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Concepts and Models for Drug Permeability
Studies
Related titles
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and Delivery
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Woodhead Publishing Series in Biomedicine:
Number 79

Concepts and Models for

Drug Permeability Studies
Cell and Tissue Based In Vitro Culture
Models

Edited by

Bruno Sarmento

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Contents

List of contributors xi
List of figures xv
List of tables xxi

1 Introduction 1
Bruno Sarmento
1.1 Introduction 1
References 2

2 Importance and applications of cell- and tissue-based in vitro

models for drug permeability screening in early stages
of drug development 3
Miguel Ángel Cabrera Pérez, Marival Bermejo Sanz,
Victor Mangas Sanjuan, Marta González-Álvarez,
Isabel González Álvarez
2.1 Introduction 3
2.2 General considerations 4
2.3 Drug transport 4
2.4 Permeability–absorption models 13
2.5 Methods for permeability calculation 19
2.6 Standardization of protocols for in vitro methods 19
2.7 The “three Rs” principle 22
2.8 Biosecurity systems 24
References 24

3.1 Cell-based in vitro models for buccal permeability studies 31

Neha Shrestha, Francisca Araújo, Bruno Sarmento,
Jouni Hirvonen, Hélder A. Santos
3.1.1 Introduction 31
3.1.2 Physiology of the buccal mucosa 32
3.1.3 Different in vitro models 33
3.1.4 Conclusions 38
References 38

3.2 Cell-based in vitro models for gastric permeability studies 41

Tiago dos Santos, Bianca N. Lourenço, João Coentro, Pedro L. Granja
3.2.1 The stomach as a natural barrier to absorption 41
3.2.2 Gastric drug delivery 43
vi Contents

3.2.3 Cellularized models of gastric permeability 48

3.2.4 Conclusions 51
Acknowledgments 52
References 52

3.3 Cell-based in vitro models for intestinal permeability studies 57

Carla Pereira, Joana Costa, Bruno Sarmento, Francisca Araújo
3.3.1 Anatomy and physiology of human small intestine 57
3.3.2 Mechanisms of transport 59
3.3.3 Intestinal barriers 60
3.3.4 Intestinal in vitro models 61
3.3.5 Validation studies 73
3.3.6 Conclusions 75
References 75

3.4 Cell-based in vitro models for nasal permeability studies 83

Flávia Sousa, Pedro Castro
3.4.1 Introduction 83
3.4.2 Nasal primary cell culture models 84
3.4.3 Immortalized nasal cell lines 88
3.4.4 Nasal permeability studies 92
3.4.5 Conclusions 97
References 97

3.5 Cell-based in vitro models for pulmonary permeability studies 101

Fernanda Andrade, João Albuquerque, Ana Vanessa Nascimento
3.5.1 Introduction 101
3.5.2 Mechanisms involved in pulmonary absorption of drugs 102
3.5.3 Cell-based models of immortalized cells 104
3.5.4 Primary cell cultures 107
3.5.5 Conclusions 109
References 109

3.6 Cell-based in vitro models for vaginal permeability studies 115

Ingunn Tho, Natasa 
Skalko-Basnet
3.6.1 Introduction 115
3.6.2 Anatomy of the female genital tract and mucosa 116
3.6.3 Human primary cells 117
3.6.4 Immortalized human cells forming monolayers (bi-/tri-layers) 121
3.6.5 Commercially available three-dimensional culture
of nontransformed human vaginal-ectocervical epithelial cells 124
3.6.6 Concluding remarks 126
References 126
Contents vii

3.7 Cell-based in vitro models for ocular permeability studies 129

Teófilo Vasconcelos, Sara Baptista da Silva, Domingos Ferreira,
Manuela Pintado, Sara Marques
3.7.1 Introduction 129
3.7.2 Ocular anatomy 130
3.7.3 Ocular pharmacokinetics in the anterior segment 131
3.7.4 Ocular pharmacokinetics in the posterior segment 133
3.7.5 In vitro eye cellular models for drug permeability 134
3.7.6 Conclusions 147
References 148

3.8 Cell-based in vitro models for dermal permeability studies 155

Francisca Rodrigues, Maria Beatriz P.P. Oliveira
3.8.1 Introduction 155
3.8.2 Human skin and dermal permeability 156
3.8.3 Drug permeability in in vitro models 157
3.8.4 Reconstructed dermal equivalents 161
3.8.5 Reconstructed full-thickness models 163
3.8.6 Conclusions and future perspectives 165
References 165

3.9 Cell-based in vitro models for studying blood–brain

barrier (BBB) permeability 169
Maria João Gomes, Bárbara Mendes, Susana Martins,
Bruno Sarmento
3.9.1 Blood–brain barrier: structure, importance, and difficulties
to overcome 169
3.9.2 BBB in vitro models 171
3.9.3 Permeability of drugs: how to screen and study 183
3.9.4 Comparison of BBB models 184
Acknowledgments 185
References 185

4.1 Tissue-based in vitro and ex vivo models for buccal

permeability studies 189
Pedro Castro, Raquel Madureira, Bruno Sarmento,
Manuela Pintado
4.1.1 Introduction 189
4.1.2 Porcine buccal mucosa 190
4.1.3 Diffusion cells 194
4.1.4 Permeation assay using porcine buccal mucosa 196
4.1.5 Tissue integrity and viability assessment 197
4.1.6 Porcine esophageal mucosa 198
4.1.7 Conclusions and future prospects 199
References 199
viii Contents

4.2 Tissue-based in vitro and ex vivo models for intestinal

permeability studies 203
Rute Nunes, Cátia Silva, Luise Chaves
4.2.1 Introduction 203
4.2.2 Current tissue-based methodologies for intestinal
permeability studies 208
4.2.3 Animal versus human intestinal tissue 225
4.2.4 In vivo versus in vitro correlations 226
4.2.5 New trends in permeability studies using tissue-based
models 227
4.2.6 Conclusions 229
References 230

4.3 Tissue-based in vitro and ex vivo models for nasal

permeability studies 237
Alejandro Sosnik
4.3.1 Brief description of the structure of the nose 237
4.3.2 Nasal administration of drugs 238
4.3.3 Limitations of in vivo models 241
4.3.4 In vitro models of nasal permeability 242
4.3.5 Ex vivo models of nasal permeability 243
4.3.6 Conclusions 249
References 249

