Drug Targeting Organ-Specific Strategies

XII List of Contributors

Grietje Molema

Groningen University Institute for Drug Exploration (GUIDE)

Department of Pharmacokinetics and Drug Delivery and

Department of Pathology and Laboratory Medicine

Medical Biology Section Tumor Immunology Laboratory Ant. Deusinglaan 1

9713 AV Groningen the Netherlands [e-mail]

Frits Moolenaar

Groningen University Institute for Drug Exploration (GUIDE)

Department of Pharmacokinetics and Drug Delivery

Ant. Deusinglaan 1

9713 AV Groningen the Netherlands

[e-mail]

Ricardo Mutuberria

Maastricht University

Department of Pathology

PO Box 616

6200 MD Maastricht

the Netherlands

Peter Olinga

Groningen University Institute for Drug Exploration (GUIDE)

Department of Pharmacokinetics and Drug Delivery

Ant. Deusinglaan 1

9713 AV Groningen the Netherlands [e-mail]

Klaas Poelstra

Groningen University Institute for Drug Exploration (GUIDE)

Department of Pharmacokinetics and Drug Delivery

Ant. Deusinglaan 1

9713 AV Groningen the Netherlands [e-mail]

Johannes H. Proost

Groningen University Institute for Drug Exploration (GUIDE)

Department of Pharmacokinetics and Drug Delivery

Ant. Deusinglaan 1

9713 AV Groningen the Netherlands [e-mail]

S. Ramakrishnan

University of Minnesota

Health Science Center Department of Pharmacology 321 Church Street Minneapolis

Minnesota 55455 USA

[e-mail]

Daisy W. J. van der Schaft

Maastricht University/

University Hospital Maastricht

Department of Internal Medicine

Tumor Angiogenesis Laboratory

Peter Debyelaan 25

6202 AZ Maastricht

the Netherlands

[e-mail]

Astrid J. Schraa

Groningen University Institute for Drug Exploration (GUIDE)

Department of Pathology and Laboratory Medicine

Medical Biology Section Tumor Immunology Laboratory Hanzeplein 1

9713 GZ Groningen the Netherlands [e-mail]

Yuichi Sugiyama

University of Tokyo

Graduate School of Pharmaceutical Sciences

7-3-1 Hongo

Bunkyo-ku Tokyo 113-0033 Japan

[e-mail]

List of Contributors

XIII

Kokichi Suzuki

Meiji Seika Kaisha Ltd

760 Moro-oka-cho

Kohoku-ku

Yokohama City 222-0002

Japan

Willem R. Verweij

Groningen University Institute for Drug Exploration (GUIDE)

Department of Pharmacokinetics and Drug Delivery

Ant. Deusinglaan 1

9713 AV Groningen the Netherlands [e-mail]

Dick de Zeeuw

Groningen University Institute for Drug Exploration (GUIDE)

Department of Clinical Pharmacology Ant. Deusinglaan 1

9713 AV Groningen the Netherlands [e-mail]

Drug Targeting Organ-Specific Strategies. Edited by G. Molema, D. K. F. Meijer Copyright © 2001 Wiley-VCH Verlag GmbH ISBNs: 3-527-29989-0 (Hardcover); 3-527-60006-X (Electronic)

Contents

Preface	. . . .	. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	V
Foreword . . .		. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	VII
List of Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .			XXI
1	Drug Targeting: Basic Concepts and Novel Advances
	Grietje Molema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		1
1.1	Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		1
1.2	Carriers used in Drug Targeting . . . . . . . . . . . . . . . . . . . . . . . . . .		2
	1.2.1	Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	2
	1.2.2	Monoclonal Antibodies and Fragments . . . . . . . . . . . . . . . . .	3
	1.2.3	Modified (Plasma) Proteins . . . . . . . . . . . . . . . . . . . . . . .	4
	1.2.4	Soluble Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	4
	1.2.5	Lipoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	6
	1.2.6	Microspheres and Nanoparticles . . . . . . . . . . . . . . . . . . . . .	6
	1.2.7	Polymeric Micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . .	7
	1.2.8	Cellular Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	7
1.3	Intracellular Routing of Drug–Carrier Complex . . . . . . . . . . . . . . . .		8
	1.3.1	Passive Versus Active Drug Targeting . . . . . . . . . . . . . . . . . .	8
	1.3.2 Lysosomes as a Cellular Target Compartment . . . . . . . . . . . . .		8
	1.3.3	Cytoplasmic Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . .	9
	1.3.4	Nuclear Targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	9
	1.3.5	Mitochondrial Targeting . . . . . . . . . . . . . . . . . . . . . . . . .	10
1.4	Drug Targeting Strategies in the Clinic . . . . . . . . . . . . . . . . . . . . . .		10
	1.4.1 Liposome Based Therapies in the Clinic . . . . . . . . . . . . . . . .		11
	1.4.2 Monoclonal Antibody Therapies in the Clinic . . . . . . . . . . . . .		11
	1.4.3 Monoclonal Antibody Based Targeting Strategies in the Clinic . . . .		13
	1.4.4 Other Drug Targeting Strategies in the Clinic . . . . . . . . . . . . .		13
1.5	Vaccination Strategies for Enhanced Immunity . . . . . . . . . . . . . . . . .		15
1.6	Drug Targeting as a Research Tool to Study Disease . . . . . . . . . . . . . .		16

