PHASCI 2973 No.

of Pages 53, Model 5G

19 March 2014

European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx

1

Contents lists available at ScienceDirect

European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier.com/locate/ejps

6
7

3 In vivo methods for drug absorption – Comparative physiologies, model

4 selection, correlations with in vitro methods (IVIVC), and applications for
5 formulation/API/excipient characterization including food effects
8 Q1 Erik Sjögren a, Bertil Abrahamsson b, Patrick Augustijns c, Dieter Becker d, Michael B. Bolger e,
9 Marcus Brewster f, Joachim Brouwers c, Talia Flanagan g, Matthew Harwood h, Christian Heinen i,
10 Rene Holm j, Hans-Paul Juretschke k, Marlies Kubbinga i,l, Anders Lindahl m, Viera Lukacova e,
11 Uwe Münster n, Sibylle Neuhoff h, Mai Anh Nguyen i, Achiel van Peer f, Christos Reppas o,
12 Amin Rostami Hodjegan p, Christer Tannergren b, Werner Weitschies q, Clive Wilson r,
13 Patricia Zane s, Hans Lennernäs a, Peter Langguth i,⇑
14 a
Department of Pharmacy, Uppsala University, Uppsala, Sweden
15 b
AstraZeneca R&D, Mölndal, Sweden
16 c
Drug Delivery and Disposition, KU Leuven, Leuven, Belgium
17 d
Vivo Drug Delivery GmbH, Wollerau, Switzerland
18 e
Simulations Plus Inc., Lancaster, CA, USA
19 f
Janssen Research and Development, Beerse, Belgium
20 Q2 g
AstraZeneca Alderley Park, UK
21 h
Simcyp Ltd., Sheffield, UK
22 i
Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Mainz, Germany
23 j
H. Lundbeck A/S, Valby, Denmark
24 k
Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany
25 l
National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands
26 m
Medical Products Agency, Uppsala, Sweden
27 n
Bayer Pharma AG, Wuppertal, Germany
28 o
National and Kapodistrian University of Athens, Faculty of Pharmacy, Athens, Greece
29 p
University of Manchester, Manchester, UK
30 q
Center of Drug Absorption and Transport, University of Greifswald, Greifswald, Germany
31 r
University of Strathclyde, Glasgow, UK
32 s
Sanofi US, Bridgewater, NJ, USA

33
34
a r t i c l e i n f o a b s t r a c t
3
4 6
8
37 Article history: This review summarizes the current knowledge on anatomy and physiology of the human gastrointesti- 49
38 Received 22 July 2013 nal tract in comparison with that of common laboratory animals (dog, pig, rat and mouse) with emphasis 50
39 Received in revised form 15 February 2014 on in vivo methods for testing and prediction of oral dosage form performance. A wide range of factors 51
40 Accepted 17 February 2014
and methods are considered in addition, such as imaging methods, perfusion models, models for predict- 52
41 Available online xxxx
ing segmental/regional absorption, in vitro in vivo correlations as well as models to investigate the effects 53

Abbreviations: ABC, ATP-Binding Cassette; An, absorption number; API, active pharmaceutical ingredient; AUC, area under the curve; BCRP, breast cancer resistant protein;
BCS, biopharmaceutic classification system; BDDCS, biopharmaceutics drug disposition drug classification system; BE, bioequivalence; CYP, cytochrome P450; D0, dose
number; Dn, dissolution number; DDI, drug–drug interactions; DSC, differential scanning calorimetry; EMA, European Medicines Agency; fabs, fraction of dose absorbed;
FaSSIF, Fasted state simulated intestinal fluid; FDA, Food and Drug Administration (USA); GI, gastrointestinal; GIT, gastrointestinal tract; GITT, GI transit times; GST,
glutathione-S-transferase; HPbCD, hydroxypropyl-b-cyclodextrin; HPMC, hydroxypropyl methylcellulose; IMMC, interdigestive migrating motor complex; IR, immediate
release; IVIVC, in vitro in vivo correlation; IVIVR, in vivo in vitro relationship; LC–MSMS, Liquid chromatography–tandem mass spectrometry; MAD, maximum absorbable
dose; mDDI, metabolic drug–drug interactions; MR, modified release; MRI, magnetic resonance imaging; MRP2, multidrug-resistance associated protein 2; NMP, N-methyl-2-
pyrrolidon; OrBiTo, oral biopharmaceutic tools; PBPK, physiologically-based pharmacokinetic; PD, pharmacodynamics; Peff, effective permeability; PEG, polyethylene glycol;
P-gp, P-glycoprotein; PK, pharmacokinetics; QbD, quality by design; SITT, small intestinal transit time; SLC, solute carrier superfamily; SLS, sodium lauryl sulfate; SULT,
sulfotransferase; tDDI, transporter drug–drug interactions; TIM, TNO intestinal model; TPGS, D-a-tocopheryl polyethyleneglycol 1000 succinate; UGT, uridine 5’-diphosphate
glucuronosyl transferases; XRD, X-ray diffraction.
⇑ Corresponding author. Address: Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudingerweg 5, 55099 Mainz, Germany. Tel.: +49 6131
392 5749; fax: +49 6131 392 5021.
E-mail address: langguth@uni-mainz.de (P. Langguth).

http://dx.doi.org/10.1016/j.ejps.2014.02.010
0928-0987/Ó 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
 PHASCI 2973 No. of Pages 53, Model 5G
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2 E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx

42 Keywords: of excipients and the role of food on drug absorption. One goal of the authors was to clearly identify the
43
54 Q3 In vivo model selection gaps in today’s knowledge in order to stimulate further work on refining the existing in vivo models and
44 Biopharmaceutic characterization
55 demonstrate their usefulness in drug formulation and product performance testing.
45 Bioavailability
56
46
Ó 2014 Elsevier B.V. All rights reserved.
Drug product development
57
47

59

Trade names used and their chemical names
Acconon EÒ Polyoxypropylene 11 stearylether or Polyoxypropyl-
ene 15 stearylether
Capmul PG8Ò Propyleneglycol monocaprylate
Cremophor RH40Ò (Kolliphor RH40Ò) Macrogolglycerol hy-
droxystearate
Cremophor ELÒ (Kolliphor ELÒ) Polyoxy 35 hydrogenated castor
oil
Eudragit EÒ Butylated methacrylate copolymer
Imwitor 742Ò Caprylic/capric glycerides
LabrasolÒ Caprylocaproyl macrogol-8 glycerides
MethocelÒ Methylcellulose and hydroxypropylcellulose poly-
mers
MiglyolÒ Triglycerides, medium chain
PluronicÒ (Poloxamer) Ethylene oxide, propylene oxide block
copolymer
SoftigenÒ Macrogol 6 caprylic/capric glycerides
SolutolÒ Macrogol 15 hydroxystearate
Tween 20Ò (Polysorbate 20) PEG(20)sorbitan monolaurate
Tween 40Ò (Polysorbate 40) PEG(20)sorbitan monopalmitate
Tween 80Ò (Polysorbate 80) PEG(20)sorbitan monooleate

60
61 1. Introduction mulations of biopharmaceutic classification system (BCS) class I 102
drugs, but are of greater influence for APIs from BCS classes II to 103
62 Optimized and robust in vivo performance of an oral drug prod- IV, since their in vivo performance relies to a greater extent on 104
63 uct is of crucial importance for its successful clinical application. the characteristics of their formulation. Therefore, it is obvious that 105
64 Yet the lengthy drug development process provides little opportu- for targeted, designed pharmaceutical products, the dosage form 106
65 nity for optimization of the therapeutic product in humans. The type, its composition in terms of identity and quantity of pharma- 107
66 subject of this review is a part of the Oral Biopharmaceutical Tools ceutical excipients, the manufacturing process and the quality pro- 108
67 (OrBiTo) project within the Innovative Medicines Initiative (IMI) cess parameters need to be defined and optimized. Quality by 109
68 framework, a pre-competitive collaboration between pharma design (QbD) approaches require the definition of a design space 110
69 industry, academia and specialist technology companies. It aims for the raw material characteristics, the manufacturing process 111
70 to enhance understanding of how orally-administered drugs are and its process conditions to assure predefined bioperformance 112
71 absorbed from the gastrointestinal tract and to apply this of the manufactured product based on an appropriate design space. 113
72 knowledge to develop new in vitro tests and in silico models that Current biopharmaceutical tools in the pharmaceutical devel- 114
73 will better predict the performance of oral formulations in humans. opment rely on in vitro dosage form characterization, whole animal 115
74 This review does not deal with drug absorption from the oral cavity studies in mice, rats, dogs, pigs and/or monkeys and studies in 116
75 and the esophagus although they represent parts of the GI-tract. In healthy human subjects and sometimes in patients. While studies 117
76 this review, in vivo methods for drug absorption, their comparative in human subjects can provide highly relevant information, they 118
77 physiologies, correlations with in vitro methods (IVIVC), and appli- face various complexities including their justification by an Ethics 119
78 cations for formulation/API/excipient characterization including Committee with additional limits in terms of throughput and cost. 120
79 food effects are covered. Attention has been paid to also point Therefore, in vivo studies in animals are sometimes preferred and 121
80 out the gaps in todays knowledge. human studies frequently are done to confirm and translate pre- 122
81 Pharmaceutical product characteristics, e.g. particle size, shape dictions to the human situation created from the other methods. 123
82 and physical form of the active pharmaceutical ingredient (API) in Often animal studies yield species differences and pose the ques- 124
83 its dosage form are influencing its performance in the fasted and tion of which animal species is most representative for humans. 125
84 fed as well as in health and diseased states. Thus, API availability Underlying reasons for these species-specific findings include dif- 126
85 and its pharmacokinetics (PK) and/or –dynamics (PD) are highly ferences in anatomy and physiology of the GI tract as described 127
86 dependent on the APIs presentation to the body by the formulation in detail in the following sections. Not only might the species be 128
87 and the interaction with the changing local physiological condi- less representative, but also the study setup like dosage regimen, 129
88 tions in the GI-tract (GIT). Such local conditions may change as a amount water/meal, chewing, etc. does frequently not reflect the 130
89 result of co-medication. For example, the use of proton pump intended daily practical use of the dosage form in humans. 131
90 inhibitors increases the gastric pH which may affect dissolution Nevertheless, pharmaceutical development relies on these studies, 132
91 of acids and bases. Additional factors such as solubility, absorption, since animals represent intact organisms necessary to simulate the 133
92 metabolism and disposition characteristics of the API come into complex interplay of drug dissolution, permeation and metabolism 134
93 play and may determine the variability of the PK and PD responses. and they are used in legally mandatory toxicology studies and drug 135
94 Biopharmaceutical factors related to the in vivo performance of disease models such as achlorhydria. Furthermore it should be 136
95 the dosage form in the respective preclinical model such as pig, pointed out that the potential knowledge acquired from animal 137
96 dog, rat, mouse and also in humans, i.e. GI-motility, GI-transit, studies should always be measured towards the intrinsic value of 138
97 mechanical stress, effects of food, enzymatic or pH-related degra- the animal. Ethical issues in terms of animal sacrifice, discomfort 139
98 dation of API but also excipients, API release profile and API and pain must always be considered. These aspects were acknowl- 140
99 absorption in various GI segments, and the direct influence of some edged by Russel and Burch in their principle of 3Rs, Replacement, 141
100 excipients on drug metabolism and transport are still insufficiently Reduction and Refinement (Russell and Burch, 1959). The essence 142
101 understood. Most of these factors have little or no impact for for- of the principle to, replace the use of animals with alternative 143

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
 PHASCI 2973 No. of Pages 53, Model 5G
19 March 2014

E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx 3

144 non-animal assays whenever possible, reduce the number of ani-

145 mals by optimized study designs and to refine the methodologies
146 to minimize stress and pain is now internationally endorsed by
147 the scientific community. Strong efforts in many disciplines have
148 been made according to the 3Rs principle in the last decades and
149 are for pharmaceutical investigations covered in the EU directive
150 2010/63/EU on the protection of animals used for scientific
151 purposes.
152 Likewise in vitro methods have been used to predict effects in
153 animals and in humans and, in order to demonstrate their predict-
154 ability, in vitro in vivo correlations (IVIVC) have been recommended
155 and are included in EMA and FDA guidelines. Thus, when robust
156 and accurate predictability can be demonstrated by an in vitro
157 method in conjunction with a validated IVIVC, a bioequivalence
158 (BE) study can be waived on the basis of a dissolution test. But
159 IVIVCs are frequently limited, to this point, to certain modified re-
160 lease (MR) dosage forms and often fail due to unknown reasons,
161 which may either be attributed to non-physiologic (poorly biorel-
162 evant) in vitro test conditions such as volume and composition Fig. 1. Fluid balance in the human gastrointestinal tract. Adapted from P.E. Paulev,
163 and static environment in the compendial dissolution tests that G. Zubieta-Calleja. Textbook in Medical Physiology and Pathophysiology: Essentials
and Clinical Problems, second ed. Copenhagen, 2004 (ISBN 87-984078-0-5).
164 also do not take permeability and/or metabolism into consider-
165 ation or to insufficient systems characterization, in terms of other
166 presystemic factors that influence absorption and systemic avail-
167 ability of the API in vivo. The simulation programs applied today volume and individual physiology (Chial et al., 2002; Geliebter, 205
168 are fairly simplistic and may poorly reflect various physiological 1988; Geliebter and Hashim, 2001). 206
169 aspects important for GI drug absorption and often are not suffi- The actual volume of the gastric content is the sum of meal vol- 207
170 cient accurate (Poulin et al., 2011). ume, fasting gastric volume, cumulative saliva and gastric secre- 208
171 It is the concept of the ‘integrated picture’ reflected by the key tion minus gastric emptying (Goetze et al., 2009; Kwiatek et al., 209
172 biopharmaceutical parameters related to the API, its immediate re- 2009). The total gastric volume has been shown to be larger than 210
173 lease (IR) or MR dosage form and the human and animal systems the consumed meal volume up to 3 h after intake of a light meal 211
174 characteristics that the authors strive to present in this review. (Burton et al., 2005). During digestion, gastric juice is produced 212
175 This will assist in the definition of the current status and identify with a total daily secretion volume in the range of 2–3 L. In the 213
176 the gaps that are the focus of research performed in the OrBiTo fasted state, an unstimulated secretion rate of about 1 mL/min oc- 214
177 project. curs which increases after meal intake to rates of 10 mL/min up to 215
50 mL/min (Versantvoort et al.). Another source of gastric filling 216
volume is saliva with an also stimulation dependent flow rate of 217
178 2. Human GI characterization
up to 10 mL/min and a total daily secretion volume of about 218
1–1.6 L per day (Engelen et al., 2003; Gaviao et al., 2004; Pedersen 219
179 The basic design of the gut is a long muscular tube with special-
et al., 2002). Due to moderate peristaltic mixing, gastric contents 220
180 ized areas for digestion and storage, supplied by arteries and
are not homogenously distributed. Typically, a lipid layer is located 221
181 drained by veins and a lymphatic trunk, all supported in a sheath
on top of the gastric fluid due to the lower density of fat compared 222
182 of connective tissue below the thorax, termed the mesentery.
to that of water. However, subject posture and ingestion order 223
183 Functionally, the gut is divided into a preparative and primary stor-
influence the location of the lipid layer (Chang et al., 1968; Kunz 224
184 age region (mouth and stomach), a secretory and absorptive region
et al., 2005). Solid high density particles accumulate in the more 225
185 (the midgut), a water reclamation system (ascending colon) and fi-
distal parts of the stomach due to their higher gravity, where they 226
186 nally a waste-product storage system (the descending and sigmoid
may be ground by a moderate antral milling activity. Gastric emp- 227
187 colon) as is illustrated in Fig. 1. Based on the luminal environment
tying occurs as a decanting process of the watery phase with small 228
188 and the nature of the tissue change along the GIT, only the small
suspended particles and emulsion droplets (Keinke et al., 1984). 229
189 intestine is structured to allow for maximal absorption. Important
The flow properties of gastric contents range from Newtonian 230
190 factors for GI drug absorption include the free luminal
flow for pure water towards non-Newtonian, pseudoplastic flow 231
191 concentration of API, the effective area, interaction with luminal
behavior showing shear thinning, i.e. lower viscosity with shear 232
192 particles and transit time. A summary of key anatomical and phys-
forces acting on the contents in the presence of solid particles 233
193 iological parameters of the human GIT is given in Table 1.
(Dikeman et al., 2006; Marciani et al., 2000; Mudie et al., 2010; 234
Takahashi and Sakata, 2002, 2004). Estimated values for the viscos- 235
194 2.1. Stomach physiology ity of the gastric contents are in the range of 10–2000 mPa s (Abra- 236
hamsson et al., 2005). 237
195 Stomach physiology and its impact on drug dissolution have Stomach motility is characterized by two different gastric mo- 238
196 recently been reviewed (Koziolek et al., 2013a,b). The stomach is tor patterns that originate from pacesetter cells located at the 239
197 divided into three functional parts. The fundus region acts together greater curvature of the corpus. In the fasted state, the interdiges- 240
198 with the middle part of the stomach (corpus) as a storage compart- tive migrating motor complex (IMMC) occurs that enables the 241
199 ment. In the distal part (antrum), food particles are milled, sieved emptying of non-digestible objects from the gastric lumen during 242
200 and finally emptied through the pylorus. The size of the stomach phases of high intensity with maximum pressures in the pyloric re- 243
201 depends largely on the filling status. Under fasting conditions, gion of up to 300 mbar (Cassilly et al., 2008; Khosla and Davis, 244
202 the stomach is mostly empty containing only approx. 10–50 mL 1990). One IMMC front moves from the proximal stomach to the 245
203 of gastric juice as well as some gas. After meal intake, stomach fill- ileum every 1–2 h (Sarna, 1985; Vantrappen et al., 1977). The 246
204 ing volume may increase to 1 L or more, depending on the ingested IMMC is interrupted by meal ingestion as the digestive motor 247