4.4 Tissue-based in vitro and ex vivo models for pulmonary

permeability studies 255
Ana Costa, Fernanda Andrade
4.4.1 Introduction 255
4.4.2 Lung physiology and tissue biology 256
4.4.3 Isolated perfused lung 260
4.4.4 Conclusions 267
References 267

4.5 Tissue-based in vitro and ex vivo models for vaginal

permeability studies 273
Alexandra Machado, José das Neves
4.5.1 Introduction 273
4.5.2 Vaginal permeability and absorption 274
4.5.3 In vitro 3D organotypic models 275
4.5.4 Ex vivo mucosal models 283
4.5.5 Conclusions 300
Acknowledgments 300
References 301
Contents ix

4.6 Tissue-based in vitro and ex vivo models for ocular

permeability studies 309
Christian Kölln, Stephan Reichl
4.6.1 Introduction 309
4.6.2 Requirements for a valid corneal cell culture model
for in vitro drug absorption studies 309
4.6.3 Methods to obtain corneal cells 310
4.6.4 Methods to verify cultivated cell layers in the construct 311
4.6.5 3D reconstructed cornea models 311
4.6.6 Discussions 320
4.6.7 Conclusions 321
References 321

4.7 Tissue-based in vitro and ex vivo models for dermal

permeability studies 325
Isabel Almeida, Paulo Costa
4.7.1 Introduction 325
4.7.2 Structure and function of the skin 326
4.7.3 Mechanisms of skin absorption 327
4.7.4 Mathematical principles of skin absorption 328
4.7.5 Conducting in vitro dermal absorption tests 330
References 338

4.8 Tissue-based in vitro and ex vivo models

for blood–brain barrier permeability studies 343
Malgorzata Burek, Ellaine Salvador, Carola Y. Förster
4.8.1 Introduction 343
4.8.2 Structure and function of BBB 343
4.8.3 Cerebral microvessels and their characteristics 345
4.8.4 Methods for cell isolation and immortalization 346
4.8.5 Cell-based in vitro BBB models and their properties
necessary for drug permeability estimation 346
4.8.6 Immortalized endothelial cell lines 347
4.8.7 Static and dynamic models of BBB compared 349
4.8.8 Measurements of drug permeability 350
4.8.9 Conclusions and future developments 351
References 351

5 Correlation between cell- and tissue-based in vitro models

for drug permeability screening with in vivo situation:
modeling and functional extrapolation 357
Paulo Paixão, Luís Gouveia, José Augusto Guimarães Morais,
Nuno Silva
5.1 Introduction 357
5.2 Empirical correlations 357
x Contents

5.3 Physiologically based pharmacokinetic models 361

5.4 Conclusions 367
References 368

Index 373
List of contributors

Jo~
ao Albuquerque University of Porto, Porto, Portugal
Isabel Almeida University of Porto, Porto, Portugal
Fernanda Andrade Laboratory of Pharmaceutical Technology, Faculty of
Pharmacy, University of Porto, Porto, Portugal; Institute for Bioengineering of
Catalonia (IBEC), Barcelona, Spain
Francisca Araujo Division of Pharmaceutical Chemistry and Technology, Faculty
of Pharmacy, University of Helsinki, Helsinki, Finland; INEB—Instituto de
Engenharia Biomédica, Universidade do Porto, Porto, Portugal; ICBAS—Instituto de
Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal
Marival Bermejo Sanz Universidad Miguel Hernandez, San Juan de Alicante,
Alicante, Espa~
na
Malgorzata Burek Department of Anaesthesia and Critical Care, University of
Wurzburg, Wurzburg, Germany

Miguel Angel Cabrera Pérez Universidad Central Marta Abreu de Las Villas,
Santa Clara, Villa Clara, Cuba; Universidad Miguel Hernandez, San Juan de
Alicante, Alicante, Espa~na
Pedro Castro CBQF—Centro de Biotecnologia e Química Fina—Laboratorio
Associado, Escola Superior de Biotecnologia, Universidade Catolica Portuguesa/
Porto, Porto, Portugal
Luise Chaves UCIBIO/REQUIMTE—Laboratory of Applied Chemistry,
University of Porto, Porto, Portugal
Jo~
ao Coentro INEB—Instituto de Engenharia Biomédica, Universidade do Porto,
Porto, Portugal; Faculdade de Engenharia da Universidade do Porto (FEUP), Porto,
Portugal; Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do
Porto, Porto, Portugal
Ana Costa Instituto de Engenharia Biomédica (INEB), University of Porto, Porto,
Portugal; CESPU, Instituto de Investigaç~ao e Formaç~ao Avançada em Ciências e
Tecnologias da Sa
ude, Gandra PRD, Portugal
Joana Costa INEB—Instituto de Engenharia Biomédica, Universidade do Porto,
Porto, Portugal; FEUP—Faculdade de Engenharia da Universidade do Porto, Porto,
Portugal
xii List of contributors

Paulo Costa University of Porto, Porto, Portugal

Sara Baptista da Silva Faculty of Pharmacy, University of Porto, Porto,
Portugal; CBQF—Centro de Biotecnologia e Química Fina—Laboratorio
Associado, Escola Superior de Biotecnologia, Universidade Catolica Portuguesa/
Porto, Porto, Portugal
José das Neves INEB—Instituto de Engenharia Biomédica, Universidade do Porto,
Porto, Portugal; Instituto de Investigaç~ao e Inovaç~ao em Saude, Universidade do
Porto, Porto, Portugal
Tiago dos Santos INEB—Instituto de Engenharia Biomédica, Universidade do
Porto, Porto, Portugal
Domingos Ferreira Faculty of Pharmacy, University of Porto, Porto, Portugal
Carola Y. F€
orster Department of Anaesthesia and Critical Care, University of
Wurzburg, Wurzburg, Germany
Isabel Gonz 
alez Alvarez Universidad Miguel Hernandez, San Juan de Alicante,
Alicante, Espa~
na
Marta Gonz 
alez-Alvarez Universidad Miguel Hernandez, San Juan de Alicante,
Alicante, Espa~
na
Luís Gouveia iMed.UL, Faculty of Pharmacy, University of Lisbon, Lisbon,
Portugal
Pedro L. Granja INEB—Instituto de Engenharia Biomédica, Universidade do
Porto, Porto, Portugal; Faculdade de Engenharia da Universidade do Porto (FEUP),
Porto, Portugal; Instituto de Ciências Biomédicas Abel Salazar (ICBAS),
Universidade do Porto, Porto, Portugal
Jouni Hirvonen Division of Pharmaceutical Chemistry and Technology, Faculty
of Pharmacy, University of Helsinki, Helsinki, Finland
Maria Jo~ ao Gomes INEB—Instituto de Engenharia Biomédica, Universisdade do
Porto, Porto, Portugal; ICBAS—Instituto de Ciências Biomédicas Abel Salazar,
Universidade do Porto, Porto, Portugal; Instituto de Investigaç~ao e Inovaç~ao em
Saude, Universidade do Porto, Porto, Portugal
Christian K€
olln Institut f€
ur Pharmazeutische Technologie, Technische Universit€at
Braunschweig, Braunschweig, Germany
Bianca N. Lourenço INEB—Instituto de Engenharia Biomédica, Universidade do
Porto, Porto, Portugal; Faculdade de Engenharia da Universidade do Porto (FEUP),
Porto, Portugal
Alexandra Machado INEB—Instituto de Engenharia Biomédica, Universidade do
Porto, Porto, Portugal; Instituto de Investigaç~ao e Inovaç~ao em Saude, Universidade
do Porto, Porto, Portugal
List of contributors xiii