XVI	Contents
1.7	Challenges in Drug Targeting Research . . . . . . . . . . . . . . . . . . . . .	18
1.8	Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	19
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		20

2	Brain-Specific Drug Targeting Strategies
	Ulrich Bickel, Young-Sook Kang, Jörg Huwyler . . . . . . . . . . . . . . . . .			23
2.1	Introduction . .		. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	23
2.2	Overview of Central Nervous System Diseases . . . . . . . . . . . . . . . . .			23
	2.2.1	Neurodegenerative Diseases . . . . . . . . . . . . . . . . . . . . . . .		23
		2.2.1.1	Alzheimer Disease (AD) . . . . . . . . . . . . . . . . . . .	23
		2.2.1.2	Parkinson’s Disease . . . . . . . . . . . . . . . . . . . . . .	24
	2.2.2	Cerebrovascular Disease . . . . . . . . . . . . . . . . . . . . . . . . .		25
	2.2.3	Brain Tumours . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		25
	2.2.4	HIV Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		25
2.3	BBB Biology and Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . .			26
	2.3.1	Physiological Transport Systems . . . . . . . . . . . . . . . . . . . . .		28
		2.3.1.1	Nutrient Carriers Versus Diffusion-mediated Uptake . . .	28
		2.3.1.2	Efflux Systems . . . . . . . . . . . . . . . . . . . . . . . . .	29
		2.3.1.3	Receptorand Absorptive-mediated Uptake . . . . . . . .	29
	2.3.2 Techniques for Measurement of Brain Uptake . . . . . . . . . . . . .			31
		2.3.2.1	In Vivo Methods . . . . . . . . . . . . . . . . . . . . . . . .	31
		2.3.2.2	In Vitro Models . . . . . . . . . . . . . . . . . . . . . . . . .	34
2.4	Drug Delivery Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .			34
	2.4.1 Small Molecule Drug Delivery . . . . . . . . . . . . . . . . . . . . . .			35
	2.4.2	Macromolecular Drug Delivery . . . . . . . . . . . . . . . . . . . . .		36
		2.4.2.1	Intraventricular Route . . . . . . . . . . . . . . . . . . . . .	36
		2.4.2.2	Intraparenchymal Route . . . . . . . . . . . . . . . . . . . .	37
		2.4.2.3	Convective Flow . . . . . . . . . . . . . . . . . . . . . . . .	39
		2.4.2.4 Delivery by Barrier Disruption . . . . . . . . . . . . . . . .		39
		2.4.2.5	Vector-mediated Delivery . . . . . . . . . . . . . . . . . . .	40
		2.4.2.6 Pharmacological Effects of Chimeric Peptides . . . . . . .		43
		2.4.2.7	Chimeric Peptide Radiopharmaceuticals . . . . . . . . . . .	46
	2.4.3 Liposomes as Drug Carriers . . . . . . . . . . . . . . . . . . . . . . .			47
		2.4.3.1 Conventional Liposomes and Small Molecules . . . . . . .		47
		2.4.3.2	Brain Targeting Using Immunoliposomes . . . . . . . . . .	47
		2.4.3.3 Drugs of Interest for Targeting to the Brain . . . . . . . . .		48
2.5	Conclusions . .		. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	49
References . .		. . . . .	. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	50

