Oligonucleotide Therapeutics

Stanley T. Crooke Isis Pharmaceuticals, Inc. Carlsbad, California

n»rger's Medicinal Chemistry and Drug Discovery Sixth Edition, Volume 2: Drug Development Edited by Donald J. Abraham ISBT 0-471-37028-2 © 2003John Wiley & Sons, Inc.

Contents

1 Introduction, 116 1.1 Definition, 116

2 History, 117

2.1 Polynucleotides, 117

2.2 The Antisense Concept, 117

2.3 Strategies to Induce Transcriptional Arrest, 118

2.4 Ribozymes, 119

2.5 Combinatorial Approaches to Oligonucleotide Therapeutics, 120

2.6 The Medicinal Chemistry of Oligonucleotides, 120

3 Proof of Mechanism, 120

3.1 Factors That May Influence Experimental Interpretations, 120

3.1.1 Oligonucleotide Purity, 120

3.1.2 Oligonucleotide Structure, 121

3.1.3 RNA Structure, 121

3.1.4 Variations in In Vitro Cellular Uptake and Distribution, 121

3.1.5 Binding and Effects of Binding to Nonnucleic Acid Targets, 121

3.1.6 Terminating Mechanisms, 122

3.1.7 Effects of "Control Oligonucleotides", 122

3.1.8 Kinetics of Effects, 122

3.2 Recommendations, 122

3.2.1 Positive Demonstration of Antisense Mechanism and Specificity, 122

4 Molecular Mechanisms of Antisense Drugs, 123

4.1 Occupancy-Only-Mediated Mechanisms, 123

4.1.1 Inhibition of 5'-Capping, 123

4.1.2 Inhibition of Splicing, 123

4.1.3 Translational Arrest, 124

4.1.4 Disruption of Necessary RNA Structure, 124

4.2 Occupancy-Activated Destabilization, 125

4.2.2 Inhibition of 3'-Polyadenylation, 125

4.2.3 Other Mechanisms, 126

4.3 Activation of RNase H, 126

4.4 Activation of Double-Strand RNases, 128

5 Antisense Technology as a Tool for Gene Functionalization and Target Validation, 128

6 Characteristics of Phosphorothioate Oligonucleotides, 129

6.1 Hybridization, 129

6.2 Interactions with Proteins, 129

6.3 Pharmacokinetic Properties, 132 6.3.1 Nuclease Stability, 132

6.4 In Vitro Cellular Uptake, 132

6.5 In Vivo Pharmacokinetics, 133

6.5.1 Aerosol Administration, 134

6.5.2 Topical Administration, 134

6.5.3 Summary, 134

6.6 Pharmacological Properties, 135

6.6.1 Molecular Pharmacology, 135

6.6.2 in Vivo Pharmacological Activities, 136

6.7 Clinical Activities, 138

6.7.1 ISIS 2302,138

6.7.2 ISIS 3521,138

6.7.3 G3139,139

6.7.4 ISIS 2503,139

6.7.5 ISIS 5132,139

6.8 Toxicology, 139

6.8.2 Genotoxicity, 139

6.8.3.1 Acute and Transient Toxicities, 141

6.8.3.2 Subchronic Toxicities, 142

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