Raquel Madureira CBQF—Centro de Biotecnologia e Química Fina—Laboratorio

Associado, Escola Superior de Biotecnologia, Universidade Catolica Portuguesa/
Porto, Porto, Portugal
Victor Mangas Sanjuan Universidad Miguel Hernandez, San Juan de Alicante,
Alicante, Espa~
na
Sara Marques CIBIO/InBIO-UP—Centro de Investigaç~ao em Biodiversidade e
Recursos Genéticos, University of Porto, Campus Agrario, Vair~ao, Portugal
Susana Martins University of Southern Denmark, Odense, Denmark
Barbara Mendes INEB—Instituto de Engenharia Biomédica, Universidade do
Porto, Porto, Portugal; Instituto de Investigaç~ao e Inovaç~ao em Saude, Universidade
do Porto, Porto, Portugal
José Augusto Guimar~ aes Morais iMed.UL, Faculty of Pharmacy, University of
Lisbon, Lisbon, Portugal
Rute Nunes INEB—Instituto de Engenharia Biomédica, University of Porto, Porto,
Portugal
Maria Beatriz P.P. Oliveira Requimte, Faculty of Pharmacy, University of Porto,
Porto, Portugal
Paulo Paix~
ao iMed.UL, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal
Carla Pereira INEB—Instituto de Engenharia Biomédica, Universidade do Porto,
Porto, Portugal; FEUP—Faculdade de Engenharia da Universidade do Porto, Porto,
Portugal
Manuela Pintado CBQF—Centro de Biotecnologia e Química Fina—Laboratorio
Associado, Escola Superior de Biotecnologia, Universidade Catolica Portuguesa/
Porto, Porto, Portugal
Stephan Reichl Institut f€
ur Pharmazeutische Technologie, Technische Universit€at
Braunschweig, Braunschweig, Germany
Francisca Rodrigues Requimte, Faculty of Pharmacy, University of Porto, Porto,
Portugal; Fourmag Lda, Parque Industrial do Cruzeiro, Moreira de Conegos, Portugal
Ellaine Salvador Department of Anaesthesia and Critical Care, University of
Wurzburg, Wurzburg, Germany
Hélder A. Santos Division of Pharmaceutical Chemistry and Technology, Faculty
of Pharmacy, University of Helsinki, Helsinki, Finland
Bruno Sarmento Instituto de Investigaç~ao e Inovaç~ao em Saude, Universidade do
Porto, Porto, Portugal; INEB—Instituto de Engenharia Biomédica, Universidade do
Porto, Porto, Portugal; CESPU, Instituto de Investigaç~ao e Formaç~ao Avançada
em Ciências e Tecnologias da Saude, Instituto Superior de Ciências da Saude-Norte,
Departamento de Ciências Farmacêuticas, Gandra-PRD, Portugal
xiv List of contributors

Neha Shrestha Division of Pharmaceutical Chemistry and Technology, Faculty of

Pharmacy, University of Helsinki, Helsinki, Finland
Catia Silva CESPU, Instituto de Investigaç~ao e Formaç~ao Avançada em Ciências e
Tecnologias da Sa
ude, Gandra PRD, Portugal
Nuno Silva iMed.UL, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal

Natasa Skalko-Basnet University of Tromsø The Arctic University of Norway,
Tromsø, Norway
Alejandro Sosnik Technion-Israel Institute of Technology, Technion City, Haifa,
Israel
Fl
avia Sousa CESPU, Instituto de Investigaç~ao e Formaç~ao Avançada em Ciências
e Tecnologias da Sa
ude, Instituto Superior de Ciências da Saude-Norte, Gandra PRD,
Portugal
Ingunn Tho University of Oslo, Oslo, Norway
Ana Vanessa Nascimento University of Porto, Porto, Portugal; IINFACTS,
Instituto de Investigaç~ao e Formaç~ao Avançada em Ciências e Tecnologias da Saude,
CESPU, Cooperativa de Ensino Superior Politecnico e Universitario, Gandra PRD,
Portugal
Te
ofilo Vasconcelos BIAL, Portela & Ca , Trofa, Portugal; Instituto de Ciências
Biomédicas Abel Salazar, University of Porto, Porto, Portugal; Institute of Biomedical
Engineering (INEB), University of Porto, Porto, Portugal
List of figures

Figure 3.1.1 (a) Schematic representation of the three-dimensional oral mucosa.

