riederer, P., The Glycine Binding Site of the NMDA Receptor: Involvement in Neurodegeneration and New Approach for Neuroprotection. J Neural Transm Suppl 1994, 43, 53-57. 44. Ransom, R. W.; Stec, N. L., Cooperative Modulation of [3H]MK-801 Binding to the N-Methyl-D-Aspartate Receptor-Ion Channel Complex by L-Glutamate, Glycine, and Polyamines. J Neurochem 1988, 51, (3), 830-836. 45. Williams, K.; Romano, C.; Molinoff, P. B., Effects of Polyamines on the Binding of [3H]MK-801 to the N-Methyl-D-Aspartate Receptor: Pharmacological Evidence for the Existence of a Polyamine Recognition Site. Mol Pharmacol 1989, 36, (4), 575-581. 46. Araneda, R. C.; Zukin, R. S.; Bennett, M. V., Effects of Polyamines on NMDA-Induced Currents in Rat Hippocampal Neurons: A Whole-Cell and Single-Channel Study. Neurosci Lett 1993, 152, (1-2), 107-112. 47. Williams, K., Modulation and Block of Ion Channels: A New Biology of Polyamines. Cell Signal 1997, 9, (1), 1-13. 48. Westbrook, G. L.; Mayer, M. L., Micromolar Concentrations of Zn 2+ Antagonize NMDA and GABA Responses of Hippocampal Neurons. Nature 1987, 328, (6131), 640-643. 171

PAGE 172

49. Traynelis, S. F.; Hartley, M.; Heinemann, S. F., Control of Proton Sensitivity of the NMDA Receptor by RNA Splicing and Polyamines. Science 1995, 268, (5212), 873-876. 50. Williams, K.; Romano, C.; Dichter, M. A.; Molinoff, P. B., Modulation of the NMDA Receptor by Polyamines. Life Sci 1991, 48, (6), 469-498. 51. Turecek, R.; Vlcek, K.; Petrovic, M.; Horak, M.; Vlachova, V.; Vyklicky, L., Jr., Intracellular Spermine Decreases Open Probability of N-Methyl-D-Aspartate Receptor Channels. Neuroscience 2004, 125, (4), 879-887. 52. Cu, C.; Bahring, R.; Mayer, M. L., The Role of Hydrophobic Interactions in Binding of Polyamines to Non NMDA Receptor Ion Channels. Neuropharmacology 1998, 37, (10-11), 1381-1391. 53. Stromgaard, K.; Mellor, I., AMPA Receptor Ligands: Synthetic and Pharmacological Studies of Polyamines and Polyamine Toxins. Med Res Rev 2004, 24, (5), 589-620. 54. Downward, J., The Ins and Outs of Signalling. Nature 2001, 411, (6839), 759-762. 55. Seger, R.; Krebs, E. G., The Mapk Signaling Cascade. Faseb J 1995, 9, (9), 726-735. 56. Flamigni, F.; Facchini, A.; Capanni, C.; Stefanelli, C.; Tantini, B.; Caldarera, C. M., p44/42 Mitogen-Activated Protein Kinase Is Involved in the Expression of Ornithine Decarboxylase in Leukaemia L1210 Cells. Biochem J 1999, 341 (Pt 2), 363-369. 57. Pegg, A. E., Polyamine Metabolism and Its Importance in Neoplastic Growth and a Target for Chemotherapy. Cancer Res 1988, 48, (4), 759-774. 58. Pegg, A. E.; Madhubala, R.; Kameji, T.; Bergeron, R. J., Control of Ornithine Decarboxylase Activity in Alpha-Difluoromethylornithine-Resistant L1210 Cells by Polyamines and Synthetic Analogues. J Biol Chem 1988, 263, (22), 11008-11014. 59. Persson, L.; Holm, I.; Heby, O., Regulation of Ornithine Decarboxylase mRNA Translation by Polyamines. Studies Using a Cell-Free System and a Cell Line with an Amplified Ornithine Decarboxylase Gene. J Biol Chem 1988, 263, (7), 3528-3533. 60. Ruchko, M.; Gillespie, M. N.; Weeks, R. S.; Olson, J. W.; Babal, P., Putrescine Transport in Hypoxic Rat Main Pasmcs Is Required for P38 Map Kinase Activation. Am J Physiol Lung Cell Mol Physiol 2003, 284, (1), L179-186. 61. Bello-Fernandez, C.; Packham, G.; Cleveland, J. L., The Ornithine Decarboxylase Gene Is a Transcriptional Target of C-Myc. Proc Natl Acad Sci U S A 1993, 90, (16), 7804-7808. 172

PAGE 173

62. Selvakumaran, M.; Liebermann, D.; Hoffman, B., The Proto-Oncogene c-myc Blocks Myeloid Differentiation Independently of Its Target Gene Ornithine Decarboxylase. Blood 1996, 88, (4), 1248-1255. 63. Auvinen, M.; Paasinen, A.; Andersson, L. C.; Holtta, E., Ornithine Decarboxylase Activity Is Critical for Cell Transformation. Nature 1992, 360, (6402), 355-358. 64. Moshier, J. A.; Dosescu, J.; Skunca, M.; Luk, G. D., Transformation of NIH/3T3 Cells by Ornithine Decarboxylase Overexpression. Cancer Res 1993, 53, (11), 2618-2622. 65. Clifford, A.; Morgan, D.; Yuspa, S. H.; Soler, A. P.; Gilmour, S., Role of Ornithine Decarboxylase in Epidermal Tumorigenesis. Cancer Res 1995, 55, (8), 1680-1686. 66. Tabib, A.; Bachrach, U., Role of Polyamines in Mediating Malignant Transformation and Oncogene Expression. Int J Biochem Cell Biol 1999, 31, (11), 1289-1295. 67. Bettuzzi, S.; Davalli, P.; Astancolle, S.; Pinna, C.; Roncaglia, R.; Boraldi, F.; Tiozzo, R.; Sharrard, M.; Corti, A., Coordinate Changes of Polyamine Metabolism Regulatory Proteins During the Cell Cycle of Normal Human Dermal Fibroblasts. FEBS Lett 1999, 446, (1), 18-22. 68. Studzinski, G. P., Oncogenes, Growth, and the Cell Cycle: An Overview. Cell Tissue Kinet 1989, 22, (6), 405-424. 69. Harper, J. W.; Adams, P. D., Cyclin-Dependent Kinases. Chem Rev 2001, 101, (8), 2511-2526. 70. Johnson, D. G.; Walker, C. L., Cyclins and Cell Cycle Checkpoints. Annu Rev Pharmacol Toxicol 1999, 39, 295-312. 71. Stevenson, L. M.; Deal, M. S.; Hagopian, J. C.; Lew, J., Activation Mechanism of Cdk2: Role of Cyclin Binding Versus Phosphorylation. Biochemistry 2002, 41, (26), 8528-8534. 72. Oredsson, S. M., Polyamine Dependence of Normal Cell-Cycle Progression. Biochem Soc Trans 2003, 31, (2), 366-370. 73. Scorcioni, F.; Corti, A.; Davalli, P.; Astancolle, S.; Bettuzzi, S., Manipulation of the Expression of Regulatory Genes of Polyamine Metabolism Results in Specific Alterations of the Cell-Cycle Progression. Biochem J 2001, 354, (Pt 1), 217-223. 74. Fredlund, J. O.; Johansson, M. C.; Dahlberg, E.; Oredsson, S. M., Ornithine Decarboxylase and S-Adenosylmethionine Decarboxylase Expression During the Cell Cycle of Chinese Hamster Ovary Cells. Exp Cell Res 1995, 216, (1), 86-92. 75. Millward, M. J.; Joshua, A.; Kefford, R.; Aamdal, S.; Thomson, D.; Hersey, P.; Toner, G.; Lynch, K., Multi-Centre Phase II Trial of the Polyamine Synthesis Inhibitor 173

PAGE 174

SAM486A (CGP48664) in Patients with Metastatic Melanoma. Invest New Drugs 2005, 23, (3), 253-256. 76. Wallick, C. J.; Gamper, I.; Thorne, M.; Feith, D. J.; Takasaki, K. Y.; Wilson, S. M.; Seki, J. A.; Pegg, A. E.; Byus, C. V.; Bachmann, A. S., Key Role for p27 Kip1 Retinoblastoma Protein Rb, and MYCN in Polyamine Inhibitor-Induced G(1) Cell Cycle Arrest in MYCN-Amplified Human Neuroblastoma Cells. Oncogene 2005. 77. Wolff, A. C.; Armstrong, D. K.; Fetting, J. H.; Carducci, M. K.; Riley, C. D.; Bender, J. F.; Casero, R. A., Jr.; Davidson, N. E., A Phase II Study of the Polyamine Analog N 1 ,N 11 -Diethylnorspermine (DENSPM) Daily for Five Days Every 21 Days in Patients with Previously Treated Metastatic Breast Cancer. Clin Cancer Res 2003, 9, (16 Pt 1), 5922-5928. 78. Kramer, D. L.; Vujcic, S.; Diegelman, P.; Alderfer, J.; Miller, J. T.; Black, J. D.; Bergeron, R. J.; Porter, C. W., Polyamine Analogue Induction of the p53-p21 WAF1/CIP1 -Rb Pathway and G1 Arrest in Human Melanoma Cells. Cancer Res 1999, 59, (6), 1278-1286. 79. Schipper, R. G.; Penning, L. C.; Verhofstad, A. A., Involvement of Polyamines in Apoptosis. Facts and Controversies: Effectors or Protectors? Semin Cancer Biol 2000, 10, (1), 55-68. 80. Ha, H. C.; Woster, P. M.; Yager, J. D.; Casero, R. A., Jr., The Role of Polyamine Catabolism in Polyamine Analogue-Induced Programmed Cell Death. Proc Natl Acad Sci U S A 1997, 94, (21), 11557-11562. 81. Debenedette, M.; Olson, J. W.; Snow, E. C., Expression of Polyamine Transporter Activity During B Lymphocyte Cell Cycle Progression. J Immunol 1993, 150, (10), 4218-4224. 82. Laitinen, J.; Stenius, K.; Eloranta, T. O.; Holtta, E., Polyamines May Regulate S-Phase Progression but Not the Dynamic Changes of Chromatin During the Cell Cycle. J Cell Biochem 1998, 68, (2), 200-212. 83. Donato, N. J.; Rotbein, J.; Rosenblum, M. G., Tumor Necrosis Factor Stimulates Ornithine Decarboxylase Activity in Human Fibroblasts and Tumor Target Cells. J Cell Biochem 1991, 46, (1), 69-77. 84. Yanagawa, K.; Yamashita, T.; Yada, K.; Ohira, M.; Ishikawa, T.; Yano, Y.; Otani, S.; Sowa, M., The Antiproliferative Effect of HGF on Hepatoma Cells Involves Induction of Apoptosis with Increase in Intracellular Polyamine Concentration Levels. Oncol Rep 1998, 5, (1), 185-190. 174