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
 Table 1
dx.doi.org/10.1016/j.ejps.2014.02.010
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
Comparison of the gastrointestinal tract of humans and animals with respect to anatomy and physiological parameters of relevance for drug absorption studies.
Parameter Human
pH fasted Stomach 1–3.5 (Dressman et al., 1990; Hila
et al., 2006; Simonian et al., 2005)
SI 6.0–7.0 (Duodenum) 6.0–7.7
(Jejunum) 6.5–8.0 (Ileum) (Brener
et al., 1983; Ferraris et al., 1983;
Lennernäs, 2007c) (46–48)
Li 5.5–6.5 (Cecum) 5.5–7.5 (Colon asc.)
7.0–8.0 (Colon desc)
pH fed Stomach 3.0–6.0 (Kararli, 1995; Simonian
et al., 2005)
SI 5.0–5.5 (Duodenum) 5.0–6.5
(Jejunum) Similar to fasted (Ileum)
(Brener et al., 1983; Davies and
Morris, 1993; Persson et al.,
2005)>4 h after meal: fasted pH
LI n.a.
Transit time fasted Stomach 10–15 min (t1/2) for liquids 0–2 h for
indigestible solids (Brener et al.,
1983; Davis et al., 1986)
SI 3–4 (Davis et al., 1986)
Li 8.0–18.0 (Davis et al., 1986)
Transit time fed Stomach Liquid: Rapid but slower than the
same liquid in fasted state (Brener
et al., 1983) Digestible solids: Very
rapidly for particles <2 mm (99%
emptying in 0.5–3 h) (Davis et al.,
1986) Rapidly for size <7–10 mm;
larger particles held for many h
(DeSesso and Jacobson, 2001)
SI 3–4 (Davis et al., 1986)
Li n.a.
Length SI 7 m (post mortem) (Dressman, 1986; 2.5–4.1 m (de Zwart, 1999)
Ferraris et al., 1983) 3.0–5.0 (in vivo)
(Hofmann et al., 1983)
Length Li 1.5 (DeSesso and Jacobson, 2001)
Canine
1.5–6.8 (values from various studies) 1.2–4.0 (Hossain et al., 1990) LR
(Arndt et al., 2013) 0.3–1.7 (Oberle and Das, 1994) MP
6.1–7.6 (de Zwart, 1999; Kalantzi
et al., 2006; Sutton, 2004)
6.5–unspecified dosing conditions
(de Zwart, 1999)
Up to neutral – depends heavily on
the pH of the ingested food, due to
minimal gastric acid output
6.1 (Kalantzi et al., 2006) 5.5–7.2
(Duodenum/Ileum) (Sutton, 2004)
6.5–unspecified dosing conditions) 6.1–6.6 (Merchant et al., 2011) LR
(de Zwart, 1999)
Solution: 2–76 min Solid 7  20 mm: 1–28 days* (Hossain et al., 1990) LR
1.2 h (t1/2) (Reppas et al., 1991;
Sutton, 2004)
60–111 min (Sutton, 2004) <1–3 days* (Hossain et al., 1990) LR
Shorter than humans based on
absorption data (Sutton et al., 2006)
unspecified dosing conditions) and
length (Kararli, 1995)
Time to phase III activity: 5.4–13.3 h
(Sutton, 2004)
Jejunal: 150–180 min (Sutton, 2004)
Shorter than humans based on
absorption data (Sutton et al., 2006)
unspecified dosing conditions) and
length (Kararli, 1995)
Cecum: 8 cm Colon: 34–60 cm (de
Zwart, 1999)
4
19 March 2014
PHASCI 2973
Pig Rat Mouse
Landrace (LR)
Minipig (MP)
4–5 (glandular region); 7 (anterior 4.0 (McConnell et al., 2008a)
region) (Davies and Morris, 1993;
Kararli, 1995)
7–8 (Oberle and Das, 1994) MP 4.5–7.5 (Davis and Wilding, 2001; 5.0 (McConnell et al., 2008a)
Lennernäs and Regårdh, 1993)
n.a. n.a. 4.7 (McConnell et al., 2008a)
4.4 (Merchant et al., 2011) LR 3.6 3.8–5.0 (Davies and Morris, 1993) 3.0 (McConnell et al., 2008a)
(Oberle and Das, 1994) MP
E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx
Duo: 4.7–6.1 Jej: 6.0–6.5 Ile:6.3–7.2 6.5–7.1 (Davies and Morris, 1993) 4.8 (McConnell et al., 2008a)
(Braude et al., 1976; Merchant
et al., 2011) LR
6.6–6.9 (Davies and Morris, 1993) 4.5 (McConnell et al., 2008a)
15–30 min (t1/2) (Langguth et al., n.a.
1994) 5–65 min (t1/2) (Maerz et al.,
1994)
3–4 h (Davis and Wilding, 2001; n.a.
Lennernäs and Regårdh, 1993)
<1–3 days* (Hossain et al., 1990) LR 10–11 h based on a total GI transit n.a.
time of 15 h (DeSesso and Jacobson,
2001)
Solution/pellets 1.4–2.2 tablet 1.5– n.a. 1 h (Padmanabhan et al., 2013)
6.0 (Davis et al., 2001; Wilfart et al.,
2007) LR
3–4 h (Davis et al., 2001; Wilfart n.a. 1–2 h (Hamada et al., 1999;
et al., 2007) LR Padmanabhan et al., 2013)
24–48 h (Davis et al., 2001; Wilfart n.a. 3 h (Padmanabhan et al., 2013)
et al., 2007) LR
No. of Pages 53, Model 5G
840–900 cm (34–63 cm/kg) 102–148 cm (Kararli, 1995) 40.2 cm (Ogiolda et al., 1998)
(Kurihara-Bergstrom et al., 1986;
Suenderhauf and Parrott, 2013) MP
470–2000 cm (17–19 cm/kg)
(Bergman et al., 2009; Merchant
et al., 2011) LR
323 cm (11 cm/kg) (Glodek and 26–26 cm (Kararli, 1995) 8.3 cm (Ogiolda et al., 1998)
Oldigs, 1981; McRorie et al., 1998)
MP 436 cm (4.3 cm/kg) (Merchant
et al., 2011) LR
 Table 1 (continued)
dx.doi.org/10.1016/j.ejps.2014.02.010
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with

19 March 2014
PHASCI 2973
Parameter Human Canine Pig Rat Mouse
Landrace (LR)
Minipig (MP)
Bile concentration SI 2.0–10 mM (fasted) (Hofmann et al., Fasted state: Approx. 10 mM (values 42–55 mM (Juste et al., 1983) LR 33.5–61.3 mM (fasted) (Staggers n.a.
1983; Persson et al., 2005) 8.0 (fed) from various studies, (Arndt et al., et al., 1982) 17–18 mM (fasted)
(Persson et al., 2005) 10–20 mM 2013) 2.4–9.4 mM (Kalantzi et al., (Kararli, 1995)
(after meal) (Hofmann et al., 1983) 2006) Fed state: 12.8–18.0 mM Compared to man higher BS/PL ratio
(Kalantzi et al., 2006) Most abundant but PL concentration similar to man
is taurocholic acid (Holm et al.,
2013b)
Metabolic activities Phase I CYP3A4, 2C9, 2C19, 2D6, 2J2 (see also Different than in humans (Haller See Table 3 CYP related activities (Takemoto CYP1a1, 1b1, 2b10, 2b19, 2b20,
Table 2) et al., 2012) et al., 2003) 2c29, 2c38, 2c40, 2e1, 3a11,
In general not correlated to humans 3a13, 3a16, 3a25, 3a44 (Komura
and Iwaki, 2008; Zhang et al.,
2003)

E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx

Phase II UGT, SULT, GST Different than in humans (Haller
et al., 2012)
Major drug P-gp, MRP2, BCRP, PepT1, OATP Peptide transporter-1 (PEPT1,
transporters SLC15A1, organic cation transporter-
1 (OCT1, SLC22A1), BCRP), and
multidrug resistance-associated
protein 1 (MRP1, ABCC1) resemble
the human tissue distribution (Haller
et al., 2012)
Permeabilities Reference Higher than human for low
permeability drugs (e.g. (Fotaki et al.,
2005))
Water volumes Stomach <50 mL (fasted) Up to 1 L (fed) (Chial Similar to humans especially for dogs
et al., 2002; Geliebter, 1988; >20 kg (Martinez et al., 2002)
Geliebter and Hashim, 2001)
SI Water pockets (Schiller et al., 2005) No specific data–water flux in fasted
upper GI is similar with humans
(Reppas et al., 1991)
Li Negligible (Schiller et al., 2005) n.a.
UGT, SULT, GST
P-gp, BCRP, MRP2, OATP
Less than in humans
Wetmass: 250 g (Merchant et al.,
2011) LR
Wetmass: 500 g (Merchant et al.,
2011) LR
wetmass: 750 g (Merchant et al.,
2011) LR
b-Glucuronidase, sulfate conjugation, UGT (Komura and Iwaki, 2011)
glucuronidation, N-acetylation
In general not correlated to humans
Similar transporter expression
patterns as in humans (Cao et al.,
2006)
Less than in humans, good
correlation
2.4 mL (Takashima et al., 2013)
3.0–4.6 mL (Takashima et al., 2013)
n.a.
P-gp (Holmstock et al., 2013)
Similar to human (Escribano
et al., 2012)
0.37–0.71 mL (McConnell et al.,
2008a)
0.81 mL (McConnell et al.,
2008a)
0.6 mL (McConnell et al., 2008a)

n.a.: not available.

*
Measured using nondisintegrating formulations.

No. of Pages 53, Model 5G

5
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6 E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx

248 activity is initiated. The intensity of the gastric pressure waves is 1.2, it is generally accepted that bicarbonate secretion is important, 310
249 typically lower in the fed state than during phases of high intensity raising the pH in the sub-mucin layer. The contribution of 311
250 whilst fasting (Ouyang et al., 1989). occasional duodenal-gastric reflux must also not be neglected as 312
251 Little in vivo data on gastric flow is available due to the exper- it produces transient neutralisation as illustrated in Fig. 2. Even 313
252 imental difficulties for these measurements. However, Boulby within a subject, it can also be appreciated that a wide range of 314
253 et al. observed peak velocities of 2–8 cm/s (Boulby et al., 1999). gastric pH with time occurs. The data also reflect the observation 315
254 Computer simulations based on computational fluid dynamics that in the stomach, the pH in the fundus will typically be one 316
255 have also been performed but reported a rather broad range of esti- pH unit higher than in the pyloric antrum. In the fed stomach, 317
256 mated values (Ferrua et al., 2011; Pal et al., 2004). Under postpran- the sampling device can find itself in pockets of acid or in the food 318
257 dial conditions liquids may probably also be cleared within a few mass (Vo et al., 2005). Twenty-four h radio-telemetry data show 319
258 min from the stomach due to a mechanism called ‘‘Magenstrasse’’ the daily excursions in pH, with very short periods at pH 1, tran- 320
259 (Pal et al., 2007). sient rises to up to pH 5 and slow recovery to baseline as illustrated 321
260 As for hydrodynamics, data on intragastric mechanical condi- in Fig. 3. The daily intake of food causes rises in pH, with fatty 322
261 tions are highly variable. It seems, however, that the antral grind- meals causing a sustained rise in proximal gut pH, which may be 323
262 ing forces represent the highest shear forces acting on solids in the important if a heavy meal is taken at night. This occurs rarely in 324
263 fed stomach with grinding force values in the range of 0.2–1.89 N clinical trials but may be fairly common in the western world in 325
264 (Kamba et al., 2000; Marciani et al., 2001a). the general population. Treatment with proton pump inhibitors re- 326
265 During digestion only liquids and small suspended particles are duces the number of pockets of acid (detected by pull through) and 327
266 delivered to the small intestine whilst larger particles are retained increases their pH (Vo et al., 2005). Increased gastric pH due to 328
267 by gastric sieving (Meyer et al., 1981). Due to the diversity of the therapy with proton pump inhibitors or H2 receptor blockers 329
268 relevant food parameters it is not possible to define a clear cut- may be of concern for enteric coated dosage forms and is also dis- 330
269 off size (Khosla and Davis, 1990; Newton, 2010; Siegel et al., 1988). cussed as a possibly quite often overseen source for a reduced bio- 331
270 Liquids are emptied according to first-order kinetics with emp- availability of drugs with a strongly pH dependent solubility 332
271 tying rates that are influenced by both caloric content and meal profile. For example, this has been recently reviewed for a number 333
272 composition. Ranges are reported from 2 to 4 mL/min, however, of oral anticancer drugs (Budha et al., 2012). Besides the changes in 334
273 initial emptying rates may reach values of up to 10 to 40 mL/min gastric pH, proton pump inhibitors also reduce stomach secretions 335
274 (Faas et al., 2001; Indireshkumar et al., 2000; Kong and Singh, (Babaei et al., 2009; Nishina et al., 1996). This anti-secretory effect 336
275 2008; Kwiatek et al., 2009). Comparable high emptying rates are may also contribute to a reduced bioavailability of poorly soluble 337
276 also observed after ingestion of water (non-caloric liquids) under compounds. 338
277 fasting conditions (Oberle et al., 1990). Solid particles are emptied
278 according to a biphasic pattern. 2.4. GIT transit and motility 339

