Nitric Oxide Donors: For Pharmaceutical and Biological Applications

Nitric Oxide Donors Edited by Peng George Wang, Tingwei Bill Cai, Naoyuki Taniguchi

Further Titles of Interest

O. Kayser, R. H. Müller (Eds.)

C.-H. Wong (Ed.)

Pharmaceutical Biotechnology

Carbohydrate-based Drug Discovery

Drug Discovery & Clinical Applications

2 Volumes

2004 ISBN: 3-527-30554-8

J.-C. Sanchez, G. L. Corthals, D. F. Hochstrasser (Eds.)

Biomedical Application of Proteomics 2004 ISBN: 3-527-30807-5

2003 ISBN: 3-527-30632-3

H. Buschmann, T. Christoph, E. Friderichs, C. Maul, B. Sundermann (Eds.)

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G. Müller, S. Petry (Eds.)

Lipases and Phospholipases in Drug Development From Biochemistry to Pharmacology 2004 ISBN: 3-527-30677-3

Nitric Oxide Donors For Pharmaceutical and Biological Applications

Edited by Peng George Wang, Tingwei Bill Cai, Naoyuki Taniguchi

Editors Professor Dr. Peng George Wang 876 Biological Sciences Building 484 W. 12th Avenue The Ohio State University Columbus, OH 43210, USA Dr. Tingwei Bill Cai 876 Biological Sciences Building 484 W. 12th Avenue The Ohio State University Columbus, OH 43210, USA Professor Dr. Naoyuki Taniguchi Department of Biochemistry Graduate School of Medicine Osaka University Medical School 2-2 Yamadoka, Suita Osaka 565-0871, Japan

All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate. Library of Congress Card No.: applied for British Library Cataloguing-in-Publication Data: A catalogue record for this book is available from the British Library. Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the Internet at http://dnb.ddb.de c 2005 WILEY-VCH Verlag GmbH & Co KGaA,
Weinheim Printed on acid-free and chlorine-free paper All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Composition Steingraeber Satztechnik GmbH, Dossenheim Printing Strauss GmbH, Mörlenbach Bookbinding Litges & Dopf Buchbinderei GmbH, Heppenheim Printed in the Federal Republic of Germany ISBN-13 978-3-527-31015-9 ISBN-10 3-527-31015-0

V

Contents

Part 1 Chemistry of NO Donors 1

1

1.1 1.1.1 1.1.2 1.2 1.3 1.3.1 1.3.2 1.4 1.4.1 1.4.2 1.5 1.5.1 1.5.1.1 1.5.1.2 1.5.1.3 1.5.1.4 1.5.2 1.5.2.1 1.5.2.2 1.5.3 1.5.4 1.5.5 1.5.6 1.6

NO and NO Donors 3 Tingwei Bill Cai, Peng George Wang, and Alvin A. Holder Introduction to NO Biosynthesis and NO donors 3 Nitric Oxide Synthases 3 Chemistry of Reactive Nitrogen Species 6 Classification of NO Donors 7 New Classes of NO Donors under Development 9 Nitroarene 9 Hydroxamic Acids 10 Development of NO-Drug Hybrid Molecules 10 Nitrate Hybrid Molecules 11 Furoxan Hybrid Molecules 13 New Therapeutic Applications of NO Donors 14 NO Donors against Cancer 15

Diazeniumdiolates (NONOates) as Promising Anticancer Drugs 15 The Synergistic Effect of NO and Anticancer Drugs 17 NO-NSAIDs as a New Generation of Anti-tumoral Agents 17 Other NO Donors with Anticancer Activity 18 NO against Virus 19 HIV-1 Induces NO Production 19 Antiviral and Proviral Activity of NO 21 Inhibition of Bone Resorption 22 Treatment of Diabetes 23 Thromboresistant Polymeric Films 23 Inhibition of Cysteine Proteases 24 Conclusion 24 References 26

Nitric Oxide Donors Peng George Wang, Tingwei Bill Cai, Naoyuki Taniguchi (Eds.) c 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Copyright
ISBN: 3-527-31015-0

VI

Contents

2

2.1 2.1.1 2.1.2 2.1.3 2.1.4 2.1.5 2.1.6 2.1.7 2.2 2.3

3

3.1 3.2 3.2.1 3.2.2 3.2.3 3.2.4 3.3 3.3.1 3.3.2 3.3.3 3.3.4 3.4 3.4.1 3.4.2 3.4.3 3.5 3.5.1 3.5.2 3.5.2.1 3.5.2.2 3.5.3 3.5.4 3.5.4.1 3.5.4.2 3.5.4.3 3.5.5

Organic Nitrates and Nitrites 33 Roger Harrison Organic Nitrates 34

Direct Chemical Reaction between Organic Nitrates and Thiols 35 Glutathione-S-transferase 36 Cytochrome P-450-dependent Systems 37 Membrane-bound Enzyme of Vascular Smooth Muscle Cells 38 Xanthine Oxidoreductase 38 Mitochondrial Aldehyde Dehydrogenase 40 Tolerance 42 Organic Nitrites 44 Conclusions 45 References 47

N-Nitroso Compounds 55 Arindam Talukdar, Peng George Wang Introduction 55 N-Nitrosamines 56 Synthesis of Nitrosamines 58

Physical Properties and Reactions of N-Nitrosamines 59 Structure–Activity Relationship of N-Nitrosamines 61 Application of N-Nitrosamines 62 N-Hydroxy-N-nitrosoamines 63 Biologically Active N-Hydroxy-N-nitrosamine Compounds 64 Synthesis of N-Hydroxy-N-nitrosamines 66 Properties of N-Hydroxy-N-nitrosamines 68 Reactivity of N-Hydroxo-N-nitrosamines 70 N-Nitrosimines 72 Mechanism of Thermal Reaction of N-Nitrosoimine 73 Properties of N-Nitrosoimines 74 Synthesis of N-Nitrosoimines 75 N-Diazeniumdiolates 75 Mechanism of NO Release 76 Synthesis of N-Diazeniumdiolates 77 Ionic Diazeniumdiolates 79 O-derivatized Diazeniumdiolates 79 Reactions of N-Diazeniumdiolates 79 Clinical Applications 80 Reversal of Cerebral Vasospasm 80 Treatment of Impotency 81 Nonthrombogenic Blood-contact Surfaces 81 Future Directions 81 References 83

Contents

4

4.1 4.1.1 4.1.1.1 4.1.2 4.1.2.1 4.1.2.2 4.1.2.3 4.1.2.4 4.1.2.5 4.2 4.2.1 4.2.2 4.2.3 4.3

5

5.1 5.1.1 5.1.2 5.1.3 5.1.4 5.2 5.3 5.4

6

6.1 6.1.1 6.1.1.1 6.1.1.2 6.1.1.2.1 6.1.1.2.2 6.1.1.2.3 6.1.1.2.4 6.1.1.3 6.1.1.4

The Role of S-Nitrosothiols in the Biological Milieu 91 Bulent Mutus Structure and Cellular Reactivity of RSNOs 91 RSNO Structure 91 Enzymatic Consumption of RSNOs 92 Formation of RSNOs in the Biological Milieu 93 Nitrite Mediated 93 NO Mediated 93 NO Oxidation Products Mediated 93 Metalloprotein Mediated 95 Transnitrosation 98 Postulated Physiological roles of RSNOs 99 Regulation of Blood Flow by HbSNO 99

Regulation of Ventilatory Response in the Brain by RSNOs 100 Role of RSNOs in Platelet Function 100 Conclusion 102 References 103

Metal–NO complexes: Structures, Syntheses, Properties and NO-releasing Mechanisms 109 Tara P. Dasgupta, Danielle V. Aquart Iron Complexes 110 Nitroprusside 110 Iron Porphyrin Nitrosyls 114 Dinitrosyl Complexes (DNICs) 116 Iron–Sulfur Cluster Nitrosyls 117 Ruthenium Complexes 118 Other Metal Nitrosyls 121 Conclusion 122 References 123

The NO-releasing Heterocycles 131 Alberto Gasco, Karl Schoenafinger Heterocyclic N-oxides 131 Furoxans 131 General Properties 132 Synthesis 134 Dimerisation of nitrile oxides 134

Dehydrogenation of á-dioximes (glyoximes) 135 Action of nitrogen oxides on olefins 136 Other methods 137 NO-release 137 Biological Actions 140

VII

VIII

Contents

6.1.1.4.1 6.1.1.4.2 6.1.1.4.3 6.1.1.5 6.1.2 6.1.2.1 6.1.2.2 6.1.2.3 6.1.2.4 6.1.3 6.1.3.1 6.1.3.2 6.1.3.3 6.2 6.2.1 6.2.1.1 6.2.1.2 6.2.1.3 6.2.1.4 6.2.2 6.2.2.1 6.2.2.2 6.2.2.3 6.3

7

7.1 7.2 7.2.1 7.2.1.1 7.2.1.2 7.2.2 7.2.2.1 7.2.2.2 7.2.2.3 7.3 7.3.1 7.3.2 7.3.3 7.4

Condensed furoxans 140 Furoxan sulfones and carbonitriles 141 Furoxancarboxamides 144 NO-donor Hybrid Furoxans 145 3,4-Dihydro-1,2-diazete 1,2-dioxides (1,2-diazetine 1,2-dioxides) 147 Generalities 147 Synthesis 148 NO-release 149 Biological Properties 150 Other Heterocyclic N-oxides 151 4H-pyrazol-4-one 1,2-dioxides (pyrazolone N,N-dioxides) 152 2H-1,2,3-triazole 1-oxides 153 1,2,3,4-Benzotetrazine 1,3-dioxides and 1,2,3-Benzotriazine 3-oxides 153 Mesoionic Heterocycles 154 Sydnonimines 155 General Properties 155 Synthesis 156 NO-release 157 Biological Properties 161 Mesoionic Oxatriazoles 163 Synthesis 164 NO-release 165 Biological Properties 167 Other Heterocyclic Systems 168 References 170

C-Nitroso Compounds, Oximes, N-Hydroxyguanidines and N-Hydroxyureas 177 S. Bruce King Introduction 177 C-Nitroso Compounds 177 Alkyl and Aryl C-Nitroso Compounds 177 Syntheses and Properties 177 NO-releasing Mechanisms 178 Acyl C-Nitroso Compounds 179 Syntheses and Properties 179 NO-releasing Mechanisms 180 Structure–Activity Relationships 181 Oximes 182 Syntheses and Properties 182 NO-releasing Mechanisms 184 Structure–Activity Relationships 185 N-Hydroxyguanidines 186

Contents

7.4.1 7.4.2 7.4.3 7.5 7.5.1 7.5.2 7.5.3

Syntheses and Properties 186 NO-releasing Mechanisms 187 Structure–Activity Relationships 188 N-Hydroxyureas 189 Syntheses and Properties 189 NO-releasing Mechanisms 191 Structure–Activity Relationships 193 References 195

Part 2 NO Donors’ Applications in Biological Research 201

8

Vasodilators for Biological Research 203 Anthony Robert Butler, Russell James Pearson

8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9 8.10 8.11

NO-donor Drugs for Biological Research 203 Sodium Nitrite (NaNO2 ) 203 S-Nitrosothiols 204 Metallic Nitrosyls 209 Sodium Nitroprusside (Na2 [Fe(CN)5 NO] ⋅ 2H2 O) 209 Organic Nitrates 212 Organic Nitrites 215 NONOates 216 NO Inhalation; NO Gas as an NO Donor 219 Sydnonimines 222 Conclusion 225 References 226

9

NO Donors as Antiplatelet Agents 233 Anna Kobsar, Martin Eigenthaler Introduction 233

9.1 9.2 9.2.1 9.2.1.1 9.2.1.2 9.2.1.3 9.2.1.3.1 9.2.1.3.2 9.2.1.3.3 9.2.1.3.4 9.2.1.3.5 9.2.1.3.6 9.2.1.3.7 9.2.2

Molecular Mechanisms of NO-mediated Platelet Inhibition 233 cGMP-dependent NO Signaling Mechanisms 234 Regulation of cGMP Levels 235 Effector Sites of cGMP 236 cGMP-PK I Substrates in Platelets 237 Inositol triphosphate (IP3 ) receptor 237 Rap 1b 238 Vasodilator stimulated phosphoprotein (VASP) 238 Heat shock protein hsp27 239 LASP 239 Thromboxane A2 (TxA2 ) receptor 240 Phosphodiesterase PDE5 240 cGMP-independent NO Signaling Mechanisms 240

IX

X

Contents

9.3 9.3.1 9.3.1.1 9.3.1.2 9.3.1.3

9.3.2 9.3.3 9.3.4 9.3.4.1 9.3.4.2 9.3.4.3 9.3.4.4 9.3.4.5 9.3.4.6 9.3.4.7 9.3.5 9.3.5.1 9.3.6 9.3.6.1 9.3.6.2 9.3.7 9.3.7.1 9.3.7.2 9.3.8 9.4 9.4.1 9.4.2

9.5 9.6 9.7

10

10.1 10.2

Effects of Different Groups of NO Donors on Platelets 241 Diazeniumdiolates 241 DEA/NO (Sodium 2-(N,N-diethylamino)-diazenolate-2-oxide) 241 DETA NONOate ((Z)-1-[N-(2-Aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate) 241 MAHMA NONOate ((Z-1-[N-Methyl-N-[6-(N-methylammoniohexyl) amino]]diazen-1ium-1,2-diolate) 242 Sodium Nitroprusside (SNP) 242 Molsidomine (3-Morpholino-sidnonimine; SIN-1) 242 S-Nitrosothiols 243 SNAP (S-Nitroso-N-acetyl-d,1-penicillamine) 243 SNVP (S-Nitroso-N-valerylpenicillamine) 243 GSNO (S-nitroso-glutatione) 243 CysNO (S-Nitrosocysteine) 244 SNAC (S-Nitroso-N-acetyl-cysteine) 244 HomocysNO (S-Nitrosohomocysteine) 244 RIG200 (N-(S-Nitroso-N-acetylpenicillamine)-2-amino-2-deoxy1,3,4,6, tetra-O-acetyl-beta-d-glucopyranose) 244 Organic Nitrates 245 GTN (Glyceryl Trinitrate, Nitroglycerin, NTG) 245 Mesoionic Oxatriazole Derivatives 245 GEA-3162 (1,2,3,4-Oxatriazolium, 5-amino-3-(3,4-dichlorphenyl)-, cloride) 245 GEA-3175 (1,2,3, 4-Oxatriazolium, -3-(3-chloro-2-methylphenyl)-5[[(4-methylphenyl) sulfonyl]amino]-, Hydroxide Inner Salt) 246 Other NO Donors 246 OXINO (Sodium trioxdinitrate or Angel’s Salt) 246 B-NOD (2-Hydroxy-benzoid acid 3-nitrooxymethyl-phenyl ester) 246 l-Arginine (l-Arg) 246 Activators of Soluble Guanylyl Cyclase 247 YC-1 (3-(5′-Hydroxymethyl-2′-furyl)-1-benzyl indazole) 247 BAY 41-2272 (3-(4-Amino-5-cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)1H-pyrazolo [3,4-b]pyridine) 247 cGMP Analogs 247 Inhaled NO and Platelet Inhibition 248 Conclusion 248 References 249

Control of NO Production 255 Noriko Fujiwara, Keiichiro Suzuki, Naoyuki Taniguchi Introduction 255 Structure of Nitric Oxide Synthase 256

Contents

10.3 10.4 10.4.1 10.4.2 10.5 10.5.1 10.5.1.1 10.5.1.2 10.5.2 10.5.3 10.5.4 10.5.5 10.6 10.7 10.8 10.8.1 10.8.2 10.9

NO Formation 257 l-Arginine and l-Arginine Derivatives 257 Inhibitors 258 Substrates 259 Non-amino Acid Inhibitors and Non-amino Acid Substrates 261 Guanidine 262 Inhibitor 262 Substrates 262 Isothiourea (ITU) 265 Amidine 267 Cyclic Amidines are Potent iNOS Selective Inhibitors 268 Indazole 269 Inhibition of NOS Function Targeted towards Cofactors 270 Regulators of NOS Gene Expression 270 NO Formation by an NOS-independent Pathway 271 Oxime 272 Hydroxyurea 272 Summary 273 References 274

Part 3 Clinical Applications of NO Donors 283

11

11.1 11.2 11.3 11.4 11.5 11.6 11.7

12

12.1 12.2 12.3

Nitric Oxide Donors in Cardiovascular Disease 285 Martin Feelisch, Joseph Loscalzo Introduction 285

Clinical Cardiovascular Applications of NO Donor Therapy – Past and Present 285 Pharmacological Cardiovascular Mechanism of Action of NO Donors 288 Clinically Available NO Donors: Structures and Mechanism of Action 290 Nitrate Tolerance 293 Is Nitrate Therapy Associated with Adverse Vascular Effects? 295 Conclusions 295 References 297

Nitric Oxide Donors as Anti-platelet Agents for Thromboembolic Disorders: Clinical Status and Therapeutic Prognosis 299 David R. Janero, David S. Garvey Introduction 299 Human Platelets, Thromboembolic Disorders, and NO 300 Nitrovasodilators 307

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Contents

12.3.1 12.3.2 12.3.3 12.4 12.4.1 12.5 12.5.1 12.6 12.7 12.8

Glyceryl Trinitrate, Nitroglycerin (GTN) 307 Isosorbide Dinitrate (ISDN) and Isosorbide Mononitrate (ISMN) 311 Sodium Nitroprusside (SNP) 312 Oxatriazolium NO Donors 314 Sydnonimines 314 Nitrosothiol NO Donors 316 S-Nitroso-glutathione (GSNO) 316 l-Arginine {S(+)-2-Amino-5-[(aminoiminomethyl)amino]pentanoic acid} (l-arg) 318 NCX-4016 [2-Acetoxybenzoate 2-(1-nitroxy-methyl)-phenyl ester] 319 Conclusion and Future Prospects 320 References 323

13

NO and Gene Regulation 329 Jie Zhou, Bernhard Brüne

13.1 13.2 13.2.1 13.2.2 13.3 13.3.1

Formation of NO and RNI-signaling 329 p53 Regulation under the Impact of RNI 331 Basic Considerations: p53 Phosphorylation and Mdm2 Binding 331 Molecular Mechanisms of RNI-evoked p53 Stabilization 332 HIF-1á Regulation under the Impact of RNI 333 Lessons from Hypoxia: Basic Considerations of HIF-1á Stability Regulation 333 Stability Regulation of HIF-1á by NO/RNI in Normoxia versus Hypoxia 335 RNI, p53 and HIF-1 in Tumor Biology 337 Conclusions 339 Abbreviations 341 References 342

13.3.2 13.4 13.5

14

Nitric Oxide and Central Nervous System Diseases 347 Elizabeth Mazzio, Karam F. A. Soliman

14.1

General Overview – Gaining Control over Various NOS Enzymes that Concurrently Contribute to Degenerative CNS Diseases 347 Signaling Controls – Neuronal NOS: TYPE-I 349 Neurotransmission 349 Neuronal Calcium Homeostasis 351 Signaling Controls, Endothelial NOS: Type-3 352 EDRF/Vascular Tone 352 eNOS, Cyclic AMP/GMP Regulation 353 Signaling Controls, Inducible NOS: Type-2 354 Inflammation, Microglia and Astrocytes 354 Stress Activated and Extra-cellular Kinases 355 Cyclic AMP/Protein Kinase a 356

14.2 14.2.1 14.2.2 14.3 14.3.1 14.3.2 14.4 14.4.1 14.4.2 14.4.3

Contents

14.4.4 14.4.5 14.5 14.5.1 14.5.2 14.5.3 14.5.4

Cyclic AMP–Phosphodiesterase Inhibitors 358 Peroxisome Proliferator-activated Receptor-gamma 358 The Neurotoxicity of NO 359 Oxidative Stress 359 Mitochondrial Impairment 361 Permeability Transition Pore Complex, Apoptosis 363 Excitotoxicity, Poly(ADP-ribose)-polymerase-1 365 References 369

Index 385

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XV

Preface The discovery of the physiological and pathophysiological roles of nitric oxide (NO) during the 1980s was one of the most surprising and exciting developments in biological research. NO exhibits a broad range of biological activities. Thus, it comes as no surprise that, as far back as 1992, the editors of the journal Science called NO the molecule of the year, and in 1998, three scientists, R.F. Furchgott, L.J. Ignarro, and F. Murad, were awarded the Nobel Prize in physiology and medicine for their contribution to elucidating the role of nitric oxide in the functions of living organisms. As a simple diatomic free radical, NO is generally considered to represent the biologically important form of the endothelium-derived relaxing factor (EDRF). Cellular NO is almost exclusively generated via the oxidation of L-arginine, which is catalyzed by nitric oxide synthetases (NOS). Under physiological conditions, NO directly activates soluble guanylate cyclase (sGC) to transform guanosine triphosphate (GTP) into cyclic guanosine monophosphate (cGMP), followed by kinase-mediated signal transduction. The endogenous formation of NO plays a key role in many bioregulatory systems, including smooth muscle relaxation, platelet inhibition, neurotransmission, and immune stimulation. Due to the instability and inconvenient handling of aqueous solutions of authentic NO, there is increasing interest in using compounds capable of generating NO in situ. These compounds are called NO donors, or NO releasing agents. Glyceryl trinitrate (GTN) may be the most well known NO donor. Although the use of GTN for medicinal purposes dates back more than 150 years, little had been revealed about its physiological mechanism of action before the 1980s. It is well known that the epoch-making invention realized by Alfred Nobel in 1863 paved the way for controlled detonation of GTN. Therefore, when Nobel’s physician recommended GTN as a treatment of his angina pectoris, Nobel wrote: “Isn’t it the irony of fate that I have been prescribed N/G 1 [nitroglycerine] to be taken internally! They called it Trinitrin, so as not to scare the chemist and the public.” Nobel would not have found it ironic if he had known that it was NO, released from GTN in vivo, that helps relieve angina. In addition to organic nitrates, many other chemicals can be transformed into NO in vitro or in vivo. Due to the diversity of NO donor structures, the pathway for each class of compounds to generate NO could differ significantly, e.g., enzymatical vs. Nitric Oxide Donors Peng George Wang, Tingwei Bill Cai, Naoyuki Taniguchi (Eds.) c 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Copyright
ISBN: 3-527-31015-0

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Preface

non-enzymatical, reductive vs. oxidative, etc. As each class of compounds offers distinct biochemical properties, this allows us to choose a compound that best meets the demands of specific investigations. Insufficient NO production causes serious medical problems. Many diseases such as hypertension, atherosclerosis and restenosis involve the deficiency of NO production. Therefore, a compound that can release NO under specific conditions can be used therapeutically to palliate NO underproduction. In fact, the best known NO donor, glyceryl trinitrate, has been used for over a century to relieve acute attacks of angina pectoris. In 1998, Carl Djerassi published a book entitled “NO”, where he plotted the success of a biotech company producing NO donor compounds to treat male impotence. In reality, NO donor compounds have a variety of biomedical applications. Our latest search using the keyword “nitric oxide donor” at ScienceFinder revealed that there are 2,880 published research papers on NO donors. More importantly, there have been 105 US and world patents on the applications of NO donors in the treatment of cardiovascular diseases, central nervous systems diseases, diseases related to immunity, physiological disorders and many other medical situations. Besides supplementation of NO in a situation where a NO insufficiency may underlie the pathology, NO donors can also regulate NO-based physiological pathways, i.e., male erectile dysfunction, and improve drug safety and efficacy, such as gastrointestinal toxicity of non-steroidal anti-inflammatory drugs. Since the mid-1980s, the development of new NO donors has offered several advantages over the previous NO donors, such as spontaneous releasing NO, donating NO under controlled rates, and even targeting NO to certain tissues. The current trends in NO donor development include discovery of new NO donors, finding novel applications of old NO donors, development of NO-drug hybrids and site-specific delivery of NO. Although a number of reviews and books on NO have been published, we felt that there was a need to publish a comprehensive text addressing the basic principles of all aspects of NO donors. This book is not only an informative resource for basic scientists in the NO field, but also for all clinicians and biologists interested in the applications of NO donors. This 14-chapter book is divided into three sections ranging from the basic chemistry of NO donors to clinically applied science. The first seven chapters present a review of medicinal chemistry of all classes of NO donors. The next three chapters continue to discuss the application of NO donors and NO inhibition in biological research. The final four chapters of the book address other important issues on biological functions of NO donors. Integrating internationally recognized authors for each chapter was not an easy job. We really appreciate the help from all these hard-working authors. We are also grateful to the editors at Wiley-VCH – without their continuous support this project would never have been possible. We would like to sincerely thank faculty members, postdoctoral fellows, graduate and undergraduate students who have contributed so much in Wang’s and Taniguchi’s laboratories on nitric oxide research. These people are Libing Yu, Zhengmao Guo, Andrea McGill, Johnny Ramirez, Jun Li, Ming Xian, Adam Janczuk, Yongchun Hou, Vladislav Telyatnikov, Yingxin Zhang, Xuejun Wu, Alvin A. Holder, Qiang Jia, Zhong Wen, Xiaoping Tang, Xinchao Chen, Jaime Martin Franco, Mingchuan Huang, Dongning Lu, Arindam Talukdar, Noriko

Preface

Fujiwara, Satoshi Kazuma, and Yasuhide Miyamoto. P. George Wang acknowledges the continuing funding support (NIH 54074) over the past ten years from the National Institute of Health on the development of nitric oxide donors. Naoyuki Taniguchi was supported by the 21st Center of Excellence Program funded by the Ministry of Education, Culture, Sports, Science and Technology, Japan. December 2004

Peng George Wang Tingwei Bill Cai Naoyuki Taniguchi

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List of Contributors

Danielle V. Aquart Department of Chemistry University of the West Indies Mona, Kingston 6 Jamaica

Tara P. Dasgupta Chemistry Department University of the West Indies Mona Campus, Kingston 7 Jamaica

Bernhard Brüne University of Kaiserslautern Faculty of Biology Department of Cell Biology Erwin-Schroedinger-Straße 13/4 67663 Kaiserslautern Germany

Martin Eigenthaler Universität Würzburg Medizinische Poliklinik Klinikstr. 6–8 97070 Würzburg Germany

Anthony Robert Butler University of St Andrews Bute Medical School The Bute Building St Andrews Fife KY16 9TS Scotland United Kingdom Tingwei Bill Cai The Ohio State University Department of Chemistry 100 W 18th Ave Columbus, OH 43210 U.S.A.

Martin Feelisch Boston University School of Medicine 650 Albany, X304 Boston, MA 02118 U.S.A. Noriko Fujiwara Department of Biochemistry Hyogo College of Medicine 1-1 Mukogawa-cho Nishinomiya Hyogo 663-8501 Japan David S. Garvey NitroMed Inc. 125 Spring St. Lexington, MA 02421 U.S.A.

Nitric Oxide Donors Peng George Wang, Tingwei Bill Cai, Naoyuki Taniguchi (Eds.) c 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Copyright
ISBN: 3-527-31015-0

XX

List of Contributors

Alberto Gasco Faculty of Pharmacy University of Turin Via Pietro Giuria 9 10125 Turin Italy Roger Harrison Department of Biology and Biochemistry University of Bath Claverton Down Bath, BA2 7AY United Kingdom Alvin A. Holder Colorado State University Department of Chemistry Fort Collins, CO 80523 U.S.A. David R. Janero NitroMed Inc. 125 Spring St. Lexington, MA 02421 U.S.A. Kiyomi Kikugawa School of Pharmacy Tokyo University of Pharmacy and Life Science 1432-1 Horinouchi Tokyo 192-0392 Japan S. Bruce King Wake Forest University Department of Chemistry Salem Hall Winston-Salem, NC 27109 U.S.A.

Anna Kobsar Universität Würzburg Institut für Klinische Biochemie und Pathobiochemie Josef-Schneider-Str. 2 97080 Würzburg Germany Joseph Loscalzo Boston University School of Medicine 715 Albany Street, E113 (Dept. Medicine) W507 (Whitaker CVI) Boston, MA 02118 U.S.A. Elisabeth Mazzio College of Pharmacy and Pharmaceutical Sciences Florida A & M University Tallahassee FL 32307 U.S.A. Bulent Mutus Chemistry and Biochemistry University of Windsor Essex Hall, 401 Sunset Avenue Windsor, Ontario N9B 3P4 Canada Russell James Pearson University of St Andrews School of Chemistry Purdie Building St Andrews Fife KY16 9ST Scotland United Kingdom Karl Schoenafinger Synthetic Medicinal Chemistry Aventis Pharma Deutschland Industriepark Hoechst 65926 Frankfurt am Main Germany

List of Contributors

Karam F. A. Soliman College of Pharmacy and Pharmaceutical Sciences Florida A & M University Tallahassee FL 32307 U.S.A.

Naoyuki Taniguchi Department of Biochemistry Graduate School of Medicine Osaka University Medical School, 2-2 Yamadoka, Room B-1, Suita Osaka 565-0871 Japan

Keiichiro Suzuki Department of Biochemistry Hyogo College of Medicine 1-1 Mukogawa-cho Nishinomiya Hyogo 663-8501 Japan

Peng George Wang The Ohio State University Faculty Departments of Chemistry and Biochemistry 484 W 12th Avenue, Columbus, OH 43210 U.S.A.

Arindam Talukdar The Ohio State University Department of Chemistry and Biochemistry 484 W 12th Avenue Columbus, OH 43210 U.S.A.

Jie Zhou University of Kaiserslautern Faculty of Biology Department of Cell Biology Erwin Schroedinger Straße 13/4 67663 Kaiserslautern Germany

XXI

Part 1 Chemistry of NO Donors

3

1

NO and NO Donors Tingwei Bill Cai, Peng George Wang, and Alvin A. Holder

Nitric oxide (NO), a magic free radical gas molecule, has been shown to be involved in numerous physiological and pathophysiological processes. Among its diverse functions, NO has been implicated in the relaxation of vascular smooth muscle, the inhibition of platelet aggregation, neurotransmission (Viagra reverses impotence by enhancing an NO-stimulated pathway), and immune regulation [1]. It was named the molecule of the year in 1992 by Science and was the subject of the Nobel Prize in 1998. NO has limited solubility in water (2–3 mM), and it is unstable in the presence of various oxidants. This makes it difficult to introduce as such into biological systems in a controlled or specific fashion. Consequently, the development of chemical agents that release NO is important if we are to target its bioeffector roles to specific cell types for biological and pharmacological applications. Based on our comprehensive review of NO donors [2], this chapter focuses on recent progress and current trends in NO donor development and novel applications which are not covered by the following chapters.

1.1

Introduction to NO Biosynthesis and NO donors 1.1.1

Nitric Oxide Synthases

Endogenous NO is produced almost exclusively by l-arginine catabolism to l-citrulline in a reaction catalyzed by a family of nitric oxide synthases (NOSs) [3]. In the first step, Arg is hydroxylated to an enzyme-bound intermediate Nù -hydroxy-l-arginine (NHA), and 1 mol of NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) and O2 are consumed. In the second step, NHA is oxidized to citrulline and NO, with consumption of 0.5 mol of NADPH and 1 mol of O2 (Scheme 1.1). Oxygen activation in both steps is carried out by the enzyme-bound heme, which derives electrons from NADPH. Mammalian NOS consists of an N-terminal oxyNitric Oxide Donors Peng George Wang, Tingwei Bill Cai, Naoyuki Taniguchi (Eds.) c 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Copyright
ISBN: 3-527-31015-0

4

1 NO and NO Donors

H2N

NH2 NH

H2N

NH

1 NADPH

O

N OH

COO

Arginine

NH

0.5 NADPH

N O

O2 H2O

O2 H2O H3N

NH2

H3N

COO

H2N

N w-Hydroxyarginine

COO

Citrulline

Nitric Oxide

Scheme 1.1 Endogenous synthesis of nitric oxide.

genase domain that binds iron protoporphyrin IX (heme), 6-(R)-tetrahydrobiopterin (H4 B) and Arg, and a C-terminal reductase domain that binds FMN (flavin mononucleotide), FAD (flavin adenine dinucleotide), and NADPH, with a calmodulin binding motif located between the two domains. To be active, two NOS polypeptides must form a homodimer. The reductase domains each transfer NADPH-derived electrons, through FAD and FMN, to the heme located in the adjacent subunit. Three distinct isoforms of NOS have been identified – neuronal, macrophage and endothelial types, and each is associated with a particular physiological process (Table 1.1). Constitutive endothelial NOS (eNOS or NOS III) regulates smooth muscle relaxation and blood pressure; constitutive neuronal NOS (nNOS or NOS I) is involved in neurotransmission and long-term potentiation; the NO produced from inducible NOS (iNOS or NOS II) in activated macrophage cells acts as a cytotoxic agent in normal immune defense against microorganisms and tumor cells. The constitutive isoforms (nNOS and eNOS) require added Ca2+ and calmidulin for activity and produce a relatively small amount of NO, while the inducible isoform (iNOS) has tightly bound Ca2+ and calmodulin, and produces a relatively large amount of NO. Tab. 1.1: Properties of NOS isoforms.

NOS nNOS (NOS-I) iNOS (NOS-II) eNOS (NOS-III)

Locations Brain, spinal cord, peripheral Macrophages, other tissues Endothelium

Characteristics Constitutive, Ca2+ dependent Inducible, Ca2+ independent Constitutive, Ca2+ dependent

Major Biological Functions Neuromediator Host defender, cytotoxic Vasodilator tone modulator

The first step of an NOS catalyzed reaction is a “classical” P450-dependent Nhydroxylation of a guanidine, except for the involvement of H4 B. As shown in Scheme 1.2, Fe(III)heme 1 first accepts one electron to give Fe(II)heme 2, which binds O2 to produce ferrous-dioxy heme 3. The second electron from H4 B reduces 3 to peroxyiron 4. Arg donates a proton to 4 to facilitate O–O bond cleavage to generate an oxo-iron (IV) cation radical species 5, which then rapidly hydroxylates the neutral guanidinium to NHA [4]. The second step of NOS oxidation is a greater challenge to enzymologists since there is no direct analogy in other systems. A variety of proposed reaction steps can be

1.1 Introduction to NO Biosynthesis and NO donors

FMNH2

FMNH .