PAGE 175

85. Manchester, K. M.; Heston, W. D.; Donner, D. B., Tumour Necrosis Factor-Induced Cytotoxicity Is Accompanied by Intracellular Mitogenic Signals in Me-180 Human Cervical Carcinoma Cells. Biochem J 1993, 290 (Pt 1), 185-190. 86. Penning, L. C.; Schipper, R. G.; Vercammen, D.; Verhofstad, A. A.; Denecker, T.; Beyaert, R.; Vandenabeele, P., Sensitization of TNF-Induced Apoptosis with Polyamine Synthesis Inhibitors in Different Human and Murine Tumour Cell Lines. Cytokine 1998, 10, (6), 423-431. 87. Lindsay, G. S.; Wallace, H. M., Changes in Polyamine Catabolism in HL-60 Human Promyelogenous Leukaemic Cells in Response to Etoposide-Induced Apoptosis. Biochem J 1999, 337 (Pt 1), 83-87. 88. Seiler, N.; Dezeure, F., Polyamine Transport in Mammalian Cells. Int J Biochem 1990, 22, (3), 211-218. 89. Igarashi, K.; Kashiwagi, K., Polyamine Transport in Bacteria and Yeast. Biochem J 1999, 344 Pt 3, 633-642. 90. Dean, M.; Hamon, Y.; Chimini, G., The Human ATP-Binding Cassette (ABC) Transporter Superfamily. J Lipid Res 2001, 42, (7), 1007-1017. 91. Tomitori, H.; Kashiwagi, K.; Asakawa, T.; Kakinuma, Y.; Michael, A. J.; Igarashi, K., Multiple Polyamine Transport Systems on the Vacuolar Membrane in Yeast. Biochem J 2001, 353, (Pt 3), 681-688. 92. Uemura, T.; Kashiwagi, K.; Igarashi, K., Uptake of Putrescine and Spermidine by Gap1p on the Plasma Membrane in Saccharomyces Cerevisiae. Biochem Biophys Res Commun 2005, 328, (4), 1028-1033. 93. Dot, J.; Lluch, M.; Blanco, I.; Rodriguez-Alvarez, J., Polyamine Uptake in Cultured Astrocytes: Characterization and Modulation by Protein Kinases. J Neurochem 2000, 75, (5), 1917-1926. 94. Porter, C. W.; Miller, J.; Bergeron, R. J., Aliphatic Chain Length Specificity of the Polyamine Transport System in Ascites L1210 Leukemia Cells. Cancer Res 1984, 44, (1), 126-128. 95. Kakinuma, Y.; Hoshino, K.; Igarashi, K., Characterization of the Inducible Polyamine Transporter in Bovine Lymphocytes. Eur J Biochem 1988, 176, (2), 409-414. 96. Seiler, N.; Delcros, J. G.; Moulinoux, J. P., Polyamine Transport in Mammalian Cells. An Update. Int J Biochem Cell Biol 1996, 28, (8), 843-861. 97. Mccann, P. P.; Tardif, C.; Mamont, P. S., Regulation of Ornithine Decarboxylase by ODC-Antizyme in HTC Cells. Biochem Biophys Res Commun 1977, 75, (4), 948-954. 175

PAGE 176

98. Palanimurugan, R.; Scheel, H.; Hofmann, K.; Dohmen, R. J., Polyamines Regulate Their Synthesis by Inducing Expression and Blocking Degradation of ODC Antizyme. Embo J 2004, 23, (24), 4857-4867. 99. Sakata, K.; Kashiwagi, K.; Igarashi, K., Properties of a Polyamine Transporter Regulated by Antizyme. Biochem J 2000, 347 Pt 1, 297-303. 100. Suzuki, T.; He, Y.; Kashiwagi, K.; Murakami, Y.; Hayashi, S.; Igarashi, K., Antizyme Protects against Abnormal Accumulation and Toxicity of Polyamines in Ornithine Decarboxylase-Overproducing Cells. Proc Natl Acad Sci U S A 1994, 91, (19), 8930-8934. 101. Seiler, N., Thirty Years of Polyamine-Related Approaches to Cancer Therapy. Retrospect and Prospect. Part 2. Structural Analogues and Derivatives. Curr Drug Targets 2003, 4, (7), 565-585. 102. Karl, P. I.; Friedman, P. A., Competition between Paraquat and Putrescine for Accumulation by Rat Lung Slices. Toxicology 1983, 26, (3-4), 317-323. 103. Porter, C. W.; Bergeron, R. J.; Stolowich, N. J., Biological Properties of N 4 -Spermidine Derivatives and Their Potential in Anticancer Chemotherapy. Cancer Res 1982, 42, (10), 4072-4078. 104. Toninello, A.; Via, L. D.; Di Noto, V.; Mancon, M., The Effects of Methylglyoxal-Bis(Guanylhydrazone) on Spermine Binding and Transport in Liver Mitochondria. Biochem Pharmacol 1999, 58, (12), 1899-1906. 105. Porter, C. W.; Cavanaugh, P. F., Jr.; Stolowich, N.; Ganis, B.; Kelly, E.; Bergeron, R. J., Biological Properties of N 4 and N 1 ,N 8 -Spermidine Derivatives in Cultured L1210 Leukemia Cells. Cancer Res 1985, 45, (5), 2050-2057. 106. Thomas, T.; Thomas, T. J., Polyamine Metabolism and Cancer. J Cell Mol Med 2003, 7, (2), 113-126. 107. Bergeron, R. J.; Neims, A. H.; Mcmanis, J. S.; Hawthorne, T. R.; Vinson, J. R.; Bortell, R.; Ingeno, M. J., Synthetic Polyamine Analogues as Antineoplastics. J Med Chem 1988, 31, (6), 1183-1190. 108. Heby, O., Ornithine Decarboxylase as Target of Chemotherapy. Adv Enzyme Regul 1985, 24, 103-124. 109. Osterman, A. L.; Brooks, H. B.; Rizo, J.; Phillips, M. A., Role of Arg-277 in the Binding of Pyridoxal 5'-Phosphate to Trypanosoma Brucei Ornithine Decarboxylase. Biochemistry 1997, 36, (15), 4558-4567. 176

PAGE 177

110. M.L.Morgan, D., Polyamine Protocols. Humana Press: 1998. 111. Persson, L.; Wallstrom, E. L.; Nasizadeh, S.; Dartsch, C.; Jeppsson, A.; Wendt, A.; Holmgren, J., Regulation of Mammalian Ornithine Decarboxylase. Biochem Soc Trans 1998, 26, (4), 575-579. 112. Jackson, L. K.; Brooks, H. B.; Osterman, A. L.; Goldsmith, E. J.; Phillips, M. A., Altering the Reaction Specificity of Eukaryotic Ornithine Decarboxylase. Biochemistry 2000, 39, (37), 11247-11257. 113. Eliot, A. C.; Kirsch, J. F., Pyridoxal Phosphate Enzymes: Mechanistic, Structural, and Evolutionary Considerations. Annu Rev Biochem 2004, 73, 383-415. 114. He, Y.; Shimogori, T.; Kashiwagi, K.; Shirahata, A.; Igarashi, K., Inhibition of Cell Growth by Combination of Alpha-Difluoromethylornithine and an Inhibitor of Spermine Synthase. J Biochem (Tokyo) 1995, 117, (4), 824-829. 115. Bey, P.; Danzin, C.; Van Dorsselaer, V.; Mamont, P.; Jung, M.; Tardif, C., Analogues of Ornithine as Inhibitors of Ornithine Decarboxylase. New Deductions Concerning the Topography of the Enzyme's Active Site. J Med Chem 1978, 21, (1), 50-55. 116. Metcalf, B., Danzin, Jung, Casera & Vevert, Catalytic Irreversible Inhibition of Mammalian Ornithine Decarboxylase (E.C. 4.1.1.17) by Substrate and Product Analogues. J Am Chem Soc 1978, 100, 2551-2553. 117. Pegg, A. E.; Mcgovern, K. A.; Wiest, L., Decarboxylation of Alpha-Difluoromethylornithine by Ornithine Decarboxylase. Biochem J 1987, 241, (1), 305-307. 118. Samejima, K.; Raina, A.; Yamanoha, B.; Eloranta, T., Purification of Putrescine Aminopropyltransferase (Spermidine Synthase) from Eukaryotic Tissues. Methods Enzymol 1983, 94, 270-276. 119. Raina, A.; Pajula, R. L.; Eloranta, T., Purification of Spermidine Aminopropyltransferase (Spermine Synthase) from Bovine Brain. Methods Enzymol 1983, 94, 276-279. 120. Pegg, A. E.; Mccann, P. P., S-Adenosylmethionine Decarboxylase as an Enzyme Target for Therapy. Pharmacol Ther 1992, 56, (3), 359-377. 121. Sturman, J. A., Effect of Methylglyoxal Bis (Guanylhydrazone) (MGBG) in Vivo on the Decarboxylation of S-Adenosylmethionine and Synthesis of Spermidine in the Rat and Guinea Pig. Life Sci 1976, 18, (8), 879-886. 122. Regenass, U.; Mett, H.; Stanek, J.; Mueller, M.; Kramer, D.; Porter, C. W., CGP 48664, a New S-Adenosylmethionine Decarboxylase Inhibitor with Broad Spectrum Antiproliferative and Antitumor Activity. Cancer Res 1994, 54, (12), 3210-3217. 177