279 2.2. Intestinal surface area In normal physiology, a balance exists between propulsive, 340
peristaltic movements and mixing contractions. This is controlled 341
280 The surface area of the gut is commonly regarded as a long mus- by signalling between external nerves, especially the vagus, by 342
281 cular tube, which is increased by foldings, and by small intestinal intestinal short-range pathways and through the plexii. Local re- 343
282 villi and microvilli. Based on static morphology, several workers sponses also occur and may cause spasm. 344
283 have calculated the apparent mucosal surface area of the small The fasting and fed patterns of GI motility are distinct and have 345
284 intestine after removal, fixation and staining to be approximately been examined extensively in vivo (Szurszewski, 1969). In the fed 346
285 2.2 m2 (Wilson, 1967). These histological measurements could mode, contractions travel down the wall of the stomach, originat- 347
286 not take account of the microvilli and their presence complicates ing below the fundus and forming an annular ring, the pyloric 348
287 the estimation of surface area since on scanning electron micros- cylinder. Towards the pylorus, the walls collapse, squeezing the 349
288 copy they are seen to be a tightly packed array. It appears that contents through a partially closed sphincter and causing retropul- 350
289 for nutrition, there is an excess capability and only the top of the sion of larger debris back into the body of the stomach. The mech- 351
290 villus may be utilized. In addition, the simple static concept is mis- anism sieves the contents, retaining larger objects for further 352
291 leading. The foldings change dynamically with transit of food and grinding and is a major determinant of the gastric emptying and 353
292 the microvilli break off to form mixed micellar phases near the onset of drug absorption for any pharmaceutical solid dosage form. 354
293 apical boundary. The effective surface area is highly dynamic and Gastric sieving is also the cause for the retention of large 355
294 is affected by nutritional status, exposure to noxious agents and non-disintegrating dosage forms in the stomach which lasts until 356
295 by the luminal viscosity. complete emptying of caloric contents and the appearance of the 357
IMMC. A clear cut-off dimension for the particle size which may 358
296 2.3. The pH of the GIT allow emptying from the stomach during digestion cannot be given 359
(Newton, 2010). A particle size below 2 mm is usually considered 360
297 In the fasted state the gastric pH value of healthy adults is re- as small enough for emptying during fed state motility. Tablets 361
298 ported to be within pH 1 to pH 3 (Dressman et al., 1990; Hila with a size of 3 mm have clearly been demonstrated to be retained 362
299 et al., 2006; Simonian et al., 2005). In elderly patients and also as with food (Podczeck et al., 2007). Disintegrating objects, near the 363
300 a function of ethnic difference, various degrees of achlorhydria sphincter are emptied as a series of pulses. This wave of contrac- 364
301 have been reported (Charman et al., 1997). After meal intake, the tion travels from the stomach to the terminal ileum and then 365
302 pH of the gastric content is increased to various degrees. For in- vanishes. 366
303 stance, after intake of the FDA standard breakfast, the maximum In the fasted state IMMC consists of four phases that are charac- 367
304 stomach pH was reached within the first 5 min and pH decreased terized by distinct intensity and duration. The IMMC front moves 368
305 to values below pH 3 not before 56 ± 42 min. Due to regional differ- from the proximal stomach to the ileum and restarts afterward. 369
306 ences in the presence of acid secreting glands, pH gradients in the These cycles repeat until food is ingested and a complete cycle lasts 370
307 stomach contents have been observed (Simonian et al., 2005). for approximately 1–2 h. Most of all the powerful contractions that 371
308 Although compendial estimates of the pH of the stomach simu- are characteristic for phase III activity (the so-called housekeeping 372
309 lated by simple media have varied over the years from pH 1 to pH waves) serve as a general cleansing allowing the emptying of large 373

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
 PHASCI 2973 No. of Pages 53, Model 5G
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E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx 7

Fig. 2. Distal and proximal 24 h pH-monitoring in human.

pressure events can result in an altered in vivo drug release profile 388
or even possibly affect release mechanisms from MR dosage forms. 389
The small intestinal transit time (SITT) is in the range of 3–4 h and 390
appears not to be dependent upon the dosage form (Davis et al., 391
1986). However, this value is probably a reflection of the typical 392
feeding regimen used in clinical trials as small intestinal transit 393
is triggered by food intake via a mechanism known as gastro-ileo- 394
caecal reflex (Fadda et al., 2009; Schiller et al., 2005). Movement of 395
dosage forms through the small intestine is characterized by typi- 396
cally short episodes of transport where peak velocities of up to 397
50 cm/s may be reached (jet propulsion) and phases of rest (Weits- 398
chies et al., 2005). Typically, dosage forms spend most of the total 399
transit time at rest in the small intestine, typically in the terminal 400
ileum (McConnell et al., 2008b). Under fasting conditions, dosage 401
forms are not necessarily in permanent contact with intestinal 402
water (Schiller et al., 2005). 403
Interestingly, small particles move faster than large particles in 404
Fig. 3. pH-profile recorded by a telemetric IntelliSite capsule in a healthy volunteer colon and due to the extremely variable composition and viscosity 405
during GI transit. Capsule ingestion occurred under fasting conditions. (Data kindly
provided by Medimetrics.)
of colonic contents, hydrodynamic properties are extremely diffi- 406
cult to predict (Abrahamsson et al., 1996; Arkwright et al., 2013; 407
Follonier and Doelker, 1992; Wilson, 2010). 408

374 non-digestible solids like enteric coated tablets or monolithic 2.5. Presence of GI fluids 409
375 extended release tablets from the stomach (Cassilly et al., 2008;
376 Khosla et al., 1989). The stomach is emptied for small particles un- Body fluid balance is primarily regulated by renal mechanisms, 410
377 der fasted state with emptying times from approx. 15 min to more by which the body conserves electrolytes and achieves acid–base 411
378 than 3 h and can be grouped into immediate and rapid, delayed but balance. GI fluids are produced by saliva, gastric and intestinal 412
379 rapid, delayed and slow, and interruptive emptying (Locatelli et al., secretions, pancreatic secretions and ingested fluids. The small 413
380 2009). intestine is a very efficient absorber of water, which has a high 414
381 As discussed in several reviews, the GI luminal conditions effective permeability (Peff) in the jejunum in vivo, approximately 415
382 change as a function of the specific GI site (Varum et al., 2010; 2  104 cm (Fagerholm et al., 1995). The half-time of gastric emp- 416
383 Weitschies et al., 2010; Wilson, 2010). The intraluminal pressure tying of water is approximately 10–15 min or less. After emptying 417
384 following the passage through the pyloric sphincter and ileocaecal into the small intestine, water is quickly taken up into the systemic 418
385 valve may reach values of up to 300 mbar (Cassilly et al., 2008; circulation. In magnetic resonance imaging (MRI) images, residual 419
386 Dinning et al., 1999). Furthermore, similar high pressure ampli- water can only be seen in a few pockets along the small intestines 420
387 tudes are also recorded in the colon (Rogers et al., 1989). Such high (Schiller et al., 2005). Free water is rarely seen in the colon (Schiller 421

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
 PHASCI 2973 No. of Pages 53, Model 5G
19 March 2014

8 E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx

422 et al., 2005). Also in relation to postprandial response, bile, bicar- confined to measurements undertaken in Beagle dog intestinal 469
423 bonate, electrolyte composition, etc. play an important role in dos- regions (Heikkinen et al., 2012). Therefore, there are opportunities 470
424 age form disintegration (coating), dissolution, solubilization and to expand on this technique to enhance our understanding of abso- 471
425 absorption of the API. lute protein abundance of numerous CYP isoforms in man. 472
The intestinal expression of intestinal phase II enzymes; uridine 473

426 2.6. Abundance of drug metabolizing enzymes 50 -diphosphate glucuronosyl transferases (UGT), sulfotransferase 474
(SULT) and glutathione-S-transferase (GST) also deserve consider- 475

427 It has been proposed that the intestinal mucosa is the most sig- ation (Coles et al., 2002; Ritter, 2007; Trdan Lusin et al., 2011). 476

428 nificant extra-hepatic site for drug metabolism (Lin and Lu, 2001). Notably, there are fewer studies characterizing the expression 477

429 The intestine possesses the potential to considerably impact drug and activity of phase II enzymes in the intestine as compared to 478

430 bioavailability (Yang et al., 2007). In order to predict first-pass ef- CYPs. The review by Ritter (2007) highlights the interest towards 479

431 fects in the intestinal wall, it is necessary to characterize the re- studies utilizing mRNA-gene expression of UGT’s in numerous 480

432 gion-specific expression of enzymes along the GIT. This is intestinal segments (Ritter, 2007). Ritter asserts that the lack of 481

433 particularly relevant to the development of mechanistic physiolog- specific antibodies, or specific probe substrates for individual 482

434 ical-based intestinal models that are capable of assessing the UGT isoforms, in addition to the difficulty in purifying/synthesizing 483

435 contribution of these enzymes to intestinal metabolism, portal vein these full length recombinant membrane anchored proteins (Milne 484

436 availability and intestinal-based DDI (Jamei et al., 2009; Pang and et al., 2011), hampers the characterization of quantitative protein 485

437 Chow, 2012). Given that the variability in metabolism can be con- activity relationships in tissues. Absolute protein abundance quan- 486

438 siderable between individuals (Lampen et al., 1996, 1995), identi- titation of intestinal UGT’s by LC–MSMS proteomic techniques 487

439 fying the basis of these differences, i.e., the variability in have been used to quantify numerous UGT isoforms in intestinal 488

440 abundance and or activity of the enzymes responsible for the microsome samples (Harbourt et al., 2012; Smith et al., 2011). 489

441 metabolism of a drug may be crucial (Gertz et al., 2011; Proctor However, the levels of these enzymes along the length of the intes- 490

442 et al., 2004; von Richter et al., 2004). tine are yet to be determined. 491

443 The enzymes most extensively expressed in the small intestine The quantification of absolute levels of SULT enzymes in the 492

444 are the cytochrome P450’s (CYPs) (Paine et al., 2006; Thelen and intestine is limited to a single documented study (Riches et al., 493

445 Dressman, 2009). Studies of the human intestine have shown that 2009). Determination of SULT expression in cytosolic fractions 494

446 the CYP isoform with the highest specific content was CYP3A4. It from duodenal samples (n = 6) was undertaken by quantitative 495

447 has been reported that average CYP3A4 abundances (65–70 immunoblotting and identified 3 out of 5 SULT isoforms under 496

448 nmol/total gut) incorporated into minimal intestinal models, such study (SULT1A3, 1B1 and 1E1) and found that their abundance in 497

449 as the Qgut model, were appropriate (Yang et al., 2007). However, the duodenum was greater than that for liver, kidney and lung 498

450 the differential expression of CYP3A along the length of the small cytosolic fractions. Further data for the expression of SULT’s in 499

451 intestine (Table 2) in a fully mechanistic compartmental model is other intestinal regions is yet to be established. 500

452 crucial for a development of any MR dosage form (Darwich et al., Additional enzymes such as epoxide hydrolase and aldehyde 501

453 2010; Jamei et al., 2009). oxidases have been shown to be expressed in the small intestine 502

454 There are conflicting views of the expression and functional rel- (de Waziers et al., 1990; Moriwaki et al., 2001). Characterization 503

455 evance of the metabolic contribution of CYPs in the colon (Cana- of their abundance and activity in human intestinal cytosolic or 504

456 paro et al., 2007; Lennernäs, 2007b). Canaparo et al. suggested S9 fractions will be required to facilitate the development of strat- 505

457 that CYP3A isoforms are expressed in the colon. But an absence egies in order to predict the impact of the activity of these enzymes 506

458 of CYP3A4 expression in certain samples has been demonstrated on bioavailability. 507

459 within this study, highlighting the potential variability for CYP3A4 Determining the transient levels of an enzyme is critical for the 508

460 expression in the colon between individuals. accurate prediction of DDI’s involving mechanism (time)-based 509

461 In addition to the CYP3A family, the CYP2C, 2D and 2J family of inhibition and induction. Enzyme levels are governed by the bal- 510

462 enzymes are also expressed in the gut mucosa (Paine et al., 2006; ance of the processes of de novo protein synthesis and degradation 511

463 Zhang et al., 1999). The relative contributions to the overall intes- and thus, when these processes are in equilibrium, enzyme levels 512

464 tinal CYP content of (CYP3A4, 2C9, 2C19, 2D6, 2J2) were measured are at steady-state (Yang et al., 2008). This balance may be 513

465 in 31 small intestinal donors (Paine et al., 2006), with details of disturbed, for example, when the rate of enzyme synthesis with 514

466 region-specific expression provided in (Table 2). Reports utilizing a concomitant absence of change or a decrease in degradation rate, 515

467 liquid chromatography–tandem mass spectrometry (LC–MSMS) leads to a rise in enzyme levels (‘induction effect’). There are a host 516

468 proteomic techniques to determine CYP abundance, are at present of in vitro and in vivo methods to determine enzyme turnover in 517

Table 2
Segmental distribution of intestinal CYP enzymes in human.