H4B

O2

O2

FeIII

FeII

S

S

S

1

2

3

NH2 R . + H4B N + N H H H _ O O

FeII

NH2 R

NH2 R

N H N H _ OH O

4

OH

N

N H

FeIII

FeIV +. S 5

FeII S

5

S

1

Scheme 1.2 The first step of NOS reaction.

roughly summarized in three mechanisms (Scheme 1.3). The popular Mechanism I was proposed by Marletta and modified by Ingold and others [5, 6], a superoxoiron(III)heme intermediate 6 abstracts the hydrogen atom of the NHA to furnish an iminoxy radical 8, which upon nucleophilic attack by the hydroperoxoiron(III)heme 7 on its carbon generates NO and citrulline. This mechanism, however, appears not to be supported by the crystal structure analysis of the NOS-NHA complex [7–9] or by a recent spectral study [10]. The second mechanism was proposed by Groves (Mechanism II), where the NOS-catalyzed aerobic oxidation of NHA occurs via a radical-type auto-oxidation process [11, 12], i.e., NHA is oxidized by the Fe(III) heme to generate an iminoxyl radical 8, which tautomerizes to the á-nitroso radical 12. Insertion of a dioxygen molecule between 12 and Fe(II) heme forms an energetic á-nitrosoperoxy Fe(III) heme intermediate that decomposes to generate NO [13, 14]. However, direct ligation of NHA to heme iron has been precluded by the X-ray crystallographic data [7–9]. The third mechanism, proposed by Silverman and others [15–18], mainly inMechanism I NH2 R

N H

NH2 8

NH2

N OH

R

O 2 , e-

N H

R

O

N O

H

O FeIII

H2N R

N O O H

N H

N H

N H

O

O

FeIII

6

R

H

O

O

FeIII

NH2

N O

OH

9

FeIII

7

+ NO

10

FeIII

Mechanism II NH2

NH2 R

N H

R

N OH -H+

N H

N O

FeIII

N H

N H III

Fe

R

OH O2 , e

-

N H O

N O

NH2 OH

R

N H O

Fe

O2

R

H2N

N O

12

6

Fe

Scheme 1.3 The second step of NOS reaction.

NH2

III

R +

-H

16

R

Fe

III

18

+

NO

FeIV 14

13

N N H O

NH2 O-

FeIII

R

N H

O

+ NO

O-

O

O

O O

H2 N

17

-

O

NH2 N H

O

N O

N H

R

N H O FeIII

15 H

N O H O

O III

N O

N H

8

11

N H

NH2

R

FeII

NH2

NH2 R

N H

FeIII

Mechanism III

NH2

R

19

FeIII

20

6

1 NO and NO Donors

volves the oxidation of the nitrogen on the protonated N-hydroxyguanidino moiety (Mechanism III). It was suggested that the initial N–H bond cleavage by superoxoiron(III)heme 6 generates a radical cation intermediate 15, which, upon heterolysis of the O–H bond, gives the iminoxy radical 17. The nucleophilic attack of peroxoiron(III)heme 18 on 17 gives an intermediate similar to 13, which decomposes to NO and citrulline. More recently, Stuehr has emphasized the involvement of H4 B in the second step of the NOS reaction [19–21]. 1.1.2

Chemistry of Reactive Nitrogen Species

One of NO’s major biological actions is to activate guanylate cyclase directly to generate cyclic guanosine monophosphate (cGMP) as an intracellular second messenger, followed by kinase-mediated signal transduction. In another pathway, NO undergoes oxidation or reduction in biological systems to convert to many different reactive nitrogen species (RNS). It can react with molecular oxygen (O2 ), superoxide anion (O2 −• ) or transition metals (M) to produce RNS such as N2 O3 , NO2 , NO2 − , NO3 − , peroxynitrite (OONO− ), and metal-nitrosyl adducts (Route A, Scheme 1.4) [22, 23]. Among these RNS, peroxynitrite stands out as an important species [24, 25]. The reaction between NO and O2 −• produces peroxynitrite at a diffusion controlled rate [26–28]. Peroxynitrite is a strong oxidizing and nitrating species that causes molecular damage leading to disease-causing cellular dysfunction [29, 30]. NO can also be rapidly oxidized by oxygen, superoxide or transition metals to nitrosonium (NO+ ) which reacts with nucleophilic centers such as ROH, RSH and RR′NH to produce RO–NO, RS–NO or RR′N–NO, respectively (Route B, Scheme 1.4) [31, 32]. These products subsequently undergo other reactions to exhibit their biological effects. In addition, NO also undergoes a one-electron reduction to produce nitroxyl (NO− ) (Route C, Scheme 1.4). The reducing potential of this reduction is approximately +0.25 V [33]. Nitroxyl converts rapidly to N2 O under physiological conditions. Other competing reactions RONO ROH

RSNO RSH

NO+

L-Arg

RR'-NO RR'NH oxidation

NH2OH

NOS

RSH

reduction

NO

B H2O NO2-

H2O2 ONOO-

M M-NO

NO-

N2O

C M

A O2-

M-NO

ONOO-

M NO2-

O2

NO2 / N2O3

O2-

NO3( NO2- )

O2

NO NO2-

ONOO-

O2

H2O

RS-NO

NO2- / NO3-

Scheme 1.4 Oxidation and reduction of reactive nitrogen species.

M M-NO

1.2 Classification of NO Donors

of nitroxyl include addition to thiol groups (singlet NO− ) to generate NH2 OH, and reaction with oxygen (triplet NO− ) to form peroxynitrite (OONO− ). Nitroxyl has also been proven to exhibit many biological functions [34], such as vasodilatation [35–37] and cytotoxicity [38–40].

1.2

Classification of NO Donors

Intensive research on the biological functions of NO and other reactive nitrogen oxide species demands exogenous sources of NO donors as research tools and pharmaceuticals. Since the mid-1980s, the development of new NO donors has offered several advantages over the previous NO donors, such as spontaneous release of NO, donation of NO under controlled rates, and even the targeting of NO to certain tissues. The structural dissimilarities of the diverse NO donors have led to remarkably varied chemical reactivities and NO-release mechanisms. Generally NO donors release NO through three kinds of mechanisms. The first route is that donating NO spontaneously, which releases NO through thermal or photochemical self-decomposition of e.g. S-nitrosothiols, diazeniumdiolates, oximes. The second route is that releasing NO by chemical reactions with acid, alkali, metal and thiol. Organic nitrates, nitrites and syndnonimines give NO though this mechanism. The third route is enzymatic oxidation where NO donors, for example, N-hydroxyguanidines, need metabolic activation by NO synthases or oxidases for NO release. Some NO donors release NO by more than one route, e.g. organic nitrates can also generate NO by enzymatic catalysis. Classification of all NO donors could be confusing, since all nitrogen– oxygen-bonded compounds have the potential to decompose, be oxidized, or be reduced to produce reactive nitrogen species. However, similar chemical structures usually have a similar NO-releasing mechanism, so all current NO donors and their pathways of NO generation are summarized in Table 1.2 according to their chemical classification. Many medicines may work by an NO-dependent mechanism. Recent studies have shown that angiotensin-converting enzyme (ACE) inhibitors (i.e. Enalapril, Captropril, Cilazapril) improve endothelium-dependent vasodilator responsiveness [41–43]. ACE inhibitors inhibit the degradation of bradykinin, thereby augmenting NO production. Another calcium channel blocker, amlodipine, also releases NO from blood vessels, and kinins mediate the generation of NO [44]. These new findings give a good explanation for the cardioprotective effects of these drugs. Furthermore, estrogen, statins (HMG-CoA reductase inhibitor) and essential fatty acids have the ability to augment NO synthesis [45, 46]. All of the above molecules do not have structural moieties which can release NO directly, so they can be called NO stimulators, and they are not discussed in this book. Currently used NO donors will be introduced in the following chapters.

7

8

1 NO and NO Donors Tab. 1.2: Current major classes of NO donors.

Chemical Class

Representative Compounds

Pathway of NO Generation Non-enzymatic

Enzymatic

Thiols

Cyt-P450, GST, etc

Hydrolysis, transnitrosation, thiols, light, heat Light, thiols, reductants, nucleophiles

Xanthine oxidase, etc

OH− , light

Cyt-P450 related enzyme

Light, heat

Peroxidases

Thiols, light

?

Spontaneous, enhanced by thiols, light, metal ions

Unknown enzymes

Light, heat

?

Spontaneous, thiols

?

Thiols

Unknown enzyme

Thiols

?

Spontaneous, enhanced by light, oxidants, pH>5

Prodrugs require enzymatic hydrolysis

Spontaneous, O2 /FeIII -porphyrin

Cyt-P450

ONO2

Organic nitrate

O2NO ONO2 H3C O

Organic nitrite

H 3C

Metal-NO complex

Na2 [Fe(CN)5 (NO)]•2H2 O

NO

H3C

HO NO

N-Nitrosamine

N Me OH

N-Hydroxyl nitrosamine

O- NH4+

N

A membranebound Enzyme

N O Me

+N _

Nitrosimine

N

N

Nitrosothiol

O

N

O

N

AcHN S

O

CO2H

C-Nitroso compound Diazetine dioxide

O2N

N

R1 R2

N

R3

N

+ _O

R4

R

+ _

N

N

O

O Ar

+

N

Oxatriazole5-imine

O-

+

R

Furoxan & benzofuroxan

O

N

_

N

NH.HCl

O

NH

Syndonimine

O

N

R2

Oxime

N

+

_N

O

R1 NOH

O2 N

CONH2

1.3 New Classes of NO Donors under Development Tab. 1.2 (continued)

Chemical Class

Representative Compounds

Pathway of NO Generation Non-enzymatic

Enzymatic

Anto-oxidation enhanced by metal ions

Catalase/H2 O2

Oxidants

NOS, Cyt-P450

H2 O2 /CuZn-SOD or ceruloplasimin, H2 O2 /Cu2+ , heme-containing proteins

Peroxidase

?

Guanylate cyclase

H

Hydroxyamine

N

OH

H

NH

N-Hydroxyguanidine & guanidine

HO N H

CO2H

N H

NH2 O

Hydroxyurea

H2N

NH OH

O OH

Hydroxamic acid

N H

1.3

New Classes of NO Donors under Development

Different types of NO donors will be discussed in the other chapters except for the following two classes. 1.3.1

Nitroarene

6-Nitrobenzo[á]pyrene (6-nitroBaP) was found to release NO under visible-light irradiation, while no such photodegradation was observed with other nitrated BaPs, such as 1- and 3-nitroBaPs [47]. It can induce DNA strand breaks upon photoirradiation. NO is generated from 6-nitroBaP via 6-nitriteBaP, which is produced from 6-nitroBaP by an intramolecular rearrangement mechanism (Scheme 1.5). This finding may be useful for the development of a new type of photochemically triggered NO donors. NO hν

NO2

NO2

6-nitroBaP

6-nitriteBaP

Scheme 1.5 Photochemical reaction of 6-nitroBap.

O 6-Oxy-BaP radical

9

10

1 NO and NO Donors

1.3.2

Hydroxamic Acids

Hydroxamic acids [general formula RC(O)N(R′)OH] have been used as inhibitors of peroxidases [48], ureases [49] and matrix metalloproteinases, and as anti-hypertensive, anti-cancer, anti-tuberculous and antifungal agents [50, 51]. Although some of these bioactivities are attributed to the chelating ability of the hydroxamate group, the hypotensive effects are due to their ability to release NO [52]. Experiments have shown that hydroxamic acids can transfer NO to ruthenium(III) and cause vascular relaxation in rat aorta by activation of the iron-containing guanylate cyclase enzyme. Of the hydroxamic acids investigated, benzohydroxamic acid (Fig. 1.1) showed higher NO releasing ability than aceto-, salicyl-, and anthranilic hydroxamic acids.

O N H

OH Fig. 1.1 Benzohydroxamic acid.

1.4

Development of NO-Drug Hybrid Molecules

An innovative approach to harnessing the beneficial properties of NO is to attach an NO-releasing moiety to an existing drug (Fig. 1.2). Different hybrid compounds can offer various drug actions with synergistic effects, with reduced toxicity and side effects. Several pharmaceutical companies are actively engaged in this research area. A series of compounds are currently in the Phase-I or Phase-II clinical study. For example, Nicox in France (www.nicox.com) has developed the NO-releasing derivative of acetylsalicyclic acid, NCX-4016, which is claimed to be able to overcome the major drawback associated with the use of aspirin as a pain reliever [53]. NCX-4016 also shows a broader mechanism than aspirin and can inhibit additional inflammatory mediators [54]. NitroMed in Boston (www.nitromed.com) has reported that nitrosylated á-adrenoreceptor antagonists moxisylate (S-NO-moxisylate) had lower toxicity and fewer adverse side effects in the treatment of erectile dysfunction [55].

General:

Drug

NO CH3

OAc O O NCX-4016

Fig. 1.2 NO-drug conjugates.

CH3

O

CH3

ONO2

N

O

H3C H3C

CH3

S-NO-Moxisylyte

SNO CH3

1.4 Development of NO-Drug Hybrid Molecules

1.4.1

Nitrate Hybrid Molecules

Recently, the combination of a nitrate moiety with another bioactive substructure in a single molecule has received particular attention. Nitric-oxide-releasing nonsteroidal anti-inflammatory drugs (NO-NSAIDs) are chemical entities obtained by adding a nitroxyalkyl moiety via an ether linkage to a conventional NSAID, such as NO-aspirin (NCX-4016), a prototype NO-NSAID. Because the use of NSAIDs is associated with significant gastro-intestinal toxicity, the development of safer NSAIDs, NO-NSAIDs, has been demonstrated to be rational and successful [56]. NO-NSAIDs inhibit inflammation via cyclo-oxygenase (COX)-dependent and independent NO-related effects. The mechanism can be explained by a general feature of NO, its capability to modify proteins that contain cysteine residues by causing the S-nitrosation of thiol groups in the enzyme catalytic center [57]. It has been proved that NCX-4016 causes the S-nitrosation and inhibition of interleukin(IL)-1â converting enzyme(ICE)-like cysteine proteases (caspases) involved in pro-IL-1â and pro-IL-18 processing, which are pivotal in the pro-inflammatory cytokine hierarchy. The NO-NSAIDs are not only devoid of gastro-intestinal toxicity, but are also more effective anti-inflammatory drugs than their parent compounds (Scheme 1.6). A similar compound, B-NOD, showed a similar activity, and does not affect blood pressure [58]. NCX-4215 and NCX-456 are designed according to the same principle. NO-NSAIDs are currently undergoing clinical Phase II trials. NO releasing

O O

NO

Inhibition of caspase

O O

Anti-inflammatory

ONO2

effect

NCX-4016 Hydrolysis Acetyl salicyclic acid

Inhibition of prostaglandin-forming COXs

Scheme 1.6 Anti-inflammatory mechanism of NO-NSAIDs.

NCX-1000 (Fig. 1.3) is another prodrug obtained by adding a nitroxybutyl moiety to ursodeoxycholic acid (UDCA), a steroid that is selectively metabolised by hepatocytes [59]. Because NCX-1000 is almost exclusively metabolised in the liver, NO is delivered directly to hepatic cells. It has been demonstrated that NCX-1000 protects against liver damage induced by Con A in mice by modulating the live-resident immune system, and reduces portal pressure in cirrhotic rats. This drug may provide a novel therapy for the treatment of patients with portal hypertension or immunomediated liver injury [60]. Using a similar approach, NCX-1015, a nitro-prednisolone, was designed and showed NO-releasing ability in biological fluids [61]. It was more

11

12

1 NO and NO Donors

O OH O O

O

ONO2

OH O

4

O

O

ONO2

O ONO2

NH2

NCX-4215

NCX-456

B-NOD

O O

O

4

ONO2

O O

O H

OMe

HO

H

H

ONO2

H H

HO

O OH

H

OH

H

O NCX-1000

NCX-1015

Fig. 1.3 Structures of NO-NSAIDs.

potent than prednisolone in acute and chronic inflammation. NO not only synergizes with the glucocorticoid moiety to produce the anti-inflammatory effect, but also counteracts the osteoclast activity of prednisolone, which causes a major side-effect of glucocorticoid drugs. Moreover, NCX-1015 showed significant bronchodilating activity [62]. As inhaled NO has been suggested as a useful therapy to induce airway dilation [63–65], NO-linked steroids may be a very effective therapy for airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). The potential synergism between NO and steroids locally released in a damaged tissue, may reduce the therapeutically effective doses of steroids and prevent the side-effects of steroids. The combination of beta-blockers with nitrovasodilators is an efficient therapeutic approach in coronary heart disease. Therefore, nitration of beta-blockers could produce an NO donor, while keeping its beta adrenergic receptor blocking effects. The S-S enantiomer of a metoprolol derivative named PF9404C (Fig. 1.4) was synthesized accordingly [66]. Pharmacological and biochemical experiments showed that when in contact with living cells, PF9404C can generate substantial amounts of NO, leading to cyclic GMP formation and vasorelaxation. Unlike rapid NO donors, PF9404C produces a slowly developing and sustained relaxation of the vessel. Its beta-blocking potency is close to that of S(-)-propranolol, four-fold higher than metoprolol. When beta-blockers alone are administered to hypertensive patients, the total peripheral resistance remains higher than in normotensives. If PF9404C is used in hypertensive patients, the NO actions, including potent vasodilating actions, inhibition of leukocyte–endothelial cell interaction, as well as platelet adherence and aggregation and vascular smooth muscle cell proliferation, may exert beneficial effects in hypercholesterolemia and ischaemic heart injury. Thus, PF9404C exhibits antihypertensive

1.4 Development of NO-Drug Hybrid Molecules

O OH

O

N H

O OH

ONO2

N H

O

ONO2 Fig. 1.4 Structures of â-blocker NO hybrid.

and cardioprotective actions through a double mechanism, NO donation and betablockade. Several reports indicate the involvement of superoxide in the mediation of tolerance [67–69]. Based on these reports, a bifunctional superoxide dismutase-mimic NO donor was designed by Haj-Yehia’s group [70]. The nitrate ester was incorporated into a nitroxide such as 3-hydroxymethyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (HMP) by its conversion into 3-nitratomethyl-PROXYL (NMP) (Scheme 1.7). HMP is a stable, metal-independent, low-molecular weight SOD-mimic with excellent cellpermeability. So NMP is the first compound that can simultaneously generate NO and destroy superoxide. This may lead to novel nontolerance-inducing nitrovasodilators. OH Nitric / sulfuric acid N O

-5 °C, 20 min

HMP

ONO2 N O NMP

Scheme 1.7 Preparation of NMP.

1.4.2

Furoxan Hybrid Molecules

H2 -antagonists are a class of drugs used in the management of peptic ulcer and gastric-acid-related disorders. Since they fail to trigger protection against gastric damage induced by NSAIDs, it would be useful to combine the antisecretory activity of H2 -receptor antagonists with the NO-dependent gastroprotective effect in the same molecule. Some new H2 -antagonist-containing NO-donor moieties have been synthesized [71]. The H2 -antagonistic substructures were derived from lamtidine and tiotidine, respectively. NO-releasing moieties were chosen from phenysulfonyl furoxan, nitrates and nitrosothio functions. The experimental results showed that only the hybrid compounds were able both to antagonize histamine effects on guinea pig papillary muscle and to display in vivo antisecretory and gastroprotective action. The best results were obtained with the lamtidine/furoxan hybrid structure, such as

13

14

1 NO and NO Donors

H2-Antagonistic Substructure

NO-Donor Moiety

Spacer

CN N N

O

3N

N H

H

O 2

SO2Ph N

21

O

N

O

Fig. 1.5 General structure of H2 -antagonist/NO hybrids and example compound.

21 (Fig. 1.5), while others hybrids showed ambiguous results. These compounds could be the prototypes of a new class of drugs, which may be useful in the therapy of gastric hypersecretion combined with inflammatory disorders. Nicorandil is an antianginal drug, which has the properties of both K+ channel openers and NO donors [72]. Structurally, it is a nicotinamide derivative with a nitrate group in its chemical structure (Fig. 1.6). The hybrid molecules of furoxan and nicorandil derivatives may achieve an ideal cardiovascular drug with good selectivity, efficiency and low toxicity [73]. A series of hybrid drugs designed by linking the furoxan ring to nicorandil analogues was investigated. Several of these compounds had good vasodilatory activity [74]. Compound 22 was further tested for its hypotensive effects in anaesthesized rats, and was able to significantly reduce blood pressure 3 h after administration. Its hypotensive effects could prevail for a further 3 h. These preliminary results indicate that the furoxan-nicorandil derivatives are a useful lead in the design of NO-donor compounds for hypertension. O

O N H

2 ONO2

N

O N

N N

H3CO Nicorandil

CH3 O

N

O

22

Fig. 1.6 Structures of Nicorandil and its hybrid.

1.5

New Therapeutic Applications of NO Donors

Due to the numerous possible reactions and related biological consequences, inappropriate overproduction of NO can cause a series of disease states such as a variety of neurodegeneration diseases including inflammation, rheumatic disease, septic shock, diabetes mellitus, and cerebral ischemia. Therefore development of isoformspecific NOS inhibitors to regulate NO synthesis has been an active research area.

1.5 New Therapeutic Applications of NO Donors

On the other hand, insufficient NO production also causes serious medical problems. Many diseases such as hypertension, atherosclerosis and restenosis involve a deficiency of NO production. Therefore, a compound that can release NO under specific conditions can be used therapeutically to palliate NO underproduction. In fact, the best known NO donor, glyceryl trinitrate, has been used for over a century to relieve acute attacks of angina pectoris. In 1998, Carl Djerassi published a book titled “NO”, where he plotted the success of a biotech company producing NO donor compounds to treat male impotence [75]. Currently, NO donor compounds have a variety of biomedical applications. Though current understanding of NO physiology and pathology seems incomplete, the largely indirect, correlative information suggests that both NO excess and insufficiency can elicit tissue injury and diseases. So far the most purported NO-insufficiency diseases are cardiovascular. The oldest NO donor drug, glyceryl trinitrate, has been used as a vasodilator since 1879. Besides supplementation of NO in a situation where an NO insufficiency may underlie the pathology, NO donors can also regulate an NObased physiological pathway, i.e., male erectile dysfunction, and improve drug safety and efficacy, i.e., gastrointestinal toxicity of nonsteroidal anti-inflammatory drugs. In addition to cardiovascular disorders [76], nerve system diseases [77] and inflammation [78], the benefit of NO in many other diseases has now been recognized. 1.5.1

NO Donors against Cancer

NO produced by activated macrophages plays an important role in modulating the host defense mechanism against tumor cells [79, 80]. Several in vitro studies have also shown that NO donors are cytotoxic to tumor cells leading to apoptosis, mainly involving changes in mitochondrial permeability transition and release of cytochrome c from the mitochondria [81]. NO released from inducible NO synthase inhibits metastasis at a higher level, but at a lower concentration, NO may cause induction of NO resistance and permit the growth of tumor cells [82]. Although there are reports indicating the genotoxicity of NO, exposure of whole cells to NO donors resulted in no appreciable mutations as compared to alkylating agents [83]. The reason is that large amounts of NO are required to generate DNA alterations and the formation of other reactive species and nitrosated DNA from NO is limited. There are also numerous defense mechanisms, such as ascorbate and glutathione, as well as intraand intercellular consumption of NO, which limit the mutation of DNA [84]. The dual role of NO in carcinogenesis is very confusing, so further studies are still needed to clarify it [85]. 1.5.1.1

Diazeniumdiolates (NONOates) as Promising Anticancer Drugs

Diazeniumdiolate compounds have already shown anti-leukemia activity [86]. However, these NO donors release NO systemically and cause severe side effects on the vascular system, so their therapeutical use has been limited. Upon modulation at the

15

1 NO and NO Donors

16 OO N O2N

N+

F

N

NO2

CH3

N

N

O

O

O

N

HN O

O JS-K

S

N

N N O

HOOC

5-FU/NONOate

NO

O

O

OH O

HO HO HO

H N

HN O

S-Nitrosocaptopril

S

CH3 CH3 CH3 NO

Man-1-SNAP

Fig. 1.7 Anticancer NO donors.

oxygen with nitro-aromatic substituents, these derivatives can release NO in target cells after a hydrolytic or enzymatic action. JS-K (Fig. 1.7), an example of O-protected diazeniumdiolate developed by the US National Cancer Institute (NCI), has attracted much attention [87]. JS-K can be attacked by the nucleophilic thiol group of glutathione (GSH), with the formation of the Meisenheimer complex; then the NONOate moiety leaves, and at physiological condition, the NONOate decomposes to release NO (Scheme 1.8). The aryl moiety of JS-K is bonded to the thiol group of GSH to give S-(2,4-dinitrophenyl)glutathione (DNP-SG). The drug kills, or slows the growth, of cancer cells without harming healthy cells [88]. For example, acute myeloid leukemia (AML) is the most common and deadly form of leukemia. Tests with the drug showed it triggered the destruction of AML cells grown in vitro. In the HL-60 human myeloid leukemia assay system, the IC-50 of JS-K is 0.5 ìM, while the IC-50 of a chemotherapeutic agent, daunorubicin, is 0.01 ìM. It also slowed the growth of AML cells in mice. In other tests on cell cultures, JS-K did the same, but to a lesser extent, in prostate, colon, and breast cancer cells. It also inhibited the growth of prostate cancer cells in mice. JS-K is found to react with glutathione S-transferases (GST), which help pump foreign substances out of certain cells. GSTs help the liver get rid of toxic substances in blood, but they also help cancer cells resist chemotherapy drugs. When GSTs in cancer cells interact with JS-K, there are two anticancer effects: GST activity is inhibited, making the cells less resistant to chemotherapy drugs, and NO is released. O HO

N H

O

S

JS-K + GSH -O

NH2

H N O

O-

O

N+

N N+ O-

O OH

NO2

HO

O

N H

O

NH2

H N S

OH O

O NO2

N N

O O

NO2

JS-K Meisenheimer complex -O

N

ON+

DNP-SG

N N

O 2 NO

+

HN

N O

Scheme 1.8 Mechanism of JS-K.

O

pH 7.4 O 4-Carbethoxy-PIPERAZI/NO

1.5 New Therapeutic Applications of NO Donors

It has been shown that JS-K inhibited cell growth with concomitant activation of mitogen-activated protein kinase (MAPK) members, ERK, JNK, p38 and their downstream effectors, c-Jun and AP-1 [89]. Inhibitors of these MAPK pathways abrogated the growth inhibitory actions of JS-K. In addition to the actions of JNK as a kinase for c-Jun, it was shown that c-Jun is also an ERK target. Furthermore, JS-K generated NO in culture and NO inhibitors antagonized both MAPK induction and the growth inhibitory effects of JS-K. These results suggest two possible mechanisms for the mediation of JS-K growth inhibitory actions, namely, NO-induction of MAPK pathway constituents as well as possible arylation reactions. The data support the idea that prolonged MAPK activation by JS-K action is important in mediating its growthinhibitory actions. In 2003, NCI accepted JS-K into its Rapid Access to Intervention Development (RAID) program, which tries to speed the development of new cancer therapies. Work done so far by the University of Utah within the RAID program has shown that JS-K is active against a broad spectrum of cancer cells. JS-K thus represents a promising platform for novel growth inhibitory analog synthesis. 1.5.1.2

The Synergistic Effect of NO and Anticancer Drugs

The 5-fluorouracil (5-FU) and NONOate conjugates (Fig. 1.7) were prepared and their cytotoxicity was tested [90]. The median effect doses of the conjugates for DU145 and HeLa cancer cell lines were 2–4-fold lower than that of 5-FU. In another study by Wink et al., the cytotoxicity of cisplatin was enhanced about 60-fold after NONOate pretreatment for 30 min [91]. The enhancement of cytotoxicity of 5-FU/NONOate conjugates and cisplatin-NONOate combination has shown that there is a synergistic effect between anticancer drugs and NO. Another study by Jia et al. demonstrated that the cytotoxicity of Taxol was enhanced by S-nitrosocaptopril (Fig. 1.7) [92]. This effect is primarily mediated via the increased influx of Taxol by NO into intracellular compartments, while NO-induced cytotoxicity cannot be excluded. In another separate study, researchers found organic nitrates can also increase the efficiency of cytostatic therapy and retard the development of drug resistance [93]. The combined therapy results in significant increase in life span and number of survivors among mice bearing leukemia P388 and L-1210. Comparative studies of organic nitrates and a similar compound in which ONO2 moieties were replaced by OH groups demonstrated that the presence of NO2 is required for adjuvant activity of compounds and confirmed that NO modifies the antitumor effects of cytostatics. It was also shown that the NO donor retards the development of drug resistance to cyclophosphamide. 1.5.1.3

NO-NSAIDs as a New Generation of Anti-tumoral Agents

Non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, were utilized primarily to protect from inflammation. Other biological effects were also found gradually, for example, induction of apoptosis [94, 95], stimulation of immune activity

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1 NO and NO Donors

[96], and inhibition of angiogenesis [97]. Studies of patients with familial adenomatous polyposis (FAP) have demonstrated a reduction by approximately 50% of colonic adenomas or colorectal cancer among patients while using aspirin [98]. The growing evidence suggests that tumor inhibition may be mediated by at least two major cellular events. These involve the ability of NSAIDs to maintain the equilibrium between proliferation and apoptosis rates in colonocyte, and their inhibitory effect on angiogenesis [99, 100]. The main problem with the regular use of NSAIDs is the occurrence of side effects such as the increased risk of gastrointestinal bleeding and the developments of ulcers [101]. In recent years, NO-NSAIDs, such as NO-aspirin (NCX 4016) (Fig. 1.2), have been developed as “safe” NSAIDs [102]. NO-aspirin is 2500–5000-fold more potent than traditional aspirin in inhibiting the growth of colon cancer cells in vitro [103]. The corresponding test in mice also confirmed the strong inhibitory effect of NO-aspirin in intestinal carcinogenesis and suggests that NONSAIDs merit further evaluation as chemopreventive agents against colon cancer [104]. A detailed study showed that NO-aspirin inhibits â-catenin/T cell factor (TCF) signaling in colon cancer cells by disrupting the nuclear â-catenin/TCF association, whereas aspirin has no affect [105]. Two clinical trials of aspirin for the prevention of cancer were published in March 2003 [106, 107]. In one study, 635 patients with a recent history of adenomas received either the placebo or 325 mg of aspirin per day. A colonoscopy was performed on 81% of the patients after at least one year. One or more adenomas were found in 17% of patients in the aspirin group compared to 27% in the placebo group. Another study gave a similar result. These two studies indicate that daily use of aspirin is associated with a significant reduction in the incidence of colorectal adenomas in patients with previous colorectal cancer. If aspirin therapy is stopped, the reduction in the risk of adenomas dissipates. Also, NSAIDs may decrease the incidence of carcinomas of the esophagus, stomach, breast, lung, prostate, urinary bladder and ovary [108]. The clinical use of these agents is limited to patients with FAP. Due to the clear protective effect of aspirin, NO-NSAIDs can be a good alternative, which can give the beneficial effects of both NO and NSAIDs. In March 2003, NCI awarded a grant to the University of Michigan to conduct a clinical trail of NO-releasing aspirin (NCX-4016). This placebo-controlled study will assess the pharmacokinetics of different doses of NCX 4016 in patients at risk of colon cancer. 1.5.1.4

Other NO Donors with Anticancer Activity

As well as NONOates, other NO donors also showed anticancer activity independently. Sodium nitroprusside (SNP), a metal-NO complex, showed cytotoxic effects on the cells of some patients with malignant lymphoma (ML), acute myelocytic leukemia (AML) or chronic myelomonocytic leukemia (CMMoL), but not with multiple myeloma [109]. SNP and cytosine arabinoside (Ara-C) did not share the drug resistance. Interestingly, SNP had no effect on lymphocytes of healthy volunteers. These results suggest that SNP has an anti-tumor effect on human hematological malignant cells.

1.5 New Therapeutic Applications of NO Donors

A series of sugar-S-nitrosothiols (sugar-SNAPs), for example, glucose-1-SNAP, have shown promising pharmacokinetic properties [110]. These compounds were designed based on the observation that facilitated transport of monosacharrides in mammalian cells was accomplished by the glucose transporter family of transmembrane properties. They were constructed from an aglycone unit conjugated with a mono- or oligosaccharide. Compared to SNAP, sugar-SNAPs had higher stability and slower NO-releasing properties in aqueous solution. Glucose-SNAPs were more cytotoxic than SNAP. The enhanced cytotoxicity of glucose-1-SNAP and glucose-2SNAP may be related to their affinity for glucose transporters present on plasma membranes, but relative experiments have not yet been done. Another possible explanation is that glucose-SNAP binds to glucose transporters and decomposes to release NO, then NO causes the apoptosis of cancer cells. Recently, mannose-SNAPs were also developed, for example Man-1-SNAP (Fig. 1.7). The cytotoxicity of Man-1SNAP was just as potent as that of glucose-SNAP [111]. Hydroxamic acid derivatives, which belong to a new class of NO donors, have been shown to inhibit the matrix metalloproteinases (MMPs) [112]. MMPs are a family of zinc-dependent endopeptidases, which play a critical role in multiple steps in the metastatic cascade and facilitate neoangiogenesis. Numerous hydroxamic acids, such as marimastat, have been developed, that bind the zinc atom in the active catalytic domain of MMPs. During a randomized Phase III trial, comparing marimastat with placebo in patients with metastatic breast cancer, marimastat was not associated with an improvement in progression-free survival or overall survival. Other studies also indicated no benefit for MMP inhibitors when used either in combination with chemotherapy or sequentially after first-line chemotherapy in a variety of cancers [113]. Currently, many pharmaceutical companies have suspended clinical development of this kind of agent. 1.5.2

NO against Virus 1.5.2.1

HIV-1 Induces NO Production

It was found that the HIV envelope glycoprotein in vitro increases the production of NO by human monocyte-derived macrophages [114]. NO production is increased in patients who have AIDS [115], and the increased concentrations of nitrite in AIDS patients with opportunistic infections is caused by T gondii, Pneumocystis carinii, Mycobacterium tuberculosis, and Mycobacterium avium, whereas nitrite concentrations are normal in symptom-free patients. It was also confirmed that there was increased production of NO in the sera of children with HIV-1 infection, and of circulating cytokines, such as interleukin 1â, tumor necrosis factor á, and interferon ã. It is postulated that rises in the concentrations of these cytokines may represent a substantial stimulation of NO production [116]. In contrast, it has been shown that there was no altered endogenous nitrate formation in eight patients with AIDS, most of whom had opportunistic infections [117]. It has also been noted that there were high

19

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1 NO and NO Donors

nitrite and nitrate concentrations in 39 patients with AIDS, especially in those with lower CD4 cell counts, whereas in symptom-free patients no such increase was seen [118]. However, AIDS patients with opportunistic infections were not selected for assessment of NO production. Groeneveld and colleagues [119] have shown that serum nitrate concentrations are higher in symptom-free HIV-1-infected patients than in healthy individuals. NO production was measured in vitro from peripheral blood leucocytes of HIV-1-infected patients after measuring nitrite concentrations from peripheral blood mononuclear cells and polymorphonuclear leucocytes [120, 121]. An increase in nitrite production in AIDS patients, especially in those with opportunistic infections, was also seen. There is substantial induction of the iNOS gene in primary cultures of human monocyte-derived macrophages, concomitant with the peak of virus replication, and exposure to low concentrations of NO donors results in a significant increase in HIV-1 replication [122]. Acute infection of macaques with a pathogenic strain of the simian immunodeficiency virus increased gene expression of iNOS in mononuclear cells obtained from bronchoalveolar lavage [123]. At the time of systemic viral load peak, NO production was greatly raised in the monkeys [123]. The activated lung macrophages of neonatal rats produced significantly more NO than did those of infant and adult rats [124]. Since HIV-1 infection in neonates progresses to AIDS more rapidly than does infection in later life in human beings, these investigators speculate that excessive NO may explain the rapid progression of HIV-1 infection to AIDS during infancy. The concentrations of nitrite or nitrate in the sera of patients infected with HIV-1 are substantially raised, especially in those with low CD4 cell counts [118]. However, during HIV-1 infection, it is difficult to find out whether the NO production is attributable to virus replication or to opportunistic infections, or both. In vitro there is a substantial rise in nitrite concentrations from blood mononuclear cells and polymorphonuclear leucocytes from patients with AIDS, especially in those with neurological disorders and pulmonary disease caused by intracellular opportunistic pathogens [121]. Interestingly, the serum concentrations of nitrate are positively correlated with plasma and cell-associated viral loads, which suggests that HIV-1 may induce NO synthesis in vivo [119]. However, the results clearly show that there is a close relation between viral replication and iNOS expression or peaks of plasma nitrate in the absence of any opportunistic infections, in either in macaques or infected patients [119, 122, 123]. NO acts as an autocrine factor that mediates HIV-1 replication; as at the molecular level, NO seems to stimulate long-terminal repeat-mediated transcription [125]. It was noted that exogenous NO increases replication of HIV-1 T-tropic isolates in primary T cells or T-cell lines, and inhibitors of iNOS partly block HIV-1 replication, especially that induced by tumor necrosis factor á [125]. The contrasting effects of exogenous NO, particularly NO donors, may depend on the type of NO donors, their releasing kinetics, and the dose used in the study design.