PAGE 178

123. Casero, R. A., Jr.; Pegg, A. E., Spermidine/Spermine N 1 -Acetyltransferase--the Turning Point in Polyamine Metabolism. Faseb J 1993, 7, (8), 653-661. 124. Persson, L.; Pegg, A. E., Studies of the Induction of Spermidine/Spermine N 1 -Acetyltransferase Using a Specific Antiserum. J Biol Chem 1984, 259, (20), 12364-12367. 125. Matsui, I.; Pegg, A. E., Effect of Inhibitors of Protein Synthesis on Rat Liver Spermidine N-Acetyltransferase. Biochim Biophys Acta 1981, 675, (3-4), 373-378. 126. Wallace, H. M.; Fraser, A. V.; Hughes, A., A Perspective of Polyamine Metabolism. Biochem J 2003, 376, (Pt 1), 1-14. 127. Desiderio, M. A., Opposite Responses of Nuclear Spermidine N 8 -Acetyltransferase and Histone Acetyltransferase Activities to Regenerative Stimuli in Rat Liver. Hepatology 1992, 15, (5), 928-933. 128. Seiler, N., Catabolism of Polyamines. Amino Acids 2004, 26, (3), 217-233. 129. Wallace, H. M.; Mackarel, A. J., Regulation of Polyamine Acetylation and Efflux in Human Cancer Cells. Biochem Soc Trans 1998, 26, (4), 571-575. 130. Seiler, N.; Bolkenius, F. N.; Knodgen, B., Acetylation of Spermidine in Polyamine Catabolism. Biochim Biophys Acta 1980, 633, (2), 181-190. 131. Seiler, N., Oxidation of Polyamines and Brain Injury. Neurochem Res 2000, 25, (4), 471-490. 132. Raina, A.; Pajula, R. L.; Eloranta, T., A Rapid Assay Method for Spermidine and Spermine Synthases. Distribution of Polyamine-Synthesizing Enzymes and Methionine Adenosyltransferase in Rat Tissues. FEBS Lett 1976, 67, (3), 252-255. 133. Lamond, S.; Wallace, H. M., Polyamine Oxidase Activity and Growth in Human Cancer Cells. Biochem Soc Trans 1994, 22, (4), 396S. 134. Dogan, A.; Rao, A. M.; Hatcher, J.; Rao, V. L.; Baskaya, M. K.; Dempsey, R. J., Effects of MDL 72527, a Specific Inhibitor of Polyamine Oxidase, on Brain Edema, Ischemic Injury Volume, and Tissue Polyamine Levels in Rats after Temporary Middle Cerebral Artery Occlusion. J Neurochem 1999, 72, (2), 765-770. 135. Webber, M. M.; Chaproniere-Rickenberg, D., Spermine Oxidation Products Are Selectively Toxic to Fibroblasts in Cultures of Normal Human Prostatic Epithelium. Cell Biol Int Rep 1980, 4, (2), 185-193. 178

PAGE 179

136. Calcabrini, A.; Arancia, G.; Marra, M.; Crateri, P.; Befani, O.; Martone, A.; Agostinelli, E., Enzymatic Oxidation Products of Spermine Induce Greater Cytotoxic Effects on Human Multidrug-Resistant Colon Carcinoma Cells (Lovo) Than on Their Wild-Type Counterparts. Int J Cancer 2002, 99, (1), 43-52. 137. Holtta, E., Oxidation of Spermidine and Spermine in Rat Liver: Purification and Properties of Polyamine Oxidase. Biochemistry 1977, 16, (1), 91-100. 138. Suzuki, O.; Matsumoto, T.; Oya, M.; Katsumata, Y., Metabolism of Acetylpolyamines by Monoamine Oxidase, Diamine Oxidase and Polyamine Oxidase. Biochim Biophys Acta 1981, 677, (2), 190-193. 139. Binda, C.; Mattevi, A.; Edmondson, D. E., Structure-Function Relationships in Flavoenzyme-Dependent Amine Oxidations: A Comparison of Polyamine Oxidase and Monoamine Oxidase. J Biol Chem 2002, 277, (27), 23973-23976. 140. Vujcic, S.; Diegelman, P.; Bacchi, C. J.; Kramer, D. L.; Porter, C. W., Identification and Characterization of a Novel Flavin-Containing Spermine Oxidase of Mammalian Cell Origin. Biochem J 2002, 367, (Pt 3), 665-675. 141. Brunton, V. G.; Grant, M. H.; Wallace, H. M., Mechanisms of Spermine Toxicity in Baby-Hamster Kidney (BHK) Cells. The Role of Amine Oxidases and Oxidative Stress. Biochem J 1991, 280 (Pt 1), 193-198. 142. Segal, J. A.; Skolnick, P., Spermine-Induced Toxicity in Cerebellar Granule Neurons Is Independent of Its Actions at NMDA Receptors. J Neurochem 2000, 74, (1), 60-69. 143. Seiler, N.; Duranton, B.; Raul, F., The Polyamine Oxidase Inactivator MDL 72527. Prog Drug Res 2002, 59, 1-40. 144. Duranton, B.; Holl, V.; Schneider, Y.; Carnesecchi, S.; Gosse, F.; Raul, F.; Seiler, N., Cytotoxic Effects of the Polyamine Oxidase Inactivator MDL 72527 to Two Human Colon Carcinoma Cell Lines SW480 and SW620. Cell Biol Toxicol 2002, 18, (6), 381-396. 145. Seiler, N.; Knodgen, B.; Gittos, M. W.; Chan, W. Y.; Griesmann, G.; Rennert, O. M., On the Formation of Amino Acids Deriving from Spermidine and Spermine. Biochem J 1981, 200, (1), 123-132. 146. Hougaard, D. M.; Houen, G.; Larsson, L. I., Regulation of Gastric Mucosal Diamine Oxidase Activity by Gastrin. FEBS Lett 1992, 307, (2), 135-138. 147. Illei, G.; Morgan, D. M., Serum Polyamine-Oxidase Activity in Spontaneous Abortion. Br J Obstet Gynaecol 1982, 89, (3), 199-201. 179

PAGE 180

148. Sharmin, S.; Sakata, K.; Kashiwagi, K.; Ueda, S.; Iwasaki, S.; Shirahata, A.; Igarashi, K., Polyamine Cytotoxicity in the Presence of Bovine Serum Amine Oxidase. Biochem Biophys Res Commun 2001, 282, (1), 228-235. 149. Tabor, C. W.; Tabor, H.; Bachrach, U., Identification of the Aminoaldehydes Produced by the Oxidation of Spermine and Spermidine with Purified Plasma Amine Oxidase. J Biol Chem 1964, 239, 2194-2203. 150. Houen, G.; Bock, K.; Jensen, A. L., HPLC and NMR Investigation of the Serum Amine Oxidase Catalyzed Oxidation of Polyamines. Acta Chem Scand 1994, 48, (1), 52-60. 151. Houen, G.; Struve, C.; Sondergaard, R.; Friis, T.; Anthoni, U.; Nielsen, P. H.; Christophersen, C.; Petersen, B. O.; Duus, J. O., Substrate Specificity of the Bovine Serum Amine Oxidase and in Situ Characterisation of Aminoaldehydes by NMR Spectroscopy. Bioorg Med Chem 2005, 13, (11), 3783-3796. 152. Crichton, R., Inorganic Biochemistry of Iron Metabolism, 2nd Edition. 2nd ed.; John Wiley & Sons, Ltd: 2001; p 326. 153. Walter, P. B.; Knutson, M. D.; Paler-Martinez, A.; Lee, S.; Xu, Y.; Viteri, F. E.; Ames, B. N., Iron Deficiency and Iron Excess Damage Mitochondria and Mitochondrial DNA in Rats. Proc Natl Acad Sci U S A 2002, 99, (4), 2264-2269. 154. Boveris, A., Biochemistry of Free Radicals: From Electrons to Tissues. Medicina (B Aires) 1998, 58, (4), 350-356. 155. Sakac, V.; Sakac, M., [Free Oxygen Radiacals and Kidney Diseases--Part I]. Med Pregl 2000, 53, (9-10), 463-474. 156. Shinar, E.; Rachmilewitz, E. A., Oxidative Denaturation of Red Blood Cells in Thalassemia. Semin Hematol 1990, 27, (1), 70-82. 157. Bertoncini, C. R.; Meneghini, R., DNA Strand Breaks Produced by Oxidative Stress in Mammalian Cells Exhibit 3'-Phosphoglycolate Termini. Nucleic Acids Res 1995, 23, (15), 2995-3002. 158. Muller, K.; Gurster, D., Hydroxyl Radical Damage to DNA Sugar and Model Membranes Induced by Anthralin (Dithranol). Biochem Pharmacol 1993, 46, (10), 1695-1704. 159. Davies, K. J.; Goldberg, A. L., Proteins Damaged by Oxygen Radicals Are Rapidly Degraded in Extracts of Red Blood Cells. J Biol Chem 1987, 262, (17), 8227-8234. 160. Bergeron, R. J.; Wiegand, J.; Mcmanis, J. S.; Weimar, W. R.; Park, J. H.; Eiler-Mcmanis, E.; Bergeron, J.; Brittenham, G. M., Partition-Variant Desferrithiocin Analogues: Organ Targeting and Increased Iron Clearance. J Med Chem 2005, 48, (3), 821-831. 180