		Contents	XVII
3	Pulmonary Drug Delivery: Delivery to and Through the Lung
	Anne H. de Boer, Grietje Molema, Henderik W. Frijlink . . . . . . . . . . . . .		53
3.1	Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		53
3.2	The Respiratory Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		55
	3.2.1 Lung Capacities and Pulmonary Ventilation . . . . . . . . . . . . . .		55
3.3	Lung Deposition and Particle Size . . . . . . . . . . . . . . . . . . . . . . . .		57
3.4	Drug Absorption via the Lung . . . . . . . . . . . . . . . . . . . . . . . . . .		58
	3.4.1 Systemic Delivery of Peptides and Proteins . . . . . . . . . . . . . .		60
3.5	Devices for Therapeutic Aerosol Generation . . . . . . . . . . . . . . . . . .		63
	3.5.1	Nebulizers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	64
	3.5.2	Metered Dose Inhalers . . . . . . . . . . . . . . . . . . . . . . . . . .	65
	3.5.3	Dry Powder Inhalers . . . . . . . . . . . . . . . . . . . . . . . . . . .	65
3.6	Formulations for Inhalation Products . . . . . . . . . . . . . . . . . . . . . .		67
	3.6.1	Formulations for Nebulizers . . . . . . . . . . . . . . . . . . . . . . .	67
	3.6.2 Formulations for Dry Powder Inhalers . . . . . . . . . . . . . . . . .		67
	3.6.3 Formulations for Peptides and Proteins . . . . . . . . . . . . . . . . .		69
3.7	Variables and Interactions in Dry Powder Inhalation . . . . . . . . . . . . . .		73
3.8	Airflow Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		74
3.9	Inspiratory Pressure and Relevant Flow Parameters . . . . . . . . . . . . . .		75
	3.9.1 Measurement of the Inspiratory Flow Curve . . . . . . . . . . . . . .		77
3.10	In Vitro Particle Size Analysis and Deposition Measurements . . . . . . . . .		78
3.11	In Vitro and In Vivo Deposition Efficacy of Inhalation Systems . . . . . . . .		80
3.12	Targeting Drugs to the Lungs via the Bloodstream . . . . . . . . . . . . . . .		81
3.13	Final Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . .		82
References . .		. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	83

4	Cell Specific Delivery of Anti-Inflammatory Drugs to Hepatic Endothelial
	and Kupffer Cells for the Treatment of Inflammatory Liver Diseases
	Barbro N. Melgert, Leonie Beljaars, Dirk K. F. Meijer, Klaas Poelstra . . . . .		89
4.1	Introduction . .	. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	89
4.2	The Liver . . .	. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	89
	4.2.1 The Parenchymal Cell (PC) . . . . . . . . . . . . . . . . . . . . . . .		91
	4.2.2 The Sinusoidal Endothelial Cell (SEC) . . . . . . . . . . . . . . . . .		91
	4.2.2.1	Receptor-mediated Endocytosis . . . . . . . . . . . . . . .	92
	4.2.2.2	Phagocytosis and Transcytosis . . . . . . . . . . . . . . . . .	93
	4.2.2.3	Regulation of the Inflammatory Process by SECs . . . . .	93
	4.2.3 The Kupffer Cell (KC) . . . . . . . . . . . . . . . . . . . . . . . . . .		93
	4.2.3.1	Receptor-mediated Endocytosis . . . . . . . . . . . . . . .	94
	4.2.3.2	Phagocytosis . . . . . . . . . . . . . . . . . . . . . . . . . .	94
	4.2.3.3 Regulation of the Inflammatory process by the KC . . . . .		94
	4.2.4 The Hepatic Stellate Cell (HSC) . . . . . . . . . . . . . . . . . . . . .		95