1.5 New Therapeutic Applications of NO Donors

1.5.2.2

Antiviral and Proviral Activity of NO

Antiviral effects of NO are known for several viruses, including murine poxvirus, herpes simplex virus, Epstein-Barr virus, coxsackievirus, and influenza virus [126, 127]. Virus infection induces directly or indirectly (through interferon ã production) overproduction of NO because of localized iNOS expression in the area of infection [128]. Many pathological effects of NO are thought to be produced via its interaction with oxygen radicals, producing peroxynitrite [129]. Since the antiviral effects of NO do not require immune recognition of infected cells, and since NO can pass readily into cells, it provides a useful early defence against viral infections before the development of a specific immune response; thus NO may be a host response modulator rather than a simple antiviral agent. Viral infections against which NO and its derivatives are thought to have inhibitory effects include DNA and RNA viruses, such as poliovirus, Japanese encephalitis virus, mouse hepatitis virus, vesicular stomatitis virus, herpes simplex virus type 1, vaccinia virus, and Epstein-Barr virus [126]. NO may inhibit an early stage in viral replication, and thus prevent viral spread, promoting viral clearance and recovery of the host. The earliest host response to viral infections is non-specific and involves induction of cytokines, especially tumor necrosis factor á and interferonã. These cytokines are potent inducers of iNOS, which generates large amounts of endogenous NO [130]. Thus, NO could be a vital factor in inducing the host’s innate immunity to control the initial stages of viral infections. Despite the protective effect of NO against various viral infections, workers in several studies have shown a harmful role of NO in many systems. NO seems to play a part in the development of pneumonia caused by influenza virus [128], in the pathogenesis in mice of tick-borne encephalitis flavivirus infection [131], and in worsening the course of the murine myocarditis caused by coxsackievirus B3 [132]. In addition, pneumonia in mice induced by herpes simplex virus type 1 could be suppressed by the inhibitor of iNOS [133]. The issue of whether NO acts as an inhibitor of viral replication or as a harmful agent, therefore, remains unanswered. This issue is particularly evident in HIV-1 infection, since NO seems to act as a double-edged sword in the pathogenesis of HIV-1. The antiretrovirus properties of NO were shown in mice infected with Friend leukemia virus, a murine retrovirus. NO produced by NO-generating compounds or activated macrophages inhibits viral replication in fibroblast cultures, and is involved in defens against this murine retrovirus in vivo [134]. It was also reported that NO donors can inhibit HIV-1 replication in human monocytes through induction of iNOS [135]. The life cycle of many viruses, including retroviruses, depends on viral proteases that cleave viral glycoproteins into individual polypeptides, and these enzymes are necessary for viral replication. NO can inactivate coxsackievirus [136]. Since cysteine proteases are critical for the virulence and replication of many viruses, nitrosation of viral cysteine proteases may be a mechanism of antiviral host defense. NO mediates nitrosation of cysteine and aspartyl proteases of HIV-1, and it was suggested that this

21

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mechanism may have a role in the inhibition of HIV-1 replication [137]. Later it was shown that NO donors inhibit the HIV-1 reverse transcriptase activity, through the modulation of the catalytic activity of cysteine enzymes [138]. The modulation mechanism of the HIV-1 reverse transcriptase activity may be relevant for the development of new strategies for inhibition of HIV-1 replication. NO has complex roles in immunological host responses against viruses, and especially against HIV-1 infection. In HIV-1 infection, NO cannot be rigidly classified as an anti-inflammatory or proinflammatory molecule, but it can be deemed a true inflammatory mediator. Many studies support a proviral effect of NO in HIV-1 infection, mainly based on stimulation of viral replication, and on toxic effects on various cells, including central nervous system cells, via oxidative injury that may cause cellular and organ dysfunction, and immunosuppression and immunopathology, especially in the central nervous system. In several studies on the antiviral effects of NO on HIV-1 infection, the proviral or antiviral effects of NO seem to be strictly related to the active production of NO during HIV-1 infection. The universal speculative interpretation of the dichotomous effect of NO is that overproduction of this substance, especially in the primary infection and in late stages of the disease, leads to active viral replication with consequent harmful effects on the course of the disease. Conversely, low production of NO may cause a reduction in or inhibition of HIV-1 replication, especially during the symptomless stage of the disease, or during treatment with highly active combined antiretroviral drugs. Recently, it has been found that NO donors inhibit HIV-1 replication in acutely infected human peripheral blood mononuclear cells (PBMCs), and have an additive inhibitory effect on HIV-1 replication in combination with 3′-azido-3′-deoxythymisylate (AZT) [139, 140]. S-nitrosothiols (RSNOs) inhibit HIV-1 replication at a step in the viral replicative cycle after reverse transcription, but before or during viral protein expression through a cGMP-independent mechanism. In the latently infected U1 cell line, NO donors and intracellular NO production stimulate HIV-1 reactivation. These studies suggest that NO both inhibits HIV-1 replication in acutely infected cells and stimulates HIV-1 reactivation in chronically infected cells. Thus, NO donors may be useful in the treatment of HIV-1 disease by inhibiting acute infection, or reactivating a latent virus. 1.5.3

Inhibition of Bone Resorption

NO is recognized as a mediator of bone cell metabolism, where it regulates osteoblast and osteoclast activity [141–143]. Osteoporosis, which frequently occurs in postmenopausal women, is a systemic skeletal disease associated with abnormal bone resorption. Addition of NO or NO donors to osteoclasts in vitro results in a reduction in bone resorption, whereas NO synthase inhibitors increase bone resorption, both in vitro and in vivo. Further research has shown that NO reduces bone resorption, via inhibition of the cysteine protease cathepsin K, which is believed to be a key protease in bone resorption. Most of the NO donors, i.e., nitroglycerin, 3-

1.5 New Therapeutic Applications of NO Donors

morpholinosydnonimine (SIN-1), S-nitrosothiols, sodium nitroprusside (SNP), have an IC50 value for cathepsin K from 0.01 ìM to about 1000 ìM. So NO donors may be a new generation of therapeutic agents for inhibiting the bone resorption activity of osteoclasts. There is also evidence suggesting that prostaglandin plays an important role as a regulator of bone remodelling in response to various stimuli, such as cytokines, sex hormones, and mechanical loading [144, 145]. Moreover, both cytokine-induced activation of NO and prostaglandin E2 (PGE2 ) pathways may act in a concerted manner to influence bone cell activity and bone turnover [146, 147]. So the combination of cyclooxygenase (COX) inhibitors and NO donors would exert more potent modulatory effects on bone turnover and bone mass. A report from NiCox indicated that flurbiprofen nitroxybutylester (HCT1026) was significantly more efficacious than the parent compound, flurbiprofen, at inhibiting osteoclast formation and bone resorption in vitro and prevented ovariectomy-induced bone loss in vivo [148]. HCT1026 may be of clinical value in the prevention and treatment of inflammatory diseases such as rheumatoid arthritis, which are characterized by joint inflammation as well as periarticular and systemic bone loss. 1.5.4

Treatment of Diabetes

SIN-1, a non-enzymatic NO donor, has been reported to inhibit insulin release in isolated pancreatic islets [149]. However, another report showed that l-arginine, an NO donor, could stimulate glucose-induced insulin secretion from the pancreas of diabetic rats [150]. Further studies showed that non-enzymatic NO donors such as SIN-1, sodium nitrite, sodium nitropusside, and S-nitroso-N-acetyl-dl-penicillamine (SNAP), increased insulin sensitivity through stimulation of NO production in the liver [151]. It was found that, besides the known vascular effect, enzymatic NO donors, such as organic nitrates, also have a hypoglycaemic/antihyperglycaemic effect. A pharmaceutical combination for the treatment and prevention of diabetes mellitus was invented, comprising at least one enzymatic NO donor and optional antidiabetic active ingredients [152]. The basis of the invention is the recognition of a new insulinsensitizing effect and synergism using NO donors and conventional antidiabetic drugs. 1.5.5

Thromboresistant Polymeric Films

Hydrophobic polymer materials that slowly release NO can be used on the surface of medical devices. Many medical devices suffer from the surface adhesion of blood platelets. To minimize this thrombogenic effect, blood thinners such as heparin, coumarin, and aspirin are often used. However, systemic administration of antiplatelet agents could increase the risk of uncontrolled bleeding elsewhere in the body. In contrast, biocompatible polymer films would solve this problem [153]. It is possible to create polymeric surfaces that mimic the inner surface of a blood vessel by

23

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locally releasing NO. Such in situ NO generation would inhibit platelet adhesion and activation on the polymer surface. Because of its short-life in blood (R=2°C>R =1°C. Substituent-mediated homolytic mechanisms for RSNO decomposition have been implicated. However, a recent computational study (Barberger et al., 2001) has discounted the homolysis-route for S–NO decomposition since it was determined that bond dissociation enthalpies (BDEs) activation parameters were essentially independent of substituent structure (31–32kcalmol−1 ) and predicted to be too high to occur at physiologically relevant temperatures. For example, the predicted half-life with respect to homolytic S–N cleavage was 2.1years at 37°C. The decomposition of these compounds was then tested experimentally. The RSNO decomposition was rapid and followed zero-order kinetics and, contrary to other reports, (Gow et al., 1997; Singh et al., 1996) thiols stabilized RSNO decomposition and changed the decomposition kinetics to first order, with NO and disulfide as the only products. The authors concluded that bimolecular, processes such as RSNO-thiol adducts, RSNO dimerization and catalytic processes involving metal ions, various oxygen species, and enzymes could be responsible for RSNO-decomposition and reactivity in vivo. 4.1.1.1

Enzymatic Consumption of RSNOs

The enzymes superoxide dismutase (Jourd’heuil et al., 1999; Johnson et al. 2001; Romeo et al., 2003), xanthine oxidase (Trujillo et al., 1998) protein disulfide isomerase (Ramachandran et al., 2001; Rautri and Mutus, 2004), thioredoxin reductase (Nikitovic and Holmgren, 1966; Jensen et al., 2001) ã-glutamyl transpeptidase (Askew et al., 1995; Lipton et al., 2001) glutathione-dependent formaldehyde dehydrogenase (GDFDH) (Liu et al., 2001) have been shown to metabolize RSNOs in vitro. A potential in vivo role for RSNO catabolism has only been demonstrated with GDFDH in yeast, mice and E. coli. In the wild type of these organisms RSNOs are found in the protein fractions with trace amounts in the cytosol. However, in knock-out organisms made deficient in GDFDH cytosolic both protein-RSNO and cytosolic-RSNO levels were elevated (Liu et al., 2001).

4.1 Structure and Cellular Reactivity of RSNOs

4.1.2

Formation of RSNOs in the Biological Milieu 4.1.2.1

Nitrite Mediated

The principal in vitro route to the formation of RSNOs is through the reaction of nitrous acid or protonated nitrite (HNO2 ) with thiols (Eq. (1)). Since the pKa of HNO2 is 3.37, this reaction is unlikely to occur in cells and tissues where the pH is maintained at 7.4. (1) HONO + RSH → RSNO + H2 O However, this reaction can and has led to errors in the measurement of RSNOs in biological fluids when the samples are improperly buffered to avoid the HNO2 -route to S-nitrosation (Tsikas, 2003). Enzymatic conversion of nitrite to RSNOs has been reported. Glutathione-Stransferase catalyzed generation of RSNOs from organic nitrites was initially demonstrated in rat liver microsomes (Ji et al., 1996). Subsequently, this activity has been identified in the rat heart and lung GSTs (Akerboom et al., 1997). 4.1.2.2

NO Mediated

NO reacts directly with thiols in vitro to yield RSNOs only in the absence of oxygen (Gow et al., 1997). Therefore, this reaction, Scheme 4.1, is unlikely to occur in biological systems. 4.1.2.3

NO Oxidation Products Mediated

The in vivo RSNO-producing reactions resulting from NO oxidation and related products are summarized in Scheme 4.1. The reaction between NO and O2 giving rise to N2 O4 [a] is third order and, as a result, highly concentration dependent. NO and O2 are low polarity compounds that are more soluble in hydrophobic compartments of cells, such as membranes and protein hydrophobic domains, than in the aqueous phase (Wink et al., 1993; Lui et al., 1998; Nedosparov et al., 2000). NO2 • formed from the homolysis of N2 O4 [b], reacts with NO giving rise to N2 O3 [c] a powerful S-nitrosating agent. Hydrophobic phase, N2 O3 could then selectively S-nitrosate thiols in the proximity of the hydrophobic–aqueous interface [d]. N2 O3 in aqueous environments is rapidly hydrolyzed to nitrite. N2 O3 -dependent S-nitrosation has been demonstrated in the case of serum albumin (Nedospasov et al., 2000; Rafikova et al., 2002), in membranes (Liu et al., 1998) and in the protein-disulfide isomerase dependent transfer of NO-equivalents from extracellular RSNOs to the cytosol (Ramachandran et al., 2001).

93

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4 The Role of S-Nitrosothiols in the Biological Milieu

•

[b’]

N=O + O=O

Scheme 4.1

Other postulated routes (Jourd’heuil et al., 2003) to RSNO formation include the reaction between NO and O2 to yield NO2 • via a second-order reaction. NO2 • and thiolate anion, RS− , react giving rise to thiyl radical, (RS• ) [e]. RS• then reacts with NO to yield RSNO [ f ]. The reaction between RS• and RS− can also be the source of non-enzymatic generation of superoxide anion (O2 •− ) [g], [h]. O2 •− reacts with NO to produce peroxynitrite (ONOO− ) [i] (Szabo, 2003). Thiols react with ONOOH to form RSNOs [k] (van der Vliet et al.,1998). Intracellular and extracellular RSNO metabolism has been studied in LPS activated macrophages (Zhang and Hogg, 2003). This study showed that ∼0.02% of the NO produced in response to LPS, (detected as NO2 − ) was converted to cytosolic RSNOs and that all of the RSNOs detected were in the large molecular weight (>3K) protein fraction and were very stable to denitrosation (t1/2 ∼3h). These authors also showed that the molecular specie(s) responsible for S-nitrosation is freely diffusible and has to be transported to the cell surface before internal S-nitrosation could take place. In a recent comprehensive study, Feelish and coworkers (Bryan et al., 2004) determined the concentrations of RSNOs, N-nitrosamines (RNNOs) NO2 − , NO3 − , heme nitrosyl (NO-heme) in plasma, RBCs, as well as brain, heart, liver, kidney, lung, and aortic tissues of rats. Furthermore, the levels of these analytes were monitored under conditions of eNOS inhibition, hypoxia and redox state. The emerging picture was that RSNOs were detected in all of the tissues examined in comparable levels to NO-

4.1 Structure and Cellular Reactivity of RSNOs

heme species which are known to turn on guanylate cyclase and turn off cytochrome c oxidase and cytochrome P450 activities. The RBCs contained the most RSNOs (∼250nM). The next largest RSNO concentration was in aorta (∼100nM) followed by liver and kidney (∼35nM), heart and lung (∼8–10nM) and plasma (∼1nM). In all of these tissues, the RNSO were associated with the protein fraction. Upon eNOS inhibition, [RSNOs], [RNNOs], and [NO3 − ], underwent an initial transient increase followed by depletion. The transient increase was explained as follows: as NO levels fall subsequent to eNOS inhibition and approach [O2 •− ], ONOO− will be formed under diffusion controlled rates (Scheme 4.1 [i]) which can lead to rapid RSNO formation (Scheme 4.1 [k]) or decompose to yield NO2 • plus OH• or be converted to NO3 − . NO2 • can yield RS• (Scheme 4.1 [e]) which in turn can react with NO to generate RSNOs (Scheme 4.1 [ f ]). The depletion of most of the NO-related metabolites indicated that these species are dependent on eNOS-generated NO. Another fascinating finding of this study was the rapid potentiation of RSNO formation accompanying hypoxia. The concentration of RSNOs in the brain increased by ∼250% 10min after a hypoxic episode. The conclusion of the authors was that these responses were too rapid to occur via the N2 O3 route (third-order process) but instead could only take place via the peroxynitrite- NO2 • route that generates RS• (Scheme 4.1 [e]) which can directly react with NO to form RSNOs. This study is the first to demonstrate that S-nitrosation takes place in all tissues and cells and is a dynamic process that can occur in vivo at similar rates to heme nitrosation. In addition, RSNOs were shown to be as labile as heme-nitrosyl species in that they were rapidly formed and destroyed in response stimuli such as hypoxia and tissue redox status. 4.1.2.4

Metalloprotein Mediated

The multi-copper carrying enzyme ceruloplasmin (CP), found in large amounts in liver and nervous tissues, has been shown to convert NO to RSNOs. The proposed mechanism involves the binding of NO to the CP type I Cu-sites. The NO is then oxidized to NO+ and transferred to RS− giving rise to RSNO (Innoue et al., 1999). Perhaps the most physiologically important metalloprotein to be S-nitrosated by NO is hemoglobin (Hb). The S-nitrosation of Hb-Cys 93 by NO was first demonstrated by Stamler and coworkers in 1996 (Jia et al., 1996; Stamler et al., 1997). To this day HbSNO remains controversial with respect to its mechanism of formation and physiological relevance. The wealth of data on HbSNO formation has given rise to two broad interpretations that either support the hypothesis that the S-nitrosation of Hb-Cys 93 is redox catalyzed by the heme and is under allosteric control or that it is N2 O3 or NO2 • -mediated without the involvement of the hemes. The main point of argument with the formation of HbSNO is that in vitro the exposure of NO to oxyhemoglobin (HbFe(II)-O2 ) results in the production of methemoglobin (HbFe(III)) plus nitrate (Eq. (2)). NO + HbFe(II)-O2

→

HbFe(III) + NO3 −

(2)

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96

4 The Role of S-Nitrosothiols in the Biological Milieu

Since the rate constant for this reaction is estimated to be ∼9×107 M−1 s−1 (Eich et al., 1996; Herold et al., 2001) the contention is that this reaction would predominate in vivo, thus minimizing the formation of HbSNO. In the following section the evidence for and against a role of the heme in HbSNO formation is discussed. In their initial report, Stamler and co-workers (Jia et al., 1996) showed that exposure of Hb to NO resulted in the S-nitrosation of C93 in the â-chains. The reported yield upon exposure of Hb to 10-fold molar excess of NO, was 1mol S–NO per tetramer. Given that there are 2 C 93/tetramer this is a 50% yield. Hb-SNO formation was accompanied by the conversion of oxyHb (HbFe(II)O2 ) to metHb (HbFe(III)). Hbâ C93 S-nitrosation could also be accomplished by exposure to RSNOs (GSNO or CysNO). The rates RSNO-dependent Hb-S-nitrosation was ∼10-fold larger in oxyHb than in deoxy-Hb. Conversely, the rate of spontaneous decay of deoxy-Hb-SNO was ∼20-fold larger than oxy-Hb-SNO. An explanation for this differential reactivity was presented in a subsequent study (Stamler et al., 1997) where protein modeling data based on the X-ray structures of Hb in T and R states indicated that in OxyHb the SNO of Cys â 93 is protected from solvent. In contrast, in deoxyHb the SNO is highly exposed to solvent. The implication was that the NO+ on Cys â93-S–NO could be transferred to thiols in RBC and eventually effluxed to induce vasodilation under conditions of low O2 saturation. Gow and Stamler (Gow and Stamler, 1998) looked more carefully at Hb-SNO chemistry under more physiological conditions i.e. at NO to Hb ratios ranging from 1:100 to 10:1. They reported that at NO:Hb ratios of ∼1:20 NO is bound to the hemes. As O2 is slowly introduced the NO is transferred from the Heme to the âC93 thiol and O2 Hb-SNO is converted to the R state. They also presented spectral evidence that in the absence of oxygen, at low NO:Hb ratios (1:2. The authors proposed that when Heme-Fe(II)-NO is in close proximity to âC93-SH, NO is directly transferred to yield SNO• H. They further proposed that O2 would then act as an electron acceptor to yield RSNO plus superoxide. Heme-Fe(II)-NO + C93 SH •

C93 -SNO H + O2 +

⇔ →

C93 -SNO• H HONS-C93 + O2

(3) •−

(4)

Alternatively they suggested that NO might be transferred from the heme to the C93SH. Interestingly, the maximum yield of Hb-SNO formed in this study was >HA− >>H2 A (H2 A = ascorbic acid) to release NO. Similar results were obtained by Wanat et al. [81] for the complex ion [Fe(CN)5 (NO2 )]3− . The outer sphere reduction of SNP by ascorbate involves three clear stages with NO being released in the last stage. The reaction is catalyzed by alkali metal ions which act as a glue between two negatively charged ions, nitroprusside and ascorbate. The formation of this ion triplet [82] seems to be a prior requirement for outer sphere electron transfer between two negatively charged ions [83, 84]. Since the catalysis by metal ions increases with cationic size, the partial deaquation mechanism explains the observed catalytic efficacy sequence for M+ (M+ =Li+ 1000 > 1000

eNOS

0.71

269

270

10 Control of NO Production

10.6

Inhibition of NOS Function Targeted towards Cofactors

Since NOS isoforms require a number of cofactors and/or prosthetic groups, such as FAD, FMN, NADPH, BH4 and calmodulin, when the cofactors cannot function or cannot bind, NOS enzymes are inactive. The flaboprotein inhibitors, diphenyleneiodonium and several of its analogs, inhibit NOS activity by their irreversible effects on the FAD binding site [90]. Calmodulin binding and activation may be an interesting target for the selective inhibition of different NOS isoforms. It was found that a calmodulin inhibitor, such as calmidazolium, W-7 and fendiline inhibit the calmodulin-dependent isoforms, nNOS and eNOS but not iNOS [91]. Kondo et al. reported that a point mutated plant calmodulin M144V, SCaM-1, acts as an antagonist for nNOS activation [92]. Since BH4 analogues bind to the BH4 binding site, pteridine-based compounds have been reported as NOS inhibitors. Among a series of tested compounds, the 4-amino analogue of BH4 , 5,6,7,8-tetrahydro-6(d-thero-1,2-dihydroxypropyl)pterine, was a potent inhibitor of the recombinant rat nNOS both in vitro (Ki = 13 nM) and in vivo [93]. The heme prosthetic group is also required for the catalytic activity of NOSs and is likely responsible for oxygen activation in a manner similar to that of other heme-containing monooxygenases such as cytochrome P450 [94]. Carbon monoxide (CO) is an extremely good ligand for ferrous (Fe2+ ) hemes and is a potent inhibitor of NOS activity [95, 96]. Other heme ligands such as KCN, miconozole [97] and even NO itself [98] were also reported to be capable of NOS inhibition. Since it is well known that imidazoles act as inhibitors of various heme-containing proteins by binding to the heme group [99], imidazole derivatives also inhibit all NOS isoforms [78].

10.7

Regulators of NOS Gene Expression

Aminoguanidine and aminoethyl-ITU, which are weaker iNOS selective inhibitors, inhibited iNOS expression in the macrophage cell line J774.2 cells stimulated by LPS [60]. N-Acetyl-5-hydroxytryptamine (N-acetylserotonin), an inhibitor of sepiapterin reductase in BH4 synthesis, inhibited the expression of iNOS, but not iNOS activity, both in a cultured macrophage cell line RAW264.7 stimulated by LPS and in vascular smooth muscle cells stimulated by interleukin-1â [100]. Moreover, the novel quinazoline derivatives (Fig. 10.12) inhibited the induction of iNOS mRNA in RAW264.7 cells stimulated by LPS (Fig. 10.13A). Surprisingly, however, they enhanced the induction of iNOS mRNA in vascular smooth muscle cells stimulated by interleukin-1â in an opposite manner (Fig. 10.13B) [101]. Although the mechanism by which these agents inhibit or enhance iNOS gene expression remains unclear, the regulation of NO production at the gene expression level may provide new insights in the development of selective inhibitors and selective enhancers of iNOS in various tissues.

10.8 NO Formation by an NOS-independent Pathway

DIQ

H 3C

4-(1,1-dimethyl-1,2-methoxyethylamino) -2-(imidazol-1-yl)-quinazoline dihydrochloride

CH3

O CH3

NH N

N

N

N

IMT 2-(imidazol-1-yl)-4-(2-methoxyethylamino) -5,6,7,8-tetrahydroquinazoline dihydrochloride

O CH3

NH N

N

N

N

IPE 2-{2-[2-(imidazol-1-yl)-5-methylthieno [2,3-d]pyrimidine-4-ylamino]ethoxy} CH3 ethanol dihydrochloride

O NH

OH N

S

N

N

N

Fig. 10.12 Structure of

quinazoline derivatives.

A RAW264.7 cells (-) (-)

(-)

LPS (10 ng ml-1) DIQ IMT IPE 5 50 5 50 5 50

iNOS

B VSMC

IL-1β (10 ng ml-1)

(-) (-)

(-)

DIQ 5 50

5

IMT 50

5

IPE 50

Fig. 10.13 Effects of the quinazoline

iNOS

derivatives on iNOS gene expression in RAW264.7 cells (A) and vascular smooth muscle cells (B).

10.8

NO Formation by an NOS-independent Pathway

It has been found that oxime (Fig. 10.4d) and hydroxyurea (Fig. 10.4f) as well as Nhydroxyguanidine (Fig. 10.4e) also release NO via an NOS-independent pathway as described below.

271

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10 Control of NO Production

10.8.1

Oxime

Cytochrome P450s have been found to catalyze the oxidative cleavage of C=N bonds of compounds containing a –C=NOH (oxime) function, such as NHA, N-hydroxyguanidine, amidoxime, ketoximes, and aldoximes, producing the corresponding derivatives and NO in vitro [62, 102]. The oxidase function of cytochrome P450s is similar to that of NOSs, both of which utilise the same prosthetic groups (NADPH, FAD, FMN and heme-thiolate). However, cytochrome P450s do not bind BH4 , which is only present in NOSs, and the reactions are not selective in terms of substrates. In addition, the oxidation of N-hydroxyguanidine, amidoximes, and ketoximes by microsomal cytochrome P450s, but not NOSs, is strongly inhibited by superoxide dismutase (SOD), indicating that cytochrome P450s require O2 •− for the oxidation [62]. It has been reported that O2 •− directly oxidizes N-hydroxy-l-arginine [103, 104], and that O2 •− efficiently oxidizes amidoximes with formation of the corresponding amides and nitriles, in addition to nitrogen oxides [102]. The BH4 free-iNOS-catalyzed oxidation of N-hydroxyguanidines is also strongly inhibited by SOD. In addition, the oxidation of N-p-chlorophenyl-N′-hydroxyguanidine by NADPH and O2 in the presence of BH4 free-iNOS led to the formation of the corresponding urea and cyanamide, in addition to nitrite and nitrate. The oxidation of NHA by BH4 -free-iNOS in the presence of NADPH and O2 also leads to the formation of N-cyanoornithine and citrulline [105]. Thus, the oxidation of oximes by cytochrome P450 and BH4 -free-iNOS show similar characteristics (reviewed by Mansuy and Boucher [106]). However, the rate of the reactions was markedly lower (in the 1–10 min−1 range) than iNOS in the presence of BH4 (in the 50–500 min−1 range). 10.8.2

Hydroxyurea

Hydroxyurea (Fig. 10.4f) was first synthesized in 1869 and has been used in the treatment of a variety of cancers and sickle cell disease for a long time. However, the mechanism of the beneficial functions of this agent has remained unclear. Hydroxyurea inhibits ribonucleotide reductase by quenching the catalytically essential tyrosyl free radical of the enzyme [107], and increases the levels of fetal hemoglobin [108, 109]. A variety of experiments have shown an increase in iron nitrosyl hemoglobin, nitrite, and nitrate as a result of the administration of hydroxyurea. These results and the molecular structure of hydroxyurea, which contains an N–O bond, suggest that the release of NO during the oxidative metabolism of hydroxyurea may explain its effects. An EPR study demonstrated that chemical oxidation of hydroxyurea with hydrogen peroxide or copper(II) sulfate in aqueous dimethyl sulfoxide produces NO and nitroxyl, the electron reduced form of NO [110]. Biological oxidants, such as iron- or copper-containing enzymes and proteins also convert hydroxyurea to NO [111]. Moreover, horseradish peroxidase catalyzes the formation of NO from hydroxyurea in the presence of hydrogen peroxide [112]. Huang et al. synthesized a variety of hydroxyurea derivatives and demonstrated that the structure of the hydroxyurea

10.9 Summary

derivative directly controls both the ability of the compound to generate NO and iron nitrosyl hemoglobin (HbNO) and the rate of NO release [113]. The development of new hydroxyurea-based NO donors may be a benefit in the treatment of sickle cell disease and cancers.

10.9

Summary

Nitric oxide (NO), a free radical, is a ubiquitous signaling molecule that is involved in both basic physiological functions and various pathological conditions. NO is produced by three distinct NO synthase (NOS) isoforms, which play distinct roles in a variety of tissues. The up/down regulation of NO release in pathological conditions is a target for drug development. Because of the complexity of the actions of NO from the various isoforms of NOS, the development of isoform-specific or cellselective inhibitors and substrates would be desirable. In the early days, l-arginine analogues such as l-NMA and l-NNA were studied and used as NOS inhibitors. Since l-arginine is regarded as a derivative of guanidine, various guanidine derivatives, such as aminoguanidine, N-alkyl-N′-hydroxyguanidines and N-alkyl-S-isothioureas have been developed as NOS inhibitors and NOS substrates. The focus of this chapter is on the length and structure of the alkyl chains of N-alkyl-N′-hydroxyguanidines and N-alkyl-S-isothioureas suitable for binding to the active site in NOS isoforms. Several reports indicate that when the alkyl substituents are too small, too large or too bulky, the ability as an NOS substrate and as an NOS inhibitor decreases significantly. X-ray crystal structure analyses of the active site in NOS isoforms have made it easy to develop new drugs for targeting the active site. In addition, compounds targeting the specific co-factor requirement of NOS isoforms, and compounds regulating the NOS gene expression have also been developed for the up/down control of NO formation. NO production by a NOS-independent pathway, including the oxidation of hydroxyurea and oxime by cytochrome P450 and peroxidase, or super oxide anion radical, represents a new approach for the regulation of NO formation. The control of NO formation at will by inhibitors or substrates specific for each NOS isoform promises to contribute to a further understanding of the NOS mechanism and therapy for various diseases that are associated with NO.

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72 Li, H., Shimizu, H., Flinspach, M.,

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Jamal, J., Yang, W., Xian, M, Cai, T., Wen, E. Z., Jia, Q., Wang, P. G., Poulos, T. L., The novel binding mode of N-alkyl-N’-hydroxyguanidine to neuronal nitric oxide synthase provides mechanistic insights into NO biosynthesis, Biochemistry 41 (2002), 13868–13875 Szabo, C., Southan, G. J., Thiemermann, C., Beneficial effects and improved survival in rodent models of septic shock with S-methylisothiourea sulfate, a potent and selective inhibitor of inducible nitric oxide synthase, Proc. Natl. Acad. Sci. USA 91 (1994), p. 12472–12476 Thiemermann, C., Ruetten, H., Wu, C. C., Vane, J. R., The multiple organ dysfunction syndrome caused by endotoxin in the rat: attenuation of liver dysfunction by inhibitors of nitric oxide synthase, Br. J. Pharmacol. 116 (1995), p. 2845–2851 Tracey, W. R., Nakane, M., Basha, F., Carter, G., In vivo pharmacological evaluation of two novel type II (inducible) nitric oxide synthase inhibitors, Can. J. Physiol. Pharmacol. 73 (1995), p. 665–669 Garvey, E. P., Oplinger, J. A., Furfine, E. S., Kiff, R. J., Laszlo, F., Whittle, B. J., Knowles, R. G., 1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo, J. Biol. Chem. 272 (1997), p. 4959–4963 Southan, G. J., Szabo, C., Connor, M. P., Salzman, A. L., Thiemermann, C., Amidines are potent inhibitors of nitric oxide synthases: preferential inhibition of the inducible isoform, Eur. J. Pharmacol. 291 (1995), p. 311–318 Salerno, L., Sorrenti, V., Di Giacomo, C., Romeo, G., Siracusa, M. A., Progress in the development of selective nitric oxide synthase (NOS) inhibitors, Curr. Pharm. Des. 8 (2002), p. 177–200 Ogawa, D., Shikata, K., Matsuda, M., Okada, S., Usui, H., Wada, J., Taniguchi, N., Makino, H.,

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Part 3 Clinical Applications of NO Donors

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Nitric Oxide Donors in Cardiovascular Disease Martin Feelisch, Joseph Loscalzo 11.1

Introduction

Nitric oxide donors comprise a heterogeneous group of different chemical classes of compounds that either decompose spontaneously or are metabolized in cells and tissues to generate nitric oxide (NO). As diverse as the chemistries of the individual agents and the pathways that lead to NO formation from them are, so are the differences in their pharmacodynamic, pharmacokinetic, and toxicological properties. Several extensive reviews on this topic are available in the literature [1], and some of these aspects are dealt with in other chapters of this volume. A common feature of all of these compounds is that they can relax isolated blood vessels in vitro (hence, the older designation, “nitrovasodilators”) and, depending on their mode and rate of biotransformation, are principally capable of enhancing blood flow and lowering blood pressure in vivo. Only a select few compounds are in clinical use today, however, and all these drugs had been introduced into medical practice long before the discovery of NO as a biological signaling molecule. The present chapter is designed to give a brief overview of the history as well as the current use of NO donors in cardiovascular disease, including a brief account of the pharmacological profile and pathways of biotransformation of the major NO-generating drugs currently in use.