Enzyme Duodenum Jejunum I Jejunum II Ileum I Ileum II Ileum III Ileum IV Total intestinal abundance (nmol)
Segmental abundance (nmol)
CYP2C9 1.78 3.51 3.51 1.02 1.02 1.02 1.02 12.9
CYP2C19 0.21 0.41 0.41 0.12 0.12 0.12 0.12 1.5
CYP2D6 0.11 0.22 0.22 0.06 0.06 0.06 0.06 0.8
CYP2J2 0.19 0.38 0.38 0.11 0.11 0.11 0.11 1.4
CYP3A4 9.11 18.03 18.03 5.26 5.26 5.26 5.26 66.2
CYP3A5 3.38 6.7 6.7 1.95 1.95 1.95 1.95 24.6

Intestinal CYP abundances are from Paine et al. (2006) and Paine et al. (1997) (Paine et al., 2006, 1997). In Paine et al. (2006), CYP abundances were relative expressions,
therefore the abundances of CYP2C9, 2C19, 2D6, and 2J2 were calibrated against CYP3A total intestinal abundances.
The expression of the active CYP3A5 enzyme is phenotypic and dependent on an individual carrying at least one CYP3A5⁄1 allele. Within a Caucasian population, this occurs at
a frequency of approximately 10–20% (Wrighton et al., 1989). There is evidence that there is also a correlation between the abundance of CYP3A4 and CYP3A5 in liver,
however, a reciprocal relationship has yet to be verified in gut samples (Barter et al., 2010).

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
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E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx 9

518 hepatic systems (Yang et al., 2008). For the intestine, the situation saturation, giving rise to the non-proportional increase in exposure 582
519 is different because enterocyte stem cells arising from the crypt by increasing drug transfer across the enterocyte apical membrane. 583
520 base migrate towards the villus tip, maturing and differentiating In addition to ABC transporters, transporter proteins belonging to 584
521 into fully functional enterocytes as they migrate. This migratory the solute carrier superfamily (SLC) operate to facilitate transport 585
522 process can take between 1 and 10 days and is likely to be more ra- across the apical and basal enterocyte membranes by binding 586
523 pid than enzyme turnover. Thus, turnover is likely to be governed and co-transporting counter ions by symport or antiport mecha- 587
524 by the enterocytes sloughing into the intestinal lumen rather than nisms (Grandvuinet et al., 2012; Koepsell et al., 2007). The impact 588
525 the intrinsic turnover of the enzyme. Enterocyte CYP turnover of SLC transporters on drug absorption is dependent on their mem- 589
526 information can be estimated indirectly in studies administering brane location and the direction into which they operate, i.e. where 590
527 grapefruit juice. Oral ingestion of grapefruit juice leads to a selec- they transfer the substrate across the membrane in which they re- 591
528 tive and irreversible inhibition of intestinal enzymes without side. For example, pro-drug strategies have utilized transporter 592
529 affecting the hepatic enzymes (Schmiedlin-Ren et al., 1997; Won function to enhance drug absorption by targeting the SLC intestinal 593
530 et al., 2012). Studies using this design with subsequent oral dosing uptake transporter (oligopeptide transporter PepT1). This was 594
531 of a probe substrate such as midazolam have been utilized to esti- shown to augment drug absorption and increase bioavailability 595
532 mate a CYP3A enterocyte turnover half-life of 23 h (Greenblatt (Varma et al., 2010; Weller et al., 1993; Steffansen et al., 2005). 596
533 et al., 2003). Intestinal transporter–substrate interactions can be complex with 597
534 There is significant scope to expand our current knowledge of flux mediated by transporters on both membrane poles bi-direc- 598
535 Phase I and II enzyme abundances in the gut. Further data should tionally as demonstrated for estrone-3-sulfate (Rolsted et al., 599
536 be obtained on the region-specific expression of Phase I and II en- 2011). 600
537 zymes, and the development of quantitative immunoblot and LC– For many years there was a reliance on determining transporter 601
538 MSMS proteomic techniques. This will enhance knowledge within protein expression by semi-quantitative immunoblotting or quan- 602
539 this field, particularly where isoform specific antibodies or full titative mRNA-gene expression approaches. These studies have 603
540 length protein standards are not available. As more studies with employed blot densitometry techniques to evaluate relative 604
541 increasing sample numbers become available analyses will focus expression differences between samples after calibration against 605
542 on evaluating the inter-individual variability in abundance, to- a reference ‘housekeeper’ protein. Meta-analyses evaluating the 606
543 gether with defining abundance–activity relationships to provide regional heterogeneity of intestinal transporter expression from 607
544 more mechanistic approaches for in vitro–in vivo extrapolation multiple literature sources, using immunoblot densitometry and 608
545 (IVIVE). mRNA-based expression, have been undertaken to incorporate 609
expression-based transporter functionality into mechanistic intes- 610
546 2.7. Abundance of membrane drug transporter tinal models (Badhan et al., 2009; Bolger et al., 2009; Darwich et al., 611
2010; Harwood et al., 2013). It is common when using relative 612
547 Passive membrane diffusion processes, where molecules pass expression techniques to normalize the expression of transporters 613
548 across membranes driven by concentration gradients play a along the intestine to a single reference compartment, i.e. the prox- 614
549 significant role in the absorption of many drugs (Lennernäs, imal ileal or jejunal segments (Bolger et al., 2009; Darwich et al., 615
550 2007b; Sugano et al., 2010). Yet, there is considerable evidence that 2010; Harwood et al., 2013). It appears there is no consensus as 616
551 many drugs can interact with transporter proteins expressed in to the relationship of the regional-specific P-gp expression imple- 617
552 enterocyte membranes along the intestine to either facilitate or mented across these models, which is likely to result from the data 618
553 reduce absorption rate (Varma et al., 2010). There is therefore a and statistical methodologies incorporated into the meta-analyses. 619
554 necessity to accurately gauge the intestinal region-specific expres- In addition, the usage of mRNA-gene expression assumes a direct 620
555 sion of these transporters in order to predict the potential impact correlation to protein activity, which may not be the case for cer- 621
556 of these transporters on the GI drug absorption and dosage form tain transporters (Berggren et al., 2007). 622
557 development (Giacomini et al., 2010). The reliance on relative expression techniques to the quantita- 623
558 Epithelial cells including enterocytes are polarized and contain tion of transporter expression owes much to the challenges in puri- 624
559 two functionally distinct membrane domains at either pole of the fying full length recombinant integral membrane proteins, and the 625
560 cell. The apical (luminal) membrane contains specific constituents lack of development of antibodies specific to transporter isoforms 626
561 that are structurally and functionally distinct to those at the basal to act as standards for quantitative immunoblotting and enzyme 627
562 (serosal-blood) side at the opposite pole of the cell. This includes linked immunosorbent (ELISA) assays (Ohtsuki et al., 2011). Over 628
563 the differential expression of transporter proteins in each mem- the last several years, development of targeted proteomic 629
564 brane. The most thoroughly characterized transporter proteins techniques has enabled the absolute quantification of integral 630
565 are those that are members of the ATP-Binding Cassette (ABC) membrane proteins such as transporters, using ‘heavy’ labeled iso- 631
566 superfamily. Transporters such as P-glycoprotein (P-gp, MDR1, topes as internal standards (Kamiie et al., 2008; Li et al., 2008). Two 632
567 ABCB1), multidrug-resistance associated protein (MRP2, ABCC2) recently published studies have utilized these techniques to quan- 633
568 and breast cancer resistant protein (BCRP, ABCG2) are expressed titate transporter abundances in 5 jejunal and 7 ileal mucosal sam- 634
569 on the apical membrane of the enterocyte. They transport drugs ples (Groer et al., 2013; Oswald et al., 2013). The abundance of 635
570 against the prevailing concentration gradient by binding them PepT1 was shown to be substantially higher than OATP2B1, P-gp, 636
571 from the intracellular milieu or inner membrane leaflet and secret- MRP2 and BCRP, however data from a greater number of samples 637
572 ing (effluxing) these molecules into the intestinal lumen resulting is required to confirm regional-specific expression of these trans- 638
573 in an absorption limitation (Aller et al., 2009). The impact of intes- porters. In addition, quantitative immunoblotting techniques have 639
574 tinal efflux transporters is expected to be at its greatest when the been employed to measure the absolute abundance of P-gp, MRP2 640
575 drug’s passive diffusion across the apical membrane enterocyte is and BCRP in frozen duodenal tissues obtained from a human tissue 641
576 low (Darwich et al., 2010). In the clinical setting, the b1-adrenocep- bank (Tucker et al., 2012). In these assays, the mucosal surface was 642
577 tor antagonist talinolol demonstrated a dose-dependent increase scraped and subsequently homogenized with an ensuing 643
578 in systemic exposure after oral administration in humans. In vitro differential centrifugation procedure. P-gp, MRP2 and BCRP in total 644
579 mechanistic studies determined that the likely mechanism for this membrane fractions from 14 samples were measured by quantita- 645
580 phenomenon was the interaction with P-gp (Wetterich et al., tive immunoblotting using s-tagging technology as an internal 646
581 1996). As the dose increases, the efflux function of P-gp reaches standard (Karpeisky et al., 1994). Within the duodenal membrane 647

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
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10 E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx

648 fractions, the transporter abundances in order from highest to low- Table 3
649 est were; BCRP > P-gp > MRP2. This is in conflict with duodenal Presence of CYP isoenzymes in landrace and Göttingen minipig (modified from Helke
and Swindle, 2013 (Helke and Swindle, 2013).
650 expression data from meta-analyses incorporating data using rela-
651 tive expression techniques that are normalized to the proximal Human CYP Landrace Göttingen minipigs
652 jejunum (Harwood et al., 2013). These analyses show a reversal 1A1 +
653 of expression in the order: MRP2 > P-gp > BCRP. The accurate 1A2 + +
654 implementation of expression data within intestinal models is crit- 2A6 + +
2B6 +
655 ical to ensure the models proximity to the in vivo milieu to provide 2C9 + 
656 the basis for predicting the impact of transporters on ADMET. 2D6 + 
657 There is a need to undertake further studies quantifying the 2E1 + +
658 region-specific abundances of a variety of ABC and SLC transporters 3A4 + +
659 that reside on the apical and basal enterocyte membranes and their +: indicated presence/activity of enzyme.
660 variability between individuals in numerous populations. In : indicates no presence/activity of enzyme.
661 addition, it would also be valuable to quantify the transporters
662 OST-a and OST-b (OST-A & OAT-B) located on the basal enterocyte
663 membrane (Ballatori et al., 2005; Grandvuinet and Steffansen,
664 2011) and cadherin-17/human peptide transporter 1 (CDH17, Canine gastric pH in the fasted state varies along the length of 708
665 HPT1) which functions as a peptide transporter (Dantzig et al., the stomach. Anterior gastric pH has a pH of 5.5 and drops to 709
666 1994). 3.4 in the posterior stomach (Smith, 1965). Perhaps it is 710
667 Many fundamental questions remain to be elucidated regarding important to mention that the most effective way to achieve a 711
668 accurately quantitating the levels of these proteins within tissues. consistently low gastric pH in fasting dogs is to administer 712
669 Loss of proteins throughout practical workflow is inevitable. Strat- 0.1 mol/l HCl-KCL buffer 15 min before the dosage form. 713
670 egies to counter these losses, or the use of recovery factors, to esti- Similarly, to elevate the gastric pH reproducibly (in the fed 714
671 mate the ‘true’ or ‘intrinsic’ tissue abundance levels could be state), omeprazole 1 mg/kg should be administered intravenously 715
672 utilized. For intestinal samples, a reasonably pure enterocyte yield at least 90 min before oral administration of the dosage form 716
673 is essential as contamination with connective tissue, i.e. lamina (Polentarutti et al., 2010). 717
674 propria, and underlying submucosal tissue will dilute the sample. With regards to the fasted small intestine, all along the length of 718
675 This is likely to lead to under-estimation in transporter abundance. the small intestine the pH increases from pH 6.2 to about 7.5 719
676 Moreover, expression of transporter proteins in any intestinal (Kararli, 1995). This was confirmed by Kalantzi et al. (2006) who 720
677 layers or red blood cells other than the enterocytes will lead to reported a fasted pH of 7.1. The buffer capacity has been measured 721
678 contamination and may bias abundances. It has been speculated 1.4–4.2 mM/pH and the osmolarity 62–207 mOsmol/kg, both low- 722
679 that different methods to obtain membrane fractions may be er than in humans (Kalantzi et al., 2006). The most abundant bile 723
680 subject to their digestion and thus quantitation may lead to differ- salt is taurocholic acid (Holm et al., 2013b). The phospholipid in 724
681 ences in abundance levels between studies (Prasad et al., 2013). the canine intestine contains 94.5% phosphatidylcholine and 5.5% 725
682 Equally, the protocols employed to reduce, alkylate and digest phosphatidylethanolamine (Alvaro et al., 1986). 726
683 proteins or the internal standard peptides used as surrogates for The type and pattern of contractions of the canine GIT in the 727
684 protein quantitation could influence the endpoint quantitation fasted state are similar to those of humans. Phase III (housekeeping 728
685 (Balogh et al., 2012). Therefore, it has been proposed that to wave) of the IMMC lasts for about 20 min in both species and oc- 729
686 elucidate whether there are any methodological biases leading to curs every 100–110 min (Itoh and Takahashi, 1981; Yamada 730
687 differences in endpoint abundances, matched protein samples et al., 1995). In contrast, agitation intensity is higher in the GIT 731
688 should be processed across laboratories using their own in-house of dogs (Katori et al., 1995). In the fed state, the pattern of contrac- 732
689 techniques where valid comparisons can take place (Rowland tions in jejunum is similar although probably more intense 733
690 Yeo et al., 2013). (Schemann and Ehrlein, 1986). 734
Gastric emptying of liquids in the fasting state has been 735
reported to be similar or faster compared with those in humans 736
691 3. Canine GI characterization
(Ehrlein and Prove, 1982; Gupta and Robinson, 1988; Hinder and 737
Kelly, 1977; Reppas et al., 1991). In the fed state, the gastric emp- 738
692 3.1. Anatomical considerations
tying rate of liquids is slower compared to humans (Nishiyama 739
et al., 1996). The gastric emptying of solids in the fasting state is 740
693 Detailed description of the anatomy of the canine GI tract can
size-dependent like in man and occurs at similar or faster rates 741
694 be found in various previously published reviews (de Zwart,
(Aoyagi et al., 1992; Gruber et al., 1987). Solid meals are emptied 742
695 1999; Kararli, 1995) and a summary is provided in Table 1. The
at slower rates than in humans (Dressman, 1986; Meyer et al., 743
696 canine stomach is anatomically similar to that of humans, e.g., vol-
1981; Meyer et al., 1979). Transit times of the small and large 744
697 ume of 0.5–1 L (living beagle) (Martinez et al., 2002). Dogs possess
intestine vary with the size of the dog. In beagles, they are about 745
698 a well developed small intestine, which is consistent with a diet
half of that of humans (Davies and Morris, 1993). 746
699 that is low in fiber but high in fat and protein, and a relatively sim-
Information on intraluminal metabolic activity in the canine 747
700 ple colon. Major differences, compared with humans, include the
GIT has been very limited (de Zwart, 1999; Martinez et al., 2002). 748
701 shorter small intestine, especially for small dogs, and the much
A recent study showed that the degradation of three ester prodrugs 749
702 shorter large intestine, about one-fourth of human colon (de Zwart,
in jejunal fasted state contents collected from Labradors and 750
703 1999; Kararli, 1995).
healthy humans was similar (Borde et al., 2012). However, the 751
enzymatic capacity of luminal contents in dogs was higher than 752
704 3.2. GI characteristics in humans, which is in line with the higher protein levels measured 753
in the canine luminal contents. Also, compared to the activity in 754
705 Some GI characteristics are presented in comparison with luminal contents, the hydrolase activity in small intestine micro- 755
706 data from other species in Table 3. An extended review on somes seemed to be lower in dogs but higher in humans (Borde 756
707 canine GI physiology is available (Smeets-Peeters et al., 1998). et al., 2012). 757

