Drug Absorption Studies (eBook)

Contents 6
Preface 16
Contributors 18
Perfused Organ Level/In Situ Techniques 24
Models for Skin Absorption and Skin Toxicity Testing 25
Abstract 25
Keywords: 25
Abbreviations 25
1.1. Introduction 26
1.2. Structure and Function of the Skin 27
1.2.1. Anatomical Structure of Human Skin 27
1.2.2. Biological Activity of the Skin 28
1.2.3. Skin Appendages 28
1.2.4. Skin Absorption Pathways 29
1.3. Strategies for Skin Invasion Testing Classified According to Their Resemblance of the In Vivo Situation 30
1.3.1. In Vivo Studies Using Pharmacodynamic Response 31
1.3.2. In Vivo Dermatopharmacokinetic Approach 31
1.3.3. In Vivo Dermal Microdialysis 32
1.3.4. Perfused Skin Models 33
1.3.5. In Vitro Skin Permeation Studies 34
1.3.6. In Vitro Skin Penetration Studies 38
1.4. Testing on Skin Toxicity 40
1.4.1. Skin Sensitization 40
1.4.2. Skin Irritation and Corrosion 43
1.4.3. Skin Phototoxicity 45
References 46
Models of the Small Intestine 56
Abstract 56
Keywords: 56
Abbreviations 56
2.1. Introduction 57
2.2. Theoretical Models Describing the Gastrointestinal Absorption of Drugs 59
2.2.1. Estimating Drug Absorption Trends from Physiochemical Characteristics 59
2.2.2. General Model Describing Gastrointestinal Absorption 62
2.2.3. The Effective Permeability Coef.cient 63
2.2.4. Estimating Effective Intestinal Permeability Coefficient Using a Mass Balance Approach 64
2.2.5. Using Peff to Estimate the Extent of Absorption 66
2.3. In Situ Models 68
2.3.1. Intestinal Perfusion Techniques 68
2.3.2. Intestinal Perfusion with Venous Sampling Models 72
2.3.3. The Isolated and Vascularly Perfused Intestinal Models 75
2.3.4. Mesenteric Lymph Duct Cannulated Anaesthetised Rat Model 76
2.3.5. Anaesthetised Large Animal Model 77
2.4. In Vivo Models 77
2.4.1. Cannulated Conscious Rat Models 77
2.4.2. Cannulated Conscious Large Animal Model 79
2.4.3. Single-Pass Perfusion in Conscious Dog/Pig–Loc-I-Gut 81
2.4.4. Single-Pass Perfusion in Conscious Humans—Loc-I-Gut 82
2.5. Discussion 84
2.5.1. Standardisation and Validation Criteria in Peff Determination 84
2.5.2. Choice of in Situ Versus in Vivo Models 86
2.5.3. Choice of Animal Species 87
References 90
Drug Absorption from the Colon In Situ 99
Abstract 99
Keywords: 99
Abbreviations 99
3.1. Introduction 100
3.2. In Situ Rat Colon Model for Absorption Evaluation 101
3.3. Permeability Characteristics of the Rat Colonic Membrane 103
3.3.1. Carrier-Mediated Transport 103
3.3.2. Passive Transport 106
3.4. Concluding Remarks 108
References 108
In Vivo and In Vitro Models for Assessing Drug Absorption Across the Buccal Mucosa 111
Abstract 111
Keywords: 111
4.1. Introduction 111
4.2. Structure and Environment of the Buccal Mucosa 112
4.2.1. Epithelial Organization 112
4.2.2. Organization of the Intercellular Domain 112
4.3. The Barriers of the Buccal Mucosa 113
4.3.1. Location of the Permeability Barrier 113
4.3.2. Chemical Nature of the Permeability Barrier 114
4.3.3. Other Permeability Barriers in the Buccal Mucosa 114
4.4. Mechanisms Involved in Oral Mucosal Absorption 116
4.4.1. Passive Diffusion 116
4.4.2. Carrier-Mediated Transport 116
4.5. Methods Employed to Assess the Permeability of the Buccal Mucosa 117
4.5.1. In Vivo Methods 118
4.5.2. In Vitro Methods 119
4.5.3. Buccal Cell Cultures 124
4.6. Concluding Remarks 125
References 125
In Situ and Ex Vivo Nasal Models for Preclinical Drug Development Studies 134
Abstract 134
Keywords: 134
Abbreviations 134
5.1. Introduction 135
5.2. Advantages of Ex Vivo and in Situ Models for Drug Absorption and Metabolism Studies 136