11.2

Clinical Cardiovascular Applications of NO Donor Therapy – Past and Present

Nitroglycerin (NTG) as the prototypic NO donor and longer-acting organic nitrates, such as isosorbide dinitrate (ISDN) and isosorbide-5-mononitrate (IS-5N), have been used for the treatment of coronary atherothrombotic disease and its complications (principally, heart failure) for many decades. These agents are primarily used for the prophylaxis and treatment of myocardial ischemia in patients with coronary artery disease, and are highly effective in relieving acute attacks of angina pectoris. Nitroglycerin itself was first proposed for the treatment of angina pectoris by William

Nitric Oxide Donors Peng George Wang, Tingwei Bill Cai, Naoyuki Taniguchi (Eds.) c 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Copyright
ISBN: 3-527-31015-0

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Murrell, who reported in 1879 that a one percent solution of the drug administered sublingually relieved angina and prevented attacks thereafter [2]. Nitroglycerin has an interesting history and is one of the few medications in current use that was discovered before the twentieth century. It is distinguished from other drugs by the fact that it was adopted by allopathic physicians from homeopathic physicians [3, 4]. Shortly after its first synthesis by the Italian scientist, Ascanio Sobrero, in 1846, Constantine Hering, a leading homeopathic physician in Germany, began exploring the possible therapeutic value of this powerful explosive by systematically investigating its effects in healthy individuals. These so-called homeopathic “provings” were precursors of present-day approaches to screening compounds for toxic and potential therapeutic effects, gradually replacing empiricism. Hering’s interest in nitroglycerin originated from Sobrero’s earlier observation that the substance produced a throbbing headache when placed on the tongue. After confirmation of this effect in volunteers, which in some individuals was achieved with less than 1/300th of a drop, and consistent with the homeopathy doctrine, simila similibus curantur (like cures like), nitroglycerin was advocated for the treatment of headache. Although Hering also shared with the medical community his observation that nitroglycerin affected the pulse, even when administered in very small quantities (suggesting a direct effect on the heart), and the compound was further characterized by a number of physiologists, it neither attracted much interest by allopathic physicians nor was it proposed for use as an antianginal agent until a related compound, amyl nitrite, was introduced in 1867 for the treatment of angina pectoris by the British physician, Thomas Lauder Brunton [5]. Brunton himself suffered from frequent attacks of angina and tested numerous compounds until he found, apparently without knowledge of nitroglycerin, that inhalation of amyl nitrite was effective in relieving his symptoms. His motivation to test this particular compound arose from investigations by Guthrie showing that amyl nitrite lowered blood pressure, and from the contemporary concept that angina was due to high arterial tension secondary to increased vasomotor tone. Interestingly, Brunton went on to demonstrate in animal experiments that nitroglycerin also lowered blood pressure, but never proposed it as a remedy for angina. The similarity of the pharmacodynamic profile of NTG with that of amyl nitrite led Murrell to investigate the former in his patients, and provided the basis for nitrovasodilator therapy of angina pectoris, the treatment of which at the time included venesection, digitalis, opium, and brandy. In parallel with these developments in medicine, the explosive properties of undiluted nitroglycerin made it a compound of prime interest for military use, as well as for the mining industry. The spontaneous explosion hazard posed a serious danger for its production, transport, and use, until the Swedish entrepreneur, Alfred Nobel, found a solution to this problem by adsorbing nitroglycerin onto porous silica, creating an easily-handled solid explosive he called dynamite, that could be produced comparatively safely in factories. By an ironic twist of fate, Nobel developed angina towards the end of his life and refused to be treated with the very substance that had brought him to so much wealth. Although control of the symptoms of acute angina pectoris had become possible with amyl nitrite and NTG, their short duration of action did not allow prophylactic

11.2 Clinical Cardiovascular Applications of NO Donor Therapy – Past and Present

treatment. This problem was addressed with the discovery of long-acting nitrates and the development of slow-release formulations and transdermal therapeutic systems. Pentaerythrityl tetranitrate (PETN) and related nitrate esters were synthesized in the 1930s and pharmacologically characterized in the following years [6], but did not gain much interest until recently (vide infra). In the early 1930s, 10 years after the discovery of insulin, several research groups intensively searched for carbohydrate analogs that were metabolized independent of insulin for use as a substitute diet for diabetics. At that time, John Krantz at the University of Maryland School of Medicine started a large-scale research program on sugar alcohols and their anhydrides, which fulfilled this criterion. One of the compounds he discovered was isosorbide, which was used as an osmotic diuretic initially. As an extension of this work, many of these polyalcohols were converted to their nitrate esters and investigated for their vasodilating properties. In the course of these studies, Krantz and colleagues discovered the long-lasting vasodilator properties of the dinitrated derivative of isosorbide and published their findings in 1939 [7]. Subsequent Swedish studies in the late 1940s demonstrated the prophylactic use of ISDN as an antianginal drug and its prolonged duration of action after oral administration; it was first marketed in Sweden in 1947 for this purpose. Independently, and unaware of these achievements, chemists of an American company resumed nitrate research work on an improved synthesis of ISDN based on Krantz’s data of the 1930s, and introduced the drug to the US market in 1959. They were dismayed to discover that ISDN had already been on the market in Scandinavia for 12 years. Soon thereafter, ISDN became available worldwide as the first long-acting, prophylactic treatment of angina pectoris. The popularity of organic nitrates on both sides of the Atlantic abruptly decreased after reports by Needleman’s group in the late 1960s showing that these compounds undergo rapid degradation in the liver, and that the parent drug could not be identified in the blood even shortly after ingestion, suggesting that nitrates were likely to be ineffective when given orally. This hypothesis clearly contradicted a large body of clinical evidence that nitrates were effective. Some years later, using more sensitive and reliable analytical methods, this interpretation was shown to be incorrect, and the clinical utility of long-acting, oral organic nitrate derivatives affirmed. In 1967, one of the pharmacodynamically active metabolites of ISDN, IS-5N, was shown to be formed in vivo, and shortly thereafter it was introduced as a novel long-acting NO donor with improved bioavailability. In addition to the treatment of angina pectoris, organic nitrates are also quite useful in patients with acute coronary syndromes. In unstable angina, intravenous nitroglycerin can relieve ischemic pain effectively, while in patients with acute myocardial infarction, it is often effective. The benefit of organic nitrates in acute coronary syndromes is not only a result of their hemodynamic benefits, but also, in part, a consequence of their antiplatelet effects [8]. This important antithrombotic action of organic nitrates is mediated by the NO-dependent activation of platelet guanylyl cyclase and impairment of intraplatelet calcium flux [9]. The limiting determinant of efficacy, especially in patients with acute myocardial infarction, is hypotension, which can complicate the hemodynamic consequences of the infarction itself, thereby offsetting the potential benefits of the improved coronary perfusion.

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More recently, some controversy over the use of NO-donors in patients with acute myocardial infarction has arisen based on two clinical trials, GISSI-3 [10] and ISIS-4 [11]. These trials investigated the adjunctive use of nitrate therapy with reperfusion therapy by thrombolysis in patients with acute myocardial infarction. The design of these trials was predicated on a meta-analysis of several small trials suggesting that intravenous nitroglycerin or oral nitrates reduced short-term mortality in infarct patients [12]. Both trials showed trends in favor of nitrate therapy; however, neither reached statistical significance. Of note, approximately 60% of patients in both placebo groups received open label nitrates at the discretion of the treating physician, rendering the final results significantly confounded by this therapeutic bias. Two other important groups of patients with cardiovascular diseases often benefit from NO donor therapies: those with hypertension and those with heart failure. Patients with hypertension, especially those with hypertensive emergencies, can often derive acute control of blood pressure from the judicious intravenous administration of the NO donor sodium nitroprusside (SNP). This compound, first reported by the Scottish chemist, Lyon Playfair, in 1846, is an inorganic complex comprising five cyanide anions and one NO molecule attached to a central iron atom. Its in vivo activity is very short-lived, ending a few seconds after the infusion is terminated, which makes it an ideal compound for controlled hypotension in hypertensive crises or in patients undergoing neurovascular surgery. Although the blood pressure-lowering effects of ISDN in hypertensive patients had been demonstrated as early as 1946, chronic administration of currently available long-acting oral NO donors to patients with chronic essential hypertension is not very effective for blood pressure control, except in cases of myocardial ischemia complicated by hypertension. The use of NO donors for the treatment of heart failure, a use originally suggested by Brunton at the end of the 19th century, has been widely recognized, beginning with the work of Johnson and Hale in the 1950s followed by the elegant work of Cohn and Franciosa in the 1970s in patients with acute severe congestive heart failure or frank pulmonary edema [13]. As a result of this work, nitroglycerin administered in combination with dopamine became an accepted treatment for these patients, substituted only many years later by more selective agents, including dobutamine and, more recently, nesiritide. These early observations in acutely ill heart failure patients were followed by the VHEFT-1 study, which compared ISDN and hydralazine with prazosin or with placebo in patients with chronic congestive heart failure [14]. In this prospective study, the ISDN-containing arm showed a clear benefit over treatment with prazosin or placebo.

11.3

Pharmacological Cardiovascular Mechanism of Action of NO Donors

The pharmacological mechanisms of action of NO donors that contribute to their benefit in coronary artery disease, congestive heart failure, and hypertension are listed in Table 11.1. These actions can be grouped into five categories: vasodilation, decrease in myocardial oxygen consumption, improvement in hemodynamic performance,

11.3 Pharmacological Cardiovascular Mechanism of Action of NO Donors Tab. 11.1: Mechanisms of action of NO donors in cardiovascular disease.

Disorder Stable angina pectoris

Unstable angina pectoris Acute myocardial infarction Congestive heart failure Systolic dysfunction

Diastolic dysfunction

Hypertension

Mechanism Decreased myocardial oxygen consumption –decreased LV end-diastolic dimension –decreased LV filling pressure –decreased LV systolic pressure –decreased PVR Increased coronary blood flow –epicardial coronary artery dilation –stenotic segment dilation –coronary collateral vessel dilation –increased subendocardial perfusion As above Antithrombotic effects As above Antithrombotic effects Improved hemodynamic performance –decreased end-diastolic dimension –decrease filling pressure –decreased systolic pressure –decreased SVR –decreased mitral regurgitation Arterial vasodilation Improved hemodynamic performance –as above –improved lusitropy Arterial vasodilation Altered hemodynamic performance –decreased filling pressure –decreased systolic pressure –decreased SVR

increase in myocardial blood flow, and antithrombotic effects [9, 15, 16]. Some of the elements within these categories overlap; however, they are detailed in the table for completeness. The hemodynamic and antianginal activities of organic nitrates are predominantly mediated by vasodilation of capacitance veins and conductance arteries, i.e., by a peripheral effect rather than by direct coronary dilator action. The reason for the preferential venodilator effect of nitrates is unclear, but may include differences in smooth muscle sensitivity and/or efficacy of biotransformation to NO. Alternatively, metabolically competent enzymes in arterial and venous smooth muscle cells may be inhibited to a different degree by endogenously produced NO. Since venous endothelial cells produce less NO than their arterial counterparts, nitrates may be more effectively metabolized to NO in the venous vascular wall. By dilating capacitance veins, preload is reduced, leading to a reduction in end-diastolic ventricular volume. These changes lead to a lowering of myocardial oxygen requirements and a favorable redistribution of blood flow in the heart with an increase in subendocardial

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myocardial perfusion. Another possible explanation for the blood flow redistribution across the left ventricular wall is that organic nitrates, unlike other NO donors, preferentially dilate large coronary arteries. To what extent this effect contributes to the effectiveness of these drugs in stable angina is unclear, but it is undoubtedly relevant to the relief of coronary vasospasm in patients with Prinzmetal angina. By dilating systemic conductance arteries, together with reducing left ventricular enddiastolic volume, afterload (the resistance against which the heart has to pump blood into the periphery) is decreased, which is another key determinant of myocardial oxygen consumption. Another important effect of organic nitrates is dilation of coronary collateral vessels, which results in improved perfusion of ischemic myocardium. Importantly, organic nitrates do not affect coronary resistance vessels (presumably owing to a lack of biotransformation to NO at this site), thereby minimizing the risk of myocardial ischemia from a coronary “steal” phenomenon (“luxury” perfusion of adequately perfused myocardium at the expense of hypoperfused segments). Amyl nitrite and molsidomine produce hemodynamic effects similar to those of organic nitrates, although headaches are less frequent. Sodium nitroprusside has a more balanced effect on the arterial and venous circulation. In contrast to organic nitrates, sodium nitroprusside also dilates small resistance vessels, which accounts for its potent hypotensive effects and for the potential for coronary steal in patients with active myocardial ischemia.

11.4

Clinically Available NO Donors: Structures and Mechanism of Action

Clinically available NO donors approved for use in the U.S. in patients with cardiovascular disease include nitroglycerin, ISDN, IS-5N, amyl nitrite, and SNP. Pentaerythrityltetranitrate (PETN) has been approved for use in the U.S. for many years, but has been largely replaced by ISDN and IS-5N. Nicorandil and molsidomine [(which is converted to the active moiety, 3-morpholinosydnonimine (linsidomine, SIN-1), in vivo)] are not approved for use in the U.S., but, like PETN, are available abroad. The chemical structures of these agents are given in Figure 11.1. The formulations of each of these vary, and include oral, sublingual, topical (ointment), transdermal (patch), buccal, and intravenous preparations. While the hemodynamic profiles of different organic nitrates are very similar, there are marked differences in the pharmacokinetic properties of the individual compounds. All nitrate esters are prodrugs that must be biodegraded to achieve the desired therapeutic effect. Biotransformation essentially requires enzymatically catalyzed denitration and reduction, with the consequent generation of nitric oxide. The partially denitrated metabolites remain pharmacologically active, albeit of lower potency compared to the parent molecules; and together with the fully denitrated alcohols, are excreted either unchanged or as glucuronide or sulfate conjugates. Despite intense research carried out for more than a century, the precise biochemical and molecular mechanisms by which organic nitrates are metabolized remain controversial. Sulfhydryl groups are probably required, either in the active center of the

11.4 Clinically Available NO Donors: Structures and Mechanism of Action CH2

O

NO2

CH

O

NO2

CH2

O

NO2

ONO2

O

ONO2

O

O

O OH

ONO2

Nitroglycerin

Isosorbide dinitrate

Isosorbide-5-mononitrate

_

CN CH3

2Na

CN Fe CN

+ CN NO

CH CH2 CH2 ONO2 CH3

2

CN

Amyl nitrite

Sodium nitroprusside

NO2 O CH2 NO2 O CH2

C

CH2 O NO2

CH2 O NO2 Pentaerythrityl tetranitrate O O ONO2 NH

N N O

N N N

N

O

N 1

Nicorandil

Mosidomine

Fig. 11.1 Chemical structures of common NO donors.

nitrate metabolizing enzyme(s), as reducing equivalents or as cofactors [17, 18]. Interestingly, virtually all thiol compounds can facilitate conversion of organic nitrates to nitrite, but only a select few concomitantly generate NO [19, 20]. The reason for this selectivity is not understood, but may offer important mechanistic clues as to the enzymatic pathway of NO formation. Thionitrates (S-nitrothiols; RSNO2 ) are thought to be the principal reaction intermediates that give rise to either nitrite or

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nitrite and NO [21]. Since Hay’s original observations on the metabolism of nitroglycerin in blood at the end of the 19th century [22], we know that organic nitrates are converted to nitrite. Inorganic nitrite and nitrate are, however, unlikely mediators of vasorelaxation, as equimolar amounts of nitrite or the fully hydrolyzed organic nitrate injected into blood do not elicit a comparable degree of vasodilation. It is, thus, generally believed that it is the NO produced during organic nitrate biotransformation that accounts for the vasorelaxant effect, although this view has recently been questioned [23, 24]. Very recent work suggests that (in ischemic tissue) nitrite can react with deoxyhemoglobin to generate NO [25]. Regardless of the nature of the active metabolite involved, biotransformation occurs in both vascular smooth muscle and endothelial cells, but is not limited to the vascular wall. In fact, metabolism of organic nitrates occurs in all tissues, with particularly high prevalence in the liver and the intestine [26]. Such extravascular metabolic events are generally believed to contribute only to inactivation (the so-called “first-pass” metabolism) of organic nitrates, but this view may not be entirely correct as the metabolism at these sites can conceivably generate longer-lived NO-adducts that may re-enter the systemic circulation to exert vascular effects distal to their site of generation. Many different enzyme systems have been proposed to be involved in organic nitrate metabolism, including certain cytochrome P450 and glutathione S-transferase isoforms, and other glutathione and NAD(P)H-dependent but poorly characterized enzymatic activities [27–29]. Nitrates may also be metabolized by xanthine oxidoreductase, although this pathway is likely to be limited to hypoxic situations as enzyme activity is inhibited by oxygen [30]. In addition to other nonspecific esterase activities, several bacterial enzymes with amino acid sequence homology to the “Old Yellow Enzyme” (the first flavin-dependent enzyme identified in yeast), as well as fungal and plant enzymes, are capable of nitrate ester degradation, which plays an important role in the removal of contaminant organic nitrates from wastewater in the industrial production process. In addition to these enzymatic pathways, organic nitrates may undergo non-enzymatic metabolism by reacting with thiol-containing biomolecules, including cysteine, glutathione, and sulfhydryl-bearing proteins, such as albumin. None of these pathways, however, has unequivocally been demonstrated to play a key role in the bioactivation of organic nitrates in the vasculature. Many of the earlier investigations in this area suffer from a lack of distinction between organic nitrate breakdown (to nitrite/nitrate and the denitrated metabolites) and activation (to NO) in that only a decrease in concentration of the parent compound or an increase in nitrite/nitrate or denitrated metabolites was measured, without direct assessment of NO and/or cGMP formation. The significance of those early studies for the bioactivation of organic nitrates, therefore, remains unclear. Other studies carried out in cultured cells or isolated tissues of non-vascular origin are equally difficult to interpret. For example, recent evidence suggests that mitochondrial aldehyde dehydrogenase may play a role in the biotransformation of nitroglycerin [24] and in the development of tolerance to organic nitrates [31], although this hypothesis remains controversial at the current time [32]. Thus, the identity of the enzyme(s) involved in the bioactivation of organic nitrates remains an unresolved issue [29].

11.5 Nitrate Tolerance

Although amyl nitrite was the first chemical entity that was specifically advocated for the treatment of angina pectoris, 135 years later we still know little about its mode of action at the molecular level. Considering the relatively high chemical reactivity and nitrosating potential of nitrite esters, which form the basis for their use in preparative organic chemistry, and their rapid reaction with nucleophiles, such as thiols, it is likely that S-nitrosothiols are active intermediates involved in blood vessel relaxation [18, 33]. In view of the high intracellular concentrations of reduced glutathione in mammalian tissues, a substantial portion of amyl nitrite that enters the cell may undergo non-enzymatic reactions. Whether or not their interaction with reactive amino moieties also produces potentially harmful N-nitrosamines is unknown. Although a full characterization of the enzymatic pathways involved in generating NO from organic nitrites and nitrates is lacking, it appears that their metabolic routes in the vasculature are distinct [34]. Whereas the majority of enzymatic breakdown of organic nitrites seems to occur in the cytosol, that of organic nitrates is associated with the membrane fraction. Owing to the high volatility of most simple alkyl nitrites, these compounds have been increasingly used as recreational drugs. In addition to prolonged hypotension, severe methemoglobinemia is a common problem observed after misuse of amyl nitrite, which can lead to acute hemolytic anemia. Despite intense study of the chemical reactivity of the inorganic NO donor SNP with a number of electrophiles and nucleophiles (in particular thiols), the mechanism of NO release from this drug also remains incompletely understood. In biological systems, both enzymatic and non-enzymatic pathways appear to be involved [28]. Nitric oxide release is thought to be preceded by a one-electron reduction step followed by release of cyanide, and an inner-sphere charge transfer reaction between the nitrosonium ion (NO+ ) and the ferrous iron (Fe2+ ). Upon addition of SNP to tissues, formation of iron nitrosyl complexes, which are in equilibrium with S-nitrosothiols, has been observed. A membrane-bound enzyme may be involved in the generation of NO from SNP in vascular tissue [35], but the exact nature of this reducing activity is unknown.

11.5

Nitrate Tolerance

Tolerance to nitrates is defined as the reduction in hemodynamic effect or the requirement for higher doses to achieve a persistent effect with continuous use in the face of constant plasma concentrations [15]. Nitrate tolerance was first described for nitroglycerin in 1888 [36]; it occurs with all organic nitrates, albeit to different extents. For reasons that are not understood, PETN appears to be the least susceptible to the development of tolerance. No, or much less, tolerance is observed with nitrite esters, such as amyl nitrite [37], molsidomine, and sodium nitroprusside. Earlier investigations suggested that a depletion of intracellular thiols is involved in tolerance development [17], but this has not been substantiated in later studies [38, 39]. As with organic nitrate bioactivation, the precise mechanism(s) involved in nitrate tolerance remain(s) unknown, but it is likely to be complex and multifactorial. Two principal

293

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mechanisms have been proposed: impaired biotransformation to NO and increased endothelial generation of reactive oxygen species such as superoxide anions. The sources of the latter likely include NAD(P)H oxidase, nitric oxide synthases, xanthine oxidases, and enzymes of the mitochondrial respiratory chain [40]. Interestingly, hydralazine may potentiate the activity of NO donors by virtue of its ability to inhibit NAD(P)H oxidases [41], the most important source of which, from the standpoint of tolerance, is the endothelial cell [42]. The increased formation of superoxide is thought to be secondary to an increase in vascular angiotensin II following activation of the renin-angiotensin system. Superoxide anion can, in turn, oxidize tetrahydrobiopterin, an essential cofactor for NO generation by nitric oxide synthases; a deficiency of this cofactor leads to so-called “uncoupling” of the enzyme, converting it from a source of NO to a source of superoxide anion. Other potential mechanisms include intracellular l-arginine depletion, also leading to an uncoupling of endothelial nitric oxide synthase, upregulation of phosphodiesterase activity, degradation of certain cytochrome P450 isoforms, plasma volume expansion, and neurohormonal activation leading to loss of the tolerance-dependent sympatho-inhibitory mechanism, blunting the response to the NO donor. Some of the confusion in this area arises from an unfortunate lack of clear distinction between the different circumstances under which a reduction in nitrate efficacy is observed. In this respect it is worth noting that the conditions induced by the bathing of isolated tissue with suprapharmacological concentrations of an organic nitrate have little to do with the clinical situation. As pointed out above, in vivo tolerance is multifactorial and, therefore, cannot be readily mimicked by biochemical manipulations of isolated vascular tissue. It is possible that the contribution of each of the individual pathways involved in nitrate tolerance differs, depending on the hemodynamic situation and the particular dose used, which would explain the high degree of variability observed with combination therapies aimed at preventing or overcoming nitrate tolerance. The success of the measures taken to counteract the development of tolerance depends critically on our understanding of the mechanism(s) involved. Of note, nitrate tolerance is often assessed by measurement of surrogate parameters, such as pulmonary capillary wedge pressure, blood pressure, or finger pulse plethysmographic parameters, rather than hard, clinically relevant endpoints, and the relevance of those surrogates for the overall antiischemic effects is not very clear. Another issue is that the extent and rate of development of tolerance may differ substantially between tissues/target organs. This point is exemplified by earlier observations in munitions workers exposed to high concentrations of nitroglycerin vapors, as well as clinical results from patients on nitrate therapy in whom the nitrate headache typically disappears over the course of 2–3 days, while hypotensive, antianginal, and antiplatelet effects persist in most cases. This issue raises a general question as to the clinical significance of nitrate tolerance, which, in general, appears to be more of an issue in Europe than in the U.S. Attempts to counteract tolerance development include the use of thiols such as N-acetylcysteine, antioxidants such as vitamin C and vitamin E, and angiotensinconverting enzyme inhibitors or angiotensin II receptor antagonists. Other approaches to decreasing the development of tolerance include intermittent therapy

11.6 Is Nitrate Therapy Associated with Adverse Vascular Effects?

with the NO donor (using a nitrate-free period every 24 hours), the use of supplemental l-arginine [43], combination therapy with hydralazine [41], and the use of folate [42, 44]. Nitrate-free intervals, while helpful in some cases, run the risk of creating the problem of inadequate antianginal protection of the patient. Simply switching to another nitrate does not resolve the problem because of marked cross-tolerance between different nitrates.

11.6

Is Nitrate Therapy Associated with Adverse Vascular Effects?

Organic nitrates have a low incidence of unwanted side-effects. Those effects known to occur more frequently, such as headache and orthostatic hypotension, are consequences of the vascular actions of NO and, for the most part, minor. Often, tolerance to those unwanted effects develops faster than that to the principal pharmacodynamic effect, and appropriate dose adjustments may alleviate the problem. Despite an impeccable safety record in hundreds of millions of patients over the course of nearly 130 years of therapeutic use, nitrate therapy has recently been suspected to affect endothelial function adversely [45], and to be associated with increased oxidative stress and impaired mitochondrial function [46]. In view of the potent antioxidant properties of NO, this conclusion at first appears counterintuitive, but may be a consequence of enhanced angiotensin II levels and is likely to occur with any vasodilator. The principal difference with NO donors is that the interaction of the NO with superoxide can lead to the formation of the potent oxidant peroxynitrite; however, neither tissue markers of peroxynitrite nor cardiac mechanical function appear to be affected during long-term nitroglycerin treatment, suggesting that organic nitrate therapy does not result in oxidative damage of the heart [47]. Moreover, recent investigations from our own laboratory demonstrate that nitrate metabolism is associated with a marked increase in the level of S-nitrosothiols, compounds known to act as storage forms of NO, conceivably conferring an additional level of protection.

11.7

Conclusions

Similar to other popular drugs such as aspirin, organic nitrates would probably not have overcome today’s regulatory hurdles to enter the clinical arena considering, for example, their mutagenic potential, the development of tolerance, and their almost instantaneous side-effects (hypotension, severe headache). These potential problems are direct consequences of the production of the endogenous messenger molecule NO, but either do not represent a concern in vivo or can be controlled by careful dose titration and choice of the optimal formulation. Sodium nitroprusside suffers from the problem of cyanide accumulation and associated toxicity during prolonged infusion. Molsidomine has been off the market in Europe for some time owing to toxicological concerns (nasopharyngeal carcinoma in rats) that arose from an effect

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later shown to be specific to rodents. Most NO donors in clinical use today were introduced into medicine in an era well before governmental regulation became very restrictive. In fact, nitroglycerin and amyl nitrite were introduced to medicine based on empiric observation without scientific proof of either mode of action or efficacy. Today, the contributions of Hering and his followers, demonstrating that a powerful explosive can be safely administered to humans, have been largely forgotten. Were it not for the peculiar doctrines of homeopathy, nitroglycerin would probably never have been proposed as a remedy for anything. In the last 15 years, safety concerns about NO-generating compounds in general, and the complexity of NO action and metabolism in particular, limited the pharmaceutical industry’s interest in developing new NO-donors. Nevertheless, some progress has been made in the last decade, in particular with “NO-enhanced medicines”, i.e., combination molecules of established drugs with an NO-releasing moiety (either an S-nitrosothiol or an organic nitrate). Several of these compounds show promising pharmacological profiles with clearly reduced untoward side-effects, but none of the lead compounds under development has been approved for clinical use at the time of this writing. An improved understanding of the biological chemistry of NO in recent years and the availability of specific biomarkers for NO in tissues are likely to have a positive impact on the development of selective and effective NO donors for novel cardiovascular applications in the near future.

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Nitric Oxide Donors as Anti-platelet Agents for Thromboembolic Disorders: Clinical Status and Therapeutic Prognosis David R. Janero, David S. Garvey

12.1

Introduction

The notion that inhibitors of human platelet activity might be useful therapeutics rests upon century-old observations [1]. The eminent pathologist Virchow, in 1860, theorized that damage to the blood vessel wall triggers aggregation of blood elements and risks occlusion of the vessel lumen. Bizzozero soon thereafter linked the adhesive properties of the small, colorless corpuscles in human blood to coagulation and clot formation (thrombosis). By 1883, the colorless corpuscles were called platelets, and their defensive physiological role in restoring vascular integrity (hemostasis) became sufficiently established such that, in 1910, hemorrhagic tendency (“bleeding time”) was proposed as a clinical index of platelet number/activity. Subsequent work has provided great insight into the life cycle, physiology, and regulation of the human platelet, lately at molecular resolution. Evolving knowledge about the human platelet has accompanied an increased appreciation of the clinical significance of thromboembolic disorders, whose pathogenesis at least partly reflects an occlusive thrombus, either attached to the blood vessel wall or circulating in the vasculature (embolus). Their efficacy and safety limitations notwithstanding, anti-platelet drugs have clear clinical outcome benefits against morbidity and mortality: they reduce the incidence of nonfatal stroke or myocardial infarction in patients at risk of occlusive vascular events [2]. Recent reviews are available for background and primary references on platelet biogenesis and function in human health and disease [3, 4] and on current plateletinhibitor drugs [2, 5]. To preface consideration of the existing clinical data regarding NO donors as anti-platelet therapy, the following section relates human platelet physiology to pathological thrombus formation and the influence of nitric oxide (NO) therein.

Nitric Oxide Donors Peng George Wang, Tingwei Bill Cai, Naoyuki Taniguchi (Eds.) c 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Copyright
ISBN: 3-527-31015-0

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12.2

Human Platelets, Thromboembolic Disorders, and NO

Human platelets are anucleate, subcellular fragments derived from megakaryocyte cytoplasm within bone marrow. Formed and released into the blood during thrombopoiesis, platelets circulate in the body for about 7–10 days as discrete entities and are replenished at the rate of ≈1011 per day. The high blood concentration (2–3×108 ml−1 ), small size (≈2–3 ìm diameter), and discoid shape of platelets ensure their efficient dissemination throughout the vasculature of the healthy human. These features and the hydrodynamic tendency of flowing blood to push platelets toward the blood vessel wall make circulating platelets highly effective monitors of the integrity of the body’s entire vascular tree [3, 6]. As a critical component of primary hemostasis, the resulting patrol system is well-engineered to fulfill its fundamental, life-saving physiological role: arrest bleeding from wounds. Since severe inherited platelet disorders in humans are very rare [7, 8], the platelet phenotype usually reflects a delicate balance between intrinsic platelet properties defined at thrombopoiesis and a stringently controlled interplay among pro- and anti-thrombotic mediators from the platelet itself, blood cells, and the blood vessel wall (particularly its endothelial lining) [3, 4, 9]. The vascular endothelium is an interface biocompatible with flowing blood. The healthy endothelial lining responds to physical and biochemical stimuli by balancing its production of smooth-muscle relaxing and contracting substances such that vascular tone and homeostasis are maintained. Likewise, the vascular endothelium contributes dynamically to the molecular control of platelet activity [10]. Healthy endothelium is inherently anti-thrombotic due to its regulated production of, predominantly, two potent, synergistic platelet inhibitors: the lipid prostacyclin (PGI2 ), an eicosanoid product of the enzymatic oxygenation of arachidonic acid, and the radical nitric oxide (NO), the gaseous product of enzymatic l-arginine (l-arg) oxidation by the constitutive endothelial nitric oxide synthase (eNOS) isoform [11]. Human platelets contain a constitutive NOS plus a NOS isoform (iNOS) induced rapidly upon platelet stimulation [12]. Although the precise in vivo contributions of various NO sources often remain elusive from clinical studies, NO from both the vascular endothelium and the platelet helps prevent thrombotic episodes and is critical to platelet quiescence and vessel patency [13, 14]. In contrast to agents that inhibit enzymatic production of platelet activators (e.g., aspirin) or bind to platelet surface receptors for agonists/adhesion molecules (e.g., GPIIb/IIIa antagonists) [2, 15], NO is a commonpathway platelet inhibitor that directly stimulates guanylate cyclase production of guanosine 3′,5′-cyclic monophosphate (cGMP), which suppresses platelet function regardless of the particular receptor-mediated agonist(s) activating the platelet [16] (Fig. 12.1). NO (and NO-derived nitrogen oxides) may also inhibit platelet function independently from the conventional, cGMP-mediated mechanism [16]. Normally, quiescent platelets freely circulate through the vasculature, reflective of the hemocompatible character of the vascular endothelium and the anti-thrombotic nature of healthy human blood vessels. Traumatic vascular damage incites a spatially and temporally coordinated platelet transformation encompassing several major, sequential phenotypic changes: platelet adhesion to subendothelial matrix components

12.2 Human Platelets, Thromboembolic Disorders, and NO

PGI2 G

PAF

R

G

ADP

R

G

R

G

Agonists / Activators

Catecholamines Thrombin Collagen

R

PI ATP

R

G

AC

+ PLP IP3 TxA2

AMP Activation protein expression (e.g., P-selectin)

+ +

[Ca++]cytosolic

storage

release

+

Ca++ stored

-

R TK

Shear

GTP

GC

+ NO

cGMP NOS

PDE

GMP

Fibrinogen binding to GPIIb/IIIa Aggregation

Thrombus formation

NO/NO donor

L-Arg

>

Ca++

Fig. 12.1 Schematic representation of human platelet activation. Platelet activators/agonists act generally through receptors R coupled to G-proteins G or kinases, such as tyrosine kinase TK. Receptor-mediated signal transduction activates phospholipases (PLP), some of which catabolize phosphoinositol (PI) to generate inositol trisphosphate (IP3 ), and others which liberate arachidonic acid substrate for eicosanoid [including thromboxane A2 (TXA2 )] production. IP3 is a positive effector, stimulating calcium (Ca++ ) release from intra-platelet stores, whereas prostayclin [prostaglandin I2 , (PGI2 )], stimulates calcium storage by binding to a specific receptor and activating adenylate cyclase (AC) to produce cAMP as second messenger. Calcium mobilization from internal platelet stores stimulates calcium entry into the platelet from the external milieu down a concentration gradient. Elevated cytosolic free calcium

Secretion/ degranulation (ADP, TxA2)

L-Arg

>

>

PDE

cAMP

stimulates the platelet, whereas a reduction in platelet cytosolic calcium decreases the level of platelet activation. Nitric oxide (NO), generated from either an exogenous NO donor or from platelet nitric oxide synthase (NOS) acting upon L-arginine (L-arg) substrate taken up from the plasma, stimulates guanylate cyclase (GC) conversion of GTP to the second messenger cGMP, which inhibits stored calcium release and external calcium entry and thus suppresses platelet activation, regardless of the agonist(s) activating the platelet. Both cAMP and cGMP are actively broken down by phosphodiesterase (PDE) enzymes to AMP and GMP, respectively. Increased platelet activity may encompass the key activation responses listed and culminate in thrombus formation. The symbols + and − denote stimulation or inhibition, respectively. Solid arrows ( ) signify enzymatic reactions; broken arrows ( ) signify effector pathways or responses.

exposed to flowing blood by the vascular insult; platelet shape change, activation, and secretion (degranulation); further platelet recruitment into the wound site; and elaboration of matrix molecules to consolidate the aggregated platelets [4, 17] (Fig. 12.2).