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758 3.3. Intestinal permeability and metabolic activity and mucosal) and transporter activities in dogs, would improve 819
the usefulness of this model in drug absorption and API/formula- 820
759 It has been estimated that the unstirred layer thickness for tion studies. 821
760 rapidly absorbed compounds is similar in dogs and humans (of
761 the order of 40 lm) (Fagerholm and Lennernäs, 1995; Levitt
4. Pig GI characterization 822
762 et al., 1990). Differences in the available surface area and in the
763 tightness of the junctions between epithelial cells have been con-
4.1. Introduction 823
764 sidered to be important for low permeability compounds (Fotaki
765 et al., 2005; He et al., 1998). There is some evidence for improved
Pigs are considered a translational model in biomedical 824
766 drug permeability through the canine small intestine as compared
research because of anatomical, physiological and biochemical 825
767 to human intestine which has been related to these factors (Sutton,
similarities to humans (Puccinelli et al., 2011; Swindle, 2007; 826
768 2004). However, good correlation of the relative bioavailabilities of
Swindle and Smith, 1998; Tissot et al., 1987). Pigs and in particular 827
769 11 compounds administered to the dog and human colon have
mini-pigs have therefore become increasingly popular as an alter- 828
770 been observed (Sutton, 2004).
native species in drug development (Bode et al., 2010; Ganderup 829
771 To date, the distribution of metabolic enzymes and membrane
et al., 2012; Helke and Swindle, 2013; Swindle et al., 2012). How- 830
772 transporters in dogs has not been comprehensively investigated.
ever, in the literature there is a disagreement on the potential use 831
773 Relevant studies have mainly focused on the expression and cata-
of pigs as an in vivo model for drug formulation research and devel- 832
774 lytic activity of metabolic enzymes in the liver (Kyokawa et al.,
opment, which needs further exploration. In order to understand in 833
775 2001; Mills et al., 2010). Less is known about the intestinal expres-
which circumstances pigs should be considered, this section 834
776 sion distribution of CYPs, UGTs, membrane transporters as well as
includes a description of pig GI physiology, metabolism and 835
777 their substrate specificities and variability in expression compared
membrane transporters and the similarity/dissimilarity to humans. 836
778 with that in humans (Bock et al., 2002; Conrad et al., 2001; Fraser
There are several breeds of minipigs, such as the Yucatan and 837
779 et al., 1997; Locuson et al., 2009; Mealey et al., 2008; Turpeinen
Göttingen, where Göttingen minipig is the most frequently used 838
780 et al., 2007).
pig strain in contemporary pharmaceutical literature (Simianer 839
781 According to a recent study (Haller et al., 2012), the gene
and Kohn, 2010). However, since considerable characterizations 840
782 expression pattern of five drug transporters in the liver and along
of the domestic landrace pig are applicable and valid for the mini- 841
783 the intestine of beagle dogs has a number of differences compared
pig assessment, this strain will also be included in the evaluation 842
784 with human data. In particular, the tissue distribution of CYP iso-
(Forster et al., 2010). 843
785 zymes and P-gp appears to be markedly different in dogs compared
786 with humans, whereas UGT1A6, peptide transporter-1 (PEPT1,
787 SLC15A1), organic cation transporter-1 (OCT1, SLC22A1, BCRP), 4.2. Anatomical considerations 844
788 and multidrug resistance-associated protein 1 (MRP1, ABCC1)
789 more closely resemble the human tissue distribution (Haller The size of the GIT regions, in relation to total body weight, in 845
790 et al., 2012). pigs is generally very similar to human. The stomach, small 846
intestine and large intestine represent approximately 0.45/0.95% 847
791 3.4. Gall bladder emptying and lymphatic transport (landrace/Göttingen minipig), 2% and 1.4%, respectively, in pigs in 848
comparison to 0.7%, 2.5% and 1.8%, respectively, in humans (Bollen 849
792 In the fasted state, canine gallbladder shows brief alternating et al., 1998; Kühn, 2001; Phuc and Hieu, 1993; Price et al., 2003). 850
793 excursions of filling and emptying with the number of emptying As in humans, the pig is monogastric and acid secretion results 851
794 events exceeding the filling events during phase II of IMMC (Abiru as a function of stimuli, such as food intake (Schubert, 2009; von 852
795 et al., 1994). In the fed state, the intensity of gallbladder contrac- Rosenvinge and Raufman, 2010). The length and diameter of the 853
796 tions is dependent on the ingested calories and meal lipid content small intestine is 470–2000 cm (17–19 cm/kg) and 2.5–3.5 cm in 854
797 (Romanski and Slawuta, 2003). The in vivo lymph cannulated landrace pigs and 832–900 cm (34–63 cm/kg) and 2 cm in minipigs 855
798 canine model has been described and used to study intestinal (Bergman et al., 2009; Glodek and Oldigs, 1981; Kurihara-Berg- 856
799 lymphatic targeting and transport. Khoo et al. (2001) have strom et al., 1986; Merchant et al., 2011; Suenderhauf and Parrott, 857
800 proposed a triple-cannulated conscious dog model, which allows 2013). In landrace pigs the large intestine (cecum:colon) is 858
801 sampling from the thoracic duct lymph as well as portal and sys- 23:413 cm (0.22:4 cm/kg) while in minipigs it is 20:303 859
802 temic blood (Khoo et al., 2001). In contrast to other animal models, (0.23:10 cm/kg), the colonic diameter is 2.7 cm (Glodek and Oldigs, 860
803 the dog model allows administration of dosage forms that are of a 1981; McRorie et al., 1998; Merchant et al., 2011; Suenderhauf and 861
804 size relevant for human administration, and it facilitates to study Parrott, 2013). 862
805 the effects of fed versus fasted states on drug absorption. However, Stomach fasting pH is highly variable (1.2–4.4) with indication 863
806 the higher concentration of bile acids in the GI chyme should be of different pH regions within the stomach while the small intes- 864
807 recognized (Persson et al., 2005). tine pH was reported to be about 7–8 (Hossain et al., 1990; Oberle 865
and Das, 1994). No information of the pH in the large intestine 866
808 3.5. Concluding remarks and gaps to be filled could be found for a confirmed fasted state. In the fasted state 867
the gastric transit time of nondisintegrating dosage forms was 868
809 Characteristics of the canine GIT, especially the luminal charac- shown to be significantly retained (1–28 days) with high variabil- 869
810 teristics of large dogs (body weight of about 30 kg), show various ity (Hossain et al., 1990). The transit times in small and large intes- 870
811 similarities with man. The most important distinctions from the tine were shown to be shorter and less variable (<1–3 days) 871
812 human GIT relate to the higher bile concentrations and higher sol- (Hossain et al., 1990). In the fed state the gastric pH was reported 872
813 ubility/dissolution of BCS Class II drugs, to the higher absorption of to be 3–4.5 and the transit was generally shorter (1–6 h) than in 873
814 BCS class III drugs, and to differences in colonic characteristics the fasted state but still highly dependent on the gastric content 874
815 (personal communication Abrahamsson; Persson et al., 2005). (Merchant et al., 2011; Oberle and Das, 1994). The small and large 875
816 There are also indications that the intersubject variability in regard intestine transit times in the fed state are less variable about 3–4 h 876
817 to GI characteristics is larger in dogs than in humans (de Zwart, and 24–48 h, respectively, and the pH ranges from 4.7 to 7.2 877
818 1999). A more thorough understanding of enzyme (both luminal (Braude et al., 1976; Davis et al., 2001; Hossain et al., 1990; 878

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in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
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12 E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx

879 Ruckenbusch and Bueno, 1976; Wilfart et al., 2007). However, the Laan et al., 2010). In other cases, the metabolic activity in the intes- 916
880 transit time is somewhat influenced by intestinal content, for an tinal wall is significant. This can be similar to human but in other 917
881 example, a high fibre diet generated a value of 26 h while other liq- cases not, e.g., the bioavailability of metoprolol is 48 ± 22% in hu- 918
882 uids and solids had a transit time of 25–49 h (van Leeuwen et al., mans compared to 3% in pigs (Holm et al., 2013a; Sandberg 919
883 2006; Wilfart et al., 2007). The water content, given as wet mass, et al., 1991; van der Laan et al., 2010). Not all human CYP isoforms 920
884 for pigs fed ad libitum has been reported to be 250 g in the stomach have an ortholog in pigs (Helke and Swindle, 2013). Comparison 921
885 and 500 g (0.25 g/cm gut length) and 750 g (1.7 g/cm gut length) in between pig and human cDNA and amino acids show high se- 922
886 the small and large intestine, respectively (Merchant et al., 2011). quence homology, though metabolic responses may vary as also 923
887 Bile is stored in a gall bladder and secreted to duodenum as in hu- demonstrated with the metoprolol example (Lu and Li, 2001; 924
888 mans. It is produced at a rate of approximately 20–50 ll/min/kg Monshouwer et al., 1998; Toutain et al., 2010). In Table 3, the 925
889 with a concentration of 42–55 mmol/L and has a similar composi- CYP enzyme activity in landrace and Göttingen minipigs can be 926
890 tion to human bile (Bergman et al., 2009; Juste et al., 1983; Nakay- found and an overview of the CYP isoenzyme involved in the 927
891 ama, 1969; Petri et al., 2006; Sjödin et al., 2008). The amplification reactions with a number of substrates are presented in Table 4, 928
892 of surface area due to folding and villi was found in landrace pig to demonstrating some differences in the CYP isoenzymes involved 929
893 be 3.7 in the small intestine and 2.5 in the large intestine (Snipes, between the two species. A comprehensive characterization of 930
894 1997). Even though a systematic investigation or compilation of abundances and substrate specificity of conjugating enzymes, 931
895 pig permeability data is absent, a number of single pass intestinal e.g., UGT’s and SULT’s, is still absent for the pig. However, in vitro 932
896 perfusion studies in situ have been completed where Peff were and in vivo studies have shown intestinal and hepatic UDP and 933
897 determined. For some of these compounds, there are human Peff SULT functionality with potential similarity to humans (Gu et al., 934
898 reference values available, for instance Peff (104 cm/s) pig:human: 2006; Rahikainen et al., 2013; Sjögren et al., 2012; Thörn et al., 935
899 fexofenadine (0.02:0.07), verapamil (1:6.8), antipyrine (0.61:5.6), 2012). 936
900 cyclosporine (0.62:1.65) (Chiu et al., 2003; Persson et al., 2008; Altogether, based upon substrates, induces, inhibitors and regu- 937
901 Petri et al., 2006; Thörn et al., 2009). For other APIs, a direct lation data, there are no major differences among CYP1A1, 1A2, 2B, 938
902 measurement of human Peff is absent, such as in the case of danazol 2E1 and 3A in pigs when compared to humans (Puccinelli et al., 939
903 (pig: 1.1  104 cm/s) (Persson et al., 2008). Methodological 2011). Hence, the pig could be a good model for human (with re- 940
904 aspects, e.g., surgery and anaesthesia, as well as species-related spect to metabolic profile) for compounds that are mainly metab- 941
905 differences in physiology have been suggested to the lower Peff olized by these CYP enzymes, However, as less is known of CYP2C, 942
906 values measured in pigs (Fagerholm et al., 1996; Petri et al., CYP2D6 and phase II metabolism in pigs, more caution should be 943
907 2006). In vitro permeability investigation using pig material is lim- taken when selecting pigs as a model for these compounds which 944
908 ited. However, there are examples of studies with excised mucosal interact with these enzymes. 945
909 tissue using the Ussing chamber technique as well as employing
910 isolated and cultured enterocytes (Bader et al., 2000; Nejdfors 4.4. GI transporters 946
911 et al., 2000; Winckler et al., 1999).
Only a limited number of references on drug transporter 947
912 4.3. GI metabolism expression and functionality in pigs and minipigs are available. 948
However, Schrickx presented an RNA and protein expression study 949
913 There are similarities between man and pig with regard to bio- on the landrace pig of P-gp, BCRP and MRP2 in various tissues 950
914 transformation, but also significant differences. E.g., pigs have very including the GIT (Schrickx, 2006). P-gp and BCRP RNA levels were 951
915 low CYP2D and CYP2C19 expression compared to man (van der found to increase along the small intestine to reach highest 952

Table 4
CYP test reactions and human and porcine isoenzymes involved.

Substrate Human Porcine Reaction Reference

Methoxyresorufin CYP1A2 CYP1A O-Demethylation 1
Ethoxyresorufin CYP1A2 CYP1A O-Demethylation 1–2
Coumarin CYP2A6 CYP2A 7-Hydroxylation 2–6
Nicotine CYP2A6 CYP2A/NI O-Oxidation 7
Benzyloxyresorufin CYP2B6 CYP2B? (NI) O-Debenzylation 8
7-Ethoxy-4-triflouromethylcoumarin CYP2B6 CYP2B? (NI) O-Debenzylation 3
CYP2B6 CYP2B? (NI) Dealkylation 9, 10
Pentoxyresurfin CYP2B6 ND Demethylation 2
Mephenytoin
Diclofenac CYP2C8/9 CYP2C9/NI 4-Hydroxylation 3, 8.16
Tolbutamid CYP2C9 NI 4-Hydroxylation 11
S-Mephenytoin CYP2C19 ND 4-Hydroxylation 2, 3, 8
Desbrisoquine CYP2D6 ND 4-Hydroxylation 12
Bufuralol CYP2D6 CYP2B 1-Hydroxylation 3, 12
Dextromethorphan CYP2D6 CYP2B O-Demethylation 12, 16
Clorzoxazone CYP2E1 CYP2E1+2A+3ª 6-Hydroxylation 2, 3, 9, 13
p-nitrophenol CYP2E1 CYP2E1 + CYP2A/NI 2-Hydroxylation 1, 14
Aniline CYP2E1 CYP2A/(NI) 4-Hydroxylation 1
Midazolam CYP3A4 CYP3A 1- and 4-hydroxylation 15
Testosterone CYP3A4 CYP3A 6b-Hydroxylation 2, 3
Nifedipine CYP3A4 CYP3A N-oxidation 2, 9

NI: specific porcine CYP isoenzyme responsible for this reaction not yet identified ND: no detectable metabolism.
(1) (Nebbia et al., 2003), (2) (Skaanild and Friis, 1999), (3) (Bogaards et al., 2000), (4) (Skaanild and Friis, 2000), (5) (Shimada et al., 1994), (6) (Pelkonen et al., 2000), (7)
(Skaanild and Friis, 2005), (8) (Myers et al., 2001), (9) (Desille et al., 1999), (10) (Behnia et al., 2000), (11) (Anzenbacher et al., 1998), (12) (Skaanild and Friis, 2002), (13)
(Wiercinska and Squires, 2010), (14) (Skaanild and Friis, 2007), (15) (Lu and Li, 2001), (16) (Thörn et al., 2011).