5.3. Limitations of Ex Vivo and in Situ Models for Drug Absorption and Metabolism Studies 138
5.4. Specific Applications of in Situ Methods in Nasal Drug Delivery Studies 139
5.4.1. Permeability Studies 139
5.4.2. Metabolism Studies 140
5.4.3. Optimization of Drug Absorption Enhancement Strategies 141
5.4.4. Nasal Drug Irritation and Tolerance 143
5.5. Specific Applications of Ex Vivo Models in Nasal Drug Delivery Studies 143
5.5.1. Permeability Studies and Characterization of Drug Absorption Pathways 143
5.5.2. Metabolism Studies 147
5.5.3. Optimization of Formulation Parameters and Drug Absorption Enhancement Strategies 148
5.5.4. Nasal Drug Irritation and Tolerance 149
5.6. Correlation Between Nasal Drug Absorption Models 149
5.7. Conclusions 151
References 152
The Isolated Perfused Lung for Drug Absorption Studies 157
Abstract 157
Keywords: 157
Abbreviations 157
6.1. Respiratory Drug Delivery 158
6.1.1. Inhaled Drug Delivery 158
6.1.2. The Lung 159
6.1.3. Drug Administration to the Lung 163
6.1.4. Drug Absorption in the Lung 164
6.1.5. Lung Model Selection for Drug Absorption Studies 165
6.2. The Isolated Perfused Lung 168
6.2.1. Principles of the Preparation 168
6.2.2. Experimental Set-up 169
6.2.3. Drug Administration to the IPL 172
6.2.4. Drug Absorption Studies Using the Isolated Perfused Lung 173
6.2.5. Developments in the IPL 176
References 177
Cellular Level—In Vitro Models of Epithelial and Endothelial Barriers 186
In Vitro Models for Investigations of Buccal Drug Permeation and Metabolism 187
Abstract 187
Keywords: 187
7.1. Introduction 187
7.2. In Vitro Studies 188
7.2.1. Isolated Buccal Tissue Mounted in Diffusion Cell Apparatus 188
7.2.2. Buccal Epithelial Cell Cultures 192
7.3. Concluding Remarks 196
References 197
In Vitro Screening Models to Assess Intestinal Drug Absorption and Metabolism 202
Abstract 202
Keywords: 202
Abbreviations 202
8.1. Introduction 203
8.1.1. General Factors In.uencing Intestinal Drug Absorption 203
8.1.2. The Intestinal Mucosa as a Physical and Biochemical Barrier to Drug Absorption 204
8.2. In Vitro Models to Study Intestinal Absorption 207
8.2.1. Membrane-Based Models (PAMPA) 207
8.2.2. Cell Culture-Based Models (Caco-2) 212
8.2.3. Ex Vivo Models (Ussing Chambers Technique) 221
8.2.4. Combination of Models 223
8.3. In Vitro Methods to Assess Intestinal Metabolism 224
8.4. Concluding Remarks 225
References 225
In Vitro Cellular Models for Nasal Drug Absorption Studies 236
Abstract 236
Keywords: 236
Abbreviations 236
9.1. Introduction 237
9.2. Structure and Physiology of the Nasal Cavity 238
9.2.1. Anatomy and Function 238
9.2.2. Physiology of Nasal Mucosa 239
9.3. Factors Affecting the Nasal Absorption 239
9.3.1. Physiological Factors 239
9.3.2. Physicochemical Characteristics of the Drugs 240
9.3.3. Effect of Formulation 240
9.4. Transport Routes of Nasal Epithelial Barrier 241
9.4.1. Transcellular Route 241
9.4.2. Paracellular Route 242
9.5. Models for Nasal Drug Transport and Absorption Studies 243
9.5.1. In Vivo Animal Models 243
9.5.2. In Vitro Excised Models 244
9.5.3. Cell Line Models 244
9.5.4. In Vitro Primary and Passaged Cell Culture Models 244
9.6. Conclusions 249
References 250
In Vitro Models of the Tracheo-Bronchial Epithelium 255
Abstract 255
Keywords: 255
Abbreviations 255
10.1. Introduction 256
10.1.1. Anatomy of the Lung Airways 256
10.1.2. Drug Delivery to the Tracheo-Bronchial Region 259
10.2. In Vitro Models of the Tracheo-Bronchial Epithelium 260
10.2.1. Primary Cell Cultures 260
10.2.2. Tracheo-Bronchial Epithelial Cell Lines 261
10.2.3. In Vitro Models of CF Airway Epithelium 262