Key Activation Responses

R

Thromboxane

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12 Nitric Oxide Donors as Anti-platelet Agents for Thromboembolic Disorders

Fig. 12.2 Schematic depiction of the role of platelets in thrombus formation. (a) Circulating platelets are kept inactive by prostaglandin (i.e., prostacyclin, PGI2 ) and nitric oxide (NO) released by vascular endothelial cells. Endothelial cells also express CD39 on their surface, which inhibits platelet activation by converting ADP – a potent platelet agonist – to AMP. (b, c) At sites of blood vessel wall injury, platelets adhere to the exposed subendothelium through interactions between collagen, von Willebrand factor, and fibronectin and their receptors on the platelet, integrin á2â1, glycoprotein Ib-IX (GP Ib-IX), and integrin á5â1, respectively. Both thrombin and ADP induce the platelet into an active conformation. (d) The activated platelet secretes ADP, platelet-derived growth factor (PDGF), and fibrinogen from

internal storage granules (“degranulation”) and releases newly-synthesized thromboxane A2 (TXA2 ). ADP and TXA2 signal circulating platelets to become activated and change shape, starting the process of platelet recruitment into the vascular injury site. (e) Glycoprotein IIb/IIIa (GP IIb/IIIa) receptors expressed on the activated platelet surface bind fibrinogen, leading to the formation of fibrinogen bridges between the platelets (“platelet aggregation”). Aggregation and the simultaneous generation of a fibrin meshwork lead to platelet thrombus or clot formation. (f) Clot retraction then leads to formation of a stable thrombus. Figure reproduced with permission from Ref. [2]. Copyright 2002 Nature Publishing Group (http://www.nature.com).

The balance of opposing pro- and anti-platelet forces determines the overall hemostatic response. Successful hemostasis is achieved when assorted signal-transduction systems, mediators, white blood cells, and platelet receptors for agonists and adhesion molecules overcome the local resistance against platelet activation to generate

12.2 Human Platelets, Thromboembolic Disorders, and NO

a stable thrombus that stops the bleeding at the injury site and allows healing to commence [18]. Platelets cannot inherently distinguish between a wounded, hemorrhagic blood vessel that has lost its integrity and an intact, but diseased, vessel predisposed toward local platelet adhesion and activation. Not surprisingly, therefore, hemostatic platelet plug formation and pathological thrombosis share many mediators and platelet responses. The platelet regulatory functions of the human vascular endothelium are impaired by pro-inflammatory and conventional cardiovascular risk factors (including hypercholesterolemia, male gender, family history, age, obesity, smoking, diabetes, hyperhomocysteinemia) and during the course of disorders including atherosclerosis, coronary artery disease, essential hypertension, the hypertension of preganacy (preeclampsia), hemolytic uremia syndrome, and thrombotic thrombocytopenic purura [19, 20, 21]. A vasoconstrictive, pro-thrombotic environment promoting occlusive platelet aggregate formation also contributes to atrial fibrillation and the failure of synthetic vessel grafts and revascularization techniques such as endarterectomy, angioplasty, and stenting [22, 23, 24]. Perhaps the most compelling illustration of the clinical significance of pathological thrombosis is the role of platelets in precipitating most acute coronary syndromes [25, 26]. Occlusive atherosclerotic plaque in coronary arteries, while eliciting stable angina, seldom causes acute unstable angina or acute myocardial necrosis (infarction). Plaque fissure, erosion, or rupture (particularly, rupture of the plaque’s protective fibrous cap) abruptly transitions the chronic, stable state into an acute coronary syndrome. Plaque disruption exposes flowing blood to several subendothelial components adhesive to platelets. At the injury site, hemodynamic forces and a sequence of platelet {adhesion →activation →secretion →recruitment →aggregation}, reminiscent of the hemostatic response, support local formation of a thrombus composed mostly of platelet aggregates in a fibrin reticulum (Fig. 12.2). When coronary arteries are narrowed or occluded by this mechanism over a sufficient period of time, nutritive blood flow to working heart muscle becomes critically compromised (ischemia), leading to acute coronary syndromes (including unstable angina and myocardial infarction) having the potential for devastating clinical manifestations (e.g., cardiac electrical instability, sudden death). Indeed, continuing platelet activation in patients with acute ischemia/infarction is associated with adverse prognosis [27]. A similar acute thrombotic scenario dominated by the platelet in other large- and medium-sized arteries (including peripheral and carotid arteries) has been implicated in the pathogenesis of debilitating peripheral vascular disease and ischemic stroke [28, 29]. Since acute cardiovascular syndromes are the main reasons for admission to coronary care units in the United States and much of the Western world, thrombosis represents a leading cause of morbidity and mortality [30]. Dysregulation of the vascular endothelium has emerged as a critical component of most thrombotic disorders [10, 21]. Often without any anatomical sign of atherosclerosis, many cardiovascular diseases express a vasomotor abnormality termed endothelial dysfunction, indexed clinically as impaired endothelium-dependent vasodilation [31]. Although its mechanism is multifactorial, endothelial dysfunction is characterized by diminished vascular NO production and/or bioavailability [32]. The

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12 Nitric Oxide Donors as Anti-platelet Agents for Thromboembolic Disorders

resultant NO-deficient state creates an imbalance in an important platelet inhibitor relative to other endothelium-derived vasoactive factors. Attenuated platelet NO production/responsiveness and abnormal circulating and urinary biomarkers for NO and platelets are also associated with many cardiovascular diseases and often correlate with coronary risk factors [10, 31, 32]. This scenario implies that endothelial dysfunction and platelet NO resistance pathologically compromise the NO-dependent, anti-thrombotic properties of the vessel wall. Thus, the principal causes of hospitalization and death in the Western world, ranging from childhood stroke to acute coronary syndromes, share a NO-related, pro-thrombotic vasodilator dysfunction. Given the fundamental role of the human platelet in the etiology of atherothrombosis, the proven efficacy of platelet-inhibitor drugs in the prevention and treatment of cardiovascular disease is unremarkable. First-generation anti-platelet agents (aspirin, ticlopidine, and clopidogrel) and the anticoagulants heparin and warfarin reduce the risk of vascular death by about 20% and of non-fatal myocardial infarction and stroke by about 30% in patients with angina, suspected acute myocardial ischemia, or a past history of myocardial ischemia, stroke, or transient ischemic attacks [2, 27, 33, 34]. In a wide range of patients at high risk of occlusive vascular disease, chronic plateletinhibitor therapy (commonly, low-dose aspirin) offers primary protection against myocardial ischemia, stroke, and death [35, 36]. But some patients with coronary artery disease, diabetes mellitus, hypercholesterolemia, and hypertension exhibit increased platelet aggregability that is not readily inhibited by, or may even be refractory to, aspirin [34, 37]. Despite aspirin treatment in patients with acute coronary syndromes or carotid artery stenosis, persistent platelet activation is associated with adverse prognosis [37]. Second-generation anti-platelet agents, such as GPIIb/IIIa antagonists, have yet to prove their general value and safety and may require intravenous administration [2, 15]. Risks, safety issues, and efficacy limitations associated with current anti-platelet/anti-thrombotic clinical strategies complicate patient management [2, 15, 34, 37]. The inability of current drugs to stem the persistence of thrombosis as a leading cause of morbidity and mortality and ameliorate the high incidence of major ischemic cardiovascular events invites new approaches toward improved anti-platelet therapy. One of these, the use of NO donors, has great intrinsic appeal from the crucial importance of NO as a physiological anti-thrombotic agent and the increasing recognition that vascular NO insufficiency is a component of various atherothrombotic diseases (vide supra). The clinical benefits of organic nitrates are attributed mainly to their vasodilator property, effected through their denitration (bioactivation) to yield NO [38]. Since 1967, nitrovasodilators of diverse chemical structures have been known to have anti-platelet effects in vitro [39], suggesting that at least some of their therapeutic benefit in diseases associated with a heightened thrombotic risk may reflect in vivo platelet inhibition. Furthermore, the anti-platelet actions of NO are uniquely diverse: NO inhibits platelet activation, aggregation, degranulation, and recruitment and promotes platelet disaggregation [13, 40]. The following sections will summarize the salient clinical data on NO donors as anti-platelet agents, organized according to the specific NO donor studied (Table 12.1). The discussion will consider only those clinical trials in which a NO donor

12.2 Human Platelets, Thromboembolic Disorders, and NO Tab. 12.1: NO donors examined in clinical trials for anti-platelet/anti- thrombotic activity.*

NO donor

Structure O

Nitroglycerine

NO2

O

O2N

O

O2N O

NO2

H O

O H

Isosorbide dinitrate O2N O

O NO2

H O

O H

Isosorbide 2-mononitrate HO

OH

H O

O H

Isosorbide 5-mononitrate

O NO2

NO CN Fe NC CN CN

2-

NC

2Na+ .2H2O

Sodium nitroprusside O

+ N N N O

3-Morpholinosydnonimine (SIN-1) O

N

Molsidomine

* References

NH2

O O

O

HO

S-Nitroso-glutathione

N

+ N N O

O

NH

N H

S N H N

O O OH

O

are cited in the text.

was administered to human subjects and at least one purported index of platelet activity was assessed. This presentation will also encompass clinical studies that have attempted to document a prognostic outcome benefit from a known platelet inhibitory NO donor, whether or not platelet activation status was assessed. By these

305

306

12 Nitric Oxide Donors as Anti-platelet Agents for Thromboembolic Disorders Tab. 12.1: (continued)

NO donor

Structure O

NH

HO

N H

NH2

l-Arginine

NH2

O O

O O

O

NO2

NCX-4016

criteria, although isolated human platelets are a convenient, homogeneous model, NO donors identified solely as in vitro inhibitors of isolated human platelets will not be dealt with e.g., [41–48] (Table 12.2). Likewise, the very limited, far from consensus, clinical data on the anti-platelet effect of NO gas e.g., [49], ingested inorganic nitrate (NO3 − ) e.g., [50], or a nitrate-rich diet [51] are not considered. It should also be noted that some NO donors have been studied in the clinic e.g., [52, 53] and are prescribed as cardiovascular medicines (e.g., pentaerithrityltetranitrate [54]) without published documentation of their potential platelet effects in a human outcome trial.

Tab. 12.2: Examples of NO donors shown to inhibit isolated human platelet activity in vitro.

NO Donor

Structure -

O

N N

DEA/NO

Reference

O N

[41] -

O

+ H 3N

O N

N N

N H

SPER/NO

O

S

[42]

CO2H

O N

NH2

N

[43]

S-NO-Captopril O HO

S

N

O

HN

S-NO-Acetyl-cysteine

O

[44]

12.3 Nitrovasodilators Tab. 12.2: (continued)

NO Donor

Structure

Reference

O HO

S

N

O

HN

S-NO-N-acetyl-penicillamine

[45]

O O

O O

HO

O NO 2

H H

21-NO-Prednisolone (NCX-1015)

H

O

[46] CN + O N

4-Phenyl-3-furoxancarbonitrile

GEA 3175

[47]

N O

Cl

+ N N N O

N O

S O

[48]

O2N O O2N O O NO2

Pentaerythrityltetranitrate

O NO2

[54]

12.3

Nitrovasodilators 12.3.1

Glyceryl Trinitrate, Nitroglycerin (GTN)

Over a century ago, empirical observation was made that organic nitrates, including glyceryl trinitrate (GTN), alleviate angina. Since then, GTN has been a mainstay therapy for angina and cardiac failure, even with the possible loss of effectiveness (tolerance) over extended dosing [55] and the risk of platelet hyperactivity in GTNtolerant patients [54]. Despite this venerable therapeutic history, the mechanism of GTN bioactivation to NO is speculative at best [38]. In 1967, some 15 years before identification of NO as a biological entity, GTN was the first nitrovasodilator shown

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12 Nitric Oxide Donors as Anti-platelet Agents for Thromboembolic Disorders

capable of inhibiting human platelet activity (i.e., aggregation) in vitro [39]. As subsequently demonstrated, the suprapharmacological concentrations required reflect the fact that (human) platelets lack a robust, intrinsic capacity for organic nitrate bioactivation to NO [56]. The 1980s saw the earliest clinical investigation of GTN’s potential to inhibit human platelet function in vivo [57–60]. Sublingual and intravenous GTN, at therapeutic doses or greater, was generally found to increase hemorrhagic tendency (i.e., prolong bleeding time) in healthy subjects and in coronary disease patients. Negligible, if any, effect of GTN administration on more specific platelet endpoints (e.g., number of circulating platelet aggregates or agonist-induced platelet aggregation in plateletrich plasma in vitro) could be demonstrated. The sole subject in these four studies who did show a marked inhibition of ex vivo platelet aggregation after one hour of high-dose GTN infusion also had the greatest hemodynamic response (i.e., bloodpressure drop). Accordingly, the prolongation of bleeding time observed in these early, uncontrolled studies likely reflected vasodilation and increased venous capacitance rather than pharmacological inhibition of platelet function [60]. Reagrdless of mechanism, the effect of therapeutic GTN on bleeding time in healthy volunteers appeared slight at best in a subsequent, controlled trial [61]. Under some conditions of blood collection and in vitro aggregometry, the inhibitory effect of GTN on agonist-induced platelet aggregation was not expressed unless exogenous thiol (e.g., N-acetyl-cysteine) was added prior to aggregometry [62]. Yet in a double-blind, randomized, controlled, cross-over trial with healthy male volunteers, transdermal GTN at doses 2–4-fold above therapeutic levels had no effect on ADP-induced platelet aggregation in whole blood, whether or not N-acetyl-cysteine had been co-administered [63]. This trial may have been confounded by the development of nitrate tolerance in its subjects. Although not conducted in a double-blind, placebo-controlled, cross-over fashion, the study of Diodati et al. represents a methodological and conceptual turning-point in the clinical investigation of GTN as an anti-platelet agent [64]. These investigators demonstrated that inhibition of human platelet aggregation by GTN is so transient and reversible that in vitro aggregometry must be conducted without delay after GTN administration. Consequently, Diodati et al. employed bedside aggregometry in whole blood, which eliminated the need for platelet-rich plasma preparation and allowed assessment of the potential anti-platelet effect of therapeutic GTN infusion in coronary artery disease patients within 30 s of phlebotomy. Under these assay conditions, GTN infusions incrementally titrated to produce no more than a 10% decrease in mean arterial blood pressure were shown to inhibit in vitro platelet aggregation by at least 50% in 8 out of 10 total subjects, regardless of platelet agonist. Notably, the anti-platelet effect was lost within 15 min post-infusion. GTN’s anti-aggregatory and hemodynamic (i.e., hypotensive) effects did not correlate, suggestive of direct platelet inhibition by GTN in vivo. Subsequent clinical studies provided additional evidence that GTN exerts antiplatelet activity in vivo and offered insight into the nature of GTN’s anti-aggregatory pharmacology. A dose–effect relationship between intravenous GTN and inhibition of platelet aggregation was uncovered in healthy male subjects, in whom plasma

12.3 Nitrovasodilators

concentrations of GTN and its metabolites correlated with the anti-aggregatory efficacy [65]. Sublingual GTN administered at a therapeutic dose to healthy subjects and to patients with stable angina and hyperaggregable platelets inhibited ADP-induced platelet aggregation by ≈35%, as evaluated within three minutes post-dosing by in vitro aggregometry in platelet-rich plasma [66]. An increase in platelet cGMP was not observed and was perhaps lost during plasma preparation. Two studies with transdermal GTN were likewise positive. In healthy subjects, an anti-platelet effect could be demonstrated by whole-blood aggregometry up to 2 h after GTN patch application, provided that a phosphodiesterase inhibitor was introduced into the blood at the time of sampling and aggregometry was carried out within 1 min of phlebotomy [67]. The lack of effect of transdermal GTN on heart rate or blood pressure in this study suggested that GTN had exerted a direct anti-platelet effect in vivo. The same dose of transdermal GTN was administered in a subsequent randomized, doubleblind, controlled trial to stable angina patients, in whom GTN was vasoactive [68]. Significant platelet inhibition was observed: in vitro whole-blood aggregation to ADP decreased by ≈25% in 70% of the patients administered GTN and in only 27% taking a placebo. In contrast, a hemodynamically effective infusion of GTN into coronary disease patients, either before or after bypass surgery, did not influence three perioperative indices of hemostasis: bleeding time, in vitro platelet adherence to glass, and in vitro clot formation [69]. More direct platelet function tests (e.g., aggregation) were not conducted. The significance of this omission is highlighted by a report that, in healthy volunteers given transdermal GTN at a hemodynamically active dose, fibrinolytic capacity was unaffected, yet ADP-induced platelet aggregation in vitro was inhibited [70]. Three subsequent GTN infusion studies were predicated on the importance of activated platelets in precipitating most acute coronary syndromes [25–27]. The first exploited the observation that rapid atrial pacing in patients with stable coronary artery disease causes platelet hyperaggregability across the coronary bed, similar to the platelet hyperresponsiveness that helps precipitate unstable angina [71]. To this intent, Diodati et al. sampled both coronary sinus and arterial blood from stable coronary disease patients at rest and up to 10 min after inception of atrial pacing, with or without a therapeutic GTN infusion causing hypotension [72]. Pacing-induced platelet activation (i.e., platelet hyperaggregability) was observed in coronary-sinus blood and was abrogated by GTN pretreatment. In another study [73], patients premedicated with aspirin (300 mg, p.o.) were administered intravenously a vasoactive GTN dose within 48 h of the onset of either acute myocardial infarction or unstable angina. Platelet activation was determined prior to GTN infusion and after GTN administration once blood pressure returned to post-infusion values as activation antigen/adhesion protein (P-selectin, GPIIb/IIIa) expression on the platelet surface. According to this virtually immediate readout, GTN infusion inhibited the platelet activation that persisted in acute coronary syndrome patients treated with aspirin. Similar acute inhibitory effects of therapeutic GTN infusion on basal and stimulated platelet adhesion-protein expression have also been documented in healthy male volunteers [74].

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Two somewhat paradoxical GTN trials remain to be considered. In one, patients within 5 days of an ischemic or hemorrhagic stroke were randomized to receive either placebo patches or a therapeutic, vasoactive dose of transdermal GTN over the ensuing 12 days [75]. Neither in vitro platelet aggregation nor platelet activation antigen expression was affected on the first or the eighth day of GTN treatment. The nitrate tolerance that developed in these subjects and their relatively late treatment after clinical presentation may help explain the negative result. In patients with non-insulin-dependent diabetes, free of cardiovascular complications, sublingual or transdermal GTN did not inhibit ADP-induced platelet aggregation in platelet-rich plasma [76]. Yet up to 12 h after dosing, GTN significantly inhibited platelet aggregation in control nondiabetics. Since antioxidants (glutathione, vitamin E) normalized the hyperaggregability of the diabetic patients’ platelets, underlying oxidative stress may have confounded GTN’s potential anti-platelet action. Can the anti-platelet properties of GTN be linked to significant prognostic impact in a positive clinical outcome study? Aside from a small, randomized, placebocontrolled trial demonstrating that sublingual GTN increased maximum walking distance in patients with peripheral vascular disease [77], two major investigations have dealt with this question. The first is a meta-analysis of the collective mortality results from seven randomized, controlled, intravenous GTN trials in patients at high mortality risk from acute myocardial infarction [78]. This analysis is quite controversial, since virtually all of the individual trials considered were insufficiently powered to detect clinical benefit, and none was designed to assess GTN’s mortality effect. Nevertheless, the meta-analysis suggested that intravenous GTN elicited an early mortality reduction of around 35% in acute myocardial infarction. The greatest impact on mortality seemed to occur during the first week of follow-up. In contrast, GISSI-3, a multi-center, randomized, placebo-controlled trial, was specifically designed and powered to assess whether early intravenous plus subsequent transdermal GTN improved cardiac function and six-week survival after acute myocardial infarction [79]. Alone or together with the ACE inhibitor lisinopril, GTN did not show any independent effect on severe ventricular dysfunction or mortality, whereas lisinopril had positive effects on both outcome measures. The negative GISSI-3 trial argues against a substantive, clinically beneficial anti-platelet effect of GTN in acute myocardial infarction, at least of the tenor that had been demonstrated for aspirin [2, 27, 33, 34]. However, the additive effect of lisinopril plus GTN in reducing mortality in the GISSI-3 trial could reflect a component of in vivo platelet inhibition by GTN. Several factors may have conspired to obscure any difference between GISSI-3 control vs. GTN-treated patients, including: a heterogeneity of response with varying infarct size; the intensive subject pre-exposure to thrombolytic therapy and aspirin; and the administration of nitrovasodilators (including intravenous GTN) to over 50% of the control subjects.

12.3 Nitrovasodilators

12.3.2

Isosorbide Dinitrate (ISDN) and Isosorbide Mononitrate (ISMN)

Along with GTN, ISDN is the organic nitrovasodilator most widely used in clinical practice for the treatment of angina, although tolerance may develop with extended dosing [55, 80]. ISDN has a longer duration of action than GTN, since its in vivo metabolites, IS-2-MN and IS-5-MN, are cleared relatively slowly [81]. By the late 1980s, it was well established that ISDN and its mononitrate metabolites, at suprapharmacological concentrations, inhibited isolated human platelet aggregation in vitro, IS-2-MN being the most potent anti-aggregatory agent of the three, and IS-5-MN, the weakest [82]. Reminiscent of the initial clinical experience on the anti-thrombotic effects of GTN, early reports constitute essentially observational studies on ISDN or ISMN infused into either healthy subjects or stable angina patients. In vitro aggregation in platelet-rich plasma was commonly used to index an in vivo platelet effect post-dosing. Despite the very weak response of isolated human platelets to ISDN or ISMN, four uncontrolled studies by DeCaterina et al. in stable angina patients demonstrated that therapeutic levels of infused ISDN, IS-5-MN, or IS-2-MN markedly inhibited subsequent adenosine diphosphate- and adrenaline-induced platelet aggregation in vitro and acutely reduced the number of circulating platelet aggregates [83–86]. An antiplatelet effect persisted up to 30 min after discontinuation of mononitrate infusion and up to 60 min after ISDN infusion. Although platelet inhibition by ISDN could be detected at hemodynamically neutral doses, the anti-platelet effects of ISDN and its mononitrate metabolites were generally dose-dependent and correlated well with the degree of blood pressure drop. At the highest doses, the platelet-inhibitory effect diminshed, presumably due to excessive vasodilation that incited a compensatory discharge of pro-aggregatory catecholamines. Indeed, catecholamine discharge may help account for the reported lack of an anti-aggregatory effect of oral IS-5-MN in stable angina patients [87]. Yet platelet sensitivity to PGI2 increased, a finding reminiscent of the correlation between PGI2 level and in vitro inhibition of platelet aggregation noted in aortic blood of ischemic heart disease patients after bolus oral ISDN administration [88]. In patients with peripheral (femoral) and coronary artery diseases, oral ISDN did not alter radiolabeled platelet deposition over the femoral atherosclerotic lesion site, but did synergize the inhibition of platelet deposition by PGE1 , a platelet-inhibitory prostaglandin [89]. These results [88, 89] implicate eicosanoid mediators in the anti-platelet activity of ISDN in vivo. Not every platelet endpoint need be sensitive to ISDN/ISMN: oral IS-5-MN administered to healthy volunteers inhibited platelet activating factor (PAF)-induced aggregation in vitro without affecting platelet secretion, plasma cGMP level, or bleeding time [90, 91]. Virtually all of the above cited studies were uncontrolled and involved acute oral or intravenous ISDN/ISMN administration followed within hours by in vitro platelet activity assessment. In contrast, Sinzinger et al. showed in an uncontrolled trial that oral ISDN (100 mg daily) administered to coronary artery disease patients over four weeks inhibited ADP-induced aggregation in platelet-rich plasma in vitro and reduced the number of circulating platelet aggregates and platelet production of thromboxane

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12 Nitric Oxide Donors as Anti-platelet Agents for Thromboembolic Disorders

B2 (TxB2 ), a vasoconstrictive platelet activator [92]. Wallen et al. reported the results of a randomized, double-blind, placebo-controlled cross-over study of 20 mg ISDN given twice daily for two weeks to ischemic heart-disease patients either at rest or after platelet aggregation induced in vivo by dynamic exercise [93]. A number of operational factors were controlled in this study to obviate potential complications from nonstandard patient compliance, diurnal variations in platelet function, and changes in subject posture that could increase plasma catecholamines and spuriously activate platelets. Although exercise potentiated platelet aggregability and secretion and ISDN had clinical effects (e.g., blood pressure lowering, headache induction), no inhibition of platelet function was seen. Three randomized, double-blind, placebo-controlled studies, one of infused ISDN in patients undergoing cardiopulmonary bypass [69], the others of ISDN [70, 94] and IS-5-MN [70] in healthy volunteers, likewise failed to show any effect of these organic nitrates on platelet function, despite significant blood pressure drops in the nitrate-treated subjects. These data make it tempting to conclude that the acute anti-platelet effects of ISDN/ISMN observed in uncontrolled studies are not readily verifiable in comparing active treatment and placebo in controlled clinical trials. Whether this difference in study design alone fully explains the contrasting efficacy findings is questionable: an uncontrolled trial documented only marginal immediate platelet inhibition by graded ISDN infusions into stable angina patients [95], whereas a randomized, placebo-controlled study involving aspirin-treated men with acute myocardial infarction demonstrated that infused ISDN reduced both in vitro platelet aggregation and adhesion [96]. An outcome-based clinical study showed that up to 16 days of transdermal ISDN treatment in four pre-eclamptic women elicited a virtually immediate and profound improvement in fetoplacental circulation [97]. The improvement might have reflected inhibition of microthrombus formation, but platelet activity was not assessed. One large, multi-center, randomized, placebo-controlled clinical trial of IS-5-MN in a wide range of low-to high-risk patients with suspected or documented acute myocardial infarction has been conducted [98]. In this so-called ISIS-4 trial, the treatment group received daily oral doses of controlled-release IS-5-MN for one month. Reminiscent of the GISSI-3 trial with GTN [79], the ISIS-4 trial offered no evidence that IS-5-MN started early in acute myocardial infarction improved survival, even in patients free of non-study nitrates, although there were clear signs (hypotension, headache) that IS-5-MN was indeed active. As with GISSI-3, most patients in the ISIS-4 trial had undergone thrombolysis. The possibility thus remains that nitrate therapy might exert a decisive anti-platelet effect in, for example, patients not eligible for thrombolysis. The ISIS-4 trial has been criticized for employing IS-5-MN, a weak NO donor [73, 82]. 12.3.3

Sodium Nitroprusside (SNP)

The nitrovasodilator sodium nitroprusside (SNP) has been used for decades to manage acute hypertensive crises and congestive heart failure complicating myocardial ischemia [99]. However, prolonged SNP administration is limited by tolerance, the

12.3 Nitrovasodilators

need for parenteral administration, and the potential toxicity of the cyanide generated upon NO release [100]. The anti-aggregatory effect of therapeutic levels of SNP on isolated human platelets was initially documented in 1974 [101]. Soon thereafter, the much greater in vitro potency of SNP as inhibitor of human platelet aggregation relative to the organic nitrovasodilators GTN and ISDN was noted [102]. This difference could most readily be rationalized by the fact that organic nitrates require bioactivation to generate NO, whereas SNP is a coordination complex that releases NO both spontaneously at physiological pH and as a consequence of tissue catabolism [103]. In uncontrolled clinical studies, Mehta and Mehta demonstrated that infused SNP titrated to produce a hemodynamic response normalized the characteristically high number of circulating platelet aggregates in heart-failure patients and dosedependently decreased (by 20–40% pre-infusion responses) ADP- or epinephrineinduced platelet aggregation in platelet-rich plasma in vitro [57, 104]. The first prospective, controlled human study of clinical doses of infused SNP on platelet function was also positive: SNP infused for blood pressure control during anesthesia prior to coronary bypass surgery acutely and dose-dependently inhibited (by up to 50%) platelet aggregation to ADP and epinephrine and concomitantly prolonged bleeding time [105]. As measured by whole-blood aggregometry in vitro, the platelet hyperaggregability induced by rapid atrial pacing in stable coronary artery disease patients was blunted acutely by therapeutic SNP infusion, whereas SNP did not affect these subjects’ platelet aggregability to ADP or thrombin prior to pacing [72]. In subjects with normal left-ventricular function undergoing elective coronary bypass surgery, a 60 min therapeutic SNP infusion prevented the up-regulation of platelet adhesion molecules up to 35–60 min post-infusion without hemodynamic effect, as documented in two randomized, prospective, placebo-controlled studies [106, 107]. Three essentially negative SNP trials must be acknowledged. In an uncontrolled trial with healthy volunteers, clinical doses of infused SNP inhibited neither platelet Pselectin expression nor ADP-induced aggregation in vitro, but did suppress epinephrine-induced aggregation up to 4 min after discontinuation of the SNP infusion [108]. This study is complicated by the fact that SNP, by markedly increasing heart rate, likely induced catecholamine release that would have primed platelet aggregation. Another study found no acute effect of therapeutic and vasoactive SNP infusion on platelet aggregation to collagen or ADP in whole blood from patients with angina, either with or without angiographic evidence of atherosclerosis [109]. A randomized, doubleblind, placebo-controlled, two-way cross-over study demonstrated that the platelet activation observed within 5 min after the start of hemodialysis was not affected by a 15 min SNP infusion delivered via the inlet of the hemodialysis device [110]. Since there was no blood-pressure effect of SNP in this study, the negative platelet result may reflect sub-optimal dosing and/or scavenging of SNP-derived NO by the erythrocyte hemoglobin in the whole-blood dialysate. Perhaps the in vivo anti-platelet effects of SNP are most likely seen when platelets are pre-activated in vivo (e.g., by plaque rupture in unstable angina patients). A meta-analysis of the collective outcome results from three randomized, controlled intravenous SNP trials suggested that intravenous SNP reduces early mortality by ≈35% in acute myocardial infarction [78]. Since each of the component trials was

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12 Nitric Oxide Donors as Anti-platelet Agents for Thromboembolic Disorders

not designed to assess SNP’s efficacy in reducing mortality, the conclusion of clinical benefit is controversial. Suggestion that SNP could have a platelet-related effect sufficient to improve clinical outcome comes from the demonstration that SNP infused during coronary bypass attenuated the associated systemic inflammatory response, an important determinant of the success of open-heart surgery [111].

12.4

Oxatriazolium NO Donors 12.4.1

Sydnonimines

The sydnonimine class of NO donors is typified by 3-morpholinosydnonimine (SIN1), which is generated from its precursor, molsidomine, mainly in the liver [112]. Molsidomine has a slower onset and longer duration of action than conventional nitrovasodilators due to its relatively slow conversion to SIN-1, whereas SIN-1 itself has a rapid onset and short duration of action [113]. NO release from SIN-1 occurs spontaneously in blood with concomitant generation of superoxide anion radical (O2 •− ) [114]. Both molsidomine and SIN-1 are vasodilators, and this effect was first exploited clinically in 1978 to alleviate angina [115]. Since molsidomine and SIN-1 do not induce tolerance or cross-tolerance with conventional nitrates, both sydnonimines received considerable attention in the 1980s for their potential to serve as tolerancefree alternatives to classic nitrovasodilators in stable angina and heart failure [113]. The overall therapeutic appeal of molsidomine and SIN-1 has since been weakened by the potential generation of the potent oxidizer peroxynitrite (ONOO− ) from SIN1 decomposition products NO and O2 •− , the frequent dosing necessitated by SIN1’s short duration of action, and concerns about the ill-understood sydnonimine mechanism of vasodilation [116]. Both molsidomine and SIN-1 have been evaluated clinically for anti-platelet effects. In a double-blind, placebo-controlled study with 12 healthy volunteers, oral administration of a 4 mg dose of molsidomine markedly attenuated platelet-activating factor (PAF)-induced platelet aggregation in vitro up to 30 min post-dosing. 24 h thereafter, molsidomine’s anti-aggregatory effect was lost [88]. In a subsequent uncontrolled study by the same group, six male and six female healthy volunteers were administered molsidomine intravenously at a dose of 60 ìg kg−1 . In eight of the ten patients evaluated, in vitro platelet activation by PAF was significantly attenuated 40 min postinfusion. Plasma levels of molsidomine, its active metabolite (SIN-1), and cGMP were measured up to 1 h following molsidomine infusion. Plasma concentrations of both molsidomine and SIN-1 dropped off rapidly after molsidomine dosing, and plasma cGMP was unchanged from pre-treatment level. The lack of a SIN-1 affect upon plasma cGMP was attributed to the target of NO activation. NO stimulates cGMP production from the intracellular (cytosolic) form of guanylate cyclase, as opposed to the membrane-bound isoform. Plasma cGMP may be a marker for the activity of the membrane-bound guanylate cyclase isoform [117].