(b) Closer view of different layers in the oral mucosa: epithelium,
fibroblasts, macrophages, capillaries, and extracellular matrix (ECM). 32
Figure 3.1.2 Filter-grown TR146 cells cultured submerged for 23 days.
Formalin-fixed paraffin-embedded section. Hematoxylin and eosin
staining demonstrates a stratified squamous epithelium-like tissue.
Original magnification
6400. Scale bar 10 mm. 34
Figure 3.1.3 Schematic representation of oral keratinocyte cultures on dead
de-epidermized dermis (DDED) supported by stainless steel grids and
filter. 36
Figure 3.2.1 Representation of the stomach mucosa showing its gastric glands
and gastric pit structures, as well as its different cell types. 42
Figure 3.3.1 Representation of the organization of the small intestine epithelium. 58
Figure 3.3.2 Sequential barriers during the transit of a drug molecule when orally
administered. 60
Figure 3.3.3 Schematic representation of the Transwell system. 62
Figure 3.3.4 Illustration of the Caco-2 monoculture model setup and culture
conditions. 63
Figure 3.3.5 Staining of co-culture day 3, Caco-2 and HT29-MTX single cultures
with alcian blue (which stains acidic mucosubstances) and eosin. 67
Figure 3.3.6 Illustration of the Caco-2/HT29-MTX co-culture model setup and
culture conditions. 68
Figure 3.3.7 Scanning electron microscopy analysis of a co-culture of Caco-2/Raji
B cells where M cells (M) were identified due to their lack of microvilli
in contrast to Caco-2 (C) cells. 68
Figure 3.3.8 Illustration of the Caco-2/Raji B co-culture model setup and
culture conditions. 69
Figure 3.3.9 Illustration of the Caco-2/HT29-MTX/Raji B co-culture model setup
and culture conditions. 70
Figure 3.3.10 Illustration of the 3D model setup comprising Caco-2, HT29-MTX,
fibroblasts, and THP-1 and culture conditions. 72
Figure 3.3.11 Assessment of the morphological and functional parameters to validate
in vitro intestinal models. 73
Figure 3.5.1 Schematic representation of absorption mechanisms in lung
epithelium. 102
Figure 3.5.2 Schematic representation of cellular culture (a), under submerged
conditions (b), and under an air–liquid interface (c). 106
xvi List of figures

Figure 3.6.1 The female genital tract: vagina, and ectocervix are composed of
stratified squamous epithelia and endocervix and uterus of single-layer
columnar epithelia. The transformation zone where two types of
epithelia meet is called the squamocolumnar junction. 116
Figure 3.6.2 Schematic description of the isolation and cultivation of human
ectocervical epithelia cells (hECE) from cervicovaginal tissue: (a) main
steps of the isolation procedure, (b) main steps of seeding on filter inserts. 118
Figure 3.6.3 Simple drawing of the permeability testing setup in the Transwell
system with epithelial cell membrane on the filter support. 120
Figure 3.6.4 Schematic representation of the dual-chamber model. 123
Figure 3.7.1 Schematic representation of the anatomy of the eye. 130
Figure 3.8.1 Morphology of human skin and different in vitro dermal permeation
models: (a) normal human skin epidermis, (b) EpiSkinÔ model,
(c) EpiDermÔ model, (d) StrataTestÔ, (e) SkinEthicÔ RHE model,
(f) EpiDerm FTÔ, and (g) EpiCSÔ (hematoxylin–eosin). 159
Figure 3.9.1 The cell associations at the BBB (Abbott, 2013). The NVU is a complex
cellular system that includes highly specialized ECs, a high concentration
of pericytes embedded in the EC basement membrane; astrocytic endfeet
associated with the parenchymal basement membrane, neurons, and
immune cells. 170
Figure 4.1.1 Scheme of Franz diffusion cell. After harvesting and preparing (c)
(tissue sample) and introducing (f) (magnetic stirrer), (a) (drug solution)
is introduced through the assembled diffusion chamber being retained
between (b) (donor compartment) and (d) (receiver compartment).
(d) should be filled with the receiver solution and preheated by inducing
the flow of water heated to 37  C through (e) (insulating jacket).
Periodically, samples may be taken from (g) (sampling port). 195
Figure 4.1.2 Scheme of Ussing diffusion cell. After harvesting and preparation, (c)
(tissue sample) is introduced in a proper compartment where the drug
solution will flow unidirectionally. (b) (Valve to adjust flow rate of
gassing) will allow control of the entrance and mixture of carbogenic
gas or nitrogen with the perfused solution in (a) (U-shaped glass tube).
(d) (Current injector) and (e) (voltmeter) allow TEER testing to assess
tissue viability. 196
Figure 4.2.1 Schematic representation of the small intestine and transport pathways
across the intestinal barrier: (a) transcellular passive diffusion,
(b) paracellular passive diffusion, (c–d) influx/efflux facilitated transport
by membrane proteins, (e) endocytosis with lysosome degradation,
and (f) transcytosis. 204
Figure 4.2.2 Schematic representation of the (a) classical Ussing chamber and
(b) Franz diffusion cell. 211
Figure 4.2.3 I. Microscopic image of Peyer’s patch (a) and non-Peyer’s patch (b) of
excised porcine tissue from the abattoir after transport to the laboratory.
Tissue deterioration was particularly evident in sections directly exposed to
the intestinal lumen. Tissue deterioration was expressed by destruction
of the functional epithelium and goblet cells layers (Ib) and by the presence
of cell agglomerates covering the remnants of villi (arrowheads). II.
Histological images of mucosal disintegration of porcine small intestine
(from freshly slaughtered piglets) at different time points and under dif-
ferent storage conditions. (a, b) Immediately after animal death; (c, d)
List of figures xvii