XVIII	Contents
4.3	Hepatic Inflammation and Fibrosis . . . . . . . . . . . . . . . . . . . . . . . .			96
4.4	Liver Cirrhosis: Causes and Therapy . . . . . . . . . . . . . . . . . . . . . . .			98
4.5	Drug Targeting to the Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . .			99
	4.5.1 Carriers Directed at SECs and KCs . . . . . . . . . . . . . . . . . . .			100
		4.5.1.1	Albumins . . . . . . . . . . . . . . . . . . . . . . . . . . . .	100
		4.5.1.2	Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . .	101
		4.5.1.3	Carriers with Intrinsic Anti-inflammatory Activity . . . . .	102
	4.5.2 Targeting to other Hepatic Cells . . . . . . . . . . . . . . . . . . . . .			103
4.6	Anti-inflammatory Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . .			103
	4.6.1	Nonsteroidal Anti-inflammatory Drugs (NSAIDs) . . . . . . . . . .		103
	4.6.2	Glucocorticosteroids . . . . . . . . . . . . . . . . . . . . . . . . . . .		104
	4.6.3	Other Anti-inflammatory Drugs . . . . . . . . . . . . . . . . . . . . .		105
4.7	Anti-fibrotic Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .			105
4.8	Testing Liver Targeting Preparations . . . . . . . . . . . . . . . . . . . . . . .			106
	4.8.1	Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		106
	4.8.2	Cellular Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . .		108
	4.8.3	Efficacy and Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . .		108
4.9	Targeting of Anti-inflammatory Drugs for the Treatment of Liver Fibrosis . .			110
	4.9.1	Targeting of NSAIDs . . . . . . . . . . . . . . . . . . . . . . . . . . .		111
	4.9.2	Targeting of Glucocorticosteroids . . . . . . . . . . . . . . . . . . . .		112
4.10	Selective Drug Delivery for the Treatment of Other Hepatic Disorders . . .			114
4.11	Conclusions . .		. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	115
References . .		. . . . .	. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	115

5Delivery of Drugs and Antisense Oligonucleotides to the Proximal Tubular Cell of the Kidney Using Macromolecular and Pro-drug Approaches

	Marijke Haas, Yukio Kato, R. Folgert G. Haverdings, Frits Moolenaar,
	Kokichi Suzuki, Dick de Zeeuw, Yuichi Sugiyama, Dirk K. F. Meijer . . . . . .		121
5.1	Introduction . .	. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	121
	5.1.1 Kidneys and their Functions . . . . . . . . . . . . . . . . . . . . . . .		121
	5.1.2 Proximal Tubular Cells and their Functions . . . . . . . . . . . . . . .		123
	5.1.3 Cellular Targets for Drug Delivery in the Kidney . . . . . . . . . . .		124
	5.1.4 Renal Pathology and the Proximal Tubular Cell for Therapeutic
	Intervention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		124
	5.1.5 Targeting to the Proximal Tubular Cell . . . . . . . . . . . . . . . . .		125
5.2	Renal Delivery Using Pro-Drugs . . . . . . . . . . . . . . . . . . . . . . . . .		126
	5.2.1 The Alkylglycoside Approach . . . . . . . . . . . . . . . . . . . . . .		126
	5.2.1.1	Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .	126
	5.2.1.2	Concept of the Alkylglycoside Approach . . . . . . . . . .	126
	5.2.1.3	Distribution of Alkylglycoside-derivatized AVP In Vivo . .	126
	5.2.1.4 Specific Binding of Alkylglycoside-derivatized AVP
		in Kidney Plasma Membranes . . . . . . . . . . . . . . . . .	129
	5.2.1.5	Structure–Kinetic Relationship Studies . . . . . . . . . . .	129

		Contents	XIX
	5.2.1.6	Identification of Target Molecules for Alkylglycosides . . .	131
	5.2.1.7 Perspectives of Renal Delivery with Alkylglycoside Vectors		131
5.2.2	The Amino Acid Pro-drug Approach . . . . . . . . . . . . . . . . . .		132
	5.2.2.1	Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .	132
	5.2.2.2 The Concept of the Amino Acid Pro-drug . . . . . . . . . .		132
	5.2.2.3 Renal Specificity of Amino Acid Pro-drug and their Effects		133
	5.2.2.4	Benefits and Limitations of the Amino Acid Pro-drug . . .	133
	5.2.2.5 The Soft Drug Concept . . . . . . . . . . . . . . . . . . . .		134
5.2.3	The Folate Pro-drug Approach . . . . . . . . . . . . . . . . . . . . . .		134
	5.2.3.1	Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .	134
	5.2.3.2 Potential Renal Selectivity of Folate Constructs . . . . . . .		134
	5.2.3.3 Benefits and Limitations of Folate . . . . . . . . . . . . . .		135