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953 expression in distal jejunum with reduced expression in ileum and (Langguth et al., 1994). Gastric emptying in the rat is mainly 1013
954 negligible amounts in the large intestine. The same expression pat- controlled by the energy content of the ingested food in a similar 1014
955 tern was observed for MRP2 with the difference that the expres- manner like in humans. For instance, the gastric emptying half-life 1015
956 sion was quite significant in the large intestine. This work also for solution with 0–6 kcal were 5–65 min (Maerz et al., 1994). The 1016
957 presented high homology between human and porcine P-gp with rat often has a higher gastric pH than that of man of about 4–5 (Da- 1017
958 regard to transcriptional, protein and functional level. The work vies and Morris, 1993; Kararli, 1995). Despite the increased gastric 1018
959 of Tang and co-workers reported an increased P-gp protein expres- secretion, fasted gastric pH is generally increased by food intake 1019
960 sion along the small intestine and also some expression in large across all species. The magnitude of gastric pH increase is highly 1020
961 intestine in the Yucatan minipig (Tang et al., 2004). Minor expres- dependent on the meal composition and a direct comparison be- 1021
962 sion in the intestine (region unknown) has also been reported for a tween species is difficult. The contents of GI fluids such as bile 1022
963 pig homolog to human OATP1A2 with high similarity both in se- salts, lipids and buffer species as well as the GI motility and pH 1023
964 quence and functionality (Yu et al., 2013). are the main factors responsible for the initial saturation. The 1024
965 No in vivo study has been published on pigs with direct secretion of bile acids (taurocholate is the major bile acid at a total 1025
966 information of the intestinal transporters, e.g., PK data presented concentration of 8–25 mM in rats) is induced endogenously by 1026
967 together with information of protein expression or genetic hetero- food intake. In rats, within 10 min the secretion of bile-pancreatic 1027
968 zygosity. However, several studies have been conducted using the juice proteins increased from 0.2 mg under fasted state with oral 1028
969 domestic landrace pig with known substrates of transporters with saline solution to 0.7 mg under fed state with oral fatty acids or 1029
970 or without transporter inhibitors, such as fexofenadine, ximelaga- sucrose solution (Hiraoka et al., 2003). Total bile acid and 1030
971 tran, rosuvastatin, verapamil and digoxin (Bergman et al., 2009; phospholipids concentration for the rat intestine is changing with 1031
972 Petri et al., 2006; Sjödin et al., 2008; Tannergren et al., 2006; Thörn segment such that saturation solubilities of compounds also 1032
973 et al., 2009). A few ex vivo studies wherein brush-border depend on the segmental fluid composition and distribution 1033
974 membrane of pig small intestine showing expression of carriers (Tanaka et al., 2012). 1034
975 of amino acids and D-glucose have also been published (Munck The GI fluids mostly control the in vivo drug solubility, along 1035
976 et al., 1995) (Maenz and Patience, 1992; Munck et al., 2000; Schar- with the volume of the co-administered water and the adminis- 1036
977 rer et al., 2002; Stevens et al., 1982). As this section demonstrates, tered dose. The pH in small intestine increases continuously from 1037
978 further investigations are needed to increase the knowledge and duodenum to terminal ileum within a similar range as humans, 1038
979 role of intestinal transporters in drug absorption in pigs. i.e., 4.5–7.5. SITT of 3–4 h in rats is also similar to humans (Davis 1039
and Wilding, 2001; Lennernäs and Regårdh, 1993). 1040
The anatomical features of the GI tract such as radius and 1041
980 4.5. Concluding remarks and gaps to be filled
length, microbial content, the hydrodynamic characteristics of 1042
volume, flow rate of the GI fluids, as well as elements closely 1043
981 The slow and variable gastric emptying of the pig is an impor-
associated with permeability for example tight junction have pre- 1044
982 tant species difference to human with high potential implications
viously been reviewed (DeSesso and Jacobson, 2001; Kararli, 1995). 1045
983 for the in vivo GI drug absorption of enteric coated or modified
Focusing on the epithelial permeability in small and large intestine, 1046
984 release formulations as well as investigations in the fed state. In
the rat has been used in several in situ single pass perfusion exper- 1047
985 contrast to gastric transit times, the transit times in small and large
iments (Cao et al., 2006; Fagerholm et al., 1996, 1997, 1999). This 1048
986 intestine are generally less variable and more comparable to that in
technique has, in several reports, shown that the rat jejunal perme- 1049
987 humans. Also, as pointed out by this summary, further
ability correlates strongly with the corresponding human jejunal 1050
988 characterization of pH and transit times in the fasted state is nec-
Peff. The Peff for passively absorbed drugs on average was 3.6 times 1051
989 essary, especially for small and large intestine and disintegrating
higher in humans compared to rats. Compounds with carrier-med- 1052
990 formulations. Further investigations, both in vitro and in vivo, for
iated absorption deviated from this relationship, which indicates 1053
991 the characterization of the intestinal permeability, metabolism
that scaling of these processes needs further consideration (Cao 1054
992 (especially mediated by CYP2C, CYP2D6 and phase II enzymes)
et al., 2006; Fagerholm et al., 1996, 1999). Mechanistic investiga- 1055
993 and transporters are also needed for an optimum application of
tions by determining human and rat jejunal permeability and at 1056
994 the pig as a pre-clinical model for drug absorption. Anatomical
the same time examining the expression levels of transporters 1057
995 and physiological data of the GI tract of pigs is summarized in
and metabolic enzymes through GeneChip techniques has found 1058
996 Table 1.
the same good correlation between human and rat permeability 1059
(R2 = 0.8), only a moderate correlation for the transporter 1060
997 5. Rat GI characterization expression levels in duodenum (R2 > 0.56), but no correlation in 1061
metabolizing enzyme levels (Cao et al., 2006). This agrees with 1062
998 In general there are important differences between the physiol- the well-established difference between the species in drug metab- 1063
999 ogy of rodents and humans such as the fact that rodents are noc- olism and oral bioavailability where a mean allometric coefficient 1064
1000 turnal animals with consequences for timing of dose and an of 0.66 was determined in a correlation of plasma clearance of 54 1065
1001 option to change day–night rhythm. Further rodents are prone to extensively metabolized drugs between humans and rats (Chiou 1066
1002 coprophagy with consequences of re-uptake of fecal excreted and Barve, 1998). On the basis of these studies, the rat in situ sin- 1067
1003 drugs. In addition, the size of rodents limits their use in studies gle-pass perfusion model is considered as the most appropriate 1068
1004 with intact dosage forms intended for human use and generally animal model for predicting human permeability and absorption 1069
1005 higher metabolism is observed in rodents as compared to humans. from the small intestine (Cao et al., 2006; Fagerholm et al., 1996, 1070
1006 Drugs are often dosed per body weight in animal studies and if 1997, 1999). In addition, the Ussing chamber with rat tissue has 1071
1007 comparing gastric volumes adjusted for body weight, the relative also been reported to be a useful model to predict human intestinal 1072
1008 gastric volume of the rat is larger than for humans (Davies and absorption based on data from both the small and large intestine 1073
1009 Morris, 1993). The gastric emptying rate of liquids in the fasted (Lennernäs et al., 1997; Ungell et al., 1998). It seems that the rat 1074
1010 state, being the most relevant factor when comparing drug absorp- colon may be useful to predict absorption of drugs intended to 1075
1011 tion rates of BCS class I compounds, is somewhat similar in rats and be used in oral MR dosage forms but this requires further valida- 1076
1012 humans, with a gastric emptying half-life around 15–30 min tion. An opportunity can be seen in the assessment of absorption 1077

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in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
 PHASCI 2973 No. of Pages 53, Model 5G
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14 E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx

1078 for BCS II, III and IV drugs in IR dosage forms using this technique and water volumes for the mouse small intestine from a variety of 1140
1079 because of the regional differences. However, even if the rat is a studies by different groups over more than a decade. 1141
1080 suitable preclinical model for GI absorption it has limitations such
1081 as body size, dietary intake difference and the shortcomings
1082 related to dosing of intact solid dosage forms. 6.2. Gastric emptying and small intestinal transit time 1142

Recently, technetium-labeled activated charcoal diethylenetri- 1143

1083 6. Mouse GI characterization aminepentaacetic acid (99mTc-Ch-DTPA) detected by single-pho- 1144

ton emission computed tomography (SPECT) was used to study 1145

1084 6.1. Anatomical considerations gastrointestinal transit times in mice (Padmanabhan et al., 2013). 1146
It was found that stomach transit time was 1 h, small intestinal 1147

1085 Although a description of the anatomy and physiology of the transit time was 1–20 h, and cecum and colon transit time was 1148

1086 mouse GI tract can be found in various previously published 3 h. In a study of the influence of salmon calcitonin on the small 1149

1087 reviews (de Zwart, 1999; Kararli, 1995; McConnell et al., 2008a; intestinal transit time (SIT) in the mouse, Hamada et al. reported 1150

1088 Ogiolda et al., 1998), the amount and quality of the data regarding the average non-inhibited SIT to be 1 h (Hamada et al., 1999). 1151

1089 biopharmaceutically relevant information in mice in particular for

1090 drug product development is much less than human and rat, 6.3. pH of the mouse small intestine 1152
1091 although the mouse has been the most extensively studied species
1092 in pharmacology. McConnell et al. investigated the pH, water content, and lym- 1153
1093 The mouse stomach is divided into a glandular and non-glandu- phoid tissue distribution in two groups of mice (McConnell et al., 1154
1094 lar portion (de Zwart, 1999). The non-glandular portion is thin-- 2008a). The first group was fasted overnight with free access to 1155
1095 walled and is a higher percentage than found in humans. The water and the second group was given free access to a low protein 1156
1096 glandular portion is thick-walled and secretes mucus, pepsinogen, (18%)/low fat (5%) standard diet and water at all times. When 1157
1097 and HCl. Ogiolda studied the size of various parts of the mouse GIT 9.17 g of mouse chow was mixed with 10 mL of water until the 1158
1098 at 8-months of age after selective breeding of 8-week old mice (Og- food pellet disintegrated, the resulting pH was 5.9. GI pH was 1159
1099 iolda et al., 1998). The body weights at 8-months of age resulted in determined after sacrificing the animal, by dissecting the GI 1160
1100 the following categories: heavy (63 g male, 58 g female), light (22 g segments, collecting and mixing the undiluted contents, and mea- 1161
1101 male, 21 g female), and randomly selected (45 g male, 41 g female). suring pH with a meter. In contrast to humans, mice had lower 1162
1102 Since most of the anatomical and physiological publications relate stomach pH (3.0) under fed conditions than fasted (4.0 ± 0.3). Un- 1163
1103 to mice that are 18–22 g we will focus on the average properties of der fasting conditions, all sections of the mouse small intestine had 1164
1104 male and female mice reported by Ogiolda for the ‘‘light’’ category. similar pH values and were slightly higher (5.0 ± 0.25) than in fed 1165
1105 McConnell reported that a 20 g mouse is comfortably full with a mice (4.8 ± 0.03). Compared to the fasted human small intestine 1166
1106 stomach volume of 0.37 mL and could expand up to 0.71 mL max- where the pH ranges from 6 to 7.4, the lower pH in the mouse 1167
1107 imum (McConnell et al., 2008a). Assuming the stomach is a sphere has implications for in vivo testing. When delivered as a solution 1168
1108 with surface area of 2.47 cm2, the radius would be 0.443 cm and formulation, acidic drugs may precipitate in the mouse small 1169
1109 the resulting spherical volume would be 0.365 mL. Thus, there is intestine but remain in solution or dissolve readily when tested 1170
1110 a good correlation between the data collected by Ogiolda for the in humans. Also, when tested in mice, the performance of pH-sen- 1171
1111 ‘‘light’’ category mouse and the physiological data of McConnell. sitive polymeric delivery formulations may not exhibit the same 1172
1112 More importantly, for estimation of mouse anatomy for a given release characteristics as expected in humans. In contrast to the 1173
1113 body weight, Ogiolda did not find statistically significant differences in normal values of pH between human and mouse, 1174
1114 differences between the ‘‘light’’, ‘‘random’’, or ‘‘heavy’’ mice when in a study of the intestinal permeabilities of the following five 1175
1115 the ratios of GI section weight/body weight, or surface area/body model drugs furosemide, piroxicam, naproxen, ranitidine and 1176
1116 weight(2/3), or length/body weight(1/3) were compared. Ogiolda amoxicillin in the in situ intestinal perfusion technique in mice, it 1177
1117 found the following additional average dimensions for the ‘‘light’’ was found that permeability was similar for the above mentioned 1178
1118 category mouse: Duodenum weight (166 mg) and length compounds when compared to human (Escribano et al., 2012). 1179
1119 (38 mm); Jejunum/Ileum weight (1156 mg) and length
1120 (364 mm); Cecum weight (305 mg) and length (31 mm); and Colon
1121 weight (550 mg) and length (83 mm). 6.4. Metabolism and transport 1180
1122 Assuming consistency and reported relationship to body weight
1123 of these GI parts, the regional anatomy for any size mouse should Information on the quantitative expression of intestinal 1181
1124 be easily estimated. In fact, these dimensions are consistent with metabolic enzymes in the mouse is not as plentiful as some other 1182
1125 Kararli who reported a total SI length of 35–45 cm for mice (Karar- preclinical species. Zhang et al. reported on mRNA expression and 1183
1126 li, 1995). In a study of the influence of lactation on processing of identity of the following Cytochrome P450 enzymes; Cyp1a1, 1b1, 1184
1127 dietary protein, Harmatz et al. reported that the average 2b10, 2b19, 2b20, 2c29, 2c38, 2c40, 2e1, 3a11, 3a13, 3a16, 3a25, 1185
1128 circumfence of the control mouse (21 g) small intestine was and 3a44 (Zhang et al., 2003). They found that Cyp3A13 was found 1186
1129 0.7 cm (Harmatz et al., 1993). The radius determined from this va- in the highest abundance in the small intestine followed by 3a11. 1187
1130 lue of circumference would be 0.11 cm and if it is assumed that the However, liver expression of 3a11 was higher than 3a13. Komura 1188
1131 small intestine is a cylinder of 36 cm long, its volume would be et al. published on species differences for in vitro and in vivo small 1189
1132 1.38 mL. Of course, the small intestine does not have a constant intestinal metabolism for several CYP and UGT substrates in mice 1190
1133 radius going from the duodenum to the distal ileum, but more (Komura and Iwaki, 2008, 2011). Mutch et al. commented on 1191
1134 accurate measurements of the radius for different segments of regional variability in ABC transporter expression in mice but less 1192
1135 the mouse intestine are not available. McConnell et al. measured information is available for other drug transporters (Mutch et al., 1193
1136 the average water content of the mouse small intestine (70%) by 2004). Finally, Holmstock et al. has reported on a transgenic mouse 1194
1137 weighing the contents before and after lyophilization and esti- model that has human CYP3A4 and P-gp for use in studying the 1195
1138 mated fasted water volume to be 0.81 mL and fed water volume inducing effects of xenobiotics on human intestinal P-gp (Holm- 1196
1139 to be 0.98 mL. Thus, there is very good agreement of total volumes stock et al., 2013). 1197