10.3. Use of In Vitro Models of Tracheo-Bronchial Epithelial Barriers in Biopharmaceutical Research 263
10.3.1. Drug Absorption Studies 263
10.3.2. Drug Metabolism Studies Using Tracheo-Bronchial Epithelial Cells 265
10.4. Concluding Remarks 269
References 270
In Vitro Models of the Alveolar Epithelial Barrier 278
Abstract 278
Keywords: 278
Abbreviations 278
11.1. Introduction 279
11.1.1. The Alveolar Epithelium 279
11.2. In Vitro Models of the Alveolar Epithelial Barrier for Drug Absorption Studies 286
11.2.1. In Vitro Models of the Alveolar Epithelial Barrier 286
11.2.2. The Use of Alveolar Epithelial Cells in Biopharmaceutical Research 289
11.3. Concluding Remarks 294
References 295
Cell Culture Models of the Corneal Epithelium and Reconstructed Cornea Equivalents for In Vitro Drug Absorption Studies 303
Abstract 303
Keywords: 303
Abbreviations 303
12.1. Introduction 304
12.2. Anatomy and Physiology of Human Cornea 305
12.3. Transcorneal Drug Absorption into the Eye 309
12.4. Corneal Cell Culture Models 310
12.4.1. Primary Corneal Cell Cultures 310
12.4.2. Immortalized Continuous Cell Lines for Corneal Epithelial Cells 311
12.5. Organotypic Equivalents 314
12.6. Concluding Remarks 320
Reference 321
The Conjunctival Barrier in Ocular Drug Delivery 327
Abstract 327
Keywords: 327
Abbreviations 327
13.1. Introduction to the Ocular Surface and the Relative Contribution of the Conjunctiva 328
13.2. Trans- and Sub-Conjunctival Ocular Drug Delivery 330
13.3. An Overview of Conjunctival Disorders 332
13.4. Models for Studying Conjunctival Transport Properties 333
13.4.1. Excised Conjunctival Tissues 333
13.4.2. Primary Culture Models of Conjunctival Epithelial Cell Layers 336
13.4.3. Conjunctival Disease Models 337
13.5. Summary and Conclusions 337
References 338
Inner Blood–Retinal Barrier: Transport Biology and Methodology 341
Abstract 341
Keywords: 341
Abbreviations 341
14.1. Introduction 342
14.2. Conditionally Immortalized Cell Lines as a Novel in Vitro Inner Blood – Retinal Barrier Model (Uptake Studies) 344
14.3. In Vivo Blood-to-Retina In.ux Transport 346
14.3.1. Integration Plot Analysis 346
14.3.2. Retinal Uptake Index Method 347
14.4. In Vivo Vitreous/Retina-to-Blood Effiux Transport (Microdialysis Study) 348
14.5. Ex Vivo Transporter Gene Expression Levels at the Inner Blood – Retinal Barrier (Magnetic Isolation of Retinal Vascular Endothelial Cells) 350
14.6. Mechanism of Drug Transport at the Inner Blood – Retinal Barrier 352
14.6.1. Blood-to-Retina Influx Transport 352
14.6.2. Retina-to-Blood Efflux Transport 354
14.7. Conclusions 354
References 355
Regulation of Paracellular Permeability in Low-Resistance Human Vaginal-Cervical Epithelia 359
Abstract 359
Keywords: 359
Abbreviations 359
15.1. Introduction 360
15.2. The Ussing-Zerahn Model of Transepithelial Fluid Transport 361
15.3. Regulation of Paracellular Transport Across Secretory Epithelia 364
15.4. Regulation of Paracellular Permeability 365
15.5. Regulation of RTJ 366
15.5.1. Role of Cao 366
15.5.2. Regulation of Assembled Tight Junctions by Extracellular ATP 369
15.5.3. Estrogen Regulation of RTJ 369
15.5.4. Early Stages of Tight Junctional Disassembly 370
15.5.5. Claudin/Occludin Model of Regulation of the 371
15.6. Regulation of the RLIS 372
15.6.1. Estrogen Regulation of the 373
15.6.2. Estrogen Regulation of Actin Polymerization 374
15.6.3. Modulation of Actin Polymerization: Estrogen vis-à-vis Aging Effects 374
15.6.4. Estrogen Regulation of Cortical Myosin 375
15.6.5. Composite Effects of Estrogen on Paracellular Permeability 376
15.7. Implications of the Data for Topical Drug Delivery 377
15.8. Concluding Remarks 377
References 378