PAGE 181

161. Neilands, J. B., Siderophores. Arch Biochem Biophys 1993, 302, (1), 1-3. 162. Neilands, J. B., Siderophores: Structure and Function of Microbial Iron Transport Compounds. J Biol Chem 1995, 270, (45), 26723-26726. 163. Bergeron, R. J.; Mcmanis, J. S.; Weimar, W. R.; Wiegand, J.; Eiler-Mcmanis, E., Iron Chelators and Therapeutic Uses. In Burger's Medicinal Chemistry and Drug Discovery, 6th ed.; Wiley Interscience: 2003; Vol. 3, pp 479-561. 164. B. E. Kalinowski, L. J. L., S. L. Brantley, A. Barnes, and C. G. Pantano, X-Ray Photoelectron Evidence for Bacteria-Enhanced Dissolution of Hornblende. Geochimica et Cosmochimica Acta 2000, 64, (8), 1331. 165. Winkelmann, G., Specificity of Iron Transport in Bacteria and Fungi. In CRC Handbook of Microbial Iron Chelates, Winkelmann, G., Ed. CRC Press, Florida: 1991; p 366. 166. Wesley R. Harris, C. J. C., Stephen R. Cooper, Stephen R. Sofen,Alex E. Avdeef, James V. Mcardle, and Kenneth N. Raymond, Coordination Chemistry of Microbial Iron Transport Compounds. 19. Stability Constants and Electrochemical Behavior of Ferric Enterobactin and Model Complexes. J Am Chem Soc 1979, 101, (20), 6097-6104. 167. Vallet, V.; Wahlgren, U.; Grenthe, I., Chelate Effect and Thermodynamics of Metal Complex Formation in Solution: A Quantum Chemical Study. J Am Chem Soc 2003, 125, (48), 14941-14950. 168. Wesley R. Harris, C. J. C., Kenneth N. Raymond, Spectrophotometric Determination of the Proton-Dependent Stability Constant of Ferric Enterobactin. J Am Chem Soc 1979, 101, 2213-2214. 169. Braun, V.; Braun, M., Iron Transport and Signaling in Escherichia Coli. FEBS Lett 2002, 529, (1), 78-85. 170. Hoegy, F.; Celia, H.; Mislin, G. L.; Vincent, M.; Gallay, J.; Schalk, I. J., Binding of Iron-Free Siderophore, a Common Feature of Siderophore Outer Membrane Transporters of Escherichia Coli and Pseudomonas Aeruginosa. J Biol Chem 2005, 280, (21), 20222-20230. 171. Hanks, T. S.; Liu, M.; Mcclure, M. J.; Lei, B., ABC Transporter FtsABCD of Streptococcus Pyogenes Mediates Uptake of Ferric Ferrichrome. BMC Microbiol 2005, 5, 62. 172. Jin, B.; Newton, S. M.; Shao, Y.; Jiang, X.; Charbit, A.; Klebba, P. E., Iron Acquisition Systems for Ferric Hydroxamates, Haemin and Haemoglobin in Listeria Monocytogenes. Mol Microbiol 2006, 59, (4), 1185-1198. 181

PAGE 182

173. Bergeron, R. J.; Weimar, W. R., Kinetics of Iron Acquisition from Ferric Siderophores by Paracoccus Denitrificans. J Bacteriol 1990, 172, (5), 2650-2657. 174. Halle, F.; Meyer, J. M., Ferrisiderophore Reductases of Pseudomonas. Purification, Properties and Cellular Location of the Pseudomonas Aeruginosa Ferripyoverdine Reductase. Eur J Biochem 1992, 209, (2), 613-620. 175. Lesuisse, E.; Crichton, R. R.; Labbe, P., Iron-Reductases in the Yeast Saccharomyces Cerevisiae. Biochim Biophys Acta 1990, 1038, (2), 253-259. 176. Stintzi, A.; Barnes, C.; Xu, J.; Raymond, K. N., Microbial Iron Transport Via a Siderophore Shuttle: A Membrane Ion Transport Paradigm. Proc Natl Acad Sci U S A 2000, 97, (20), 10691-10696. 177. Neilands, J. B.; Erickson, T. J.; Rastetter, W. H., Stereospecificity of the Ferric Enterobactin Receptor of Escherichia Coli K-12. J Biol Chem 1981, 256, (8), 3831-3832. 178. Bergeron, R. J.; Elliott, G. T.; Kline, S. J.; Ramphal, R.; St James, L., 3rd, Bacteriostatic and Fungostatic Action of Catecholamide Iron Chelators. Antimicrob Agents Chemother 1983, 24, (5), 725-730. 179. Fred S. Archibald, I. W. D., Removal of Iron from Human Transferrin by Neisseria Meningitidis. FEMS Microbiology Letters 1979, 6, (3), 159-162. 180. Holbein, B. E., Enhancement of Neisseria Meningitidis Infection in Mice by Addition of Iron Bound to Transferrin. Infect Immun 1981, 34, (1), 120-125. 181. Husson, M. O.; Legrand, D.; Spik, G.; Leclerc, H., Iron Acquisition by Helicobacter Pylori: Importance of Human Lactoferrin. Infect Immun 1993, 61, (6), 2694-2697. 182. Otto, B. R.; Verweij-Van Vught, A. M.; Maclaren, D. M., Transferrins and Heme-Compounds as Iron Sources for Pathogenic Bacteria. Crit Rev Microbiol 1992, 18, (3), 217-233. 183. O'malley, S. M.; Mouton, S. L.; Occhino, D. A.; Deanda, M. T.; Rashidi, J. R.; Fuson, K. L.; Rashidi, C. E.; Mora, M. Y.; Payne, S. M.; Henderson, D. P., Comparison of the Heme Iron Utilization Systems of Pathogenic Vibrios. J Bacteriol 1999, 181, (11), 3594-3598. 184. Gomme, P. T.; Mccann, K. B.; Bertolini, J., Transferrin: Structure, Function and Potential Therapeutic Actions. Drug Discov Today 2005, 10, (4), 267-273. 185. Mason, A. B.; Halbrooks, P. J.; James, N. G.; Connolly, S. A.; Larouche, J. R.; Smith, V. C.; Macgillivray, R. T.; Chasteen, N. D., Mutational Analysis of C-Lobe Ligands of Human Serum Transferrin: Insights into the Mechanism of Iron Release. Biochemistry 2005, 44, (22), 8013-8021. 182

PAGE 183

186. He, Q. Y.; Mason, A. B.; Woodworth, R. C.; Tam, B. M.; Macgillivray, R. T.; Grady, J. K.; Chasteen, N. D., Inequivalence of the Two Tyrosine Ligands in the N-Lobe of Human Serum Transferrin. Biochemistry 1997, 36, (48), 14853-14860. 187. Schlabach, M. R.; Bates, G. W., The Synergistic Binding of Anions and Fe 3+ by Transferrin. Implications for the Interlocking Sites Hypothesis. J Biol Chem 1975, 250, (6), 2182-2188. 188. Bailey, S.; Evans, R. W.; Garratt, R. C.; Gorinsky, B.; Hasnain, S.; Horsburgh, C.; Jhoti, H.; Lindley, P. F.; Mydin, A.; Sarra, R.; Et Al., Molecular Structure of Serum Transferrin at 3.3-A Resolution. Biochemistry 1988, 27, (15), 5804-5812. 189. Jandl, J. H.; Inman, J. K.; Simmons, R. L.; Allen, D. W., Transfer of Iron from Serum Iron-Binding Protein to Human Reticulocytes. J Clin Invest 1959, 38, (1, Part 1), 161-185. 190. Lawrence, C. M.; Ray, S.; Babyonyshev, M.; Galluser, R.; Borhani, D. W.; Harrison, S. C., Crystal Structure of the Ectodomain of Human Transferrin Receptor. Science 1999, 286, (5440), 779-782. 191. Dubljevic, V.; Sali, A.; Goding, J. W., A Conserved RGD (Arg-Gly-Asp) Motif in the Transferrin Receptor Is Required for Binding to Transferrin. Biochem J 1999, 341 (Pt 1), 11-14. 192. Hemadi, M.; Kahn, P. H.; Miquel, G.; El Hage Chahine, J. M., Transferrin's Mechanism of Interaction with Receptor 1. Biochemistry 2004, 43, (6), 1736-1745. 193. Dhungana, S.; Taboy, C. H.; Zak, O.; Larvie, M.; Crumbliss, A. L.; Aisen, P., Redox Properties of Human Transferrin Bound to Its Receptor. Biochemistry 2004, 43, (1), 205-209. 194. Paterson, S.; Armstrong, N. J.; Iacopetta, B. J.; Mcardle, H. J.; Morgan, E. H., Intravesicular Ph and Iron Uptake by Immature Erythroid Cells. J Cell Physiol 1984, 120, (2), 225-232. 195. Nunez, M. T.; Gaete, V.; Watkins, J. A.; Glass, J., Mobilization of Iron from Endocytic Vesicles. The Effects of Acidification and Reduction. J Biol Chem 1990, 265, (12), 6688-6692. 196. Rao, K.; Van Renswoude, J.; Kempf, C.; Klausner, R. D., Separation of Fe +3 from Transferrin in Endocytosis. Role of the Acidic Endosome. FEBS Lett 1983, 160, (1-2), 213-216. 197. Macgillivray, R. T.; Moore, S. A.; Chen, J.; Anderson, B. F.; Baker, H.; Luo, Y.; Bewley, M.; Smith, C. A.; Murphy, M. E.; Wang, Y.; Mason, A. B.; Woodworth, R. C.; Brayer, 183