25 min after animal death; (c) stored at 0  C, (d) room temperature. Tissue
architecture was strongly affected before the permeability study began,
although normal viability values were obtained with the viability markers
used. (E, intestinal epithelium; LF, lymphoid follicle; PC, plica circularis;
TS, tela submucosa; VI, villi; arrowheads, mucosal disintegration.
Bar ¼ 100 mm). 218
Figure 4.2.4 Figure of an everted gut sac apparatus with dimensions (a) and
complete setup (b). 220
Figure 4.2.5 (a) Schematic representation of the medium-throughput system
(InTESTineÔ) developed by The Netherlands Organization for Applied
Scientific Research, in which ex vivo pig intestinal segments were
mounted. (b) Multiple devices can be arrayed at the same time. In the
figure, a 24-well setting is presented. 229
Figure 4.3.1 Butorphanol plasma levels after intravenous, intramuscular, and
nasal spray administration of a 2-mg dose. 239
Figure 4.3.2 Percent drug diffused through sheep nasal mucosa for curcumin
nanoemulsion and curcumin mucoadhesive nanoemulsion, using a
curcumin solution as reference. 247
Figure 4.3.3 In vitro ribavirin (RBV) permeation profiles across rabbit nasal mucosa
from an aqueous solution (empty circle, n ¼ 9) compared with ribavirin
powder (filled square, n ¼ 3). Data are expressed as mean  standard
error of the mean. 248
Figure 4.4.1 Morphology and thickness of epithelium in different lung regions. 257
Figure 4.4.2 Scheme of submucosal gland at the submucosa. 258
Figure 4.4.3 Schematic representation of IPL system. 262
Figure 4.5.1 The human vaginal mucosa of the fertile adult, presenting (E)
nonkeratinized stratified squamous epithelium and (LP) lamina propria
(H&E,
40). 275
Figure 4.5.2 The Gorodeski model. Effect of permeable supports on the stratification
of human ectocervical epithelial cells in vitro. Cells were cultured on
(a) solid or (b) filter supports. Cultures are shown at 12 days after
confluence. Arrowheads in (b) indicate attached envelopes (i.e., cells
after nuclei loss). Note that the envelopes have a more condensed
cytoplasm than the cells in the basal layer. The light areas between
the cells are intercellular spaces and extracellular matrix
(H&E,
400). 277
Figure 4.5.3 The EpiVaginalÔ model. H&E histological cross-sections of (a) VEC-100
tissue model containing normal human vaginal-ectocervical epithelial
cells, (b) human vaginal tissue explant, and (c) VEC-100-FT tissue
model. All tissues have nucleated basal and suprabasal cell layers
followed by layers in which nuclei are lost and cells become filled with
glycogen. 279
Figure 4.5.4 Reconstructed vaginal mucosa model developed by Sivard et al. as
observed by light microscopy after 14 days of submerged culture
on de-epidermized dermis. (a) 7 to 10 epithelial cell layers are seen
in a cryostat section stained with hematoxylin (scale bar ¼ 15 mm).
(b) Immunohistochemical labeling of Langerhans cells shows the
expression of langerin (in brown). Langerhans cells are present in the
basal and suprabasal layers of the mucosal epithelium (scale
bar ¼ 15 mm). 281
xviii List of figures

Figure 4.5.5 Vaginal mucosa reconstruction proposed by Bouschbacher et al. (2008)

and normal human vagina present similar morphology. (a) Schematic
representation of the various steps of vaginal mucosal reconstruction
(LC, Langerhans cells). (b and c) Immunohistochemical analysis of normal
human vagina and vaginal mucosa reconstructions. Collagen IV,
cytokeratin 13, involucrin, and loricrin expression is shown in (b) and
CD45 in (c). CD45þ cells are noted by black arrows. (d) Confocal
microscopy of Langerhans cells integrated in the vaginal mucosa recon-
struction. Langerhans cells precursors were labeled with CellTrackerÔ
Orange CMTMR (red) before seeding in the reconstructs. Nucleus were
labeled with 40 ,6-diamidino-2-phenylindole and reconstructs were subse-
quently analyzed by confocal microscopy. A top view of the mucosa
reconstruction is shown. Scale bars ¼ 20 mm. Open arrow shows the pol-
ycarbonate membrane containing 12 mm pores. 282
Figure 4.5.6 The HVEÔ model (H&E saffron staining). Image kindly provided by
SkinEthic/EpiSkin. 283
Figure 4.5.7 New Zealand rabbit vaginal histology. (a) Schematic diagram showing
a 10-week-old rabbit vagina after longitudinal vaginotomy. Photomicro-
graphs of cross-sections of the (b) upper, (c) middle, and (d) lower thirds
of the rabbit vagina (H&E, reduced from
40). Note that the muscle
component (arrowheads) is scarce but that sinusoidal structures (arrows)
are rich in the lower third. M, mucosal side; S, serosal side. 287
Figure 4.5.8 Histological variability of the vaginal mucosa of guinea pigs with various
phases of the estrous cycle (H&E). (a) Proestrus: the epithelium is
composed of a thick layer of mucous cells beneath which is a thinner layer
of immature stratified squamous epithelium; (b) estrus: the epithelium is
composed of a thick layer of mucous cells beneath which is a thick layer of
immature (keratinizing) stratified squamous epithelium; (c) metestrus:
mature stratified squamous epithelium overlies fibrous stroma of the
lamina propria; (d) early diestrus: immature stratified squamous epithelium
overlies fibrous stroma of the lamina propria; (e) late diestrus: many
mucous cells have developed in the stratified squamous epithelium;
(f) immature guinea pigs: stratified squamous epithelium. E, stratified
squamous epithelium; K, keratinizing epithelium; M, mucous cells;
S, fibrous stroma of the lamina propria. 288
Figure 4.5.9 Histological structure of sheep vaginal mucosa. H&E- and Masson’s
trichrome-stained sections showing the anterior and posterior vaginal
walls of (a–d) virgin, (e–h) parous, and (i–l) pregnant sheep. Asterisk
indicates blood vessels. Arrows in (g) indicate the three vaginal layers.
Scale bar ¼ 100 mm (H&E) or 250 mm (Masson’s trichrome). 290
Figure 4.5.10 Mean steady-state and mean estimated steady-state flux values of water,
17b-estradiol, r-arecoline, oxytocin, and vasopressin through human and
porcine vaginal mucosa. Hum, human vaginal mucosa; Por, porcine
vaginal mucosa. 292
Figure 4.5.11 Fluorescent confocal microscopy imaging of pig vaginal mucosa
obtained after incubation for 2 h with fluorescent nanoparticles. Green,
blue, and red signals are from nanoparticles, Hoechst 33342 (DNA), and
WGA, Alexa Fluor 594 conjugate (sialic acid/N-acetylglucosaminyl
residues at cell membranes/mucin), respectively. Scale bar ¼ 10 mm
and z-axis range is 6 mm. SE, sub-epithelium (lamina propria);
VL, vaginal lumen. 293
List of figures xix