In Vitro Models and Multidrug Resistance Mechanisms of the Placental Barrier 388
Abstract 388
Keywords: 388
Abbreviations 388
16.1. Introduction 389
16.2. Structure of the Placenta 390
16.3. Placental Transport Mechanisms 390
16.4. Available Model Systems 391
16.4.1. Perfused Placental Model 391
16.4.2. Trophoblast Tissue Preparations 392
16.4.3. Trophoblast Cultures 394
16.5. Multidrug Resistant Transporters of the Placenta 397
16.5.1. MDR1/P-Glycoprotein (ABCB1) 397
16.5.2. Multidrug Resistance-Associated Proteins 401
16.5.3. Breast Cancer Resistance Protein (BCRP/ABCG2) 403
16.6. Conclusions 406
References 406
In Vitro Models to Study Blood–Brain Barrier Function 417
Abstract 417
Keywords: 417
Abbreviations 417
17.1. Introduction 418
17.1.1. Discovery of the Blood–Brain Barrier 418
17.2. Structure and Function of the Blood–Brain Barrier 419
17.3. Relevance of the Barrier for Drug Delivery 420
17.3.1. Rapid Transport Protein Modulation 423
17.3.2. Modulation of P-glycoprotein Transcription 424
17.4. Models to Study Blood–Brain Barrier Function 425
17.4.1. Isolated Cerebral Capillaries 426
17.4.2. Brain Capillary Endothelial Cell Culture 426
17.4.3. In Silico Methods 430
17.5. Perspectives 430
References 431
High-Throughput Screening Using Caco-2 Cell and PAMPA Systems 438
Abstract 438
Keywords: 438
Abbreviations 438
18.1. Introduction 438
18.2. Caco-2 Cell Monolayer System 439
18.3. Parallel Arti.cial Membrane Permeability Assays 445
18.4. Combining Use of Caco-2 and PAMPA 446
18.5. Concluding Remarks 447
References 447
Instrumented In Vitro Approaches to Assess Epithelial Permeability of Drugs from Pharmaceutical Formulations 450
Abstract 450
Keywords: 450
Abbreviations 450
19.1. Introduction 451
19.2. Intestinal Permeability of Drugs Delivered as Solid Dosage Forms 451
19.2.1. Rationale for Connecting Dissolution and Permeation Measurements 451
19.2.2. Connecting Dissolution and Permeation Measurement in One Instrumented Setup 456
19.2.3. Critical Evaluation of the State of the Art and Further Needs 462
19.3. Permeability Assessment of Pulmonary Aerosol Formulations 463
19.3.1. Measuring Drug Transport Across Epithelial Barriers in Submersed Conditions: Ussing Chamber and Transwell R-Like Systems 465
19.3.2. Setups Allowing to Measure Drug Transport Across Pulmonary Epithelia Interfacing Air 466
19.3.3. Critical Evaluation of the State of the Art and Further Needs 470
References 470
Bioinformatics—In Silico Tools to Predict Drug Absorption 476
Modeling Transdermal Absorption 477
Abstract 477
Keywords: 477
Abbreviations 477
20.1. Introduction 478
20.2. The Skin Barrier 478
20.3. The Diffusion Equation 479
20.4. Data Analysis 479
20.4.1. QSPR Models 481
20.4.2. Non Steady-State Solutions and Morphological Models 495
20.5. Pharmacokinetic Models 496
20.6. Outlook 497
References 498
Physiologically Based in Silico Models for the Prediction of Oral Drug Absorption 504
Abstract 504
Keywords: 504
Abbreviations 504
21.1. Introduction 505
21.2. Absorption Process 506
21.2.1. Overall Absorption Process 506
21.2.2. Drug Dissolution 507
21.2.3. Drug Absorption 508
21.3. Physiologically-Based Absorption Models 509
21.3.1. General Considerations 509
21.3.2. Mixing Tank Models 510
21.3.3. Mass Balance Models 512
21.3.4. Compartmental Absorption and Transit (CAT) Models 514
21.3.5. Other Models (PK-MapTM/ PK- Sim 517
21.4. The Use and Validation of Physiologically Based Simulation Models 518
21.4.1. How Predictive are the Models? 518
21.4.2. Situations Where Less Accurate Predictions Can Be Encountered 520
21.5. Summary 523
References 524