PAGE 184

G. D.; Baker, E. N., Two High-Resolution Crystal Structures of the Recombinant N-Lobe of Human Transferrin Reveal a Structural Change Implicated in Iron Release. Biochemistry 1998, 37, (22), 7919-7928. 198. Mckie, A. T., A Ferrireductase Fills the Gap in the Transferrin Cycle. Nat Genet 2005, 37, (11), 1159-1160. 199. Harris, W. R., Estimation of the Ferrous-Transferrin Binding Constants Based on Thermodynamic Studies of Nickel(II)-Transferrin. J Inorg Biochem 1986, 27, (1), 41-52. 200. Chasteen, N. D.; Harrison, P. M., Mineralization in Ferritin: An Efficient Means of Iron Storage. J Struct Biol 1999, 126, (3), 182-194. 201. Theil, E. C., Ferritin: Structure, Gene Regulation, and Cellular Function in Animals, Plants, and Microorganisms. Annu Rev Biochem 1987, 56, 289-315. 202. Treffry, A.; Bauminger, E. R.; Hechel, D.; Hodson, N. W.; Nowik, I.; Yewdall, S. J.; Harrison, P. M., Defining the Roles of the Threefold Channels in Iron Uptake, Iron Oxidation and Iron-Core Formation in Ferritin: A Study Aided by Site-Directed Mutagenesis. Biochem J 1993, 296 (Pt 3), 721-728. 203. Cowley, J. M.; Janney, D. E.; Gerkin, R. C.; Buseck, P. R., The Structure of Ferritin Cores Determined by Electron Nanodiffraction. J Struct Biol 2000, 131, (3), 210-216. 204. Hoy, T. G.; Jacobs, A., Ferritin Polymers and the Formation of Haemosiderin. Br J Haematol 1981, 49, (4), 593-602. 205. Wixom, R. L.; Prutkin, L.; Munro, H. N., Hemosiderin: Nature, Formation, and Significance. Int Rev Exp Pathol 1980, 22, 193-225. 206. Miyazaki, E.; Kato, J.; Kobune, M.; Okumura, K.; Sasaki, K.; Shintani, N.; Arosio, P.; Niitsu, Y., Denatured H-Ferritin Subunit Is a Major Constituent of Haemosiderin in the Liver of Patients with Iron Overload. Gut 2002, 50, (3), 413-419. 207. Peters, T. J.; Selden, C.; Seymour, C. A., Lysosomal Disruption in the Pathogenesis of Hepatic Damage in Primary and Secondary Haemochromatosis. Ciba Found Symp 1976, (51), 317-329. 208. Iancu, T. C.; Neustein, H. B., Ferritin in Human Liver Cells of Homozygous Beta-Thalassaemia: Ultrastructural Observations. Br J Haematol 1977, 37, (4), 527-535. 209. Mann, S.; Wade, V. J.; Dickson, D. P.; Reid, N. M.; Ward, R. J.; O'connell, M.; Peters, T. J., Structural Specificity of Haemosiderin Iron Cores in Iron-Overload Diseases. FEBS Lett 1988, 234, (1), 69-72. 184

PAGE 185

210. Anderson, D.; Yardley-Jones, A.; Vives-Bauza, C.; Chua-Anusorn, W.; Cole, C.; Webb, J., Effect of Iron Salts, Haemosiderins, and Chelating Agents on the Lymphocytes of a Thalassaemia Patient without Chelation Therapy as Measured in the Comet Assay. Teratog Carcinog Mutagen 2000, 20, (5), 251-264. 211. Jacobs, A., Low Molecular Weight Intracellular Iron Transport Compounds. Blood 1977, 50, (3), 433-439. 212. Pollycove, M.; Maqsood, M., Existence of an Erythropoietic Labile Iron Pool in Animals. Nature 1962, 194, 152-154. 213. Wintrobe, G. R. G. A. M. M., A Labile Iron Pool. J Biol Chem 1946, 165, 397-398. 214. Glickstein, H.; El, R. B.; Shvartsman, M.; Cabantchik, Z. I., Intracellular Labile Iron Pools as Direct Targets of Iron Chelators: A Fluorescence Study of Chelator Action in Living Cells. Blood 2005, 106, (9), 3242-3250. 215. Petrat, F.; De Groot, H.; Rauen, U., Subcellular Distribution of Chelatable Iron: A Laser Scanning Microscopic Study in Isolated Hepatocytes and Liver Endothelial Cells. Biochem J 2001, 356, (Pt 1), 61-69. 216. Kakhlon, O.; Cabantchik, Z. I., The Labile Iron Pool: Characterization, Measurement, and Participation in Cellular Processes(1). Free Radic Biol Med 2002, 33, (8), 1037-1046. 217. Ioav Cabantchik, O. K., Silvian Epsztejn, Giulianna Zanninelli, and William Breuer, Intracellular and Extracellular Labile Iron Pools. In Iron Chelation TherapyAdvances in Experimental Medicine and Biology, Hershko, C., Ed. Kluwer Academic/Plenum Publishers: 2002; Vol. 509. 218. Kruszewski, M., The Role of Labile Iron Pool in Cardiovascular Diseases. Acta Biochim Pol 2004, 51, (2), 471-480. 219. Kruszewski, M., Labile Iron Pool: The Main Determinant of Cellular Response to Oxidative Stress. Mutat Res 2003, 531, (1-2), 81-92. 220. Eisenstein, R. S.; Blemings, K. P., Iron Regulatory Proteins, Iron Responsive Elements and Iron Homeostasis. J Nutr 1998, 128, (12), 2295-2298. 221. Theil, E. C., Regulation of Ferritin and Transferrin Receptor Mrnas. J Biol Chem 1990, 265, (9), 4771-4774. 222. Hentze, M. W.; Kuhn, L. C., Molecular Control of Vertebrate Iron Metabolism: mRNA-Based Regulatory Circuits Operated by Iron, Nitric Oxide, and Oxidative Stress. Proc Natl Acad Sci U S A 1996, 93, (16), 8175-8182. 185

PAGE 186

223. Papanikolaou, G.; Pantopoulos, K., Iron Metabolism and Toxicity. Toxicol Appl Pharmacol 2005, 202, (2), 199-211. 224. Cairo, G.; Pietrangelo, A., Iron Regulatory Proteins in Pathobiology. Biochem J 2000, 352 Pt 2, 241-250. 225. Samaniego, F.; Chin, J.; Iwai, K.; Rouault, T. A.; Klausner, R. D., Molecular Characterization of a Second Iron-Responsive Element Binding Protein, Iron Regulatory Protein 2. Structure, Function, and Post-Translational Regulation. J Biol Chem 1994, 269, (49), 30904-30910. 226. Pantopoulos, K., Iron Metabolism and the IRE/IRP Regulatory System: An Update. Ann N Y Acad Sci 2004, 1012, 1-13. 227. Lavaute, T.; Smith, S.; Cooperman, S.; Iwai, K.; Land, W.; Meyron-Holtz, E.; Drake, S. K.; Miller, G.; Abu-Asab, M.; Tsokos, M.; Switzer, R., 3rd; Grinberg, A.; Love, P.; Tresser, N.; Rouault, T. A., Targeted Deletion of the Gene Encoding Iron Regulatory Protein-2 Causes Misregulation of Iron Metabolism and Neurodegenerative Disease in Mice. Nat Genet 2001, 27, (2), 209-214. 228. Rouault, T. A., Post-Transcriptional Regulation of Human Iron Metabolism by Iron Regulatory Proteins. Blood Cells Mol Dis 2002, 29, (3), 309-314. 229. Miret, S.; Simpson, R. J.; Mckie, A. T., Physiology and Molecular Biology of Dietary Iron Absorption. Annu Rev Nutr 2003, 23, 283-301. 230. Fleming, R. E.; Bacon, B. R., Orchestration of Iron Homeostasis. N Engl J Med 2005, 352, (17), 1741-1744. 231. Anderson, G. J.; Frazer, D. M.; Mckie, A. T.; Vulpe, C. D., The Ceruloplasmin Homolog Hephaestin and the Control of Intestinal Iron Absorption. Blood Cells Mol Dis 2002, 29, (3), 367-375. 232. Petrak, J.; Vyoral, D., Hephaestin--a Ferroxidase of Cellular Iron Export. Int J Biochem Cell Biol 2005, 37, (6), 1173-1178. 233. Parmley, R. T.; Barton, J. C.; Conrad, M. E.; Austin, R. L.; Holland, R. M., Ultrastructural Cytochemistry and Radioautography of Hemoglobin-Iron Absorption. Exp Mol Pathol 1981, 34, (2), 131-144. 234. Parmley, R. T.; May, M. E.; Spicer, S. S.; Buse, M. G.; Alvarez, C. J., Ultrastructural Distribution of Inorganic Iron in Normal and Iron-Loaded Hepatic Cells. Lab Invest 1981, 44, (5), 475-485. 235. Uzel, C.; Conrad, M. E., Absorption of Heme Iron. Semin Hematol 1998, 35, (1), 27-34. 186

PAGE 187

236. Conrad, M. E.; Umbreit, J. N.; Moore, E. G., Iron Absorption and Transport. Am J Med Sci 1999, 318, (4), 213-229. 237. Raffin, S. B.; Woo, C. H.; Roost, K. T.; Price, D. C.; Schmid, R., Intestinal Absorption of Hemoglobin Iron-Heme Cleavage by Mucosal Heme Oxygenase. J Clin Invest 1974, 54, (6), 1344-1352. 238. Nicolas, G.; Chauvet, C.; Viatte, L.; Danan, J. L.; Bigard, X.; Devaux, I.; Beaumont, C.; Kahn, A.; Vaulont, S., The Gene Encoding the Iron Regulatory Peptide Hepcidin Is Regulated by Anemia, Hypoxia, and Inflammation. J Clin Invest 2002, 110, (7), 1037-1044. 239. Andrews, N. C., Molecular Control of Iron Metabolism. Best Pract Res Clin Haematol 2005, 18, (2), 159-169. 240. Centers for Disease Control and Prevention (http://www.cdc.gov) 241. Beard, J. L.; Dawson, H.; Pinero, D. J., Iron Metabolism: A Comprehensive Review. Nutr Rev 1996, 54, (10), 295-317. 242. Siah, C. W.; Trinder, D.; Olynyk, J. K., Iron Overload. Clin Chim Acta 2005, 358, (1-2), 24-36. 243. Feder, J. N.; Gnirke, A.; Thomas, W.; Tsuchihashi, Z.; Ruddy, D. A.; Basava, A.; Dormishian, F.; Domingo, R., Jr.; Ellis, M. C.; Fullan, A.; Hinton, L. M.; Jones, N. L.; Kimmel, B. E.; Kronmal, G. S.; Lauer, P.; Lee, V. K.; Loeb, D. B.; Mapa, F. A.; Mcclelland, E.; Meyer, N. C.; Mintier, G. A.; Moeller, N.; Moore, T.; Morikang, E.; Wolff, R. K.; Et Al., A Novel MHC Class I-Like Gene Is Mutated in Patients with Hereditary Haemochromatosis. Nat Genet 1996, 13, (4), 399-408. 244. Hanson, E. H.; Imperatore, G.; Burke, W., Hfe Gene and Hereditary Hemochromatosis: A Huge Review. Human Genome Epidemiology. Am J Epidemiol 2001, 154, (3), 193-206. 245. Lieu, P. T.; Heiskala, M.; Peterson, P. A.; Yang, Y., The Roles of Iron in Health and Disease. Mol Aspects Med 2001, 22, (1-2), 1-87. 246. Norris, R. P.; Mc, E. F., Exogenous Hemochromatosis Following Multiple Blood Transfusions. J Am Med Assoc 1950, 143, (8), 740-741. 247. Hershko, C.; Peto, T. E., Non-Transferrin Plasma Iron. Br J Haematol 1987, 66, (2), 149-151. 248. Weiss, G.; Umlauft, F.; Urbanek, M.; Herold, M.; Lovevsky, M.; Offner, F.; Gordeuk, V. R., Associations between Cellular Immune Effector Function, Iron Metabolism, and 187