Figure 4.5.12 Photomicrographs of porcine vaginal mucosa slices stained with H&E
technique and showing the morphological similarities (a and c) before
and (b and d) after freeze-thawing. Bar ¼ 100 mm for (a) and (b), 50 mm
for (c), and 30 mm for (d). 294
Figure 4.5.13 Photomicrographs of transverse sections of the anterior vaginal
mucosa of cows at different stages of the estrous cycle. (a) Proestrus
showing two to five layers of low columnar (L) to polyhedral (P) cells;
(b) estrus showing a single layer of tall, columnar mucus-producing
cells; (c) metestrus showing tall columnar mucus-secreting cells in one part
of the section (M) and several layers of polyhedral cells in another (P);
and (d) diestrus showing several layers of flattened epithelial cells
in some parts of the section (F) and several layers of polyhedral cells in
others (P). All sections stained with H&E,
260 in (a) and (b),
130 in (c)
and (d). 295
Figure 4.5.14 Comparative histology of the vaginal epithelium of rhesus and
pigtail macaques. (a) Technique used to measure epithelial thickness.
To eliminate observer bias, an electronic grid was placed on images
of vaginal epithelium and 16 measurements were taken at the
intersection of each vertical gridline and the basal lamina, taking two
measurements (thinnest and thickest) between each set of gridlines.
All measurements were taken at
100. Vaginal biopsies from the
same normal (b, c) pigtail or (d, e) rhesus macaque at day 0 (luteal
phase: b, d) and peak follicular (c, e) stage of the menstrual cycle.
Note that the keratinized layer (KE) is essentially absent in the luteal
phase of pigtail macaques. Also note that the epithelium is markedly
thinner in the luteal phase of both macaque species. 297
Figure 4.7.1 Representation of two possible routes for permeation through intact SC. 328
Figure 4.7.2 Schematic representation of Franz diffusion cell (a) and Keshary–Chien
diffusion cell (b). 334
Figure 4.7.3 Cumulative permeation patterns after finite and infinite dose
techniques. 338
Figure 4.8.1 Cell-based in vitro BBB models. (a) Monoculture. Endothelial
cells are grown on permeable insert. (b) Double co-culture.
Endothelial cells are grown on a permeable insert whereas astrocytes
are grown in the bottom of the well. (c) Triple co-culture. Endothelial
cells and astrocytes are grown on the upper and lower sides of the
permeable insert, respectively. Pericytes are grown in the bottom
of the well. 347
Figure 5.1 Relationship between the predicted logarithm of the effective human
jejunal permeability estimated by multiple linear regression and
in vivo observed values. The human jejunal permeability is predicted
by the equation log (Peff)(cm/h) ¼ 0.932 þ 0.763
log (Papp)(cm/h) þ
0.0324
RBN (Papp is determined in Caco-2 cells and RBN is
the number of rotatable bonds in the molecule) and has an
r ¼ 0.887. 359
Figure 5.2 Schematic representation of a whole-body PBPK model. Organs are
represented by compartments typically with well-stirred assumptions and
defined by physiologic volumes. Rate of drug arriving to the organ is
defined by both the drug concentration in blood and the specific blood
flow for each individual organ. Elimination organs may be defined by
either linear or saturable kinetics. 362
xx List of figures

Figure 5.3 Scheme of TAMD model. Dark blue compartments represent drug
particles in solid state; light blue compartments represent drug particles
dissolved. Stomach is represented by both states. Blue arrows represent
transit rates and its direction. Red arrows represent dissolution rate of
drug particles (Kd). Green arrows represent drug absorption rates and
vivid red arrows represent a sequenced drug transfer from enterocytes to
the portal vein, liver, and central compartment. Black arrows represent
drug transfer from central compartment into the exterior and into the
peripheral compartment, as well as from the peripheral into the central
compartment. 365
List of tables

Table 2.1 Different cell- and tissue-based in vitro models to predict drug permeability 5
Table 2.2 Summary of some physicochemical factors relevant for membrane
permeation by passive diffusion 15
Table 2.3 Equations used for permeability estimations in different methods 20
Table 3.1.1 Different types of oral mucosa with their properties 33
Table 3.2.1 Comparison of the anatomical lengths of the different organs along the
gastrointestinal tract and respective absorptive surface areas 47
Table 3.3.1 Alternatives to Caco-2 cells in drug permeability studies 65
Table 3.3.2 Cell morphology, features, and specific markers 74
Table 3.4.1 Atraumatic sampling methods to obtain human nasal epithelial cells 86
Table 3.4.2 Examples of techniques used to obtain viable primary nasal
epithelial cell cultures 89
Table 3.4.3 Example of protocol to culture serially passage nasal epithelial cells
using ALI and LCC conditions 94
Table 3.4.4 Examples of marker compounds for use in permeability studies 95
Table 3.4.5 Examples of drugs permeability studies using cell-based in vitro models 96
Table 3.5.1 Available models based on monocultures including cells of bronchial or
alveolar regions 105
Table 3.5.2 In vitro cell-based models using co-cultures of epithelial and other
cell lines (mainly cells from the immune, vascular systems, or support
cells) 105
Table 3.5.3 Molecular markers for alveolar epithelial type I and II cells 108
Table 3.6.1 Immortalized human cervicovaginal cell lines used in permeability studies 121
Table 3.7.1 List of the most relevant cell culture lines related to ocular tissues 136
Table 3.8.1 Three-dimensional models available on the market for dermal
permeation studies 160
Table 3.9.1 Qualitative comparison of principal BBB models 185
Table 4.1.1 Examples of permeability studies using porcine buccal mucosa as
tissue-based in vitro model 191
Table 4.2.1 Main factors governing intestinal permeability 207
Table 4.2.2 Comparison between in vitro methods versus in vivo assays,
and tissue-based versus cell-based 210
Table 4.2.3 Main advantages and drawbacks of Ussing chamber as a model for
drug permeability studies using excised tissues 212
Table 4.2.4 Parameters influencing experimental outcomes of diffusion chamber
assay that should be controlled and standardized 213
Table 4.2.5 Common buffers employed in drug permeability studies using intestinal
tissues and respective compositions 215
xxii List of tables