12.4 Oxatriazolium NO Donors

A double-blind, placebo-controlled study in which a 4 mg dose of molsidomine was orally administered confirmed the anti-aggregatory effects of the drug when administered intravenously. Although the anti-platelet effects were maintained 1 h after molsidomine administration, bleeding time was unaffected [89]. In an uncontrolled study in patients with obliterating atherosclerosis of the lower extremities, molsidomine and PGI2 were evaluated for potential synergistic actions on platelet and fibrinolytic activities. At the doses given, neither molsidomine nor PGI2 as a monotherapy affected either function. However, the two drugs combined enhanced the overall anti-platelet and fibrinolytic activity without concomitant potentiation of the hypotensive effects of the two compounds. The synergism observed was attributed to the complimentary mechanisms by which each drug exerts its effects. The anti-platelet activity was attributed to a NO-mediated potentiation by cGMP of the anti-aggregatory action of PGI2 -mediated cAMP production. The fibrinolytic synergism was characterized as arising from an inhibition of plasminogen inhibitor release from platelets by NO and a PGI2 mediated release of tissue plasminogen activator from endothelial cells [118]. In an uncontrolled study with stable angina patients, the vasodilator and in vitro anti-platelet effects of graded doses of infused SIN-1 were assessed. Platelet responses to either ADP or the thromboxane B2 mimetic, U-46619, indicated that SIN-1 treatment only marginally affected platelet aggregation to either agonist [92]. Another uncontrolled study of SIN-1 in the same patient population examined the effects of the drug on platelet Ca+2 handling. SIN-1 reduced cytosolic Ca+2 in unstimulated platelets by decreasing Ca+2 influx, while in stimulated platelets it attenuated Ca+2 mobilization from internal stores. Superoxide dismutase (SOD), but not catalase, potentiated these responses. Thus, the effects of SIN-1 on Ca+2 handling resembled those of NO as modulated by the simultaneous release of O2 •− . By virtue of catalase’s lack of effect in this in vitro aggregation system, the influence of SIN-1 on Ca+2 handling was shown to be independent of O2 •− conversion to H2 O2 . Since SOD scavenges O2 − and thus limits ONOO− formation, it was felt unlikely that ONOO− contributed to the platelet Ca+2 handling response to SIN-1; rather, it was concluded that SOD increases NO bioavailability [119]. Molsidomine and its NO-donor active metabolite, linsidomine, have been studied in one large-scale, placebo-controlled, double-blind outcome study involving over 4,000 subjects [120]. In this so-called ESPRIM trial, patients with acute myocardial infarction and no signs of overt heart failure were randomly assigned to receive within 24 h of symptom onset either placebo or linsidomine (1 mg h−1 intravenously for 48 h) followed by 16 mg oral molsidomine daily for 12 days. All other standard treatments,but not vasodilators, were allowed at physicians’ discretion. With the exception of increased headache frequency in the treatment group, sydnonimine treatment did not reduce 35 day or long-term mortality or affect the incidence of major or minor adverse events. Similar to the GISSI-3 trial [79] with GTN and the ISIS-4 trial with IS-5-MN [98], the sydnonimine ESPRIM trial failed to demonstrate an independent survival benefit of an anti-platelet NO donor.

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12.5

Nitrosothiol NO Donors 12.5.1

S-Nitroso-glutathione (GSNO)

S-Nitroso-glutathione (GSNO) is a naturally-occurring NO donor that can act as a physiological NO surrogate or congener in vivo [121]. Cells and platelets synthesize GSNO by as-yet ill-defined mechanisms probably involving reaction between glutathione and nitrosating nitrogen oxides (e.g., N2 O3 , N3 O4 ). Generation of NO from GSNO likely occurs through mechanisms compatible with physiological pH, oxygen tension, and redox poise: e.g., transition metal (particularly copper)-catalyzed decomposition and/or transnitrosylation (transfer of a NO+ -equivalent from GSNO to an acceptor thiol) [116]. Plasma glutathione peroxidase augments NO liberation from GSNO [122]. Another mammalian enzyme, glutathione-dependent formaldehyde dehydrogenase, may also facilitate platelet GSNO turnover, although the enzyme’s presence in platelets is unconfirmed [123]. Whether synthesized endogenously or sequestered from plasma, platelet GSNO can be mobilized during platelet activation to dampen platelet activity [124]. For over a decade, GSNO has been known to inhibit isolated human platelet activation and aggregation [125]. The mechanism of this inhibition is complex. The prevalent signal-transduction pathway for platelet inhibition by NO (liberation of NO from GSNO to activate platelet guanylate cyclase and increase platelet cGMP content) may operate along with cGMP-independent mechanisms involving platelet protein nitration and NO metabolites such as ONOO− [126, 127]. Erythrocytes efficiently scavenge GSNO-derived NO, raising doubt about GSNO’s potential to exert an anti-thrombotic effect in vivo [128, 129]. Yet de Belder and colleagues demonstrated in healthy male volunteers that GSNO infusions up to 1.5 ìg min−1 for 5 min markedly inhibited ADP-induced platelet aggregation in platelet-rich plasma in vitro at doses that only marginally influenced vascular tone [130]. These investigators also showed that a 30 min GSNO infusion into healthy female volunteers (2.5 mg maximum total dose) inhibited in vitro platelet aggregation by up to 66% without altering systemic blood pressure or pulse [131]. Subsequent studies in stable angina patients undergoing coronary angioplasty [132] and in females with severe preeclampsia [133] documented that infused GSNO (16–22 mg total dose) reduced the platelet activation-protein expression associated with these maladies. Particularly noteworthy was the finding that hemodynamically neutral GSNO doses exerted a platelet inhibitory effect in angioplasty patients pre-medicated with aspirin and GTN [132]. These studies gave rise to the concept that GSNO is a “platelet-selective” NO donor preferentially affecting platelet activation over vasodilation. In contrast, the ready bioactivation of the organic nitrates GTN, ISDN, and ISMN by the vascular endothelium, but not by the (human) platelet, predisposes these drugs toward smooth-muscle relaxation and vasodilation. Further support for this concept was provided by Langford et al.’s demonstration that both infused GTN and GSNO inhibited the systemic platelet activation in unstable angina patients af-

12.5 Nitrosothiol NO Donors

ter aspirin treatment, but GSNO was much better tolerated [73]. Likewise, infused GSNO exerted a significant, acute anti-platelet effect (reduction of P-selectin surface expression) in bypass patients without hemodynamic influence when infused after the subjects had been weaned off of cardiopulmonary bypass, though all subjects had received GTN and anticoagulant (heparin) [134]. A negative report that GSNO infused into stable angina patients during coronary bypass surgery did not alter platelet P-selectin and GPIIb/IIIa expression may have been confounded by GSNO photodegradation within the cardiopulmonary bypass circuit [135]. In a series of four small trials, Markus and colleagues studied whether intravenous GSNO affected thromboembolus formation in patients undergoing either carotid endarterectomy or angioplasty and stenting for symptomatic carotid artery stenosis. Despite routine administration of aspirin and heparin to these patients, procedurallyinduced vascular endothelial denudation incites circulating cerebral thromboemboli, the frequency of which can be quantified as a surrogate endpoint of platelet activity by noninvasive ultrasonography. Initial data demonstrated that GSNO infusion (to a maximum total dose of ≈500 nmol kg−1 or until a threshold blood-pressure drop was attained) reduced the mean embolization frequency after carotid endarterectomy by 80% [136]. The effect was rapid in onset and sustained for 6 h, although the GSNO infusion had been terminated 2 h post-endarterectomy. A follow-up randomized, double-blind, placebo-controlled trial with a larger number of patients showed that a comparable, 90 min GSNO infusion elicited a 98% reduction in the frequency of embolic signals (vs. saline-treated controls) during the first 4 h after carotid endarterectomy [137]. A significantly diminished embolization frequency was maintained up to 24 h post-surgery relative to the saline group. Consonant with these results, short-term GSNO infusion markedly and rapidly reduced the frequency of embolic signals in patients with actively embolizing symptomatic carotid stenosis who had not undergone endarterectomy [138]. No side-effects (e.g., hypotension, hemorrhage) were observed in these patients, despite the fact that the suppression of embolization by GSNO persisted over 24 h. That the anti-thromboembolic effect of GSNO was not restricted to the setting of endarterectomy could also be demonstrated by a randomized, double-blind, placebo-controlled study in which a 90 min GSNO infusion virtually eliminated embolic signals within the first 6 h after angioplasty and stenting for treatment of symptomatic, high-grade carotid stenosis [139]. Although the frequency of thromboembolism correlates with perioperative stroke risk in the clinical setting of carotid endarterectomy or angioplasty, the consistently positive results of Markus and colleagues [136–139] do not imply significant clinical benefit, for the studies were not powered for an effect on morbidity or mortality. Suggestion from this work that GSNO might have the potential for clinical outcome benefit in patients with carotid artery stenosis comes from the reduction in recurrent throboembolic events after GSNO treatment vs. placebo group noted in one study [138] and the trend toward improved overall clinical status in another [139]. A case report demonstrating that a total GSNO infusion of 7.5 mg over 90 min rapidly improved the condition of one subject with a rare pre-eclampsia variant characterized by thrombotic microangiopathy is similarly suggestive of GSNO’s potential to exert a prognostic clinical effect that is platelet-related [140].

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12.6 L-Arginine {S(+)-2-Amino-5-[(aminoiminomethyl)amino]pentanoic acid} (L-arg)

The conditionally essential amino acid l-arginine (l-arg) has varied roles in mammalian metabolism [141]. Some of l-arg’s bioactivity could modulate platelet function: l-arg is a secretagogue for insulin, an anti-thrombotic humoral factor, and serves as the physiological substrate for catalytic NO production by NOS. Human platelets internalize circulating plasma l-arg through high- and low-affinity transport systems that supply platelet NOS with substrate for NO biosynthesis, and platelet function is influenced by endogenously generated NO [142, 143]. Consequently, a substratecontrol mechanism exists by which l-arg supplementation could potentiate directly platelet NO production and inhibit platelet activity. Less direct means have also been proposed to explain how supplemental l-arg might increase bioactive NO in the vasculature and/or elicit an anti-platelet effect advantageous to vascular health [144]. Regardless of the mechanisms through which exogenous l-arg influences platelet activity, the association between impaired platelet-derived NO production and acute coronary syndromes suggests that platelet NO deficiency contributes to thromboembolic disorders [13]. Although not without side-effects at very high chronic doses, l-arg supplementation is appealing from the standpoint of safety, since the finite catalytic capacity of the NOS system and the hydrolytic activity of (vascular) arginase would tend to obviate NO overload and its adverse sequellae [141]. As shown in double-blind, placebo-controlled, randomized studies with healthy subjects, both infused [145] and oral [146] l-arg significantly inhibited (by ≈40%) ADP-induced platelet aggregation in vitro and potentiated platelet cGMP content. The effect, though, was weak: the plasma concentration of l-arg required to produce an anti-platelet effect was some 2-fold above normal, steady-state levels, and the oral anti-aggregatory l-arg dose was 4-fold greater than the usual daily l-arg intake in humans. The infused l-arg dose that effectively inhibited platelet activity (30 g total) was hypotensive and increased heart rate, whereas the oral anti-platelet dose (7 g per day over 3 days) did not affect blood pressure, suggestive of oral l-arg platelet selectivity. A comprehensive, randomized, placebo-controlled trial of infused bolus l-arg and its enantiomer (d-arg) included healthy subjects, non-insulin dependent diabetics, hypertensive subjects, and normotensives with primary hypercholesterolemia [147]. A blood-pressure drop and an acute inhibition of ADP-induced aggregation in plateletrich plasma were observed in all subjects after l-arg administration (≤5 g). Both responses to l-arg infusion closely correlated in magnitude, were weaker in noninsulin dependent diabetics and hypercholesterolemics, and declined with increasing age. Notably, d-arg did not elicit any of the l-arg effects, which were reduced by some 70% when superimposed upon ongoing, nonselective NOS inhibition with infused l-N-monomethyl-arginine (L-NMMA). Since d-arg is not a NOS substrate, and L-NMMA is a substrate-competitive NOS inhibitor, the l-arg effects observed in this study were theorized to reflect a rise in vascular NO production by eNOS. In contrast, the inhibition of platelet aggregation observed in vitro after a 5 min l-arg infusion (160 mg total dose) into healthy subjects and patients with angiographic

12.7 NCX-4016 [2-Acetoxybenzoate 2-(1-nitroxy-methyl)-phenyl ester]

atherosclerosis paralleled the anti-aggregatory effect of l-arg when added to whole human blood, suggestive of a direct effect on platelet NOS [109]. Hypercholesterolemic subjects without clinically evident cardiovascular disease and free of vasoactive or anti-platelet medications were the focus of a double-blind, randomized, placebo-controlled trial of long-term dietary l-arg supplementation (8.4 g daily for 2 weeks), which was well tolerated with no side-effects and markedly elevated circulating l-arg levels [148]. But l-arg only modestly and variably (by ≈20% on average) attenuated the platelet hyperreactivity characteristic of hypercholesterolemia, an effect that persisted 2 weeks after l-arg discontinuation in some subjects. The rigorous inclusion criteria for entry into this positive trial appear key to the modest l-arg effect observed, for a randomized, double-blind, placebo-controlled study of dietary l-arg (3 g, 3 times per day for 1 month) in coronary disease patients on standard therapy did not alter platelet activation-protein (P-selectin) expression [149]. Whether the negative result reflects the vasoactive and anti-platelet medicines (aspirin, nitrovasodilators) that had been administered as standard therapy for coronary artery disease or a suboptimal l-arg dose remains open to question. In nonmedicated, healthy male volunteers, l-arg infused at doses that normalized a NOS-inhibitor-induced increase in blood pressure did not affect platelet activation status (P-selectin expression, number of circulating platelet-leukocyte aggregates) [150]. Standard pre- and peri-operative therapy with aspirin and heparin did not appear to have a confounding effect in a randomized, double-blind, placebo-controlled l-arg trial in patients undergoing carotid endarterectomy [137]: a 90 min l-arg infusion (30 g total dose) reduced the frequency of embolic signals by 79% vs. placebo-treated group during the first 4 h after the procedure. A significant reduction in embolization was maintained up to 24 h post-surgery. Two small trials suggest that l-arg infusion could have a therapeutic effect related to in vivo platelet inhibition. Intravenous l-arg treatment (10.5 g daily for 7 days) of patients with peripheral vascular disease decreased ADP- and collagen-induced platelet aggregation in vitro, increased plasma cGMP, elongated the pain-free walking period, and improved absolute and pain-free walking distances [151]. A higher l-arg dose (16 g daily for 21 days) increased the duration and distance of pain-free walking for up to 6 weeks after treatment in patients with peripheral vascular disease [152].

12.7

NCX-4016 [2-Acetoxybenzoate 2-(1-nitroxy-methyl)-phenyl ester]

A recent trend in the pharmaceutical industry has been to harness the intrinsic tissueprotective properties of NO for improving the gastric tolerance of nonsteroidal antiinflammatory drugs (NSAIDs). This trend has led to the synthesis of hybrid, chimeric molecules containing an NSAID or aspirin moiety and a NO-donor functionality [153, 154]. One such hybrid is a NO-releasing derivative of aspirin, NCX-4016. In a doubleblind, randomized, placebo-controlled gastrointestinal safety assessment in healthy subjects, NCX-4016 (400 or 800 mg twice daily for 7 days) acted like aspirin as an inhibitor of arachidonic acid-induced platelet aggregation in vitro [155]. Whether

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this anti-platelet effect reflected NO-donor activity at all could not be determined from this study, since, for example, the des-NO-donor analog of NCX-4016 was not investigated.

12.8

Conclusion and Future Prospects

Pharmacological platelet inhibition has therapeutic value in preventing major cardiovascular events [34]. Aspirin prophylaxis reduces morbidity and mortality in patients at risk of clinical complications from occlusive vascular disease [35, 36]. Anti-platelet therapy also helps ease, but nowhere near eliminates, the growing, worldwide healthcare burden from patients with coronary risk factors, acute coronary syndromes, atherosclerosis, failed vessel grafts, and unsuccessful percutaneous revascularization attempts, with or without stenting [2, 27, 33, 34]. These and other conditions are generally characterized by a pro-thrombotic state, expressed clinically as hyperactive platelets and deficiencies in platelet- and endothelium-derived NO [10, 31, 32, 156]. The routine clinical use of NO-donor, anti-anginal nitrovasodilators in the management of ischemic syndromes, myocardial infarction, and cardiac failure [55, 80] and the pathogenic involvement of platelets in these same situations [25–27] invite the possibility that the salutary effects of nitrovasodilator drugs partly reflects an NO-based anti-platelet action over and above vasodilation. If so, then a clear therapeutic niche would exist in several high-impact cardiovascular disorders for platelet-inhibitory NO donors, especially since current anti-platelet drugs have not solved the medical problem of thrombosis. In vivo, platelet activation is inherently complex, involving many interactive, often iterative factors [3, 4, 9, 17, 18] (Figs. 12.1 and 12.2). The physiological chemistry of NO and NO-derived metabolites is profoundly influenced by their environment [157]. These factors conspire to limit severely the translational significance of laboratory demonstrations that NO donors in vitro inhibit the agonist-induced activation of isolated platelets. In vitro platelet systems might provide clinically useful data in, for example, targeting compounds or identifying anti-platelet drugs acting within the NO signal-transduction pathway (e.g., NO-dependent protein kinase modulators, guanylate cyclase activators) [158–160]. Likewise, although laboratory results with animal models of thrombosis generally support the notion that NO donors have anti-platelet effects in vivo, even the best thrombosis models can only approximate evolving thromboembolic disease in humans, during which vascular redox tone and steady-state NO background may flux dramatically [161, 162]. Reminiscent of the trend with laboratory studies, most (33 out of 43 cited above) uncontrolled clinical trials with either healthy volunteers or cardiovascular patients suggest that oral and intravenous NO donors at therapeutic doses acutely inhibit platelet activation in vivo (vide supra). Aside from their lack of long-term dosing and a placebo control group, several considerations restrict the predictive clinical value of these uncontrolled clinical studies: limited numbers of subjects; nonuniform criteria for subject entry and treatment outside of the trial; induction of adrenaline or

12.8 Conclusion and Future Prospects

catecholamine discharge in response to NO-donor induced vasorelaxation; lack of independent dosing optimization; and undocumented patient compliance. In vitro measurements of platelet activation status (e.g., agonist-induced aggregation; activation protein expression) or integrated evaluation of overall hemostasis (e.g., bleeding time) invite further complication, since the diagnostic utility of these parameters for monitoring anti-platelet strategies and predicting clinical outcomes is far from established [163]. Methodological artifacts may be compounded by the transient and rapidly reversible nature of platelet inhibition by NO, which prohibits extensive platelet studies post-dosing. Time constraints obviate examination of multiple endpoints of platelet activation status or even aggregation alone over a range of agonists and agonist concentrations [164]. The sensitivities of platelet endpoints (aggregation, adhesion protein expression, etc.) to a given exogenous NO exposure can differ markedly [165, 166], and the chemical form of NO delivery per se may modify the subsequent platelet response [167]. These factors lend an air of uncertainty as to what degree uncontrolled clinical studies can represent the effectiveness of an NO donor as an anti-thrombotic agent. Nonetheless, the aggregate study-design and methodological considerations would tend to underestimate potential platelet inhibition by an NO donor in vivo, as would the likelihood that some trial subjects with cardiovascular disease had NO-resistant platelets [168]. Many of these same factors impinge upon placebo-controlled clinical trials of NO donors and platelet activation in vivo. As opposed to open-label, uncontrolled trials, it has been somewhat more difficult to show a convincing, clear-cut difference on platelet activity between placebo and active NO-donor treatment (14 positive controlled trials out of 25 cited above). Perhaps this is because distinctions between placebo and treatment groups tend to be reduced, if not masked, by the considerable intrinsic genetic variability of the responsiveness of individuals to anti-platelet agents [169]. Drugs inconsistently administered outside of the trials may also have had an obfuscating effect: appropriate heart disease therapy includes aspirin and nitrovasodilators, which have anti-platelet effects themselves (vide supra). Nitrates administered outside of two large, randomized, multi-center, placebo-controlled trials (GISSI-3 [79], ISIS-4 [98]) may have contributed to their failure to show an independent effect of nitrovasodilator therapy on clinical outcome (i.e., survival advantage) that would at least be suggestive of in vivo platelet inhibition by an NO donor. Although in vitro platelet inhibition by a NO donor was reported as early as 1967 [39], there is no extant, clear-cut demonstration from a well-powered, randomized, controlled and blinded trial that a NO donor at therapeutic doses has in vivo antiplatelet activity sufficient to exert an independent effect upon clinical outcome. Since there is a greater likelihood for false negative than false positive results in the three such trials (GISSI-3 [79], ISIS-4 [98], ESPRIM [120]) reported so far, the possibility that NO-donor drugs could exert an independent anti-platelet effect for prognostic benefit in cardiovascular disease must still stand. The rational design of small-molecule, platelet-targeted NO donors compatible with vascular homeostasis and function that release bioactive NO only in the presence of activating platelets might yield pharmacologically attractive, safe, and tolerance-free compounds with which to verify this possibility. A site- and event-specific NO donor would be expected to prevent

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platelet aggregation and thrombus formation regardless of the agonist(s) operative and should efficiently counteract the pro-thrombotic and vasoconstrictor mediators produced by activated platelets themselves without exerting undue hemodynamic side-effects (e.g., concomitant hypotension or hemorrhage). Testing of such an agent in a properly designed and controlled clinical study would seem to be a worthwhile goal toward demonstrating an independent, anti-platelet effect of an NO donor on classical outcome measures, provided that patient entry criteria were well-defined. Potential criteria could include: standardization as to anti-platelet drug response; compromised endogenous NO bioactivity; platelets not refractory to NO and activated by a pre-existing condition such as unstable angina or post-bypass grafting; no pre-medication with platelet-active drugs. With regard to vascular devices, biocompatible, anti-thrombotic NO-donor coatings might offer ways to improve the success rate of revascularization procedures and extend their applicability [170, 171]. In the light of experimental and clinical demonstration that sustained organic nitrate use alters vascular biochemistry and function [172, 173], the paucity of data regarding the effects of long-term NO-donor administration on the human platelet is particularly noteworthy. Improved technology for monitoring platelet activity in the clinical setting would be most welcome. As for now, demonstrations that NO donors with various chemical structures acutely inhibit human platelet activity under certain clinical conditions in both healthy subjects and patients with cardiovascular disease await confirmation of their true significance by “gold-standard” validation from positive human outcome trials.

Acknowledgement

We thank Deborah Farnham for reprint assistance, Dawn Spooner for illustration assistance and Ginny Braman for technical comments.

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NO and Gene Regulation Jie Zhou, Bernhard Brüne 13.1

Formation of NO and RNI-signaling

With the discovery of the EDRF (endothelium derived relaxing factor), its identification as nitric oxide (NO) and the notion that NO is a versatile molecule beyond the vascular system, it came as some surprise that this small molecule powers signal transmission in nearly all areas of life [1, 2]. NO taught us to revise traditional thinking and to appreciate that formation of a radical stirs efficient pathophysiological signaling in biology/medicine. Signal transmission of NO is often elicited by “reactive nitrogen intermediates” (RNI) rather than the NO radical itself. The RNI comprise oxidation states and adducts of the products of nitric oxide synthase (NOS), including NO-radical (• NO), NO− and NO+ , as well as the subsequent adducts of these species such as NO2 , NO2 − , NO3 − , N2 O3 , N2 O4 , S-nitrosothiols, peroxynitrite and nitrosyl-metal complexes [3, 4]. Determination of RNI involvement in biological responses is often based on the use of compounds that mimic an endogenous response by administration of chemically diverse NO donors, by blocking RNI formation with NOS-inhibitors, or by using knockout mice that lack isotype specific NOS [5]. NO is produced by NOS that converts l-arginine to citrulline and NO [6]. Three isoenzymes known as neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) are named after the cell type from which they were first isolated and cloned. Isoenzymes show variations in terms of a high (iNOS) versus low (eNOS, nNOS) output capacity and differ with respect to their basic mechanisms of activation. The major distinction is regulation of nNOS as well as eNOS by a cytosolic calcium increase resulting in a pulsative enzyme activation versus the cytokine-inducible iNOS that produces RNI until the enzyme is degraded [7]. Biological signaling attributed to RNI is, in a first and simple approach, distinguished as either being cGMP-dependent or cGMP-independent [2]. Binding of RNI to the heme moiety of soluble guanylyl cyclase and concomitant cGMP formation constitutes the classical RNI response with broad implications for vascular medicine, at the same time acknowledging the landmark discovery of EDRF [8]. Besides the cGMP-signaling cascade that is mimicked by lipophilic cGMP analogs, alternative signaling pathways are activated by RNI via redox and additive chemistry. This may Nitric Oxide Donors Peng George Wang, Tingwei Bill Cai, Naoyuki Taniguchi (Eds.) c 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Copyright
ISBN: 3-527-31015-0

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promote covalent modification of proteins or oxidation events that do not require attachment of the NO group [3, 9]. Among these modifications, S-nitrosylation/Snitrosation [10], protein nitration, oxidation and cGMP-independent phosphorylation have increasingly attracted scientific attention as (ir)reversible post-translational protein alterations. These cGMP-independent protein modifications often elicit signal transmission that culminates in activation or suppression of genes [11–13]. Most, if not all, gene regulatory activities evoked by RNI are indirect. Up to now there is no evidence for the existence of DNA elements within promoter regions of eukaryotic genes that directly respond to RNI. Thus, to understand the signaling qualities of RNI in regulating genes we need to consider modification of transcription factors, their compartmentalization, their action as transcriptional activators or inhibitors, the stability of target mRNAs as well as transcription factor protein stability regulation. Numerous studies have analyzed the regulatory effects of RNI on transcription factors and transcriptional regulation. Although outside the main focus of this article, to summarize these primary observations we provide a table showing several transcription factors and their regulation under the impact of RNI (Table 13.1). For extensive coverage of the primary literature we refer to a number of review articles [11–14]. Often, RNI exert contradictory effects with regard to transcription factor activation. This may reflect the use of RNI with different signaling properties, different concentrations of RNI, cell-free versus intact cell systems, different types of cells, as well as the fact that activation of transcription factors is a result of complex upstream signaling cascades that themselves are targeted by RNI. For example, in resting cells RNI increase NF-êB- or AP-1-dependent gene transcription while RNI attenuate these responses in activated cells [13]. Thus, the signaling qualities of RNI depend on the biological milieu, i.e. the presence or absence of modulatory co-signals, often considered to be superoxide [15]. Along that line, the primary target of RNI as well as specific molecular modification(s) often remain unknown and different parallel versus interfering signals may be subjected to different redox control mechanisms. Considering thiol modification, an important post-translational protein alteration, and taking into account that many transcription factors share redox sensitivity based on thiol residues found in their DNA-binding domain, it is not surprising that their activity is under the control of RNI. However, a simple prediction on activation versus inhibition of gene activation as a result of RNI is still lacking. Any conditions of stress are potentially harmful to cells and require appropriate defence responses. Among other systems, the hypoxia inducible factor-1á (HIF-1á) [16–19] and the tumor suppressor protein p53 [20–22] are important transcriptional regulators that act in response to stress signals to coordinate a cellular reply by inducing growth arrest, apoptosis or adaptation [23]. Ultimately, the nature and intensity of stress signals, the cell type, and the cellular context will dictate the final outcome. Multiple levels of regulation must ensure that p53 and HIF-1á are fine-tuned to guarantee appropriate transcriptional regulation of target genes to cope with the stress situation. Interestingly, among multiple signals, hypoxia and RNI emerged as activators of both, p53 and HIF-1á.

13.2 p53 Regulation under the Impact of RNI Tab. 13.1: Transcription factors under the control of RNI. Selected examples for the regulatory

impact of RNI on prokaryotic and eukaryotic transcription factors. In a very simplistic way activation versus inhibition by RNI are indicated. Prokaryotic factors Transcription factor (TF) SoxR, OxyR Fur LAC9 Ace1

Modulation by RNI −rather uniform results showing activation −de-repression of genes that are under the control of Fur −attenuated DNA-binding in vitro −inhibition of DNA-binding in yeast

Eukaryotic notably mammalian factors NF-êB −activation in resting cells, low level RNI −inhibition in stimulated cells, high level RNI AP-1 −activation in unstimulated cells, low level RNI −inhibition in activated cells Sp1, Egr-1 (zinc finger TFs) −rather uniform inhibition −activation if Sp1 de-represses the e.g. TNFá promoter VDR, RXR (nuclear hormone R) −inhibition of DNA-binding and reporter activity PPARã −activation at low level RNI −inhibition at high level RNI NFAT −inhibition in activated NK cells HSFs −activation (HSP70 expression) p53 −activation (for details see below) HIF-1 −activation under normoxia (for details see below) −inhibition under hypoxia TF: transcription factor, R: receptor, Fur: ferric uptake regulation protein, NF-êB: nuclear factorêB, AP-1: activator protein-1, Egr-1: early growth response-1, VDR: 1á,25-dihydroxy-vitamin D3 receptor, RXR: retinoid X receptor, PPARã: peroxisome proliferator-activated receptor ã NFAT: nuclear factor of activated T-cells, HSF: heat shock factor, p53: tumor suppressor p53, HIF-1: hypoxia inducible factor-1.

13.2

p53 Regulation under the Impact of RNI 13.2.1

Basic Considerations: p53 Phosphorylation and Mdm2 Binding

p53, a prototype tumor suppressor, controls cell cycle progression and apoptosis with the notion that these functions are altered in many tumors, thus contributing to malfunction. In unstressed cells p53 exhibits an extremely short half-life and the protein amount is maintained at a low, often undetectable, level by efficient proteasomal degradation. Cellular stress, such as DNA damage, oncogene activation, or hypoxia, stabilizes p53 predominantly by post-translational modification, with the notion that a common denominator in all p53-inducing stresses may be nucleolar disruption [24]. As a transcriptional activator p53 promotes transcription not only of cell cycle regulating genes such as p21WAF1/CIP1 or murine double minute 2 (mdm2) but also apoptotic ones, exemplified by Bax or Fas, [20–22]. In addition, transcription-

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independent actions contribute to the activation of proapoptotic pathways. However, these effects are not exerted indiscriminately. Quantitative and qualitative changes endow p53 with improved capability to alter the cell phenotype, either to take care of the damage or to eliminate affected cells from the replicative pool to prevent its expansion into malignant progenitor cells [25]. It appears that the choice between induction of growth arrest versus apoptosis is defined by the balance of survival signals, i.e. the cellular context as well as the particular genotype of the cell with genetic alterations affecting (in)directly the functional status of p53 [22]. Stability regulation of p53 is regulated by Mdm2, which functions as a p53-specific E3 ubiquitin-ligase. The mdm2 gene is transcribed under the control of p53 and the Mdm2 protein binds p53 at the N-terminus to facilitate its proteasomal degradation [26–28]. Ubiquitination of p53 requires a conserved Mdm2 binding region of eight cysteine and one histidine amino acids that form two zinc-binding sites, known as a RING-finger motif. The RING-finger domain additionally facilitates nuclear export of p53 [29, 30]. Blocking p53 nuclear export attenuates Mdm2-mediated p53 degradation and concomitantly stabilizes and actives p53, [31–33]. These considerations are relevant for p53 stabilization. When UV attenuates the p53/Mdm2 interaction, ubiquitination of p53 is reduced, culminating in protein stabilization [34, 35]. Although under debate, phosphorylation of p53 at serine 15 may be needed to dissociate p53 and Mdm2 [36, 37] and phosphorylation of p53 at several sites, among others serine 15, is facilitated by RNI [38] as well as other stress conditions such as UV, IR or CdCl2 treatment [39, 40]. Besides phosphorylation, an altered compartmentalization of p53 achieved by leptomycin B, a chromosome region maintenance 1 (CRM1)/exportin1mediated nuclear export inhibitor, activated p53 [41, 42]. It is predicted that serine 15 phosphorylation of p53 following UV-treatment reduced nuclear export of p53 [43]. 13.2.2

Molecular Mechanisms of RNI-evoked p53 Stabilization

For some time it has been appreciated that iNOS- or NO donor-derived RNI stabilize p53 [44, 45]. Supporting evidence came from experiments in iNOS-deficient macrophages that failed to localize p53 to the nucleus after in vivo bleomycin exposure [46]. Upregulation of p53 targets such as p21(Waf1/Cip1) or Bax in response to RNI, supported the idea that p53 was transcriptionally active [47, 48]. Experiments in thymocytes from p53 null mice or in mutant p53 human lymphoblastoid cells revealed that these cells were less vulnerable to RNI, implying that p53 may transmit a proapoptotic RNI-response [49, 50]. RNI-accumulated p53 revealed a distinct phosphorylation pattern, predominantly at serine 15 [38, 51]. This goes along with current thinking that post-translational modification of p53 at the N- and/or C-terminus contributes to protein stability regulation. Reports on the involvement of the ataxia telangiectasia-mutated (ATM) kinase in facilitating RNI-evoked phosphorylation are controversially discussed. Wang et al. ruled out a contribution from ATM or the alternate reading frame (ARF) tumor suppressor protein in p53 accumulation [52]. Based on studies in isogenic human cell lines and MEFs from gene knockout (ATM-/- ) mice these observations were challenged by demonstrating that

13.3 HIF-1á Regulation under the Impact of RNI

serine 15 phosphorylation is ATM- and ATM- and Rad3-related (ATR)-dependent but p38- and DNA-PK-independent, although mechanisms of ATM/ATR activation by RNI remained obscure [53]. Cell fractioning and heterokaryon analysis suggested that RNI, in some analogy to leptomycin B, prevented nuclear-cytoplasmic shuttling of p53 which causes nuclear protein stabilization/activation [54]. In addition to phosphorylation, reversible down-regulation of Mdm2 by RNI may contribute to activation of p53 [52]. An initial but transient drop in Mdm2 may account for early accumulation of p53 in response to RNI, whereas in a second phase p53 remains stabilized although Mdm2 increases above controls due to transactivation of the mdm2 gene [52]. Under conditions of elevated Mdm2 expression, nuclear trapping of p53 appears as a rational explanation for p53 accumulation [55]. Fig. 13.1 schematically depicts concepts to rationalize RNI actions in accumulating p53. Concepts on attenuated nuclear-cytoplasmic shuttling of serine 15-phosphorylated p53 to account for RNI action came from heterokaryon analysis, although causation between an altered compartmentalization and serine 15-phosphorylation awaits clarification. Leptomycin B, an established inhibitor of p53 nuclear export, targets an active cysteine residue in CRM1 to attenuate nuclear export. This may apply for RNI as well, considering that RNI may nitrosylate thiol residues. Observations on the nuclear trapping of p53 were corroborated when RNI increased nuclear import and retention of p53 in neuroblastoma cells in which p53 is primarily cytoplasmatic [56]. Transactivation of p53 and serine 15 phosphorylation were noticed in cells co-cultured with RNI-releasing macrophages and were correlated to iNOS expression in ulcerative colitis. This highlights RNI generation and activation of a p53 response pathway during chronic inflammation [53]. Activation of divers signaling pathways by RNI, culminating in gene expression, has been proven by the use of DNA microarrays [57] or differential analysis of library expression [58]. Among different group of genes activated by RNI was a subpopulation of genes that specifically required p53. These data support the basic observation that RNI stabilize and activate p53 under pathophysiological conditions. However, at present it cannot be ruled out that nitrosative stress during chronic inflammation might lead to mutations within the p53 gene, thus contributing to carcinogenesis [59, 60].