PAGE 188

Disease Activity in Patients with Chronic Hepatitis C Virus Infection. J Infect Dis 1999, 180, (5), 1452-1458. 249. Horwitz, L. D., Iron-Mediated Cardiovascular Injury. In Crisp Data Base National Institutes of Health, Colorado, 2003. 250. Traore, H. N.; Meyer, D., The Effect of Iron Overload on in vitro HIV-1 Infection. J Clin Virol 2004, 31 Suppl 1, S92-98. 251. Bullen, J. J.; Rogers, H. J.; Spalding, P. B.; Ward, C. G., Iron and Infection: The Heart of the Matter. FEMS Immunol Med Microbiol 2005, 43, (3), 325-330. 252. Marx, J. J., Iron and Infection: Competition between Host and Microbes for a Precious Element. Best Pract Res Clin Haematol 2002, 15, (2), 411-426. 253. Abe, F.; Tateyma, M.; Shibuya, H.; Azumi, N.; Ommura, Y., Experimental Candidiasis in Iron Overload. Mycopathologia 1985, 89, (1), 59-63. 254. Rouault, T. A., Iron on the Brain. Nat Genet 2001, 28, (4), 299-300. 255. Hicken, B. L.; Tucker, D. C.; Barton, J. C., Patient Compliance with Phlebotomy Therapy for Iron Overload Associated with Hemochromatosis. Am J Gastroenterol 2003, 98, (9), 2072-2077. 256. Brittenham, G. M., Iron Chelators and Iron Toxicity. Alcohol 2003, 30, (2), 151-158. 257. Summers, M. R.; Jacobs, A.; Tudway, D.; Perera, P.; Ricketts, C., Studies in Desferrioxamine and Ferrioxamine Metabolism in Normal and Iron-Loaded Subjects. Br J Haematol 1979, 42, (4), 547-555. 258. Porter, J. B., Deferoxamine Pharmacokinetics. Semin Hematol 2001, 38, (1 Suppl 1), 63-68. 259. Chaston, T. B.; Richardson, D. R., Iron Chelators for the Treatment of Iron Overload Disease: Relationship between Structure, Redox Activity, and Toxicity. Am J Hematol 2003, 73, (3), 200-210. 260. Hoffbrand, A. V.; Cohen, A.; Hershko, C., Role of Deferiprone in Chelation Therapy for Transfusional Iron Overload. Blood 2003, 102, (1), 17-24. 261. Cappellini, M. D.; Cohen, A.; Piga, A.; Bejaoui, M.; Perrotta, S.; Agaoglu, L.; Aydinok, Y.; Kattamis, A.; Kilinc, Y.; Porter, J.; Capra, M.; Galanello, R.; Fattoum, S.; Drelichman, G.; Magnano, C.; Verissimo, M.; Athanassiou-Metaxa, M.; Giardina, P.; Kourakli-Symeonidis, A.; Janka-Schaub, G.; Coates, T.; Vermylen, C.; Olivieri, N.; Thuret, I.; Opitz, H.; Ressayre-Djaffer, C.; Marks, P.; Alberti, D., A Phase 3 Study of 188

PAGE 189

Deferasirox (ICL670), a Once-Daily Oral Iron Chelator, in Patients with Beta-Thalassemia. Blood 2006, 107, (9), 3455-3462. 262. Sergejew, T.; Forgiarini, P.; Schnebli, H. P., Chelator-Induced Iron Excretion in Iron-Overloaded Marmosets. Br J Haematol 2000, 110, (4), 985-992. 263. Bergeron, R. J.; Wiegand, J.; Mcmanis, J. S.; Vinson, J. R.; Yao, H.; Bharti, N.; Rocca, J. R., (S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylic Acid Polyethers: A Solution to Nephrotoxicity. J Med Chem 2006, 49, (9), 2772-2783. 264. Donovan, J. M.; Plone, M.; Dagher, R.; Bree, M.; Marquis, J., Preclinical and Clinical Development of Deferitrin, a Novel, Orally Available Iron Chelator. Ann N Y Acad Sci 2005, 1054, 492-494. 265. Bergeron, R. J.; Huang, G.; Weimar, W. R.; Smith, R. E.; Wiegand, J.; Mcmanis, J. S., Desferrithiocin Analogue Based Hexacoordinate Iron(III) Chelators. J Med Chem 2003, 46, (1), 16-24. 266. Wong, C.; Richardson, D. R., Beta-Thalassaemia: Emergence of New and Improved Iron Chelators for Treatment. Int J Biochem Cell Biol 2003, 35, (7), 1144-1149. 267. Singh, S.; Khodr, H.; Taylor, M. I.; Hider, R. C., Therapeutic Iron Chelators and Their Potential Side-Effects. Biochem Soc Symp 1995, 61, 127-137. 268. Gee, B. E., Clinical Toxicity of Iron Chelators. In Iron Chelators, David G. Badman, R. J. B., Gary M. Brittenham, Ed. The Saratoga Group: 2000. 269. Bergeron, R. J.; Cavanaugh, P. F., Jr.; Kline, S. J.; Hughes, R. G., Jr.; Elliott, G. T.; Porter, C. W., Antineoplastic and Antiherpetic Activity of Spermidine Catecholamide Iron Chelators. Biochem Biophys Res Commun 1984, 121, (3), 848-854. 270. Richardson, D. R., The Therapeutic Potential of Iron Chelators. Expert Opin Investig Drugs 1999, 8, (12), 2141-2158. 271. Buss, J. L.; Greene, B. T.; Turner, J.; Torti, F. M.; Torti, S. V., Iron Chelators in Cancer Chemotherapy. Curr Top Med Chem 2004, 4, (15), 1623-1635. 272. Murray, K. F.; Lam, D.; Kowdley, K. V., Current and Future Therapy in Haemochromatosis and Wilson's Disease. Expert Opin Pharmacother 2003, 4, (12), 2239-2251. 273. Hermes-Lima, M.; Goncalves, M. S.; Andrade, R. G., Jr., Pyridoxal Isonicotinoyl Hydrazone (PIH) Prevents Copper-Mediated in Vitro Free Radical Formation. Mol Cell Biochem 2001, 228, (1-2), 73-82. 189

PAGE 190

274. Sadrzadeh, S. M.; Nanji, A. A.; Price, P. L., The Oral Iron Chelator, 1,2-Dimethyl-3-hydroxypyrid-4-one Reduces Hepatic-Free Iron, Lipid Peroxidation and Fat Accumulation in Chronically Ethanol-Fed Rats. J Pharmacol Exp Ther 1994, 269, (2), 632-636. 275. Patt, A.; Horesh, I. R.; Berger, E. M.; Harken, A. H.; Repine, J. E., Iron Depletion or Chelation Reduces Ischemia/Reperfusion-Induced Edema in Gerbil Brains. J Pediatr Surg 1990, 25, (2), 224-227; discussion 227-228. 276. Nakamura, H.; Del Nido, P. J.; Jimenez, E.; Sarin, M.; Feinberg, H.; Levitsky, S., Age-Related Differences in Cardiac Susceptibility to Ischemia/Reperfusion Injury. Response to Deferoxamine. J Thorac Cardiovasc Surg 1992, 104, (1), 165-172. 277. Glickstein, H.; Breuer, W.; Loyevsky, M.; Konijn, A. M.; Shanzer, A.; Cabantchik, Z. I., Differential Cytotoxicity of Iron Chelators on Malaria-Infected Cells Versus Mammalian Cells. Blood 1996, 87, (11), 4871-4878. 278. Mabeza, G. F.; Biemba, G.; Gordeuk, V. R., Clinical Studies of Iron Chelators in Malaria. Acta Haematol 1996, 95, (1), 78-86. 279. Torti, S. V., Molecular Mechanisms of Iron Chelator Cytotoxicity. In Crisp Data Base National Institutes of Health, North Carolina, 2000. 280. Zheng, H.; Weiner, L. M.; Bar-Am, O.; Epsztejn, S.; Cabantchik, Z. I.; Warshawsky, A.; Youdim, M. B.; Fridkin, M., Design, Synthesis, and Evaluation of Novel Bifunctional Iron-Chelators as Potential Agents for Neuroprotection in Alzheimer's, Parkinson's, and Other Neurodegenerative Diseases. Bioorg Med Chem 2005, 13, (3), 773-783. 281. Wolfe, L. C.; Nicolosi, R. J.; Renaud, M. M.; Finger, J.; Hegsted, M.; Peter, H.; Nathan, D. G., A Non-Human Primate Model for the Study of Oral Iron Chelators. Br J Haematol 1989, 72, (3), 456-461. 282. Bergeron, R. J.; Liu, C. Z.; Mcmanis, J. S.; Xia, M. X.; Algee, S. E.; Wiegand, J., The Desferrithiocin Pharmacophore. J Med Chem 1994, 37, (10), 1411-1417. 283. Anderegg, G. R., M., Metal Complex Formation of a New Siderophore Desferrithiocin and of Three Related Ligands. J Chem. Soc., Chem. Commun. 1990, 1194-1196. 284. Bergeron, R. J.; Streiff, R. R.; Wiegand, J.; Vinson, J. R.; Luchetta, G.; Evans, K. M.; Peter, H.; Jenny, H. B., A Comparative Evaluation of Iron Clearance Models. Ann N Y Acad Sci 1990, 612, 378-393. 285. Bergeron, R. J.; Streiff, R. R.; Creary, E. A.; Daniels, R. D., Jr.; King, W.; Luchetta, G.; Wiegand, J.; Moerker, T.; Peter, H. H., A Comparative Study of the Iron-Clearing Properties of Desferrithiocin Analogues with Desferrioxamine B in a Cebus Monkey Model. Blood 1993, 81, (8), 2166-2173. 190