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
 PHASCI 2973 No. of Pages 53, Model 5G
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E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx 15

1198 6.5. Concluding remarks and gaps to be filled including the Medimetrics IntelliCap pH sampling unit (Shimizu 1258
et al., 2008) and the SmartPill pressure sensor (Cassilly et al., 1259
1199 The mouse GIT shows some similarity to human and both spe- 2008), have been applied in biopharmaceutic studies. Specific de- 1260
1200 cies share the same finger-shaped morphology of intestinal villi. tails concerning these instruments can be found in engineering 1261
1201 This is in contrast to rats with tongue-shaped villi (de Zwart, journals (McCaffrey et al., 2008). Optical systems have been used 1262
1202 1999). However, the lower pH of the small intestine might make to examine deposition in the lower gut and in the vagina 1263
1203 the mouse less attractive for formulation development than some (Henderson et al., 2007). This technology is best applied to fluores- 1264
1204 of the other preclinical species. More quantitative information on cent labels or materials that have intrinsic fluorescence, aiding 1265
1205 intestinal enzyme and transporter expression will facilitate the biopsy sampling (Muldoon et al., 2007). The IntelliCap system is 1266
1206 use of physiologically based pharmacokinetic (PBPK) models for in principle also capable of sampling GI fluids, the issue being pres- 1267
1207 in vitro/in vivo extrapolation. ervation of the integrity of the sample. 1268
1208 An overall comparison of anatomical and physiological data of
1209 the GI tract of humans and commonly used animal models is given 7.1. MRI in biopharmaceutics 1269
1210 in Table 1.
The understanding of functions of the GIT is important in the 1270
application of biopharmaceutics for the development of orally 1271
1211 7. Imaging technologies for anatomy, physiology and, dosage administered drugs. Experimental approaches have mainly relied 1272
1212 form performance on tissue sections to examine the microscopic structures within 1273
the GIT as well as telemetric measurements to understand transit 1274
1213 Imaging has proved to be a useful tool in biopharmaceutical time, gastric emptying time and pH changes along the length of 1275
1214 studies as it enables a number of important attributes related to the GIT in humans and animals. While understanding human 1276
1215 the formulation. In particular, the following information can be physiology is of the utmost importance to the development of 1277
1216 acquired: new orally acting drugs, the ability to make comparisons across 1278
typical species used in drug development, such as the rat, dog 1279
1217  Patterns of motility and the transit of material along the GIT. and pig, are useful in extrapolating from these animal species to 1280
1218  The region in which dispersion/disintegration occurs. humans. In fact any of the measurements in current in silico tools 1281
1219  Time point at which release of material occurs. for these extrapolations have embedded within them the charac- 1282
1220  The influence of feeding on dosage regimens: effects of dosing teristics of these animal species. 1283
1221 relative to a meal; influence of food components. Imaging techniques have been used frequently to understand 1284
1222  The amount of water available for dissolution. how specific compounds (new chemical entities) or probe com- 1285
1223  The separation of meal components. pounds interact with the various components of the GIT through 1286
1224  Gall bladder volumes. measurement of compound in blood, plasma or urine into which 1287
1225  In vivo erosion and release rates of MR systems and enteric the compounds find their way. The compounds are typically 1288
1226 coatings. labeled with radioactive functional atoms which can be measured 1289
1227 either based on gamma emissions (scintigraphy, e.g., Single Photon 1290
1228 When combined with other measurements, the imaging tech- Emission Tomography, or positron emission tomography) (de 1291
1229 niques become even more informative as the combination of data Kemp et al., 2010; Shoghi, 2009; Van Berkel et al., 2008). In these 1292
1230 can be used for additional elucidation of aspects such as: two cases, the radioactive nuclei, 99mTc or 18F, are short-lived iso- 1293
topes which decay rapidly with half-lives in the order of 6 h and 1294
1231  Regional absorption (e.g. extent of colonic absorption). 110 min, respectively. Moreover, newer techniques have been 1295
1232  Localization of the dosage form in the GIT relative to the plasma developed to measure tissue concentrations in thin sections (e.g., 1296
1233 concentration (sometimes termed ‘pharmacoscintigraphy’ or matrix-assisted laser desorption ionization, MALDI); however, as 1297
1234 ‘pharmacomagnetography’). with the other techniques they measure compound and metabo- 1298
1235  The unambiguous relationship between position in the GIT and lites and do not focus on microscopic or patho-physiological 1299
1236 pH for tagged radiotelemetry devices. processes. 1300
1237 Functional imaging on the small animal level has not been used 1301
1238 The use of imaging has been reviewed, sometimes at length, in frequently to date but has been applied for many years to humans. 1302
1239 several articles over the past 30 years. These articles have dealt The clinical MRI equipment is quite expensive and difficult to jus- 1303
1240 with specific technologies: gamma scintigraphy (Hardy and tify for use in small animals. Approximately 25 years ago, small 1304
1241 Wilson, 1981; Newman and Wilding, 1999; Wilding et al., 2001) animal MRI instruments were developed and recently increased 1305
1242 MRI (Marciani, 2011; Schwizer et al., 2006) and magnetic marker efforts have been made in the field (Hockings, 2006; Rudin, 2005). 1306
1243 monitoring (MMM) (Weitschies et al., 2010, 2005). MMM is also Several obstacles limit MRI technologies in pharmaceutical 1307
1244 referred as magnetic moment imaging (MMI), magnetic pill preclinical animal research: animal size called for adapting instru- 1308
1245 tracking as well as related methods such as alternating current ment dimensions and magnetic field strength; the necessity for 1309
1246 biosusceptometry (ACB) (Cora et al., 2011). Characteristics of the anesthesia and its monitoring, the control of body temperature 1310
1247 currently most used imaging modalities have recently been re- among other vital parameters, all these requirements have to be 1311
1248 viewed (Weitschies and Wilson, 2011). In most cases a separate met for a successful longitudinal, repetitive and non-destructive 1312
1249 marker compound is used in a formulation (e.g. with drill & fill application of MRI. 1313
1250 method), but this may have a different release/dissolution profile From a scientific point of view, the advantages of such 1314
1251 than the API from the non-modified formulation. non-invasive technologies include the ability to work with intact 1315
1252 A very promising recent development is the MR-based combi- animal models, much closer to human reality than any isolated cell 1316
1253 nation of 19F tracking and 1H imaging allowing real time tracking suspension system, in spite of the tremendous difficulties often 1317
1254 of one or more 19F labeled dosage forms (Hahn et al., 2011, encountered. The improved predictivity of (patho)-physiological 1318
1255 2013). In addition, the availability of new telemetric tools for data obtained in living animal models as a basis for subsequent 1319
1256 clinical gastroenterology such as the Given Imaging camera pill human studies responds to the ethical requirement of utmost 1320
1257 (Glukhovsky and Jacob, 2004) and those for investigational studies safety before human application. 1321

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
 PHASCI 2973 No. of Pages 53, Model 5G
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16 E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx

(Fig. 4). The work proposed in OrBiTo is to develop best practices 1339
for the most commonly used species, rat and Beagle dog using 1340
MRI to investigate function, water content, mucus layers (Fig. 5) 1341
and motility in these species. Assessing how these factors correlate 1342
to man with regard to formulation disintegration and dissolution, 1343
particle and macromolecule (e.g. excipients) diffusion through 1344
the unstirred water layer (i.e., the mucus compartment) and its 1345
final distribution and disposition will aid in selecting the best mod- 1346
el, reduce the number of animals needed to optimize a formulation 1347
and provide input to model builders in the other work streams. 1348

8. GI luminal concentration profiling of orally administered 1349

drugs in humans 1350

GI drug concentrations after oral administration reflect the 1351

interplay of multiple GI and biopharmaceutical processes, includ- 1352
ing the drug dose, drug release, dissolution, solubility, transit and 1353
simultaneous dilution by secretions, precipitation, degradation 1354
and mucosal permeation. Monitoring these luminal concentrations 1355
as a function of time, therefore, provides a unique insight into the 1356
intraluminal behavior of a drug and its formulation (Fig. 6). 1357

8.1. Methodology: sampling and characterizing GI fluids 1358

Fig. 4. MRI of rat abdomen. To improve delineation of the intestine a mannitol The determinations of the GI drug concentrations can be per- 1359
solution was applied per gavage. Numbers indicate organs as follows: (1) Liver, (2) formed following oral administration to healthy volunteers, where 1360
Stomach, (3) Small intestine, (4) Colon. MRI details: Bruker BioSpin 70/30: T2-
GI fluids are sampled by means of a double-lumen catheter, posi- 1361
weighted RARE-image, TR 2000 ms, MTX 256, FOV 8  6 cm; toal acquisition time,
8 m 32 s. Image kindly provided by Sanofi-Aventis. tioned either via the mouth or nose into the upper GIT. Intubation 1362
with two catheters allows the simultaneous assessment of drug 1363
1322 In vivo MRI as a tool for animal research is very much in line concentrations at two intraluminal positions: e.g., the stomach 1364
1323 with the Ethics Committee recommendations of decreasing animal and duodenum (Brouwers et al., 2007b; Walravens et al., 2011) 1365
1324 use since, usually, the chosen MRI-parameter acts as a biomarker or duodenum and upper jejunum (Brouwers et al., 2005, 2006). 1366
1325 for the interesting physiological process or function of interest Positioning of the catheters is usually monitored by means of 1367
1326 and is determined dynamically over a suitable period of time. fluoroscopy (Lennernäs et al., 1992). Drugs are administered either 1368
1327 Therefore, it is not only a snap shot of this process, but depicts orally (dosage form with water) (Brouwers et al., 2005, 2007b, 1369
1328 the development up to its final ‘‘end-point’’ with an inherently 2006; Walravens et al., 2011) or directly into the stomach by 1370
1329 higher reliability. means of a catheter (solution or suspension) (Psachoulias et al., 1371
1330 MRI in drug development has mainly addressed the under- 2011; Vertzoni et al., 2012). While ‘real-life’ administration is obvi- 1372
1331 standing of normal physiological versus pathological activity of, ously most relevant, gastric dosing may be more appropriate for 1373
1332 for example, the heart or other organs and the effects produced mechanistic purposes. To this point, Psachoulias et al. investigated 1374
1333 by drugs on that tissue. Rarely has MRI technology been used to intestinal precipitation of weakly basic drugs upon gastric empty- 1375
1334 understand the effects of excipients on gastric motility or ing by administering drug solutions directly into the stomach; this 1376
1335 formulation effects in the GIT of small animals in order to better approach neglects gastric dissolution to focus on supersaturation 1377
1336 understand the human situation. and precipitation (Psachoulias et al., 2011). Fed state conditions 1378
1337 Contrast agents are tolerated by small animals and similar to can be simulated by either a nutritional drink (Brouwers et al., 1379
1338 humans, they can be used to look into the physiology of the GIT 2007b) or a homogenized liquid meal (Vertzoni et al., 2012). 1380

Fig. 5. MRI of rat intestinal mucosa. High signal intensity rim in colon, tentatively assigned to mucus layer. Mucus thickness approximately 700 lm, requires further
validation. Numbers indicate regions as follows: (1) bladder, (2) Colon lumen, (3) Rim of mucosa. The image to the right is a magnification of the region of the colon. MRI
details: Bruker BioSpin 70/30: T2-weighted RARE-image, TR 2000 ms, MTX 256, FOV 7 cm; total acquisition time, 2 min. Image kindly provided by Sanofi-Aventis.
Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
 PHASCI 2973 No. of Pages 53, Model 5G
19 March 2014

E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx 17

Fig. 6. Gastrointestinal fluid sampling upon drug administration in humans as a unique approach to identify intraluminal processes that are key to the overall drug
absorption process and the investigation of the mucosal permeation potential of a drug.