13.3

HIF-1á Regulation under the Impact of RNI 13.3.1

Lessons from Hypoxia: Basic Considerations of HIF-1á Stability Regulation

Intracellular recognition of decreased oxygen tension, i.e. hypoxia, and an appropriate response to meet these alterations is predominantly facilitated via the oxygendependent transcription factor HIF-1 as a consequence of stability regulation and/or protein synthesis of the HIF-1á subunit. Pioneering work on erythropoietin, a classical hypoxia-responsive target gene, led to the discovery of HIF-1 [61] while more recently an integrated picture of oxygen sensing emerged [16, 19, 62–64]. HIF-1 is a

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Fig. 13.1 Stability regulation of p53 by early vs. late RNI actions. RNI by (in)directly activating ATR/ATM provoke phosphorylation of p53. In turn tetramerization of p53 and binding of the coactivator p300/CBP will provoke gene activation. This may go along with RNI-evoked nuclear retention of ubiquitinated p53 although

expression of Mdm2, a p53-downstream target gene, is increased. An early action of RNI may involve transient downregulation of Mdm2 which would be consistent with p53 stabilization due to reduced poly-ubiquitination. For details see text.

heterodimer composed of one of the three alpha subunits (HIF-1á, HIF-2á or HIF3á) and one HIF-1â subunit [65]. HIF-1â is constitutively expressed and is identical to the aryl hydrocarbon receptor (AhR), known as AhR nuclear translocator (ARNT). HIF-1, as implied by its name, is predominantly active under hypoxic conditions as a result of stabilization of the alpha subunit, i.e. HIF-1á. In the presence of oxygen the alpha subunit is unstable and is generally undetectable due to polyubiquitination by an E3-ubiquitin ligase complex that is built among other proteins by the von Hippel–Lindau protein (pVHL), followed by 26S proteasomal degradation [66, 67]. An oxygen-dependent prolyl-4-hydroxylase, similar to proline hydroxylation of collagens, covalently modifies a domain of HIF-1á known as the oxygen-dependent degradation domain (ODD) by hydroxylating proline residues 402 and 564 [68, 69]. Hydroxylases

13.3 HIF-1á Regulation under the Impact of RNI

are known as orthologs of C. elegans Egl-9, designated as PH domain-containing enzymes (PHD), i.e. prolyl hydroxylases (PHD1, PHD2, PHD3 and PHD4) [70–72]. Hydroxylated HIF-1á form hydrogen bonds with pVHL side chains which promote polyubiquitination of HIF-1á, followed by proteasomal degradation [66, 67, 73]. Enzymes require 2-oxoglutarate and iron as cofactors, thus implying why “hypoxicmimetics”, e.g. the iron chelator desferrioxamine, accumulates HIF-1á by attenuating PHD activity. Besides the transactivation domain residing in the ODD another one is found in the extreme C-terminus of HIF-1á, known as the C-terminal transactivation domain (CTAD). Hydroxylation of asparagine 803 by FHI (factor inhibiting HIF-1) within the CTAD [68, 74] renders HIF-1á unable to bind to the p300/CBP coactivator, thus preventing transactivation capabilities of HIF-1. Hypoxia attenuates Pro564/402 and Asn803 hydroxylation, which in turn provokes HIF-1á protein stabilization, HIF-1â association, coactivator recruitment, subsequent activation of HIF-1 which results in expression of those targets that contain HRE (hypoxia responsive element) sites with the core DNA sequence 5′-ACGTG-3′ [64]. Target genes are categorized according to their signaling qualities with involvements in erythropoiesis, iron homeostasis, glucose/energy metabolism, viability decisions, or vascular development/remodeling [63, 75]. HIF-1 targets are related to physiology as well as pathology and therefore it is predictable that gene products contribute not only to cell protection but also to cell pathologies, in close association with several major disease states such as ischemic cardiovascular disorders, pulmonary hypertension, stroke, pregnancy disorders or cancer [63, 75]. 13.3.2

Stability Regulation of HIF-1á by NO/RNI in Normoxia versus Hypoxia

There are several lines of independent research showing HIF-1á stabilization, HIF-1 DNA binding and HIF-1 transactivation under normoxia by RNI [18]. Experiments in a variety of human, pig or bovine cells ruled out species specific or cell type restricted effects. The use of chemically distinct NO donors such as S-nitrosoglutathione (GSNO, considered the most physiological NO donor), NOC-18 (Z-1-1[2-aminoethylamino]diazen-1ium-1,2-diolate), NOC-5 (3-(hydroxy-1-(1-methylethyl)-2-nitrosohydazino)-1-propanamine, SNAP (S-nitroso-N-acetyl-d,l-penicillamine), or others indeed suggest RNI involvement [18]. Along that line the time- and concentrationdependent effects of RNI on HIF-1á accumulation have been established by using NO donors with different half-lives [76]. RNI-evoked activation of the human vascular endothelial growth factor (VEGF) promoter under normoxia in combination with deletion and mutation analysis of the VEGF promoter indicated that the RNI-responsive cis-elements were the HIF-1 binding site and an adjacent ancillary sequence that is located immediately downstream within the HRE [77, 78]. Experiments with GSNO, a nitrosonium donor, and observations that GSNO effects are reversed by dithiothreitol, lead to the proposal that S-nitrosylation stabilizes HIF-1á [79]. Although S-nitrosation of HIF-1á is confirmed in vitro, a biological significance and any (in)direct role in HIF-1á stability regulation by RNI awaits clarification [80]. The use of NO donors often raises questions on the pathophysiological importance of

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RNI signaling with respect to relevant concentrations. This potential drawback was overcome by overexpression human iNOS, thereby accumulating HIF-1á. This supports the notion that autocrine or paracrine produced RNI are capable of stabilizing HIF-1á under normoxia [76]. Supporting evidence came from a transwell co-culture set-up of lipopolysaccharide/interferon-ã activated, and thus iNOS-derived RNI producing macrophages and tubular LLC-PK1 detector cells, showing that activated, but not resting, macrophages elicited HIF-1á accumulation in LLC-PK1 cells [81]. However, considering various intracellular targets of RNI it should not be extrapolated from these results that this is a uniform reaction, especially if the intracellular redox milieu changes, i.e. by the formation of superoxide. Interference of O2 − with RNI signaling, and vice versa, is established and understandable by the diffusion-controlled radical interaction which may redirect signaling qualities of RNI towards other species, i.e. ONOO− . Experiments with the redox cycler DMNQ (2,3-dimethoxy-1,4-naphthoquinone) to generate O2 − and/or H2 O2 (derived from superoxide dismutase-triggered conversion of O2 − to H2 O2 ) attenuated RNI-elicited HIF-1á accumulation [82], which favors the assumption that agents increasing reactive oxygen intermediates (ROI), including ONOO− , destabilize HIF-1á [83]. These observations predict that the ability of RNI to stabilize HIF-1á depends to some extent on the intracellular redox milieu and is subject to modulation by cosignals. Analogous observations are shown by seminal observations in 1998/1999 stating that carbon monoxide (CO) and RNI inhibit hypoxia-induced HIF-1á accumulation [84–86]. As mechanistic insights start to become clear it appears that HIF-1á suppressing actions under low RNI concentrations depend on the inhibition of mitochondrial respiration, since it is absent in p0 -cells and is mimicked by inhibitors of mitochondrial respiration [87]. The authors propose that destabilization of HIF-1á by RNI under hypoxia is unlikely to result from oxidative stress, i.e. ROS formation, rather correlating with inhibition of mitochondrial respiration by NO which leaves more oxygen available for PHDs, thus allowing PHD activity to be regained although oxygen tension is low [87, 88]. Alternatively, the level of free iron may change under the conditions of RNI formation and hypoxia, which again may contribute to the regulation of PHD activity. Determination of free iron and PHD activity under conditions of O2 − and RNI formation, as well as under conditions of cotreatment, will help to clarify various proposals. RNI signaling and HIF-1á accumulation is supported in several experimental systems. Cell density-induced HRE activation demands the production of RNI, which are generated by densely cultured cells as a diffusible paracrine factor [89]. In human prostate cells RNI use Ras, mitogen activated protein kinase (MAPK) and HIF-1á signaling to activate HRE which connects this pathway with survival or growth advantages of tumor cells, because inhibition of iNOS blocked production of an angiogenic activity in thioglycolate-induced peritoneal and murine RAW264.7 macrophages [90]. It can be concluded that VEGF contributes to macrophage-dependent angiogenic activity and modulation of VEGF mRNA levels in macrophages is, at least in part, under the control of the iNOS pathway. Attenuating iNOS provokes formation of antiangiogenic factors which makes RNI likely players in the regulation of macrophage-

13.4 RNI, p53 and HIF-1 in Tumor Biology

dependent angiogenic activity in vivo, in wound repair and possibly in tumor development [90]. The idea that NO donors or an active iNOS promote HIF-1á accumulation demanded mechanistic explanations considering the existing details proposed for HIF1á stability regulation. In close analogy to hypoxia, RNI have been shown to decrease ubiquitination of HIF-1á and to dissociate pVHL from HIF-1á [91]. Considering that the HIF-1á-pVHL interaction requires prolyl hydroxylation of HIF-1á raises the possibility that RNI blocks HIF-1á prolyl hydroxylation. An in vitro HIF-1á-pVHL capture assay implied dose-dependent inhibition of PHD activity by GSNO, while the association of a synthetic peptide resembling the hydroxylated ODD domain of HIF-1á with pVHL remained intact. It can be concluded that hypoxia and RNI use overlapping signaling pathways and/or modifications to evoke HIF-1á stabilization. As proposed schematically in Fig. 13.2, RNI may block PHD-activity, attenuate proline hydroxylation of HIF-1á, dissociate HIF-1á from pVHL with the consequence of protein stabilization based on decreased proteasomal degradation. It is known that RNI interact with iron (II) in heme- or non-heme-containing proteins [4], exemplified by spectroscopic studies when NO directly binds to the ferrous iron in protocatechuate 4,5-dioxygenase and catechol 2,3-dioxygenase [92] or to isopenicillin N synthase [93]. These enzymes coordinate Fe2+ in their catalytic domain in a 2-histidine-1-carboxylate facial triad which is the defining structural motif of mononuclear non-heme iron(II) enzymes [94]. HIF-1á prolyl hydroxylases (PHDs) belong to a non-heme Fe2+ -containing family of enzymes. Thus, it is rational to assume Fe2+ -coordination by RNI in the catalytic site of PHD and thus competition of RNI with oxygen. The concept of direct PDH inhibition by RNI as an explanation of HIF-1á accumulation was recently challenged by the observation that the NO donor NOC18 did not inhibit HIF-1á hydroxylation, ubiquitination, and degradation [95]. Instead, based on pulse labeling studies the authors proposed increased HIF-1á synthesis. Unfortunately, major conclusions in this study are derived from overexpression experiments of FLAG-HIF-1á and HA-pVHL and it remains open whether overexpressed proteins share regulatory features of endogenous proteins. This becomes a concern, considering that expression of FLAG-tagged HIF-1á remained constant under the impact of NOC18, and thus a difference in the HIF-1á/pVHL interaction would not be expected. However, considering the proposed role of PI3K in HIF-1á translational regulation it may turn out as an exception to the rule that in some cells PI3K is stimulated by RNI which in turn provoke HIF-1á translation regulation. This scenario has also been noticed for hypoxia [96]. Under conditions where PI3K/Akt stimulation by hypoxia and/or RNI occurs, translation control mechanisms may overlap with blocked degradation pathways in accumulating HIF-1á.

13.4

RNI, p53 and HIF-1 in Tumor Biology

The critical role that HIF-1 plays in cancer biology can be deduced from immunohistochemistry data showing elevated levels of HIF-1á in a variety of primary malignant

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Fig. 13.2 Stability regulation of HIF-1á and

activation of HIF-1 by RNI. HIF-1á is subjected to hydroxylation by PHDs and FIH that allows recruitment of pVHL, subsequent polyubiquitination and concomitant 26S proteasomal degradation. RNI attenuate PHD

as well as FIH activity under normoxia, thus abrogating HIF-1á hydroxylation. Binding of HIF-1â constitutes the active HIF-1 dimer. Subsequent binding of the coactivator p300/CBP promotes gene activation. For details see text.

tumors and/or tumor metastases with normal levels of HIF-1á in benign tumors [97]. The interior of a growing tumor becomes progressively hypoxic as its size increases, based on the notion that oxygen only diffuses around 150–200 microns from capillaries. Thus stabilization of HIF-1á is in part because of its induction through the ubiquitous pathway of oxygen sensing and signaling. In addition, tumor-specific genetic alterations, i.e. mutations involving oncogenes and tumor suppressor genes, often enhance HIF-1 expression [19, 75]. For example, loss of pVHL, PTEN or p53 tumor suppressor genes is associated with HIF-1á expression as well as the transforming potential of the v-Src oncogenes. The striking upregulation of HIF-1á in many dif-

13.5 Conclusions

ferent tumors by both physiologic and epigenetic mechanisms raises the question how HIF-1 impacts tumor biology. Apparently, HIF-1 allows metabolic adaptation to hypoxia, promotes angiogenesis, enhances survival and stimulates proliferation. Therefore, aberrant HIF-1á overexpression in brain, breast, cervical, esophageal or ovarian cancers is correlated with treatment failure and mortality. Moreover, RNI have been shown to play an important role in tumor growth and progression [98]. Expression of NOS has been demonstrated in a variety of tumors including breast, head, neck, prostate, bladder, colon, and CNS tumors such as glioblastomas [99]. RNI promote tumor growth by multiple actions such as regulating blood flow, maintaining the vasodilatory tone, promoting metastasis by increasing vascular permeability as well as affecting matrix metalloproteinases and stimulating angiogenesis. The observation that RNI share with hypoxia the ability to stabilize HIF-1á may be relevant for various aspects of tumor biology. Conversely, several reports have documented that increased production of RNI reduced tumor cell survival and induced tumor cell death [100]. At least in part, mechanisms may point to a proapoptotic mechanism being compatible with RNI-evoked p53 stabilization [44]. However, as the role of RNI in affecting apoptosis can be pro- as well as anti-apoptotic, the impact of RNI is linked to pro- as well as anti-tumor activities [101]. It appears that high levels of RNI formation, as generated via iNOS, may be cytostatic or cytotoxic, whereas low level RNI generation via constitutively active NO-synthases can have the opposite effect and promote tumor growth [102, 103]. The regulation of tumor growth by RNI represents an important new dimension in cancer research. Multiple facets of RNI signaling such as HIF-1á or p53 accumulation as well as the regulatory impact of RNI on apoptosis need consideration to determine the precise role of RNI in tumor biology and to understand contrasting observations of RNI in promoting or inhibiting the etiology of cancer.

13.5

Conclusions

Altered gene expression by RNI constitutes an integral component to explain their signal transmission capabilities. This appears relevant for RNI in coordinating inflammation, affecting proliferation, differentiation and regulating cell survival decisions. Transcriptional regulation is in the sphere of RNI actions and, among multiple transcription factors, HIF-1á and p53 are emerging regulatory targets that link RNI signaling with medical related problems of tumor biology and/or cell viability decisions. Mechanistically, we are beginning to understand how RNI mimic a hypoxic response by attenuating prolyl hydroxylase activity under normoxia and how RNI stabilize p53 by affecting protein phosphorylation and/or an altered compartmentalization of p53. Fig. 13.3 summarizes these aspects and implicates pathophysiological consequences ranging from cellular signaling, i.e. adaptation, to death and tumor biology. RNI have not been considered classical, i.e. originally identified activators of p53 or HIF-1á. This provokes the question of the relevance of RNI in activating gene

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Fig. 13.3 HIF-1á and p53 as mediators of RNI

signaling. RNI affect HIF-1á stabilization to promote its interaction with HIF-1â. Recruitment of the coactivator p300/CBP causes gene activation to elicit pathophysiological responses. RNI activate

p53, e.g. causing tetramerization and recruitment of the coactivator p300/CBP provokes gene activation to suppress cell cycle progression and/or to elicit apoptosis. HIF-1, p53 as well as direct RNI actions contribute to affect tumor pathogenesis. For details see text.

expression via p53 or HIF-1. Gene expression profiling may help to answer this question in the future as well as our search for medical symptoms associated with RNI formation and transcriptional regulation via p53 and/or HIF-1. Assuming numerous genes to be under the control of RNI, using multiple transcriptional regulators, we need to establish a hierarchy of gene activation processes that determines and allows one to predict the signaling qualities of RNI.

Acknowledgement

We apologize to researchers whose primary observations that form the basis for our current knowledge in this active field could not be cited due to space limitations, or have been acknowledged indirectly by citing review articles, only. Our work was supported by grants from Deutsche Forschungsgemeinschaft (Br 999), Deutsche Krebshilfe (10-2008-Br2) and the Sander Foundation (2002.088.1).

Abbreviations

Abbreviations

ATM ATR AhR ARNT cGMP CBP CTAD GSNO HIF-1 HRE (i)NOS MAPK Mdm2 ODD p53 PHD pVHL RNI ROI VEGF

ataxia telangiectasia-mutated ATM- and Rad3-related Aryl hydrocarbon receptor AhR nuclear translocator cyclic guanosine monophosphate CREB (cAMP-response element binding protein)-binding protein C-terminal transactivation domain S-nitrosoglutathione hypoxia inducible factor-1 hypoxia-response element (inducible) nitric oxide synthase mitogen activated protein kinase murine double minute oxygen-dependent degradation domain tumor suppressor p53 prolyl hydroxylase domain-containing protein von Hippel–Lindau protein reactive nitrogen intermediates reactive oxygen intermediates vascular endothelial growth factor

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Nitric Oxide and Central Nervous System Diseases Elizabeth Mazzio, Karam F. A. Soliman 14.1

General Overview – Gaining Control over Various NOS Enzymes that Concurrently Contribute to Degenerative CNS Diseases

Nitric oxide (NO) is an unstable gaseous radical that plays a central role in human neurological and cardiovascular function, as well as central nervous system (CNS) disease. NO is synthesized from the electron oxidation of l-arginine to l-citrulline through various isoforms of nitric oxide synthase (NOS). The NOS enzyme is an oxidoreductase that requires oxygen, heme iron, tetrahydrobiopterin, â-nicotinamide adenine dinucleotide phosphate, flavin mononucleotide and flavin adenine dinucleotide [1–4]. And, the three primary isoforms of NOS are neuronal NOS (NOSI:nNOS), inducible NOS (NOS-2:iNOS) and endothelial NOS (NOS-3:eNOS). NOS-1 and NOS-3 have distinct similarities in that both isoforms are activated by intracellular Ca2+ /calmodulin [3, 5], and the NO generated is a signaling molecule that regulates 3′,5′-cyclic guanosine monophosphate (cGMP) and protein kinase G (PKG) phosphorylation events that control autonomic, central cardiovascular and neurological functions [1, 6–9]. Nitric oxide generated by NOS-2 is markedly different from NOS 1 and 3, where its primary role resides in monocyte/macrophages assisting in destruction of infection, invasive pathogens or necrotic debris. While NO molecules generated by NOS 1 and 3 are primarily transient signaling molecules, NO synthesized by NOS-2 plays a central role in immune response to injury, where it can also contribute to the destruction of healthy tissue during chronic or acute inflammatory diseases [10, 11]. In the CNS, an inflammatory response triggered by injury, infection or neuronal necrotic debris is carried out in part by glia (microglia and astrocytes). Astrocytes respond to cytokines via signaling through stress-activated mitogen-activated protein kinases (MAPKs) that phosphorylate and activate transcription factor/DNA binding elements initiating iNOS mRNA transcription [12, 13]. Since iNOS protein expression is highly up-regulated by pro-inflammatory cytokines, CNS inflammation can yield dangerously high quantities of NO over extended periods of time, possibly posing an irreversible threat to the health and viability of post-mitotic neurons [14]. All three isoforms can diverge from their functional regulatory roles and during disease states contribute to NO mediated pathologies of the CNS. Excessive accuNitric Oxide Donors Peng George Wang, Tingwei Bill Cai, Naoyuki Taniguchi (Eds.) c 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Copyright
ISBN: 3-527-31015-0

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mulation of NO by nNOS or iNOS can prompt its reactivity with molecular oxygen (O2 ) or other reactive oxygen species (ROS) to produce a number of reactive nitrogenated species (RNS), such as dinitrogen trioxide [N2 03 ] nitrogen dioxide [NO2 ], peroxynitrite [ONOO− ], peroxynitrous acid [ONOOH], nitroxyl ions [NO− ], nitrosonium ions [NO+ ], and dinitrosyl iron complexes [15]. The accumulation of ONOO− / ONOOH can induce both nitration/nitrosylation of proteins and oxidation of lipid membrane structures, that contribute to neuronal apoptosis, loss of mitochondrial electron transport (ETC) function and degradation of nuclear DNA [16–21]. The accumulation and concentration of iNOS, nitrosylated proteins and lipid peroxidation markers in the CNS juxtaposes both aging [22] and neurodegenerative injury associated with Alzheimer’s disease (AD) [23, 24], inflammation [25], autoimmune encephalomyelitis [26], ischemia, head trauma [27, 28], spinal cord injury, [29], stroke [30], multiple sclerosis (MS) [31], amyotrophic lateral sclerosis (ALS) [32], cerebrovascular damage, traumatic brain injury [33], HIV1-related encephalitis and dementia [34], demyelinating diseases [35], cerebral malaria [36] and Parkinson’s Disease (PD) [37]. Pharmaceutical manipulation as a means to effectively combat and minimize the detrimental effects of NO can be achieved through integration of drugs that either indirectly or directly target diverse effects on 1. NOS mRNA, 2. NOS enzyme activity, 3. the conversion of NO to neurotoxic species or 4. block downstream cell signaling systems involved with NO-mediated apoptosis and necrosis. While nNOS and iNOS contribute to toxic events through overproduction of NO, conversely, it is the loss of eNOS activity that can create injurious effects to the brain. NO generated by eNOS (located in the vascular endothelium), evokes vasodilation and a deficit can initiate hypertension/vasospasms, exacerbating stroke or other CNS diseases that require abundant delivery of blood, nutrients and oxygen to damaged neurons [38, 39]. And, for this reason, existing research has generally concluded that a therapeutic approach to combat the toxicity of NO in CNS disease involves employing agents that simultaneous down-regulate iNOS and nNOS, and augment eNOS [40, 41]. This can be achieved with either selective NOS enzyme inhibitors, or by manipulating equivalent cell signaling controls that perpetuate beneficial diverse effects on various isoforms of CNS NOS. For example, a number of promising agents can concomitantly antagonize pro-inflammatory transcription of glial iNOS mRNA, increase vascular eNOS, and block cell death associated with nNOS and glutamate. Some of these include poly(ADP-ribose)-polymerase-1 (PARP-1) inhibitors [42–45], thiazolidinediones (TDs)/peroxisome proliferator-activated receptor gamma (PPAR-gamma) agonists [46–50] phosphodiesterase (PDE) inhibitors (PDE-1C, PDE4, PDE-5) [51–55] and stress-activated mitogen-activated protein kinase of 38 kDa (p38/SAPK)/c-Jun-NH2 -terminal kinase (JNK/SAPK)-inhibitors [56–60]. Another promising class of drugs to treat NO-related pathologies are superoxide dismutase (SOD) mimetics [61–64], which can rapidly dissipate superoxide (O2 − ) and prevent the formation of ONOO− which, if present, appears to be the single most deleterious RNS in the CNS. ONOO− can trigger hypertension through attenuating eNOS, induce apoptosis through opening of the mitochondrial permeability transition pore complex (PTPC), and impart global oxidative stress throughout the brain. Removing O2 − or increasing the ratio of NO/ONOO− appears to switch NO from being a neurotoxic to a

14.2 Signaling Controls – Neuronal NOS: TYPE-I

neuroprotective /vasodilatory molecule. Other important cell signaling controls that can be targeted to concurrently regulate various isoforms of NOS to provide a simultaneous anti-flammatory, vasodilatory effect and/or block the downstream toxic effects of ONOO− include: adenylate cyclase, 3′,5′-cyclic adenosine monophosphate (cAMP), cGMP, protein kinase A (PKA), tyrosine kinase, protein phosphatase, Janus tyrosine kinase, angiotensin converting enzyme (ACE), extra-cellular signal-regulated protein kinase 1/2 (ERK/1/2), ERK[1/2], p38/SAPK, JNK/SAPK, PPAR-gamma, PARP-1, bcl2 family proteins, glutathione, nicotinic acid and antagonizing the effects of inositol 1,4,5-triphosphate (IP3 ) or blocking Ca2+ voltage/ligand activated receptors. Each of these will be briefly discussed in terms of mechanism, impact and control over lethal forms of NO that contribute to CNS disease.

14.2

Signaling Controls – Neuronal NOS: TYPE-I 14.2.1

Neurotransmission

In order to gain understanding as to how to combat the detrimental effects of NO in the CNS, it is worthwhile to briefly delineate the normal function of each NOS isoform, its upstream regulatory controls and the downstream target of molecular NO. In neurons, NO produced by nNOS plays a central role in cell signaling through activation of soluble guanylyl cyclase (sGC) [65, 66]. Soluble GC is variably dispersed in CNS neurons and activated by NO through its interaction with the catalytic heme binding domain of á1 and â1 subunits, prompting a conformational change that initiates conversion of guanosine triphosphate (GTP) to cGMP [67–70]. In neurons, the accumulation of intracellular cGMP can directly control cyclic nucleotide-gated membrane ion channels [71, 72] or coordinate a multitude of diverse signaling effects through activation of PKG [73]. In general, protein kinases serve to add a negative phosphate group onto protein structures, thereby altering electrostatic charges which result in the modification of structural/functional characteristics that control open/closed or on/off signaling systems [74]. While there are many types of neuronal protein kinases, PKG specifically regulates neurotransmission and feed-back responses required for adaptation and sensitization to environmental stimuli. For this reason, the activity of nNOS in the CNS is integral to neurophysiological sensory function such as vision, learning, nocioception, antinocioception, hearing, digestion, circadian rhythms, sleeping, olfaction, memory, reproduction, neuro-endocrine function and cognition [75–82]. One of the primary functions of CNS nNOS in the regulation of sGC is control over neurotransmitter (NT) release, which coordinates communication through relaying synapses. NO can either act as a direct neuromodulator [83, 84] or its role lies in carrying out Ca2+ initiated nNOS/ NO/sGC/cGMP/PKG signaling, that regulates synaptic NT release [85–90]. The presence of NO near and or around neurons is known to induce rapid neuronal firing and augment NT release, and this is consistently

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demonstrated in studies of diverse neuronal cell type such as glutaminergic, nicotinic, gamma aminobutyric acid (GABA)-ergic, cholinergic and adrenergic neurons [86–89, 91–94]. Depending upon the type of NT released and postsynaptic modulation of permeability to select ions, NO can augment both excitatory [92, 95] or inhibitory postsynaptic potentials [96]. Although a specific mechanism defining the role for NO in NT release has not been elucidated, studies consistently demonstrate that excitatory NT receptor activation can lead to Ca2+ influx, activation of nNOS and NT release, effects that are blocked by tetrodotoxin, Ca2+ -deficient buffers, intracellular Ca2+[I] buffering agents, hemoglobin (NO trapping agent) or nNOS inhibitors such as N(g)-nitro-l-arginine methyl ester (l-NAME) or N(g)-monomethyl-l-arginine (l-NMMA) [94, 97]. While NO produced endogenously through nNOS can carry out these effects, an exogenous supply of larginine or NO donors such as S-nitrosoacetylpenicillamine and S-nitrosoglutathione can also elicit NT release, effects that are blocked by conotoxin, N-type Ca2+ channel blockers, hemoglobin, inhibitors of NOS: l-NMMDA, 7-nitroindazole, sGC: 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one] (ODQ), PKG, and are exacerbated by permeable analogues of cGMP, such as 8-bromo-cGMP and dibutyl-cGMP [92, 94, 96–99]. These findings support a clear role for Ca2+ -activated nNOS, and regulation of NT release through PKG. While there is a wealth of information suggesting a relationship between Ca2+ / NO and NT release, there is little to no information defining a specific role for PKG in regulating synaptic events at the axonal nerve terminal. Further compelling, are reports that suggest that the NO molecule itself, independent of signaling systems can potentiate Ca2+ -evoked exocytosis of NT. Calcium has a well known role in NT release through its activation of Ca2+ kinases [Ca2+ /calmodulin-dependent protein kinase CAM kinase I, II] [100] that phosphorylate the synaptic vesicle protein synapsin, reducing affinity to actin and thereby initiating release of vesicles from the active zone of nerve terminals [101, 102]. However, a fairly high concentration of nNOS is also located at the nerve terminal and 3-nitrotyrosine (3-NT) has been detected in synaptic vesicles proteins such as synaptobrevin, synaptophysin, munc-18, SNAP25, indicating that NO may play a role in both docking and priming of vesicles prior to release [103–105]. Further, 3-NT is a protein oxidative marker for ONOO− [106, 107], indicating that ONOO− is being produced in the nerve terminal apparently contributing to an unknown but powerful role in synaptic vesicle destabilization. While an exact mechanism is not known, ONOO− may augment vesicle release by irreversibly inhibiting synaptosomal, plasma and SERCA/reticulum membrane Ca2+ ATP-ase pumps, which may potentiate cytosolic Ca2+[I] , creating a lower threshold for NT receptor activation and exocytosis of synaptic vesicles [108, 109]. Moreover, in the presence of NO donors, the evoked NT release is abolished in the presence of Cu2+ /Zn2 +-SOD, O2 -scavengers, Ca2+ chelator: EGTA and hemoglobin [94, 110, 111]. These findings indicate a synergistic role between ONOO− , Ca2+ and NT release, and further suggest a pre-eminent role for O2 − in this process, although posing a questionable role for PKG. While there are many aspects to the neurotoxic effects of nNOS, which are discussed in greater detail in Section 14.5, the lethal effects appear to be directly due to

14.2 Signaling Controls – Neuronal NOS: TYPE-I

accumulation of the NO molecule itself, independent of sGC activity, cGMP or PKG [112]. In disease states, the amassing of NO produced during glutamate induced – Ca2+ – overload is lethal to neurons, where both cGMP and PKG exert a non-toxic regulatory and potentially neuroprotective role [65, 113]. Therefore, a targeted endpoint for antagonizing the direct effects of nNOS, would be upstream to the function of the enzyme, achieved by controlling Ca2+[I] . 14.2.2

Neuronal Calcium Homeostasis

The primary upstream control over nNOS is the neuronal concentration of intracellular Ca2+[I] . Typically, the control of Ca2+[I] is regulated by homeostatic efflux/influx cell membrane transport systems, such as Ca2+ ATP-ase pumps, ion channels, uniporters and H+ /Ca2+ :Na2+ exchangers. In neurons, the initial rise in Ca2+[I] can occur through the influx of extracellular Ca2+ , triggered by activation of plasma membrane voltage dependent or ligand-gated Ca2+ channels and/or the loss of Ca2+ uptake from internal storage compartments, such as the endoplasmic reticulum (ER) and mitochondria. Typically, a potent spike in Ca2+ i triggers an all or none-response in excitable type cells. However, in neurons, this can also contribute to toxicity where either depolarization or overactivation of glutaminergic ionotropic Ca2+ activated channels: N-methyl-d-aspartate (NMDA), á-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) and kainite (KA), and G protein-coupled metabotropic receptors, linked to phosphospholipase C (PLC) [114] contribute to the rapid influx of Ca2+ , the activation of nNOS and neurotoxicity. The rapid influx of Ca2+ , and phospholipase C (PLC) release of IP3 , can collectively escalate the rise in Ca2+[I] , by inducing Ca2+ -induced Ca2+ release (CICR) through activation of ER-IP3 and ryanodine sensitive Ca2+ release channels, and opening of the mitochondrial permeability transition pore complex (PTPC) initiating massive Ca2+ efflux through a reverse Na2+ /Ca2+ antiporter in the mitochondria [115, 116, 117]. Preventing the initial rise of Ca2+[I] , in a superfluous environment of glutamate is neuroprotective, and can be achieved by blocking Ca2+ entry at the NMDA receptor: MK-801, N-alkylglycines, the AMPA/kainite receptor: 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 6,7-dinitroquinoxaline-2,3dione (DNQX), 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(f)-quinoxaline (NBQX) or the metabotropic glutamate receptor: 1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) [92, 118–123]. Further, blunting the rise in intracellular Ca2+[I] , can be mediated by blocking ryanodine sensitive Ca2+ release channels (dantrolene) or with PLC inhibitors, internal ER IP3 receptor antagonists, Ca2+ chelators or calmodulin antagonists [112, 123–127]. The resting voltage of the plasma membrane also plays a critical role in establishing the propensity of glutamate to open voltage-activated Ca2+ channels. Depolarization or leakage of Na2+ ions can prompt a lower threshold for action potential, creating a hyper-excitable membrane. Depolarization reduces the affinity of Mg2+ to create a voltage-dependent block of the NMDA channel, leading to opening of the pore and a greater ligand-receptor affinity for glutamate [128]. In contrast, hyperpolarizing the membrane or increasing extracellular Mg2+ concentration can establish a tighter Mg2+ voltage-dependent block of the

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NMDA receptor, with less susceptibility to Ca2+ mediated entry by glutamate [129, 130]. In total, antagonizing direct upstream control over the nNOS isoform can be achieved in part by application of internal and external Ca2+ channel blockers, Ca2+ buffering agents, hyperpolarizing agents or selective nNOS inhibitors such as 6 or 7nitroindazole, 1-(2-trifluoromethylphenyl) imidazole or 6-phenyl-2-aminopyridines [131–133].