PAGE 191

286. Bergeron, R. J.; Wiegand, J.; Dionis, J. B.; Egli-Karmakka, M.; Frei, J.; Huxley-Tencer, A.; Peter, H. H., Evaluation of Desferrithiocin and Its Synthetic Analogues as Orally Effective Iron Chelators. J Med Chem 1991, 34, (7), 2072-2078. 287. Baker, E.; Wong, A.; Peter, H.; Jacobs, A., Desferrithiocin Is an Effective Iron Chelator in Vivo and in Vitro but Ferrithiocin Is Toxic. Br J Haematol 1992, 81, (3), 424-431. 288. Bergeron, R. J., Oral Iron Chelators Predicated on Desferrithiocin. In Crisp Data Base National Institutes Of Health, 1995. 289. Bergeron, R. J.; Weimar, W. R.; Wiegand, J., Pharmacokinetics of Orally Administered Desferrithiocin Analogs in Cebus Apella Primates. Drug Metab Dispos 1999, 27, (12), 1496-1498. 290. Bergeron, R. J.; Wiegand, J.; Mcmanis, J. S.; Mccosar, B. H.; Weimar, W. R.; Brittenham, G. M.; Smith, R. E., Effects of C-4 Stereochemistry and C-4' Hydroxylation on the Iron Clearing Efficiency and Toxicity of Desferrithiocin Analogues. J Med Chem 1999, 42, (13), 2432-2440. 291. Bergeron, R. J.; Wiegand, J.; Mcmanis, J. S.; Weimar, W. R.; Huang, G., Structure-Activity Relationships among Desazadesferrithiocin Analogues. Adv Exp Med Biol 2002, 509, 167-184. 292. Bergeron, R. J.; Wiegand, J.; Weimar, W. R.; Mcmanis, J. S.; Smith, R. E.; Abboud, K. A., Iron Chelation Promoted by Desazadesferrithiocin Analogs: An Enantioselective Barrier. Chirality 2003, 15, (7), 593-599. 293. Bergeron, R. J.; Mcmanis, J. S.; Franklin, A. M.; Yao, H.; Weimar, W. R., Polyamine-Iron Chelator Conjugate. J Med Chem 2003, 46, (25), 5478-5483. 294. Sangster, J., Octanol-Water Partition Coefficients: Fundamentals and Physical Chemistry. John Wiley & Sons: West Sussex, England, 1997; Vol. 2. 295. Bergeron, R. J.; Wiegand, J.; Weimar, W. R.; Vinson, J. R.; Bussenius, J.; Yao, G. W.; Mcmanis, J. S., Desazadesmethyldesferrithiocin Analogues as Orally Effective Iron Chelators. J Med Chem 1999, 42, (1), 95-108. 296. Bergeron, R. J.; Bharti, N.; Wiegand, J.; Mcmanis, J. S.; Yao, H.; Prokai, L., Polyamine-Vectored Iron Chelators: The Role of Charge. J Med Chem 2005, 48, (12), 4120-4137. 297. Ciechanover, A., Intracellular Protein Degradation: From a Vague Idea Thru the Lysosome and the Ubiquitin-Proteasome System and onto Human Diseases and Drug Targeting. Cell Death Differ 2005, 12, (9), 1178-1190. 191

PAGE 192

298. Cutler, D. F., Introduction: Lysosome-Related Organelles. Semin Cell Dev Biol 2002, 13, (4), 261-262. 299. Ollinger, K.; Brunk, U. T., Cellular Injury Induced by Oxidative Stress Is Mediated through Lysosomal Damage. Free Radic Biol Med 1995, 19, (5), 565-574. 300. Brunk, U. T.; Neuzil, J.; Eaton, J. W., Lysosomal Involvement in Apoptosis. Redox Rep 2001, 6, (2), 91-97. 301. Doulias, P. T.; Christoforidis, S.; Brunk, U. T.; Galaris, D., Endosomal and Lysosomal Effects of Desferrioxamine: Protection of Hela Cells from Hydrogen Peroxide-Induced DNA Damage and Induction of Cell-Cycle Arrest. Free Radic Biol Med 2003, 35, (7), 719-728. 302. Bergeron, R. J.; Mcmanis, J. S.; Liu, C. Z.; Feng, Y.; Weimar, W. R.; Luchetta, G. R.; Wu, Q.; Ortiz-Ocasio, J.; Vinson, J. R.; Kramer, D.; Et Al., Antiproliferative Properties of Polyamine Analogues: A Structure-Activity Study. J Med Chem 1994, 37, (21), 3464-3476. 303. Bergeron, R. J.; Feng, Y.; Weimar, W. R.; Mcmanis, J. S.; Dimova, H.; Porter, C.; Raisler, B.; Phanstiel, O., A Comparison of Structure-Activity Relationships between Spermidine and Spermine Analogue Antineoplastics. J Med Chem 1997, 40, (10), 1475-1494. 304. Bergeron, R. J.; Wiegand, J.; Mcmanis, J. S.; Weimar, W. R.; Smith, R. E.; Algee, S. E.; Fannin, T. L.; Slusher, M. A.; Snyder, P. S., Polyamine Analogue Antidiarrheals: A Structure-Activity Study. J Med Chem 2001, 44, (2), 232-244. 305. Bergeron, R. J.; Mcmanis, J. S.; Weimar, W. R.; Schreier, K. M.; Gao, F.; Wu, Q.; Ortiz-Ocasio, J.; Luchetta, G. R.; Porter, C.; Vinson, J. R., The Role of Charge in Polyamine Analogue Recognition. J Med Chem 1995, 38, (13), 2278-2285. 306. Bergeron, R. J.; Weimar, W. R.; Luchetta, G.; Streiff, R. R.; Wiegand, J.; Perrin, J.; Schreier, K. M.; Porter, C.; Yao, G. W.; Dimova, H., Metabolism and Pharmacokinetics of N 1 ,N 11 -Diethylnorspermine. Drug Metab Dispos 1995, 23, (10), 1117-1125. 307. Bergeron, R. J.; Merriman, R. L.; Olson, S. G.; Wiegand, J.; Bender, J.; Streiff, R. R.; Weimar, W. R., Metabolism and Pharmacokinetics of N 1 ,N 11 -Diethylnorspermine in a Cebus Apella Primate Model. Cancer Res 2000, 60, (16), 4433-4439. 308. Bergeron, R. J.; Weimar, W. R.; Luchetta, G.; Sninsky, C. A.; Wiegand, J., Metabolism and Pharmacokinetics of N 1 ,N 14 -Diethylhomospermine. Drug Metab Dispos 1996, 24, (3), 334-343. 192

PAGE 193

309. Holley, J.; Mather, A.; Cullis, P.; Symons, M. R.; Wardman, P.; Watt, R. A.; Cohen, G. M., Uptake and Cytotoxicity of Novel Nitroimidazole-Polyamine Conjugates in Ehrlich Ascites Tumour Cells. Biochem Pharmacol 1992, 43, (4), 763-769. 310. Gerald M. Cohen, P. M. C., John A. Hartley,Candrew Mather, Martyn C. R. Symonsa and Richard T. Wheelhouse, Targeting of Cytotoxic Agents by Polyamines: Synthesis of a Chlorambucil-Spermidine Conjugate. J. Chem. Soc. Chem. Commun. 1992, (4), 298-300. 311. Verschoyle, R. D.; Carthew, P.; Holley, J. L.; Cullis, P.; Cohen, G. M., The Comparative Toxicity of Chlorambucil and Chlorambucil-Spermidine Conjugate to BALB/C Mice. Cancer Lett 1994, 85, (2), 217-222. 312. Wang, L.; Price, H. L.; Juusola, J.; Kline, M.; Phanstiel, O. T., Influence of Polyamine Architecture on the Transport and Topoisomerase II Inhibitory Properties of Polyamine DNA-Intercalator Conjugates. J Med Chem 2001, 44, (22), 3682-3691. 313. Wang, C.; Delcros, J. G.; Biggerstaff, J.; Phanstiel, O. T., Synthesis and Biological Evaluation of N 1 -(Anthracen-9-ylmethyl)Triamines as Molecular Recognition Elements for the Polyamine Transporter. J Med Chem 2003, 46, (13), 2663-2671. 314. Wang, C.; Delcros, J. G.; Cannon, L.; Konate, F.; Carias, H.; Biggerstaff, J.; Gardner, R. A.; Phanstiel, I. V. O. T., Defining the Molecular Requirements for the Selective Delivery of Polyamine Conjugates into Cells Containing Active Polyamine Transporters. J Med Chem 2003, 46, (24), 5129-5138. 315. Delcros, J. G.; Tomasi, S.; Carrington, S.; Martin, B.; Renault, J.; Blagbrough, I. S.; Uriac, P., Effect of Spermine Conjugation on the Cytotoxicity and Cellular Transport of Acridine. J Med Chem 2002, 45, (23), 5098-5111. 316. Burns, M. R.; Carlson, C. L.; Vanderwerf, S. M.; Ziemer, J. R.; Weeks, R. S.; Cai, F.; Webb, H. K.; Graminski, G. F., Amino Acid/Spermine Conjugates: Polyamine Amides as Potent Spermidine Uptake Inhibitors. J Med Chem 2001, 44, (22), 3632-3644. 317. Dayani, P. N.; Bishop, M. C.; Black, K.; Zeltzer, P. M., Desferoxamine (DFO)--Mediated Iron Chelation: Rationale for a Novel Approach to Therapy for Brain Cancer. J Neurooncol 2004, 67, (3), 367-377. 318. Lovejoy, D. B.; Richardson, D. R., Iron Chelators as Anti-Neoplastic Agents: Current Developments and Promise of the Pih Class of Chelators. Curr Med Chem 2003, 10, (12), 1035-1049. 319. Kicic, A.; Chua, A. C.; Baker, E., Effect of Iron Chelators on Proliferation and Iron Uptake in Hepatoma Cells. Cancer 2001, 92, (12), 3093-3110. 193