Table 4.2.6 Common markers used to assess viability and/or function of intestinal
tissues mounted in diffusion cells 216
Table 4.2.7 Electrical resistance of membrane and PD values reported in drug
permeability studies to fulfill criteria of viability of intestinal tissues
mounted in diffusion cells 217
Table 4.2.8 Factors affecting functionality and outcome of everted sac model 222
Table 4.3.1 Interspecies comparison of nasal cavity characteristics according to
conchae complexity 241
Table 4.3.2 Excised nasal tissue used to assess the intranasal route ex vivo 244
Table 4.4.1 Advantages and disadvantages of IPL 261
Table 4.5.1 Selected examples of permeability studies performed using
EpiVaginalÔ tissues 280
Table 4.5.2 Comparison between human and pig vaginal epithelium 291
Table 4.5.3 Selected examples of ex vivo permeability studies using human
cervicovaginal mucosa 298
Table 4.6.1 Summary of reported 3D cornea constructs for in vitro permeation
studies 312
Table 4.6.2 Established 3D cornea constructs not intended for permeation studies 315
Table 4.7.1 Guidelines for in vitro dermal absorption tests 331
Table 4.7.2 Parameters of in vitro dermal absorption tests, according
to detailed guidelines 332
Table 4.8.1 Immortalized cell lines 348
Introduction
Bruno Sarmento
1
Instituto de Investigaç~ao e Inovaç~ao em Saude, Universidade do Porto, Porto, Portugal;
INEB—Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal; CESPU,
Instituto de Investigaç~ao e Formaç~ao Avançada em Ciências e Tecnologias da Saude,
Instituto Superior de Ciências da Saude-Norte, Departamento de Ciências Farmacêuticas,
Gandra-PRD, Portugal

1.1 Introduction
Prediction of human drug absorption is a major goal in the design, optimization, and
selection of drugs and drug products intended for noninvasive delivery. There are
various techniques currently employed to evaluate the extent of drug absorption in
the different phases of drug discovery and development. Screening protocols to eval-
uate drug absorption include a range of preclinical methodologies, such as in silico, in
vitro, in situ, ex vivo, and in vivo.
Animal tests are the gold standard in preclinical studies to evaluate the absorption of
drugs, although they are time consuming and expensive and may not predict drug
behavior in humans. In vitro techniques for drug permeability assessment are less labo-
rious, cheaper, and more in line with the “three Rs” ethical policies (Polli, 2008).
Furthermore, in vitro models that thoroughly simulate conditions in the human mucosa
return absorption data that can help in selecting compounds entering into clinical
development. The impact of these models rests on their potential for rapid, cost-
effective, and adequate predictability of absorption potential in humans. In some cases,
the data are suitable to support regulatory filing of a new drug application.
Cell culture models offer the advantage of a highly defined tool in which the param-
eters and conditions can be easily changed. In addition, the use of human cell lines
avoids the kind of problems that arise when using animal tissue for in vitro experi-
ments (Sarmento et al., 2012).
Nevertheless, current in vitro methods often discard the proper integrity of the
simulated mucosae, are based on the confluence of cell monolayers, and do not take
into account the role of cellecell and celleextracellular stroma interactions on the ab-
sorption mechanisms. In vitro cell-based models have been optimized, becoming more
and more complex and incorporating more than one type of cell in order to more
closely resemble in vivo tissues. Therefore, new and improved tissue-based in vitro
models estimate drug absorption in a rapid, systematic, robust, and high-throughput
way to shorten drug development time.
Ex vivo tissue experiments, on the other hand, may represent an alternative
approach to track the fate of drugs across epithelial tissues, as far as tissue viability
and integrity is maintained. In fact, they would represent the closest to human condition.

Concepts and Models for Drug Permeability Studies. http://dx.doi.org/10.1016/B978-0-08-100094-6.00001-8

Copyright © 2016 Elsevier Ltd. All rights reserved.
2 Concepts and Models for Drug Permeability Studies

Considering all methodologies, the use of in vitro or ex vivo methods inherently

creates questions about the validity of extrapolating to the in vivo situation (Pelko-
nen, Boobis, & Gundert-Remy, 2001). In vitroein vivo correlation must allow the
prediction of the in vivo pharmacokinetics of a drug based on the in vitro drug
permeation profiles.
This book is intended to be an updated compilation of the most important buccal,
gastric, intestinal, pulmonary, nasal, vaginal, ocular, renal, skin, and bloodebrain bar-
rier in vitro models for predicting the permeability of drugs. The strategy of the book
will be based on three different approaches, summarizing the most recent achieve-
ments regarding models comprising immortalized cells with an intrinsic ability to
grow in a monolayer when seeded in permeable supports, primary cells isolated
from living organisms directly cultivating barrier monolayers, or tissue-based models
constructed with cell lines and extracellular matrix, resembling the tridimensional
structure of mucosae and other biological membranes.
Each model is described regarding the protocol of seeding and conservation, as well
as applications for specific drugs, taking into account the maintenance of physiologic
characteristics and functionality of epithelium, from the simplest immortalized cell-
based monoculture to the most complex engineered-tissue models.
Moreover, the equivalence between in vitro cell and tissue models and in vivo con-
ditions is discussed, highlighting how each model may provisionally resemble
different drug absorption routes.

References
Pelkonen, O., Boobis, A. R., & Gundert-Remy, U. (2001). In vitro prediction of gastrointestinal
absorption and bioavailability: an experts’ meeting report. European Journal of Clinical
Pharmacology, 57(9), 621e629.
Polli, J. (2008). In vitro studies are sometimes better than conventional human pharmacokinetic
in vivo studies in assessing bioequivalence of immediate-release solid oral dosage forms.
The AAPS Journal, 10(2), 289e299.
Sarmento, B., Andrade, F., da Silva, S. B., Rodrigues, F., das Neves, J., & Ferreira, D. (2012).
Cell-based in vitro models for predicting drug permeability. Expert Opinion on Drug
Metabolism & Toxicology, 8(5), 607e621.
Importance and applications
of cell- and tissue-based in vitro 2
models for drug permeability
screening in early stages of drug
development


Miguel Angel Cabrera-Pérez1,2, Marival Bermejo Sanz2, Victor Mangas Sanjuan2,
Marta Gonz


alez-Alvarez 2
, Isabel Gonz


alez Alvarez 2
1
Universidad Central Marta Abreu de Las Villas, Santa Clara, Villa Clara, Cuba;
2
Universidad Miguel Hern
andez, San Juan de Alicante, Alicante, Espa~na