14.3

Signaling Controls, Endothelial NOS: Type-3 14.3.1

EDRF/Vascular Tone

While nNOS is critical to the function of neurotransmission, endothelial NOS presents itself in vascular endothelium and platelets, serving a crucial role in cardiovascular function and CNS blood flow. NO generated by eNOS induces robust vasodilation, endothelial smooth muscle relaxation, and antagonizes thrombosis/platelet aggregation [8, 9, 134]. Both NO and prostacyclin are referred to as endothelium derived relaxing factors (EDRF) and a chronic deficit of one or both can lead to hypertension, atherosclerosis, stroke and congestive heart failure [135–138]. Endothelial NOS is constitutive, augmented in the presence of l-arginine [139], regulated by Ca2+ /calmodulin and controls sGC/cGMP-dependent activation of PKG which regulates phosphorylation of proteins that initiate a vasodilatory response [8, 140]. In the cardiovascular system, the eNOS/NO/sGC/cGMP/PKG vasodilatory response is triggered by prostacyclin, bradykinin, potassium chloride, low blood pressure, low oxygen tension, acetylcholine, histamine and vasoactive intestinal polypeptide (VIP) [141–145]. Bradykinin is a key regulator and potent activator of eNOS in the circulatory system. Agents that can prevent the degradation of bradykinin such as ACE inhibitors, also evoke vascular relaxation and vasodilation [146]. ACE inhibitors provide a dual protective mechanism, in that they augment a vasodilatory response to bradykinin and attenuate formation of angiotensin II (Ang II) [147, 148], that if present yields vasoconstrictor effects by acting on endothelial AT-type 1 receptors [149]. Moreover, a reduction in Ang II can also prevent its potent role in the up-regulation of NADPH oxidase activity which generates O2 − and in the presence of NO forms a potent oxidizing and vasoconstrictor molecule, ONOO− [150, 151]. In the cardiovascular system, ONOO− is detrimental because it can directly evoke hypertension [152] and induce oxidative injury to the vessel walls, effects that are attenuated by removing O2 − [153] or exacerbated in SOD-/- knockout mice [154]. Superoxide scavengers, that are one and the same with ACE inhibitors such as captopril, can have powerful therapeutic effects with ample capacity to potentiate bradykinin and even further increase NO/ONOO− [155–158]. Increasing the ratio of NO/ONOO− in the cardiovascular system has a number of beneficial effects, and for this reason ACE inhibitors can ef-

14.3 Signaling Controls, Endothelial NOS: Type-3

fectively improve circulatory function, reduce risk of heart attack, stroke, myocardial infarction, hypertension, congestive heart failure and general mortality from cardiovascular related events [159, 160]. Alternatively, EDRF/NO can also be potentiated by administration of l-arginine or nitrates such as nitroglycerin, glycerol trinitrate, amyl nitrite and isosorbide dinitrate, that can also effectively treat cardiovascular disease, myocardial ischemia, angina pectoris and hypertensive crisis [138, 161]. 14.3.2

eNOS, Cyclic AMP/GMP Regulation

Unlike nNOS, where Ca2+ channel blockers provide neuroprotective effects, agents that increase the activity of eNOS are beneficial. While eNOS is regulated in part by Ca2+ /calmodulin in a similar fashion to nNOS, it is also highly regulated by phosphorylation at serine residues through activity of phosphatidylinositol-3-OH-kinase (PI3K/AK2) and PKA [162, 163]. The effects of PKA in the potentiation of eNOS are quite robust and independent of changes in Ca2+[I] [164]. Agents that can augment intracellular cAMP (which activate PKA), such as forskolin, 8-bromo-cAMP, beta-adrenergic receptor agonist isoproterenol and adrenomedullin also heighten eNOS activity and induce a vasodilatory response [165–167]. And, primary vasoactive substances such as bradykinin and VIP also activate eNOS through a mechanism involving phosphorylation of serine residues, effects which are blocked by inhibitors of both PKA and PI3K [163, 168, 169]. These findings suggest an upstream control over eNOS via cAMP/PKA, whereas downstream effects of NO generated by eNOS involve controlling cGMP/PKG-mediated vasodilation. These findings indicate that an elevation of both cyclic nucleotides by PDE inhibitors should synergistically potentiate vasodilation of EDRF/NO and instill beneficial effects to cardiovascular function. Likewise, sildenafil or PDE-5 inhibitors are considered promising in the treatment of coronary heart disease, where they can prevent the degradation of cGMP, evoke endothelial vasodilation and attenuate platelet activation/adhesion in patients with heart disease [54, 55, 170, 171]. Moreover, the application of PDE-1 selective inhibitor, 8-methoxymethyl-1-methyl-3-(2methylpropyl)-xanthine thwarts the degradation of cGMP and induces vasodilation, effects that are blocked by sGC inhibitor ODQ and restored by NO donors, defining a specific role for NO in regulating GS/cGMP and PKG [172]. Similar effects are observed with cAMP specific-PDE inhibitors such as the type 3 inhibitors: CI 930, cilostazol milrinone, 94120 and LY 195115, which can effectively heighten NO/EDRF in vascular tissue, thoracic aorta and arterioles effects that are blocked by NOS inhibitors. These findings corroborate a role for cAMP in regulating eNOS/NO/GS/cGMP and PKG [53, 173, 174]. The application of PDE4 inhibitors (rolipram and denbufylline), also prevents degradation of cAMP and can induce endothelial relaxation, effects that are reversed by addition of l-NMMA and restored by addition of l-arginine [174]. While these studies demonstrate that PDE inhibitors augment the function of eNOS in cardiovascular tissue, the effects of blocking degradation of AMP can also downregulate the iNOS isoform in immunocompetant cells during CNS inflammation. For example, an increase of cAMP in astrocytes, microglia and other cell types can suppress NO production, and block

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induction of NOS-2 mRNA [51,175–178]. Together, cAMP selective PDE-4 /PDE-1C inhibitors may provide powerful anti-inflammatory, vasodilatory effects and thereby antagonize NOS imbalances that contribute to CNS disease. Further, since the effects of cGMP in neurons do not appear to contribute to toxicity [65, 112, 113], PDE-5 inhibitors may yield dual benefit although there is little research on this topic. Other known factors that can evoke EDRF/NO, include the statin drugs, green tea catechins [139, 155, 162, 179], lysophospholipids [180] sauna bath therapy [181], physical exercise [182], indomethacin [183], estrogen [184, 185], glutathione [186], and vitamin C [187]. In total, increasing eNOS activity can be achieved by administration of ACE inhibitors, organic nitrites, phosphodiesterase inhibitors, antioxidants, larginine, angiotensin II receptor (AT1 ) blockers, and prostacyclin analogs such as isocarbacyclin.

14.4

Signaling Controls, Inducible NOS: Type-2 14.4.1

Inflammation, Microglia and Astrocytes

During CNS inflammation, the largest contributor to the production of NO is cells of monocyte/ macrophage lineage, including microglia and astrocytes. Under normal conditions, astrocytes provide a protective functional and regulatory role in the brain by sustaining a nourishing interstitial milieu and platform for neurons to thrive. Astrocytes aid in regulating electrolyte balance, nutrient/metabolic transport, H+ ion concentrations, and assist with neuronal repair [188]. Because astrocytes are basically the CNS housekeeper, they are equipped with robust neuroprotective apparatus. Some of these include a high intracellular concentration of reduced glutathione (GSH), vigorous antioxidant enzymes such as catalase and glutathione peroxidase (GSH-Px), the ability to synthesize glycogen as a storage fuel, to release trophic factors: nerve growth factor, brain-derived neurotrophic factor, transforming growth factor-â, fibroblast growth factor, to produce anti-inflammatory cytokines, and to provide a rapid disposal route for extra-cellular glutamate via high capacity Na+ -coupled glutamate uptake transporters (GLT1 and GLAST1) with metabolic detoxification through glutamine synthetase [188–192]. During trauma or CNS disease, astrocytes can diverge from their normal defensive function, and respond to necrotic debris by creating a toxic inflammatory response. Markers of inflammation in astrocytes consist of morphological changes evident by cell proliferation, swelling, hypertrophy, increased expression of glial fibrillary acidic protein (GFAP), iNOS, cycloxygenase (COX) and expression of immunological proteins inherent to antigen-presenting cells such as class I/II major histocompatibility complex (MHC) antigens, vascular adhesion molecules (VLA-1, VLA-2, and VLA-6), lymphocyte function-associated molecule-3 and release of pro-inflammatory mediators such as tumor necrosis factoralpha (TNF-á), interleukin 1â (IL-1â), interferon–gamma (IFN-ã), and prostaglandins [193, 194]. CNS inflammatory disease often accompanies reduced expression or func-

14.4 Signaling Controls, Inducible NOS: Type-2

tion of astrocytic GLT1 and GLAST1, leading to the accumulation of extracellular glutamate [188, 195, 196]. While there are many contributors to neurological damage during inflammation, the primary vulnerabilities posed by astrocytes with regards to NO are: the extensive production of a highly diffusible NO molecule, depletion of intracellular GSH and reduced ability to handle glutamate, which accumulates in extracellular synaptic spaces resulting in excitotoxic neuronal cell death [14, 129, 191, 197, 198]. Microglia cells (resident macrophages of the brain) also become lethal participants, demonstrating ample facility to proliferate, migrate, infiltrate to various regions of the brain and participate in active phagocytosis. Active microglia also display similar characteristics to antigen-presenting cells typical to the humoral immune response, including the ability to express complement receptors, MHC class I and II antigens, Fc gamma receptors, immunoglobulin E receptors, intercellular adhesion molecule1 (ICAM-1), VCAM-1, iNOS, COX-2, and to produce and release pro-inflammatory cytokines: TNF-á and IL-1â [199-204]. Although experimentally induced CNS inflammation in rodents is associated with expression of iNOS in both microglia and astrocytes, in the human brain, astrocytes are believed to be a primary generator of NO. Studies consistently demonstrate that adult and fetal astrocytes produce abundant NO and express iNOS mRNA; however there are discrepancies amongst findings regarding iNOS in activated human microglia [194, 205, 206]. Microglia cells can however, release pro-inflammatory cytokines such as TNF-á and IL-1â that in turn can induce expression of iNOS in astrocytes. Therefore, both cell types are lethal participants in neurodegenerative CNS disease. 14.4.2

Stress Activated and Extra-cellular Kinases

Expression of glia NOS-2 mRNA is primarily mediated by kinase signaling pathways that are initiated in response to surface contact with pro-inflammatory cytokines such as TNF-á, IL-1â, IFN-ã, S100B, or pathogenic antigenic substrates derived from bacterial, parasitic or viral agents [207–211]. While there are a number of kinase signaling cascades that control induction of iNOS in glia [212–214], primarily proinflammatory agents trigger one or more of the three central mixed lineage kinase (MLK) pathways: ERK/MAPK/1/2, p38/SAPK and JNK/SAPK [58, 209, 215–217]. MLK signaling cascades are highly complex, interconnected and classified under mitogen-activated protein kinase phosphorelay modules that control diverse signal transduction pathways within mammalian cells, in particular in response to stress [218]. Ultimately, in astrocytes, SAPK/ERKs control the pro-inflammatory response, by directly phosphorylating and activating DNA binding elements and transcription factors such as activator protein-1 (AP-1), cAMP response element binding protein (CREB), Jun-c and nuclear factor kappaâ (NF-kappaâ), that permit binding to the DNA promotor region of iNOS, initiating mRNA transcription through RNA polymerase II [12, 13, 215]. The activity of p38/SAPK is central to the genetic changes that occur in response to cytokine activated pro-inflammatory signaling in astrocytes and microglia. p38/SAPK

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enzyme inhibitors such as 4-(4-fluorophenyl)-2-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole or SB203580, exert powerful anti-inflammatory effects, blocking expression of iNOS, TNF-á and IL-1â [12, 13, 58, 219]. Moreover, SB203580 is effective in blocking iNOS nuclear transcription in several models of inflammation and in response to various types of pro-inflammatory stimuli such as bacterial/viral pathogenic antigens [209, 220]. While a substantial role for p38/SAPK and JNK/SAPK in regulating cytokine regulation of iNOS is consistently reported [12, 217, 221, 222], results have been variable in defining a role for ERK/MAPK/1/2, which is typically involved with cell growth and proliferation signaling. Use of inhibitor PD98059 has been reportedly both ineffective and effective in blocking LPS/cytokine induction of iNOS in primary astrocytes, glioma and microglia cell lines [12, 58, 223]. The activities of p38/SAPK, JNK/SAPK or ERK/MAPK/1/2 are essentially involved with phosphorylation of CREB, AP-1 and NF-kappaâ, guiding conformational changes in these proteins, that allow binding to the DNA promoter region of the iNOS gene [215, 221, 224]. While protein kinases turn on expression of genes, protein phosphatase 1 (PP1) and PP2A typically cleave a phosphate group off at serine residue 133/142 of CREB [225]. In astrocytes, both PP1 and PP2A control iNOS mRNA and NF-kappaâ DNA binding, events that are reportedly upregulated in the presence of inhibitors such as okadaic acid [226]. These findings suggest that PP1 and PP2A antagonize the function of SAPK enzymes, either directly or in competition between phosphorylation/dephosphorylation of transcription factor/binding elements in the nucleus that regulate iNOS transcription. There are also other counteracting controls to the activation of NF-kappaâ in astrocytes, such as expression and accumulation of Ikappaâ-alpha (IKA) and Ikappaâ-beta (IKB) proteins [227]. IKA and IKB are inhibitory proteins that are degraded upon phosphorylation by IKA and IKB kinases, which correspond to the upregulation of NF-kappaâ. Increased activation of IKA can inactivate NF-kappaâ translocation and antagonize association with the CREB-binding protein, which is critical in initiating iNOS transcription [215, 221, 224, 228]. It appears that some of the anti-inflammatory cytokines such as IL-4 or IL-10 mediate effects via this signaling, where they up-regulate IKA mRNA, thereby reducing NOS-2 [211]. 14.4.3

Cyclic AMP/Protein Kinase a

Other protein kinases may indirectly influence the activation of NF-kappaâ. For example, in contrast to the pro-inflammatory effects typically observed with activation of kinases, the elevation of cAMP activates PKA and blocks transcription of iNOS mRNA [51, 178, 229, 230]. Astrocytes contain a variety of NT receptors that are coupled to Gsadenylate cyclase [231] and, either activation of â-adrenergic/dopamine receptors or employing agents that increase cAMP, such as forskolin (adenylate cyclase activator), PDE inhibitors [i.e. pentoxifylline], dibutyrl cAMP, or 8-bromo cAMP can attenuate lipopolysaccharide (LPS)/cytokine activated iNOS mRNA in microglia, astrocytes and a number of other cell types [51, 176, 177, 178, 232–237]. In contrast, agents that suppress the intracellular concentration of cAMP such as H-89 and Rp-cAMP are pro-

14.4 Signaling Controls, Inducible NOS: Type-2

inflammatory and augment iNOS expression in astrocytes [51]. Although there is an abundance of evidence to support cAMP/PKA activation and suppression of iNOS mRNA, in theory, this is the opposite to what would be expected by cAMP/PKA which typically phosphorylates CREB, a regulatory promotor of transcription [238]. However, this is not the case, and studies consistently demonstrate that cAMP suppresses the transcription of pro-inflammatory proteins in diverse immuno-competent cells including astrocytes and microglia [178, 229, 230, 237, 239]. Although the mechanism of action for cAMP in mediating effects on iNOS in astrocytes has not been clearly elucidated, there are preliminary studies that indicate plausible controls. The antiinflammatory effects of cAMP may involve activation of cAMP-responsive element that initiates transcription of suppressor of cytokine signaling proteins (SOCS-1,3) [240]. In immuno-competent cells, SOCS proteins counteract cell signaling directed by pro-inflammatory cytokines such as TNF-á, IL-1â, IFN-ã, by directly blocking phosphorylation and activation of janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT)–(JAK/STAT) pathway [212]. In astrocytes, tyrosine phoshorylation of JAK2 and STAT1 alpha/beta are required for LPS/IFN-ã induction of iNOS mRNA [213]. Moreover, cytokine induction of iNOS in other type of cells are blocked by JAK2 inhibitor tyrphostin B42 [241], which also prevents degradation of IKA and blocks the translocation of the NF-kappa B p65 into the nucleus [242]. Agents that can increase intracellular cAMP, such as forskolin and dibutyryl cAMP also induce SOCS-3 mRNA protein expression in immuno-competent cells [243]. Overexpression of SOCS-3, can antagonize the pro-inflammatory effects of IL-1â, IFN-ã and block cytokine induction of iNOS [244, 245]. Ultimately, it is possible that the means by which tyrosine kinase inhibitors such as genestein [214] and adenylate cyclase activators [229], attenuate iNOS transcription in astrocytes, may merge at SOSC-3/enzyme inactivation of JAK/STAT, both preventing phosphorylation and activation of JAK [246]. There is also evidence to link cAMP to both JAK/STAT and the function of ERK/MAPK/1/2, a regulatory control of iNOS in astrocytes [58, 223]. In macrophages, ERK/MAPK/1/2, can phosphorylate STAT1alpha and thereby activate JAK2/STAT1alpha which is integral for the cytokine induction of iNOS [247]. In glia, both stress and inflammation can provoke activation of the ERK/MAPK/1/2 signaling [248, 223, 249], that is blocked by cell permeable analogues of cAMP, such as 8-CPT cAMP or other cAMP potentiating agents such as forskolin/isobutylmethylxanthine or isoproterenol [248]. These data suggest that cAMP potentiation of SOSC-3 [243] may prevent the activation of JAK/STAT [240] and block ERK1/2 activation of NFkappaâ [58, 223]. In glia, elevation in cAMP juxtaposes the elevation of IKA, and inactivation of NF-kappaâ [250]. Future research will be required to delineate a more precise mechanism for cAMP in control of iNOS. However, these findings suggest therapeutic application of selective cAMP-specific PDE inhibitors, which negatively control cytokine activation of iNOS.

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14.4.4

Cyclic AMP–Phosphodiesterase Inhibitors

Both astrocytes and microglia contain PDE-4 and PDE-1C, and both show highspecificity for cyclic AMP [178, 251–253]. As mentioned previously, PDE-1C and PDE- 4 inhibitors also have beneficial effects on cardiovascular function by augmenting endothelial NO/EDRF. While there is very little research investigating the potential use of PDE-4 inhibitors in CNS disease, its primary pharmaceutical use is well known in the treatment of chronic inflammatory diseases in the periphery, such as arthritis, chronic obstructive pulmonary disease and asthma. A large number of studies consistently show that PDE-4 inhibitors modulate anti-inflammatory effects in basophils, eosinophils, macrophages, lymphocytes and neutrophils [239, 254]. Although few studies have examined the efficacy of PDE-4 inhibitors in CNS disease, rolipram (PDE-4 I) and non-specific PDE inhibitors such as ibudilast, can suppress production and release of TNF-á, IL-1â, NO and O2 − in glial cells [52, 178, 239, 255]. In vivo, non-specific PDE inhibitors are under investigation, showing capacity to reduce neurodegeneration associated with cerebral ischemia, vascular dementia, AD, and stroke [256–259]. 14.4.5

Peroxisome Proliferator-activated Receptor-gamma

There is also evidence to support another pathway for cAMP, involving the peroxisome proliferator-activated receptor gamma (PPAR-gamma). PPAR-gamma is a nuclear hormone ligand receptor that, if activated, can induce a conformational change directly to DNA coactivator complexes (i.e. CREB-binding protein), preventing activation of NF-kappaâ, AP-1 and binding to the iNOS promoter, thereby suppressing iNOS mRNA transcription [47, 48, 49, 260–262]. In the CNS, PPAR-gamma receptors are expressed in astrocytes and throughout the brain and spinal cord [263]. Administration of lipophilic PPAR-gamma activators such as non-steroidal anti-inflammatory agents (NSAIDS) (indomethacin, fenoprofen, ibuprofen, flufenamic acid and diclofenac), the TDs (ciglitizone, rosiglitazone, troglitazone) or prostaglandin 15-deoxydelta12,14-prostaglandin J2 can gain direct access to the nucleus and exert powerful anti-inflammatory effects by directly blocking transcription of pro-inflammatory proteins [264–266]. The activation of PPAR-gamma is directly associated with the reduction of Ikappaâ kinase, elevated expression of IKA mRNA and inactivation of NF-kappaâ/ AP-1, which also corresponds to the suppression of a host of proinflammatory proteins such as iNOS, MHC-II antigen, ICAM-1, P-selectin, COX-2 and TNF-á [153, 261, 267–272]. Cyclic AMP may yield immunosuppressive effects through altering the PPAR-gamma receptor. In astrocytes, norepinephrin (NE) can induce upregulation of PPAR-gamma mRNA and protein, effects that are blocked by the â-adrenergic antagonist propranolol [273]. Similarly, NE can act on â-adrenergic receptors triggering activation of adenylate cyclase and cAMP [274], which are associated with inactivation of NF-kappaâ [51]. Future research will be required to

14.5 The Neurotoxicity of NO

delineate whether cAMP or PKA can directly upregulate genetic transcription of the PPAR-gamma receptor. While the use of PPAR-gamma agonists are widely known to treat other diseases such as cancer [275], diabetes [276, 277], arthritis and microbial sepsis [153, 269, 278], there are a growing number of studies demonstrating that these agents may protect against NO-mediated injury to the CNS [279]. In experimental models of PD, administration of pioglitazone can attenuate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced dopaminergic degeneration and loss of nigrostriatal function, effects that also correspond to reduction of iNOS, NO, inactivation of NF-kappaâ and heightened concentration of IKA [268, 280]. Moreover, PPAR-gamma ligands such as troglitazone and ciglitizone attenuate microglial activation in the presence of betaamyloid (Aâ), also corresponding to reduction of astrocyte proliferation, neurotoxicity and expression of pro-inflammatory modulators such as COX and MAC-1 [281]. PPAR-gamma agonists such as pioglitazone, also provide therapeutic advantage in animal models of experimental autoimmune encephalomyelitis, where reduction of neurological dysfunction and preserved myelin is associated with reduction of TNFá, IFN-ã, reduction in T cell proliferation and elevated concentration of IKA [282, 283]. While PPAR-gamma agonists appear to prevent neurodegeneration associated with CNS inflammatory conditions, they also augment the function of eNOS. Administration of TDs and rosiglitazone reduce blood pressure [284] and attenuate cardiac damage associated with ischemic/reperfusion-injury [50]. Interestingly, there is recent evidence to suggest that PPAR-gamma agonists also prevent excitotoxicity in neurons, independent of an inflammatory response [46]. These findings indicate significant promise for this class of drugs, having combined effects to antagonize global inflammation, iNOS, protect against excitotoxicity, while potentiating the function of eNOS. In summary, controlling pro-inflammatory cytokine signaling associated with iNOS protein expression in glia, may be achieved with NSAIDS (indomethacin, ibuprofen), cAMP-specific phosphodiesterase inhibitors, â-adrenergic agonists, PPARgamma ligands, ERK/MAPK/1/2, p38/SAPK and JNK/SAPK inhibitors, select SAPK inhibitors such as quercetin [285], tyrosine kinase inhibitors, or compounds that augment IKA, inhibit NF-kappaâ translocation, activation or DNA binding.

14.5

The Neurotoxicity of NO 14.5.1

Oxidative Stress

In the CNS, accumulation of NO generated by either iNOS or nNOS, in the presence of O2 , can produce a number of nitrogen oxide species [17, 286]. However, the formation of peroxynitrite/peroxynitrous acid (ONOO− /ONOOH) is thought to be the most critical in terms of contributing to neurodegenerative injury, where singlet NO− is innocuous and even neuroprotective [287, 288, 298, 290]. Peroxynitrite is generated by

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the chemical reactivity of superoxide (O2 − ), and NO, and once formed, can protonate to yield ONOOH that can spontaneously decompose into • NO2 and OH• [85,107]. In the CNS, low concentration of ONOO− is not toxic [291], however, during disease states the accumulation of ONOO− /ONOOH, without adequate handling capacity can be dangerous. These are potent oxidizing agents with ample capacity to destroy the integrity of lipid membrane structures and protein amino acid resides such as cysteine, glutathione, methionine, tyrosine, guanine and tryptophan [16, 18, 292]. In neurons and other biological systems, lipid/protein oxidation and nitrosylation reactions can render structural and conformational changes that lead to inactivation or malfunction of cellular constituents, contributing to nerve cell death. Some of these include: ONOO− -mediated inactivation of glyceraldehyde-3-phosphate dehydrogenase, creatine kinase and aconitase [16, 286, 293], inactivation of ETC-complex I, II, and V (F1, FO-ATPase), displacement of oxygen at cytochrome oxidase, loss of oxidative phosphorylation (OXPHOS) [20, 21, 293–295], oxidation of adenine nucleotide translocase resulting in PTPC opening and apoptosis [296, 297], hyper-activation of PARP-1 nuclear enzyme leading to depletion of energy substrates [43], loss of aerobic/anaerobic ATP production, inhibition of Ca2+ and Na2+ -K+ ATP-ase pumps [16, 108, 109], inactivation of antioxidant enzymes such as GSH-Px, catalase and SOD [16, 293, 298, 299, 300], depletion of endogenous GSH, which serves as an ONOO− detoxification system [197], and lipid membrane degradation yielding 4-hydroxynonenal (4-HNE) and nitrosoperoxo–lipid oxidation adducts [16, 292]. While the presence of ONOO− /ONOOH can trigger a surplus of deleterious events that contribute to cell death, ultimately, these center around a heightened synergy between oxidative stress created by NRS and ROS. In vitro, or in vivo, the oxidation, nitrosylation and inactivation of proteins mediated by ONOO− , NO2 , and ONOOH can lead to the accumulation of the protein oxidation marker 3-NT, easily detected by immuno-histochemical analysis [18, 301, 302]. Accumulation of 3-NT in the CNS is observed during aging, inflammation and chronic degenerative diseases such as Parkinson’s disease (PD), Alzheimer’s disease (AD), multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS) where its presence precedes, and accumulates in proximity to neurodegenerative lesions and corresponds to the loss of neurological function [303–308]. The accumulation of 3-NT is also observed tantamount to formation of protein carbonyls (a marker of ROS mediated damage) [303, 307], indicating a co-operative degenerative synergy by a number of radical species potentially including the hydroxyl radical (OH• ), hydrogen peroxide (H2 02 ), O2 − and ONOO− /ONOOH. There is ample evidence to suggest that the presence of ONOO− in the CNS, may render near complete exhaustion of normal endogenous antioxidant defenses such as alpha-tocopherol, ascorbic acid and GSH [309, 310]. Peroxynitrite can inhibit the catalytic activity of nearly all antioxidant enzymes, such as GSH-Px, glutathione reductase, catalase, glutathione S-transferase [298–300] Mn2+ -SOD and Cu2+ /Zn2+ SOD [311–313]. NO/ONOO− can also initiate the release of free Fe2+ from iron-sulfur heme centers and ferritin [286], oxidize transition metal complexes [16], and, with a deficit of H2 O2 metabolizing enzymes, escalate production of the destructive OH• radical. In total, the presence of ONOO− in the CNS creates an incredible vulnerability and platform for ROS-mediated damage, by rendering near complete annihilation

14.5 The Neurotoxicity of NO

of major endogenous cytosolic and mitochondrial antioxidant defenses. This is further substantiated by evidence demonstrating accumulation of thiobarbituric acid reactive substances (TBARS, a biomarker of lipid peroxidation) and toxic lipid peroxidative aldehyde products such as 4-HNE observed in the presence of 3-NT and protein carbonyls in CNS diseases, indicating massive oxidative stress [292, 305, 314, 315]. The effects of ONOO− in the CNS are pre-eminently dangerous because this molecule may initiate a progressive cycle that perpetuates itself. The overproduction of ONOO− can lead to the inactivation of SOD, which is required for endogenous dissolution of superoxide (O2 −• ) [313]. The molecular availability of O2 −• in biological systems is the rate-limiting factor for production of ONOO− , which is the most damaging NRS species [288–290, 318]. Moreover, during inflammatory diseases, elevated production of O2 −• can occur through mitochondrial dysfunction, NADPH oxidase or Ca2+ -activated xanthine oxidase, which propels reactivity with NO to form even more ONOO− , that can then further exacerbate the inactivation of mitochondrial and cytosolic SOD [309, 316, 317]. The inability to remove O2 −• can severely impact the lethality of the NO molecule, whereas scavenging O2 −• is extremely neuroprotective. This is consistently demonstrated in studies that show a reduction of neurological injury involving 3-NT, NO, ONOO− and 4-HNE, with administration of SOD mimetics, or in cell models and transgenic mice overexpressing Mn2+ -SOD, Cu2+ /Zn2+ -SOD or nNOS/iNOS (-/-) knock out [316–321]. In contrast, NO-mediated pathologies are exacerbated in SOD deficient (-/-) mice [322]. Therapetic value SOD/catalase mimetics such as manganese-salen complexes [62] are currently under investigation and show significant promise to reduce neurological injury associated PD [323], AD [324] aging [325], stroke [326] ALS [63] and autoimmune encelphalomyelitis [327]. While SOD mimetics can prevent the formation of ONOO− , once formed, GSH is the major detoxification system and intracellular sink for removing ONOO− [197]. Once again, the presence of ONOO− in the CNS can be very dangerous, because this molecule can also create a cyclic and progressive depletion of thiols and GSH. ONOO− can oxidize thiols by a two-electron oxidation reaction, and ONOOH can directly and irreversibly deplete intracellular GSH by a one-electron oxidation formation of thiyl radicals that can initiate a chain reaction [19, 328]. Agents that augment intracellular GSH, such as 3H-1,2-dithiole-3-thione or gamma-glutamylcysteine ethyl ester (GGCE) [329–331] can attenuate the neurotoxic effects of ONOO− . Conversely, preventing the synthesis of GSH by inhibiting GSH synthetase with buthionine sulfoximine can exacerbate cellular damage by ONOO− [329, 332]. These findings establish equally important roles for GSH and SOD, in blocking the formation of / and detoxifying ONOO− to reduce neurological injury. 14.5.2

Mitochondrial Impairment

The production of NO within or around neurons, can impart irreversible adverse affects on mitochondrial O2 -dependent cellular respiration [21, 333, 334]. Degenerative diseases that involve NO correspond to toxicity that parallels the loss of mito-

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chondrial OXPHOS, impaired ETC function and a deficit of ATP [335]. Conversely, administration of nNOS inhibitors can initiate a robust elevation in whole body O2 oxygen utilization [21], suggesting a powerful adverse influence of NO on the function of the mitochondria. The effects of NO in the CNS could be catastrophic, in particular because post-mitototic neurons must sustain aerobic production of ATP to fulfill ongoing requirements for a plethora of anabolic processes, such as protein synthesis, DNA repair, membrane voltage, neuronal trafficking, Ca2+ homeostasis, axonal/dendritic outgrowth, NT packaging and synaptic release. NO can either gain direct access to neurons through diffusion, or it can be produced intracellularly via Ca2+ activated cytosolic nNOS [336, 337]. A rise in cytosolic Ca2+ juxtaposes the activation of a Ca2+ uniporter / and sequestering of Ca2+ into the mitochondria, where it can further activate constitutive mitochondrial NOS (mtNOS), thus generating NO in close proximity to the ETC [338–340]. While defining the sequence homology of mtNOS, using cross-reactivity to selective monoclonal antibodies has not been consistently demonstrated [334, 341], the enzymatic function of mtNOS is known to require l-arginine, Ca2+ , NADPH, calmodulin, tetrahydrobiopterin and its activity is inhibited by NOS inhibitors- l-NMMA and (S)-2-amino-(1-iminoethylamino)-5- thiopentanoic acid [334, 342]. In isolated mitochondria, the presence of l-arginine can produce a dose-dependent production of NO and l-citrulline, that juxtaposes heightened production of OH• , H2 02 , O2 and ONOO− /ONOOH and a loss in OXPHOS, ATP, cell respiration, effects that are reversed in the presence of NOS inhibitors [309, 333, 334, 342–344]. These findings suggest that the detrimental effects of mtNOS on cell respiration, appear to be in the main, due to the formation of NO or ONOO− /ONOOH. The production of ONOO− in the mitochondria of neurons is highly likely, due to mtNOS being located in the same organelle that accommodates the enormous aerobic metabolic requirement for O2 . The human brain consumes approximately 20% of whole body O2 , and in the mitochondria 1–2% of the O2 consumed is converted to O2 − through autoxidation of semiquinones of ubiquinone and flavin NADHdehydrogenase [308, 345]. In the mitochondria, O2 − can react rapidly with NO to form ONOO− , or ONOO− can be formed through the reactivity of O2 with nitroxyl ions [NO− ], that are emitted upon contact of NO with ferrocytochrome c or ubiquinol [18, 293, 346]. Once formed, ONOO− , can directly produce a concentration-dependent loss of mitochondrial respiration and OXPHOS [309]. In mitochondrial isolates, addition of l-arginine or Ca2+ can also lead to the accumulation of ONOO− and ROS, concomitant to the loss of cell respiration, effects that are reversed also in the presence of SOD, uric acid, oxymyoglobin, GGCE, reduced thiols and nNOS inhibitors: NMMA or l-NAME [331, 344, 347–349]. Cultured cells that overexpress mitochondria Mn2+ -SOD appear to be more resistant to NO-mediated cell death [320]. These findings clearly suggest that the presence of O2 − is responsible for the detrimental inhibitory effects of NO on the mitochondria, through production of ONOO− . In the mitochondria, ONOO− can mediate damage to OXPHOS by nitrosylating/oxidizing tyrosine or thiol functional groups, rendering catalytic inactivation of complex I [NADH: ubiquinone oxidoreductase], complex II [succinate: ubiquinone oxidoreductase] and complex V (F1, FO-ATPase), thereby impeding ETC/ OXPHOS

14.5 The Neurotoxicity of NO

and aerobic production of ATP [15, 20, 21, 293]. HNO is equally as toxic as ONOO− , contributing to the inactivation of complex II [350]. Moreover, the NO molecule itself, is known to be a reversible inhibitor of complex IV [20, 351]. Nitric oxide has a high affinity for heme, much like its role in sGC, however in the mitochondria this can be detrimental. NO is a suffocating agent, and its mechanism is similar to carbon monoxide, where its presence competitively and reversibly inhibits cytochrome oxidase by displacing O2 at cytochromes a+a3, CuA, and CuB, thereby blocking the active site with Fe-nitrosyl adducts [a32+NO] or nitrite bound adducts [a33+CuB2 +NO2 -] [352] and raising the Km for O2 [294, 295, 353]. Interestingly, SOD, can reverse the effects of NO on the mitochondria and block the toxic effects of NO, indicating that the effects of NO on complex IV do not contribute to the lethality or collapse of cell function. Further, these findings suggest a detrimental lethal role for ONOO− that far exceeds its ability to inhibit mitochondrial respiration. 14.5.3

Permeability Transition Pore Complex, Apoptosis

The damaging effects of ONOO− on the mitochondria also initiate mitochondrialdirected apoptosis [354]. The accumulation of Ca2+ in the mitochondria can lead to activation of mtNOS and production of ONOO− which can directly trigger opening of the mitochondrial permeability transition pore complex (PTPC), and thereby initiate the controlling event in a sequel of apoptotic cell death [296, 297, 345, 348, 355]. While Ca2+ is thought to initiate opening of the PTPC, ONOO− appears to play a more pre-eminent role, because opening of the PTPC is blocked by NOS inhibitors, SOD mimetics and ONOO− scavengers during Ca2+ mediated apoptosis [348, 356, 357]. The effects of ONOO− on PTPC are catastrophic and in essence define a death sentence. Peroxynitrite can maintain the PTPC in its open state, creating a pore of permeability that allows passage of high molecular weight solutes (