5.3Renal Delivery Using Macromolecular Carriers: The Low-Molecular

Weight Protein Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	135
5.3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	135
5.3.2 Renal uptake of LMWP Conjugates . . . . . . . . . . . . . . . . . . .	137
5.3.2.1 Renal Uptake of Native LMWPs . . . . . . . . . . . . . . .	137
5.3.2.2 Renal Delivery of Naproxen–Lysozyme . . . . . . . . . . .	137
5.3.2.3 Renal Delivery of Captopril–Lysozyme . . . . . . . . . . .	138
5.3.3 Renal Catabolism of LMWP-conjugates . . . . . . . . . . . . . . . .	139
5.3.3.1 Renal Catabolism of Native LMWPs . . . . . . . . . . . . .	139
5.3.3.2 Renal Catabolism of Naproxen–Lysozyme . . . . . . . . .	141
5.3.3.3 Renal Catabolism of Captopril–Lysozyme . . . . . . . . . .	141
5.3.4 Effects of Targeted Drugs Using an LMWP as Carrier . . . . . . . .	141
5.3.4.1 Renal Effects of Naproxen–Lysozyme . . . . . . . . . . . .	141
5.3.4.2 Renal and Systemic Effects of Captopril–Lysozyme . . . .	142
5.3.5 Renal Disease and LMWP Processing . . . . . . . . . . . . . . . . .	142
5.3.6 Renal Delivery of High Doses of LMWPs . . . . . . . . . . . . . . .	143

5.3.7Limitations of the LMWP Strategy of Drug Delivery to the Kidney . 144

5.4	Renal Delivery of Antisense Oligodeoxynucleotides . . . . . . . . . . . . . .		144
	5.4.1	Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	144
	5.4.2 Mechanism of Action of Antisense Oligodeoxynucleotides . . . . . .		145
	5.4.3	Stabilization of Antisense Oligodeoxynucleotides . . . . . . . . . . .	145

5.4.4Pharmacokinetic Aspects of Antisense Oligodeoxynucleotides and

		Renal Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . .	146
	5.4.5 Cellular Uptake of Antisense Oligodeoxynucleotides . . . . . . . . .		147
	5.4.6 Metabolism and Elimination of Antisense Oligodeoxynucleotides . .		147
	5.4.7 Effects of Antisense Targeting to the Proximal Tubule . . . . . . . .		148
	5.4.8	Benefits and Limitations of Antisense Oligodeoxynucleotides . . . .	149
5.5	Drugs for Renal Targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		149
5.6	In Vitro and In Vivo Models for Renal Targeting . . . . . . . . . . . . . . . .		149
	5.6.1	In Vitro Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	149
	5.6.2	In Vivo Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	150
5.7	Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		151
References . .		. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	151

XX Contents

6A Practical Approach in the Design of Colon-specific Drug Delivery Systems

	Claudia S. Leopold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		157
6.1	Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		157
6.2	Physiological Characteristics of the Colon . . . . . . . . . . . . . . . . . . . .		157
6.3	Pathological Processes in the Colon . . . . . . . . . . . . . . . . . . . . . . .		159
6.4	Approaches to Colon-specific Drug Delivery . . . . . . . . . . . . . . . . . .		160
	6.4.1	pH-Controlled Drug Release . . . . . . . . . . . . . . . . . . . . . . .	161
	6.4.2	Enzyme-controlled Drug Release . . . . . . . . . . . . . . . . . . . .	163
	6.4.3	Time-controlled Drug Release . . . . . . . . . . . . . . . . . . . . . .	166
	6.4.4	Pressure-controlled Drug Release . . . . . . . . . . . . . . . . . . . .	167
6.5	Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		168
References . .		. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	168

7Vascular Endothelium in Inflamed Tissue as a Target for Site Selective Delivery of Drugs

	Maaike Everts, Astrid J. Schraa, Lou F. M. H. de Leij, Dirk K. F. Meijer,
	Grietje Molema	. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	171
7.1	Introduction . .	. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	171
7.2	Regulation of Immune Responses in (Chronic) Inflammation . . . . . . . . .		171
	7.2.1 Induction of an Immune Response . . . . . . . . . . . . . . . . . . .		171
	7.2.2 The Resolution of Inflammation . . . . . . . . . . . . . . . . . . . . .		173
7.3	Chronic Inflammatory Disorders . . . . . . . . . . . . . . . . . . . . . . . . .		173
	7.3.1 Pathophysiology of Chronic Inflammatory Disorders . . . . . . . . .		173
	7.3.1.1	Rheumatoid Arthritis . . . . . . . . . . . . . . . . . . . . .	173
	7.3.1.2	Atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . .	174
	7.3.1.3	Inflammatory Bowel Disease . . . . . . . . . . . . . . . . .	174
	7.3.1.4	Other Diseases . . . . . . . . . . . . . . . . . . . . . . . . .	175
	7.3.2 Angiogenesis in Chronic Inflammation . . . . . . . . . . . . . . . . .		175