2.1 Introduction
Discovery, development, and registration of a new drug is a long, labor-intensive, risky,
and extremely costly process (DiMasi, Hansen, & Grabowski, 2003; Kola & Landis,
2004). One of the main reasons for the high attrition rates in drug development is the
number of new chemical entities with poor pharmacokinetic properties (Kubinyi, 2003).
The development of high-throughput biological screening (van de Waterbeemd,
2002), genomics, and combinatorial chemistry (Seneci & Miertus, 2000) has dramat-
ically increased the number of pharmacologically active molecules with unfavorable
biopharmaceutical properties. The main challenge for pharmaceutical researchers in
drug development is to identify the major pharmacokinetic hurdles of drug candidates
and validate the “developability” of a compound in the early stages to select superior
drug candidates with the best chances of market success (Stoner et al., 2004).
Currently, several in vitro experimental models with high-throughput capacity,
cost-effectiveness, and adequate predictability of absorption potential in humans are
available for evaluating intestinal permeability and transport across different biological
membranes (Sarmento et al., 2012).
Among the most popular in vitro models for assessing permeability/absorption are
those based in artificial membranes, such as parallel artificial membrane permeability
assay (PAMPA); systems based in cells, such as Caco-2, MDCK (Madin Darby canine
kidney), etc.; and systems based on tissues. Although all these methods do not reflect
the effect of physiological factors, their financial and ethical considerations have made
possible their successful use as decision-making tools during early drug development.
In this sense the goal of this chapter is to bring a general theoretical and practical
description of the different in vitro methods used to study drug permeability by
different administration routes.

Concepts and Models for Drug Permeability Studies. http://dx.doi.org/10.1016/B978-0-08-100094-6.00002-X

Copyright © 2016 Elsevier Ltd. All rights reserved.
4 Concepts and Models for Drug Permeability Studies

2.2 General considerations

Permeability data obtained from different experimental models can be different be-
tween laboratories, even though good in vitroein vivo correlations with human data
have been obtained. Among the main factors affecting the permeability values are
the laboratory materials, source of biological material, and nonstandardized intralabor-
atory and interlaboratory assays. In order to develop suitable permeability procedures,
several criteria are required, such as the experimental protocol should be confirmed by
standardized procedures, with good relationship with human data, and where high-
permeability internal standards, markers for tissue integrity and viability, and reference
compounds have to be used (Volpe, 2010).
During the drug development process many cell- and tissue-based in vitro models
have been used to study drug permeability through different administration routes.
Table 2.1 gives a general description of the most relevant experimental procedures
for permeability studies. A better description of each permeability assay will be pro-
vided over the course of this book.
Nevertheless, although in vivo animal permeability studies better reproduce the hu-
man results, the cell- and tissue-based in vitro models provide several advantages: (1) a
lesser amount of drug is needed for the assay; (2) few or no animals are used; (3) more
compounds can be screened; (4) mechanism of transport and metabolism can be stud-
ied; (5) the analytical evaluation is more simple compared with assays in biological
fluids; and (6) these in vitro models offers reproducibility, simplicity, and a reduced
operation cost. Finally, the application of in vitro models in drug permeability studies
represents a useful screening tool for assessing the biopharmaceutical appropriateness
of new chemical entities.

2.3 Drug transport

2.3.1 Transport mechanisms
A drug must interact with its receptors at the action sites or therapeutic targets for
exerting its pharmacological or toxic effects. The essential step, therefore, after drug
administration by any extravascular route is the absorption into the systemic circula-
tion to be distributed to tissues and organs.
The barriers that the drug has to cross before reaching the blood flow are integrated
by semipermeable cell membranes in all the administration routes. Drug transport
across a cellular barrier may involve transcellular (across the cell) or paracellular
(between cells) movement. While paracellular transport is size-restricted passive diffu-
sion, transcellular transport mechanisms include passive diffusion, carrier-mediated
transport (active or facilitated diffusion), and endocytosis.
Passive diffusion involves the movement of drug molecules down a concentra-
tion or electrochemical gradient without the expenditure of energy. This transport is
not saturable, does not involve a carrier, and shows a low structural specificity
Table 2.1 Different cell- and tissue-based in vitro models to predict drug permeability
Kind of
permeability Type of assay Tissue Cell Advantages Drawbacks

Importance and applications of cell- and tissue-based in vitro models

Buccal Diffusion cells • Isolated animal Similar to human tissue; low Limited surface area; tissue
(e.g., Franz) buccal tissue cost of acquisition; no damage by mastication;
compound loss due to laborious and time-
swallowing; resistance to consuming excision
irritation and loss of procedure; lack of blood
integrity (test many types flow; high variability;
of excipients); correlation large number of tissue
with in vivo animal data samples are needed; poor
prior to human evaluation potential for automation
• Hamster pouch Good correspondence to Culture growth rate,
epithelium human buccal mucosa number of differentiated
culture cell layers, lipid
• TR146 cell composition, and the
culture optimum days in culture
• EpiOral are not well
characterized; limited
number of compounds
tested
Intestinal Brush border From animal and human Nonspecific binding;
membrane tissue; useful for predictive only for
vesicles mechanistic studies passive transport
Artificial High-throughput screening Predictive only for passive
membranes capacity; low cost; transport; membrane
(PAMPA) different lipid retention for lipophilic
compositions; good compounds; dependency
predictability on the lipid membrane
composition and pH;
presence of a thicker

5
unstirred water layer
Continued
Table 2.1 Different cell- and tissue-based in vitro models to predict drug permeability—cont'd
Kind of
permeability Type of assay Tissue Cell Advantages
Isolated intestinal Animal or human origin;
cells relatively easy to develop
Cell culture models Caco-2 High screening capacity;
derived from a human
colon adenocarcinoma;
evaluation of transport
mechanisms, absorption
enhancers, and toxicity;
reduction of laboratory
animals; the most
extensively characterized
cell-based model; short
cell culture time; good
potential for automation
and miniaturization
TC7 (Caco-2 Suitable for drug transport
subclone) and biotransformation
studies; higher enzymatic
expression
MDCK (Madin The culture time to
Darby canine confluence is shorter than
kidney) Caco-2 cells; the
6
Drawbacks
High intervariability
during cell preparation;
requires a radiolabeled
or fluorescent permeant
Lack of mucus-secreting
cells; thicker unstirred
water layer and tighter
monolayers compared to
human small intestine;
different expression of
metabolic enzymes; high
inter- and intralaboratory
Concepts and Models for Drug Permeability Studies
permeability data; long
differentiation period;
low expression of uptake
transporters; is not a
dynamic model: the
conditions of culture
influence the
performance of the cell
model
The expression of CYP3A
remains lower than the
level in human
enterocytes
Nonhuman and
nonintestinal origin;
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