In Silico Modeling for Blood–Brain Barrier Permeability Predictions 528
Abstract 528
Keywords: 528
Abbreviations 528
22.1. Introduction 529
22.2. In Silico Methods Published for Prediction of BBB Permeability 530
22.3. Summary 562
References 572
Molecular/Sub-Cellular Level — Mechanistic Tools 575
Impact of Drug Transport Proteins 576
Abstract 576
Abbreviations 576
Keywords: 577
23.1. Introduction 577
23.2. Determination and Classi.cation of Drug Transporters 578
23.3. Characteristics of Major Drug Transporters 578
23.3.1. PEPT (SLC15) 578
23.3.2. OATP (SLCO) 580
23.3.3. OCT/OCTN/OAT/URAT (SLC22) 582
23.3.4. MATE (SLC47) 584
23.3.5. ABC Transporters 584
23.4. Conclusions and Perspectives 586
References 587
Cloning and Functional Heterologous Expression of Transporters 594
Abstract 594
Keywords: 594
Abbreviations 594
24.1. Introduction 595
24.2. Cloning Techniques 595
24.2.1. Historical Overview and General Considerations 595
24.2.2. Xenopus Laevis Oocytes for Cloning of Drug Carrier 597
24.2.3. Complementation Cloning Strategies 601
24.2.4. Homology Cloning 602
24.3. Heterologous Expression Systems 605
24.3.1. Cell-Free Expression Systems 605
24.3.2. Bacterial Expression Systems 607
24.3.3. Yeast Expression Systems 608
24.3.4. Xenopus Oocytes 609
24.3.5. Insect Cell Lines 610
24.3.6. Mammalian Cell Lines 610
24.4. Concluding Remarks 613
References 613
The Pharmacology of Caveolae 615
Abstract 615
Keywords: 615
Abbreviations 615
25.1. Introduction 616
25.2. Action at Caveolae 619
25.3. Caveolae and Vesicular Drug Transport 619
25.3.1. Exploiting Caveolae for Drug Delivery 619
25.3.2. Caveolae-Linked Endocytosis and Non-Caveolae, Clathrin- Independent Endocytotsis Offer Delivery of Drugs to Novel Intracellular Targets 620
25.4. Caveolae and Cancer 621
25.4.1. Caveolin Expression 621
25.4.2. A Caveolar Mechanism of Multidrug Resistance 622
25.4.3. A Gateway to Targeted Cancer Cell Ablation 623
25.4.4. Caveolae, Folate Receptor, Receptor Clustering, and Potocytosis 624
25.4.5. Folate Conjugates and Therapeutics 625
25.5. Pharmacology at Caveolae: Presentation, Presentation, Presentation 625
References 626
Immortalization Strategies for Epithelial Cells in Primary Culture 633
Abstract 633
Keywords: 633
Abbreviations 633
26.1. Introduction 634
26.2. Cell Transformation and Immortalization Strategies 635
26.2.1. Transformation Versus Immortalization 635
26.2.2. Selecting the Appropriate Cell Line 635
26.2.3. Human Epithelial Cells as a Model for Transformation and Immortalization 636
26.2.4. Strategies to Generate Transformed and/or Immortalized Epithelial Cells 636
26.3. Protocols 638
26.3.1. Generation, Isolation, and Characterization 638
26.4. Summary 643
References 645
Binding-Uptake Studies and Cellular Targeting 657
Abstract 657
Keywords: 657
Abbreviations 657
27.1. Introduction 658
27.1.1. Design of Targeted Drug Delivery Systems 659
27.1.2. Cell Culture Models 660
27.1.3. Analytical Tools: Labeling and Detection of Targeted Drug Delivery Systems 660
27.2. The Cell–Target System Interaction 662
27.2.1. Cytoadhesion Assays 662
27.2.2. Specificity of Interaction 664
27.2.3. Binding Versus Uptake—Cytoinvasion Assays 665
27.2.4. Cytoevasion 667
27.2.5. Active or Passive Uptake? 667
27.2.6. Uptake and Intracellular Localization Within Acidic Compartments 668
27.3. Analytical Techniques 669
27.3.1. Flow Cytometry 669
27.3.2. Intracellular Localization by Means of Confocal Laser Scanning Microscopy 672
27.4. Concluding Remarks 676
References 676
Regulatory Considerations 680
Drug Permeability Studies in Regulatory Biowaiver Applications 681
Abstract 681
Keywords: 681
Disclaimer: 681