7.3.3Activation Pathways of Endothelial Cells in Chronic Inflammation . 177

7.4	Targeting Drugs to the Endothelial Cell . . . . . . . . . . . . . . . . . . . . .			179
	7.4.1 Target Epitopes on Inflammatory Endothelium . . . . . . . . . . . .			180
	7.4.2	Targeting Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		180
		7.4.2.1	Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . .	180
		7.4.2.2	Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	181
		7.4.2.3	Oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . .	182
	7.4.3	Drugs Inhibiting Endothelial Activation . . . . . . . . . . . . . . . .		182
		7.4.3.1 Inhibitors of NFκB and Other Intracellular Signalling
			Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . .	182
		7.4.3.2	Glucocorticoids, NSAIDs and Others . . . . . . . . . . . .	183
		7.4.3.3	Antisense Oligonucleotides . . . . . . . . . . . . . . . . . .	185
		7.4.3.4	Drugs that Inhibit Angiogenesis-associated Events . . . . .	186

		Contents	XXI
7.5	In Vitro Techniques for Studying Endothelial Cell Activation . . . . . . . . .		187
	7.5.1	Cell Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	187
	7.5.2	Read-out Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	187
7.6	In Vivo Animal Models for Studying Inflammation . . . . . . . . . . . . . . .		189
	7.6.1	Rheumatoid Arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . .	189
	7.6.2	Inflammatory Bowel Disease . . . . . . . . . . . . . . . . . . . . . . .	189
	7.6.3	Atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	190
	7.6.4	Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	190
	7.6.5	General Inflammation Models . . . . . . . . . . . . . . . . . . . . . .	190

7.7General Considerations and Practical Directions for Endothelial Cell

	Targeting Research . . . .			. . . . . . . . . . . . . . . . . . . . . . . . . . . . .	191
	7.7.1 The Choice of a Target Epitope . . . . . . . . . . . . . . . . . . . . .				191
	7.7.2	Disease Stage . . .		. . . . . . . . . . . . . . . . . . . . . . . . . . . .	191
	7.7.3	Drugs of Choice . .		. . . . . . . . . . . . . . . . . . . . . . . . . . . .	191
7.8	Conclusions . .		. . . . . . .	. . . . . . . . . . . . . . . . . . . . . . . . . . . .	193
References . .		. . . . .	. . . . . . .	. . . . . . . . . . . . . . . . . . . . . . . . . . . .	193
8	Strategies for Specific Drug Targeting to Tumour Cells
	Jos G. W. Kosterink, Wijnand Helfrich, Lou F. M. H. de Leij . . . . . . . . . .				199
8.1	Introduction . .		. . . . . . .	. . . . . . . . . . . . . . . . . . . . . . . . . . . .	199
8.2	Cancer Pathology . . . . . .			. . . . . . . . . . . . . . . . . . . . . . . . . . . .	199
	8.2.1 Cell Biology of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . .				199
	8.2.2	Histogenesis . . . .		. . . . . . . . . . . . . . . . . . . . . . . . . . . .	201
8.3	Currently Available Therapeutics . . . . . . . . . . . . . . . . . . . . . . . . .				201
8.4	Barriers in Tumour-directed Therapies/Strategies . . . . . . . . . . . . . . . .				202
	8.4.1 Tumour Structure and Physiology . . . . . . . . . . . . . . . . . . . .				202
	8.4.2	Physiological Barriers . . . . . . . . . . . . . . . . . . . . . . . . . . .			203
	8.4.3 Cellular and Biochemical Barriers, Multi-drug Resistance . . . . . .				203
	8.4.4	Pharmacokinetic Barriers . . . . . . . . . . . . . . . . . . . . . . . . .			204
8.5	Strategies to Deliver Drugs to Targets within the Tumour (Cells) . . . . . . .				205
	8.5.1 Monoclonal Antibody-mediated Therapeutics . . . . . . . . . . . . .				206
		8.5.1.1	Antigenic Targets . . . . . . . . . . . . . . . . . . . . . . . .		206
		8.5.1.2	Unconjugated Antibodies . . . . . . . . . . . . . . . . . . .		209
			8.5.1.2.1 Potential Disadvantages and Limitations of
				the MAb Approach . . . . . . . . . . . . . . . .	210
		8.5.1.3	Recombinant Antibodies . . . . . . . . . . . . . . . . . . .		211
			8.5.1.3.1	Recombinant DNA Technology . . . . . . . . .	211
			8.5.1.3.2 Single Chain Fv Antibody Fragments . . . . . .		211
			8.5.1.3.3	Phage Display Library . . . . . . . . . . . . . . .	212
			8.5.1.3.4	Transgenic ‘Human’ Animals . . . . . . . . . . .	212
			8.5.1.3.5 Considerations for Recombinant Antibody
				Production . . . . . . . . . . . . . . . . . . . . .	212
		8.5.1.4	Immunotoxins (ITs) . . . . . . . . . . . . . . . . . . . . . .		213