PAGE 194

320. Chua, A. C.; Ingram, H. A.; Raymond, K. N.; Baker, E., Multidentate Pyridinones Inhibit the Metabolism of Nontransferrin-Bound Iron by Hepatocytes and Hepatoma Cells. Eur J Biochem 2003, 270, (8), 1689-1698. 321. Kicic, A.; Chua, A. C.; Baker, E., The Desferrithiocin (DFT) Class of Iron Chelators: Potential as Antineoplastic Agents. Anticancer Drug Des 2001, 16, (4-5), 195-207. 322. Kicic, A.; Chua, A. C.; Baker, E., Desferrithiocin Is a More Potent Antineoplastic Agent Than Desferrioxamine. Br J Pharmacol 2002, 135, (6), 1393-1402. 323. Cory, J. G., Ribonucleotide Reductase as a Chemotherapeutic Target. Adv Enzyme Regul 1988, 27, 437-455. 324. Nyholm, S.; Mann, G. J.; Johansson, A. G.; Bergeron, R. J.; Graslund, A.; Thelander, L., Role of Ribonucleotide Reductase in Inhibition of Mammalian Cell Growth by Potent Iron Chelators. J Biol Chem 1993, 268, (35), 26200-26205. 325. Cooper, C. E.; Lynagh, G. R.; Hoyes, K. P.; Hider, R. C.; Cammack, R.; Porter, J. B., The Relationship of Intracellular Iron Chelation to the Inhibition and Regeneration of Human Ribonucleotide Reductase. J Biol Chem 1996, 271, (34), 20291-20299. 326. Seligman, P. A.; Schleicher, R. B.; Siriwardana, G.; Domenico, J.; Gelfand, E. W., Effects of Agents That Inhibit Cellular Iron Incorporation on Bladder Cancer Cell Proliferation. Blood 1993, 82, (5), 1608-1617. 327. Brard, L.; Granai, C. O.; Swamy, N., Iron Chelators Deferoxamine and Diethylenetriamine Pentaacetic Acid Induce Apoptosis in Ovarian Carcinoma. Gynecol Oncol 2006, 100, (1), 116-127. 328. Selig, R. A.; White, L.; Gramacho, C.; Sterling-Levis, K.; Fraser, I. W.; Naidoo, D., Failure of Iron Chelators to Reduce Tumor Growth in Human Neuroblastoma Xenografts. Cancer Res 1998, 58, (3), 473-478. 329. Hider, R. C.; Zhou, T., The Design of Orally Active Iron Chelators. Ann N Y Acad Sci 2005, 1054, 141-154. 330. Bergeron, R. J.; Weimar, W. R.; Muller, R.; Zimmerman, C. O.; Mccosar, B. H.; Yao, H.; Smith, R. E., Effect of Polyamine Analogues on Hypusine Content in Jurkat T-Cells. J Med Chem 1998, 41, (20), 3901-3908. 331. Goddard, J. G.; Kontoghiorghes, G. J., Development of an HPLC Method for Measuring Orally Administered 1-Substituted 2-Alkyl-3-Hydroxypyrid-4-One Iron Chelators in Biological Fluids. Clin Chem 1990, 36, (1), 5-8. 332. Beutler, E.; Hoffbrand, A. V.; Cook, J. D., Iron Deficiency and Overload. Hematology (Am Soc Hematol Educ Program) 2003, 40-61. 194

PAGE 195

333. Pearson, R. G., Hard and Soft Acids and Bases. J Am Chem Soc 1963, 85, 3533-3539. 334. Pearson, R. G., Acids and Bases. Science 1966, 151, 172-177. 335. Casero, R. A., Jr.; Wang, Y.; Stewart, T. M.; Devereux, W.; Hacker, A.; Wang, Y.; Smith, R.; Woster, P. M., The Role of Polyamine Catabolism in Anti-Tumour Drug Response. Biochem Soc Trans 2003, 31, (2), 361-365. 336. Mcgovern, K. A. Synthesis and Cellular Uptake of Polyamines and Their Derivatives. University of Florida, Gainesville, FL, 1983. 337. Aziz, S. M.; Yatin, M.; Worthen, D. R.; Lipke, D. W.; Crooks, P. A., A Novel Technique for Visualizing the Intracellular Localization and Distribution of Transported Polyamines in Cultured Pulmonary Artery Smooth Muscle Cells. J Pharm Biomed Anal 1998, 17, (2), 307-320. 338. Cullis, P. M.; Green, R. E.; Merson-Davies, L.; Travis, N., Probing the Mechanism of Transport and Compartmentalisation of Polyamines in Mammalian Cells. Chem Biol 1999, 6, (10), 717-729. 339. Soulet, D.; Covassin, L.; Kaouass, M.; Charest-Gaudreault, R.; Audette, M.; Poulin, R., Role of Endocytosis in the Internalization of Spermidine-C(2)-Bodipy, a Highly Fluorescent Probe of Polyamine Transport. Biochem J 2002, 367, (Pt 2), 347-357. 340. Tartakoff, A. M., The Golgi Complex: Crossroads for Vesicular Traffic. Int Rev Exp Pathol 1980, 22, 227-251. 341. Ladinsky, M. S.; Mastronarde, D. N.; Mcintosh, J. R.; Howell, K. E.; Staehelin, L. A., Golgi Structure in Three Dimensions: Functional Insights from the Normal Rat Kidney Cell. J Cell Biol 1999, 144, (6), 1135-1149. 342. Pelham, H. R., Traffic through the Golgi Apparatus. J Cell Biol 2001, 155, (7), 1099-1101. 343. Soulet, D.; Gagnon, B.; Rivest, S.; Audette, M.; Poulin, R., A Fluorescent Probe of Polyamine Transport Accumulates into Intracellular Acidic Vesicles Via a Two-Step Mechanism. J Biol Chem 2004. 344. Jentsch, T. J.; Stein, V.; Weinreich, F.; Zdebik, A. A., Molecular Structure and Physiological Function of Chloride Channels. Physiol Rev 2002, 82, (2), 503-568. 345. Schlichter, L. C.; Grygorczyk, R.; Pahapill, P. A.; Grygorczyk, C., A Large, Multiple-Conductance Chloride Channel in Normal Human T Lymphocytes. Pflugers Arch 1990, 416, (4), 413-421. 195

PAGE 196

346. Phipps, D. J.; Branch, D. R.; Schlichter, L. C., Chloride-Channel Block Inhibits T Lymphocyte Activation and Signalling. Cell Signal 1996, 8, (2), 141-149. 347. Puljak, L.; Kilic, G., Emerging Roles of Chloride Channels in Human Diseases. Biochim Biophys Acta 2006, 1762, (4), 404-413. 348. Kang, J. X.; Man, S. F.; Brown, N. E.; Labrecque, P. A.; Clandinin, M. T., The Chloride Channel Blocker Anthracene 9-Carboxylate Inhibits Fatty Acid Incorporation into Phospholipid in Cultured Human Airway Epithelial Cells. Biochem J 1992, 285 (Pt 3), 725-729. 349. Tanaka, H.; Matsui, S.; Kawanishi, T.; Shigenobu, K., Use of Chloride Blockers: A Novel Approach for Cardioprotection against Ischemia-Reperfusion Damage. J Pharmacol Exp Ther 1996, 278, (2), 854-861. 350. Noboru H.; Masana Y.; Nobuko S.; Yasunori M.; Mitsuaki Y., Participation of Chloride Channel for Cardioprotective Effect Induced by Isoflurane in Guinea Pig Single Cardiac Myocyte. Anesthesiology 2001, 95, A604. 351. Jiang, B.; Hattori, N.; Liu, B.; Nakayama, Y.; Kitagawa, K.; Inagaki, C., Suppression of Cell Proliferation with Induction of P21 by Cl(-) Channel Blockers in Human Leukemic Cells. Eur J Pharmacol 2004, 488, (1-3), 27-34. 352. Bortner, C. D.; Cidlowski, J. A., Apoptotic Volume Decrease and the Incredible Shrinking Cell. Cell Death Differ 2002, 9, (12), 1307-1310. 353. Suzuki, M.; Morita, T.; Iwamoto, T., Diversity of Cl(-) Channels. Cell Mol Life Sci 2006, 63, (1), 12-24. 354. Wei, L.; Xiao, A. Y.; Jin, C.; Yang, A.; Lu, Z. Y.; Yu, S. P., Effects of Chloride and Potassium Channel Blockers on Apoptotic Cell Shrinkage and Apoptosis in Cortical Neurons. Pflugers Arch 2004, 448, (3), 325-334. 355. Lang, F.; Foller, M.; Lang, K. S.; Lang, P. A.; Ritter, M.; Gulbins, E.; Vereninov, A.; Huber, S. M., Ion Channels in Cell Proliferation and Apoptotic Cell Death. J Membr Biol 2005, 205, (3), 147-157. 356. Bergeron, R. J.; Muller, R.; Bussenius, J.; Mcmanis, J. S.; Merriman, R. L.; Smith, R. E.; Yao, H.; Weimar, W. R., Synthesis and Evaluation of Hydroxylated Polyamine Analogues as Antiproliferatives. J Med Chem 2000, 43, (2), 224-235. 196

PAGE 197

BIOGRAPHICAL SKETCH Hua Yao was born on March 29, 1970, to Guo Wei Yao and Hui Min Song in Lanzhou, China. The family moved to Xian, China where she attended middle school and high school. In July of 1992, she graduated with a bachelor of science degree from Tianjin University of Science and Technology. After graduation, she worked in Xian Institute of Dairy Science for two years. In February of 1995, she came to the United States and worked as a full time biological technician in the research laboratory of Dr. Raymond J. Bergeron in the College of Pharmacy at the University of Florida. She began her PhD study in August of 2001 with Dr. Bergeron. Her graduate research emphasized applying polyamines as vectoring agents for the intracellular transport of therapeutic compounds, particularly iron chelators. She intends to pursue a career in the pharmaceutical industry where she can enhance her knowledge and share and extend her experience. 197