Abbreviations 681
28.1. Biopharmaceutics Classification System 682
28.2. Regulatory Application of the BCS 683
28.3. Permeability Measurements 685
28.4. Method Suitability 688
28.5. Conclusions 692
References 693
Appendix Lab Protocols 697
Index 698

20 Modeling Transdermal Absorption (p. 459-460)

Dirk Neumann

Abstract The human skin has long since been realized as a possible pathway for drug molecules to enter the human body. The skin, however, impedes drug absorption quite effectively, since one of its main purposes, like in all epithelial tissues, is the protection of the organism by sealing off the body from the environment. While several laboratory techniques exist to assess the migration of drug molecules into and through the skin, computational models able to predict such experimental results in a reliable manner hold obvious advantages. The interest in such models has given rise to numerous and quite different approaches, which makes it virtually impossible to list all of them. Here, a selection of computational models—divided into different classes according to their underlying concept—is presented. The chapter starts with the fundamentals outlining the properties of the skin barrier and the experimental assessment of permeability. In the next sections, the theoretical framework for each class is presented followed by a description of representative models. The chapter concludes with an outlook illustrating current and possible future trends.

Keywords: Skin permeability, Percutaneous absorption, Skin penetration, Mathematical model, Quantitative structure-activity relationships, Permeability coefficient, Human skin

Abbreviations

AIC - Akaike information criterion
ANN - Artificial neural network
COSMO - Conductor-like screening model
kNN - k-nearest-neighbor
LSER - Linear solvation energy relationship
PCA - Principal component analysis
QSPR - Quantitative structure permeability relationship
RC - Retardation coefficient
RMS - Root-mean-square

20.1. Introduction

Despite its physical properties like softness and thinness, the skin forms a formidable and surprisingly effective barrier keeping harmful substances from entering the body while at the same time reducing water loss from the inside. For systemic administration of drugs, however, pharmaceutical companies strive to find means for overcoming this barrier in a predictive manner. Here, computational models for estimating the rate and extent of drug permeation into the human body allow for increased productivity. On the other hand, prediction of transdermal drug absorption may help in risk assessment in cosmetic and agrochemical fields. Consequently, many different predictive models have been developed—which might lead to the premature presumption that the problem of predicting transdermal permeation has been fully solved. When it comes to skin diseases the skin is also a site of topical administration. Strangely enough, the number of models for predicting time-dependent concentration changes within skin layers (skin penetration) is quite small.

This chapter starts with a short introduction on the skin barrier’s properties and the methods employed for analyzing experimental data. This is followed by an overview of several selected approaches to predict steady-state diffusion through the skin. Then a few approaches that approximate the structural complexity of the skin by predicting drug diffusion in biphasic or even multiphasic two-dimensional models will be presented. Finally, the chapter concludes with a short summary of the many variables possibly influencing drug permeation and penetration.