XXII	Contents
		8.5.1.5	Monoclonal Antibody–Drug Conjugates . . . . . . . . . . .	213
		8.5.1.6	Radioimmunoconjugates . . . . . . . . . . . . . . . . . . .	215
	8.5.2	Bispecific Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . .		215
	8.5.3	Pro-drug Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		217
		8.5.3.1	Antibody-directed Enzyme Pro-drug Therapy (ADEPT) .	217
		8.5.3.2	Pro-drug Monotherapy . . . . . . . . . . . . . . . . . . . .	217
	8.5.4	(Synthetic) (co)Polymers . . . . . . . . . . . . . . . . . . . . . . . . .		218
	8.5.5	Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		220
8.6	Clinical Studies		. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	221
	8.6.1 MAb and MAb-based Constructs . . . . . . . . . . . . . . . . . . . .			221
	8.6.2	Pro-drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		224
	8.6.3	(Synthetic) (co)Polymers . . . . . . . . . . . . . . . . . . . . . . . . .		225
	8.6.4	Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .		225
8.7	Animal Models: their Predictive Value . . . . . . . . . . . . . . . . . . . . . .			226
8.8	Conclusions and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . .			226
References . .		. . . . .	. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	228
9	Tumour Vasculature Targeting
	Daisy W. J. van der Schaft, S. Ramakrishnan, Grietje Molema, Arjan W. Griffioen			233
9.1	Introduction . .		. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	233
	9.1.1 Functions of Vascular Endothelial Cells in the Body . . . . . . . . . .			234
	9.1.2	Molecular Control of Tumour Growth-related Angiogenesis . . . . .		234
		9.1.2.1 Role of Growth Factors VEGF and FGF-2 . . . . . . . . .		235
		9.1.2.2	Role of Integrins . . . . . . . . . . . . . . . . . . . . . . . .	236
		9.1.2.3 Role of the Extracellular Matrix . . . . . . . . . . . . . . .		236
		9.1.2.4 Role of Subendothelial Support Cells . . . . . . . . . . . .		236
9.2	Angiogenesis Assays and Models . . . . . . . . . . . . . . . . . . . . . . . . .			237
	9.2.1	Endothelial Cell Sources . . . . . . . . . . . . . . . . . . . . . . . . .		237
	9.2.2 Functional Assays with Endothelial Cells . . . . . . . . . . . . . . . .			238
		9.2.2.1	Cell Growth Assays . . . . . . . . . . . . . . . . . . . . . .	238
		9.2.2.2	Adhesion and Migration Assays . . . . . . . . . . . . . . .	239
	9.2.3 In Vitro Angiogenesis Assays . . . . . . . . . . . . . . . . . . . . . . .			239

9.2.4In Vivo Assays to Study Angiogenesis and Targeting of Angiogenic

		Blood Vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	240
9.3	Tumour Vasculature Targeting and Pre-clinical Experience . . . . . . . . . .		241
	9.3.1	Growth Factor Receptor Targeting . . . . . . . . . . . . . . . . . . .	243
		9.3.1.1 VEGF Receptor Targeting . . . . . . . . . . . . . . . . . . .	243
		9.3.1.2 Other Growth Factor Receptors Used for Targeting
		Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .	245
	9.3.2	Endoglin Targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . .	245
	9.3.3	Targeting Integrins to Tumour Vasculature . . . . . . . . . . . . . . .	246
	9.3.4 Tumour Vasculature-specific Blood Coagulation Induction . . . . . .		247
	9.3.5	Other Potential Targets . . . . . . . . . . . . . . . . . . . . . . . . . .	249