1381 GI fluids are sampled as a function of time, typically for a period 8.2.2. Gastric dissolution of weakly basic drugs and the role of acidic 1418
1382 of 2–4 h following administration. To minimize the effect of sam- beverages 1419
1383 pling drug on the absorption process, aspirated volumes should Posaconazole is a poorly soluble and weakly basic antifungal 1420
1384 be kept to a minimum, especially when multiple GI sites are sam- drug, administered as an oral suspension (NoxafilÒ). GI concentra- 1421
1385 pled and/or plasma concentrations are determined in parallel. At tion profiling in healthy volunteers confirmed the importance of 1422
1386 the time of sampling, enzymatic processes should be inhibited gastric dissolution for posaconazole absorption (Walravens et al., 1423
1387 (e.g. cocktail of lipase and protease inhibitors), non-dissolved 2011). In line with other weakly basic drugs, fasted state intake 1424
1388 and/or precipitated drug particles should be separated (centrifuga- of posaconazole with an acidic cola beverage significantly 1425
1389 tion or filtration) and possible further precipitation should be enhanced absorption by improving gastric dissolution. In contrast 1426
1390 avoided by diluting the particle-free sample. The observed drug to the usually accepted hypothesis, this could not necessarily be 1427
1391 concentrations will be related to characteristics of the GI fluids attributed to a reduction of the gastric pH. In normal fasted state 1428
1392 such as: pH, osmolality, viscosity, surface tension, buffer capacity conditions, the acidic beverage enhanced dissolution by prolonging 1429
1393 and concentration of bile salts, phospholipids and dietary the gastric residence time without affecting the gastric pH. 1430
1394 (digestion) products.
8.2.3. The role of intestinal precipitation in the absorption of weakly 1431
1395 8.2. Applications of GI concentration profiling in humans basic drugs 1432
Intraluminal drug precipitation from supersaturated solutions 1433
1396 8.2.1. Fosamprenavir: understanding an unexpected food effect is currently extremely difficult to predict, since the influence of 1434
1397 Fosamprenavir is a phosphate ester prodrug of the poorly GI physiology on the precipitation process has not yet been eluci- 1435
1398 soluble HIV protease inhibitor amprenavir. To investigate in vivo dated. By characterizing intestinal fluids aspirated after gastric 1436
1399 the intraluminal dephosphorylation of fosamprenavir, required administration of solutions of the weakly basic drugs ketoconazole 1437
1400 for transepithelial permeation, GI fluids and plasma samples were and dipyridamole, intestinal precipitation was recently 1438
1401 collected after administration of an IR tablet of fosamprenavir investigated for the first time in vivo (Psachoulias et al., 2011). As 1439
1402 (TelzirÒ) to healthy volunteers in the fasted and fed state expected, the intestinal fluids appeared supersaturated with both 1440
1403 (Brouwers et al., 2007b). The plasma concentrations of amprenavir drugs as a result of the solubility difference between stomach 1441
1404 demonstrated a distinct food-induced delay in absorption (mean and intestine. In contrast to previous in vitro experiments (Kos- 1442
1405 tmax increased by 2.5 h). Previous in vitro studies suggested that tewicz et al., 2004), however, the observed precipitation in vivo 1443
1406 the inhibition of fosamprenavir dephosphorylation in fed state was minimal. The obtained clinical data were used to optimize 1444
1407 conditions as a potential cause of this delay (Brouwers et al., the experimental conditions of in vitro transfer experiments for 1445
1408 2007a). However, the clinical study revealed duodenal appearance weak bases, pursuing more accurate and biorelevant prediction 1446
1409 of fosamprenavir, and not dephosphorylation, as the major of intestinal precipitation (Psachoulias et al., 2012). 1447
1410 determinant of plasma tmax. In the fed state, duodenal appearance
1411 appeared to be delayed due to an unexpected delay in gastric dis- 8.2.4. GI fluids as reference for biorelevant flux assessment 1448
1412 integration of the IR tablet of fosamprenavir. This study clearly The above mentioned examples illustrate the unique value of GI 1449
1413 illustrated the use of GI concentration profiling to identify key concentration–time profiles to elucidate intraluminal processes 1450
1414 intraluminal processes for absorption. In addition, the data were crucial for absorption. Another application involves the use of aspi- 1451
1415 used as reference to validate the TNO Intestinal Model (TIM) to rated GI fluids as a reference for the biorelevant in vitro assessment 1452
1416 predict food-dependent disintegration of IR tablets (Brouwers of drug flux across an epithelial monolayer. Flux is an informative 1453
1417 et al., 2011). measure of drug and formulation performance, since it not only 1454

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
 PHASCI 2973 No. of Pages 53, Model 5G
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18 E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx

1455 depends on intraluminal drug concentrations but also on the per-

1456 meability of the monolayer for the drug. Absorption-enabling
1457 strategies, including food- or excipient-based solubilization of lipo-
1458 philic drugs, often generate highly complex intraluminal fluids that
1459 may drastically affect the permeation potential of the drug.
1460 Simulating these effects in vitro may be challenging and requires
1461 reference fluids. For instance, upon administration of a co-solvent
1462 and surfactant-based formulation of amprenavir (AgeneraseÒ),
1463 intestinal fluids contained high amprenavir concentrations as a re-
1464 sult of solubilization by D-a-tocopheryl polyethyleneglycol 1000
1465 succinate (TPGS) (Brouwers et al., 2006). Subsequent use of these
1466 fluids as the donor medium in Caco-2 transport experiments
1467 demonstrated the interplay of multiple factors in determining
1468 the amprenavir flux: (1) increased amprenavir concentrations, (2)
1469 entrapment of amprenavir in TPGS-based micelles (reduced per- Fig. 7. Schematic illustration of experimental techniques for human intestinal
perfusion studies. (A) Open perfusion system. (B) Proximal balloon perfusion
1470 meability), and (3) inhibition of the efflux carrier, P-gp, by bile salts
system, (C) Double balloon perfusion system. Solid arrows indicate where the
1471 and TPGS. perfusate enters and leaves the intestinal segment. Generally the perfusate leaves
1472 Similarly, intestinal fluids were collected and analyzed upon the segment by force of gravity, as the fluid is collected on ice standing on the floor
1473 administration of the highly lipophilic drug danazol together with while the subject is positioned in a bed. The dotted arrow specifies the proximal-to-
1474 a heterogeneous liquid meal (Vertzoni et al., 2012). In comparison distal direction of the intestine.

1475 to a simple aqueous medium, diluted aspirates and micellar phases

1476 of aspirates significantly reduced the permeability for danazol
1477 across Caco-2 monolayers as a result of entrapment in the luminal  a triple-lumen oro-nasal tube including a mixing segment, 1515
1478 coarse and/or micellar lipid structures. In terms of flux, however,  a multi-lumen tube with a proximal occluding balloon, 1516
1479 the increased danazol concentrations in lipid structures overcom-  a multi-lumen tube (Loc-I-GutÒ) with two balloons occluding a 1517
1480 pensated for the reduced permeability. 10 cm long intestinal segment (Fig. 7), 1518
 two 20 cm adjacent jejunal segments were isolated with the 1519

1481 8.3. Future of GI concentration profiling multi-lumen perfusion catheter. 1520

1521

1482 In comparison with the use of classic PK studies that require The advantages and disadvantages of the various intestinal 1522

1483 deconvolution or modeling to simulate drug appearance in the sys- perfusion techniques are discussed elsewhere (Lennernäs, 1998). 1523

1484 temic circulation from other disposition processes, the sampling Direct determination of compound transport and metabolism 1524

1485 and analysis of GI fluids upon drug administration directly reflect through blood concentration measurements in the mesenteric 1525

1486 intraluminal drug and formulation behavior. As such, the tech- and portal vein is not possible in humans for obvious reasons. 1526

1487 nique provides unique reference data for optimization of in vitro Intestinal perfusion techniques based on drug disappearance and 1527

1488 and computational models to assess drug absorption. Especially appearance in the lumen do, however, offer great possibilities of 1528

1489 for drugs with a suboptimal absorption potential (BCS class II–IV) measuring various intestinal transport processes. Over the past 1529

1490 that rely on complex absorption-enabling strategies, e.g., solubili- 70 years different in vivo intestinal perfusion techniques have been 1530

1491 zation and supersaturation, the predictive power of existing simu- developed and the importance of such in vivo work has been 1531

1492 lation models is insufficient. It can be expected that direct clearly demonstrated (Drescher et al., 2003; Igel et al., 2007; Len- 1532

1493 assessment of intraluminal drug concentrations will play a crucial nernäs, 1998; Lennernäs et al., 1992, 1994; Modigliani et al., 1533

1494 role to resolve the performance of these strategies in the complex 1973a,b; Pfeiffer et al., 1990; Rambaud et al., 1973; von Richter 1534

1495 GI environment and to guide further optimization of models. et al., 2001). A good correlation between in vivo determined Peff 1535
and historical data on fraction of dose absorbed (fabs) for a large 1536
number of structurally diverse drugs have been established and 1537
1496 8.4. Regional absorption methodologies/in vivo and ex vivo animal reported (Lennernäs, 2007). The effects of the tube on GI physiol- Q4 1538
1497 models for characterizing segmental/regional drug absorption ogy are minimal and do not question the pharmaceutical relevance 1539
of drug absorption data collected using these perfusion methods. 1540
1498 The physicochemical properties of the API and the complex For instance, Näslund et al. showed that there was no difference 1541
1499 physiological and biochemical interactions of the GIT determine in the sensitive gastric emptying process between the following 1542
1500 the regional intestinal Peff in vivo. Variations in mucosa physiology three methods: scintigraphic, oral dosing of paracetamol tracer 1543
1501 may affect regional Peff differently depending on the transport and subsequent plasma sampling, and polyethylene glycol (PEG) 1544
1502 mechanism(s) involved (Chadwick et al., 1977a,b; Corrigan, 1997; dilution methods using intubation tubes (Näslund et al., 2000). 1545
1503 Davis and Wilding, 2001; Lennernäs, 1998; Ungell et al., 1998; Validated double balloons methods have been used for regional 1546
1504 Winiwarter et al., 2003, 1998). Intestinal Peff depends on the single-pass perfusions of the proximal jejunum and distal rectum 1547
1505 coexistence of multiple, parallel transport processes such as pas- in vivo in humans on separate occasions. The small intestinal tube 1548
1506 sive transcellular diffusion and carrier-mediated absorption and has been extensively used to examine jejunal Peff of various com- 1549
1507 carrier-mediated efflux (Sugano et al., 2010). pounds (Lennernäs, 1998; Lennernäs et al., 1992, 1994; Tannergren 1550
et al., 2003a,b). The jejunum is the major absorbing region for 1551
1508 8.4.1. Human model drugs and nutrients in most mammals. It also has the largest 1552
1509 Direct measurements of intestinal Peff and secretion of drugs in surface area and is the site of the most active carrier-mediated 1553
1510 humans are possible by regional intestinal perfusion techniques transport in the gut (Chadwick et al., 1977a,b; Collett et al., 1554
1511 (Drescher et al., 2003; Igel et al., 2007; Lennernäs, 1998; Lennernäs 1997; Hilgendorf et al., 2007; Lennernäs et al., 1992; Ungell 1555
1512 et al., 1992, 1994; Modigliani et al., 1973a,b; von Richter et al., et al., 1998). Human in vivo jejunal Peff values for 42 compounds 1556
1513 2001). In general, four different clinical perfusion principles have (31 drugs) have been determined using this technique and will 1557
1514 been employed in the human small intestine (Fig. 7): be referred to later on (Chiu et al., 2003; Fagerholm et al., 1995, 1558

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
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1559 1996, 1997, 1999; Lennernäs, 1997, 1998; Lennernäs et al., 1992, (Ungell et al., 1998). This introduces a risk that the results obtained 1624
1560 1994, 2002, 1993, 1997; Lindahl et al., 1996; Petri et al., 2006, in the animal model are not fully predictive for the situation in 1625
1561 2003; Sandström et al., 1998, 1999; Sun et al., 2002; Söderholm man. The main aspects to consider in deciding on using an animal 1626
1562 et al., 1997; Takamatsu et al., 2001, 1997; Tannergren et al., model are physiological features of the different regions of the GI 1627
1563 2004, 2003a, 2003b; Winiwarter et al., 2003, 1998). tract, such as surface area, tight junction pore size, intestinal 1628
1564 The human in vivo jejunal Peff (at pH 6.5) for fexofenadine, furo- transporters, residence times in different segments, physical and 1629
1565 semide, hydrochlortiazide and inogatran is 0.07  104, physicochemical characteristics and volumes of GI fluids, the pres- 1630
1566 0.05  104, 0.04  104, and 0.03  104 cm/s, respectively (Fag- ence of enzymes that could metabolize drugs and gut wall metab- 1631
1567 erholm et al., 1997; Tannergren et al., 2003a; Winiwarter et al., olism since these factors could directly affect regional absorption. 1632
1568 1998). The fabs for each drug is 30–41%, 40–60%, 55% and 5–10%, The dog is in many cases an acceptable model due to its similar- 1633
1569 respectively. These drugs have hydrophilic properties and their ity to man regarding anatomy, motility pattern, residence times 1634
1570 passive transcellular diffusion is expected to be low. Also, they and many secretory aspects (Dressman and Yamada, 1991; see also 1635
1571 are too large to be considered to have a significant paracellular up- canine section in this review). This is further verified by the 1636
1572 take, which is indicated by the low and incomplete fabs. A potential compilation of GI physiology data provided in Table 1. In addition, 1637
1573 explanation for the relatively high interindividual variability seen the size of dogs also allows for subsequent studies of formulations 1638
1574 in the permeability estimates is that there is a small difference developed for human use. The focus of the current work is to re- 1639
1575 between inlet and outlet concentration in the perfusate. For the view comparisons between man and dog regarding regional 1640
1576 two diuretics, there was also a strong induction of fluid flux into absorption allowing some general conclusions about suitability 1641
1577 the segment that might have affected the determination of the Peff and role of the dog model. In addition, the use of rat for prediction 1642
1578 value. of regional human drug absorption will also be briefly addressed. 1643
1579 Some human permeability data have been reported from
1580 studies in which a triple lumen tube was used to perfuse 80 cm
8.4.2. Dog model 1644
1581 segments in jejunum and ileum at a perfusion rate of 5 mL/min
Several, different methods have been applied for regional 1645
1582 (Sutcliffe et al., 1988). From these data, it can be speculated that
absorption studies in dogs including endoscopic or colonoscopic 1646
1583 the Peff values of these low permeability compounds are somewhat
methods (Sutton et al., 2006; Tajiri et al., 2010b), remote control 1647
1584 higher than that measured by the regional perfusion method with
capsules (Ishibashi et al., 1999b; Parr et al., 1999) and direct access 1648
1585 two balloons. This apparent discrepancy may be explained by pH
to the intestine through surgical access ports (Kim et al., 1994). 1649
1586 differences and/or by the open nature of the triple lumen perfusion
The most extensively published comparison between dog and 1650
1587 technique, which may have allowed absorption from a much long-
man covering the relative bioavailabilities following administra- 1651
1588 er segment than it actually was designed for (due to uncontrolled
tion in more distal parts of the GI tract, primarily the colon, versus 1652
1589 flow of perfusate in both directions). It is obvious that more explor-
administration to the proximal small intestine has been performed 1653
1590 atory in vivo studies are required in order to obtain reliable data on
by Sutton et al. (Fig. 8) (Sutton et al., 2006). Their study included 11 1654
1591 regional intestinal drug absorption. It is crucial to accurately deter-
compounds and showed a good correlation (r2 = 0.8) between dog 1655
1592 mine the regional intestinal Peff, as this information will contribute
and man regarding colonic relative bioavailability. In addition, the 1656
1593 to form the basis for the expected increase in in silico predictions of
relationship of relative colonic bioavailability between dog and 1657
1594 oral biopharmaceutics. It is suggested that it would be feasible to
man was close to 1:1. 1658
1595 use open, single-pass perfusion studies for the in vivo estimation
The study included drugs from all BCS classes. It was clear that 1659
1596 of regional intestinal Peff, but that care should be taken in the study
low permeability compounds consistently provided low relative 1660
1597 design to optimize the absorption conditions.
colonic bioavailability in both dog and man. Regarding low solubil- 1661
ity compounds with high permeability (Class II), two out of three 1662
1598 8.4.1.1. In vivo animal models for characterizing segmental/regional
compounds had relatively high colonic drug absorption in both 1663
1599 drug absorption. A MR dosage form administered after fasting con-
species. The greatest individual difference between dog and man 1664
1600 ditions reach the colon in most instances within 3–6 h (Follonier
was obtained for atenolol which had 67% relative colonic bioavail- 1665
1601 and Doelker, 1992). Thus, if longer duration of drug release and
ability in dog and only 15% in man. A similar investigation was 1666
1602 absorption is desired, drug absorption in colon is a prerequisite.
performed within AstraZeneca using dogs with colonic fistulas 1667
1603 In addition, for compounds with incomplete absorption in the
for direct administration to this site where a corresponding 1668
1604 small intestine, e.g., BCS class III, IV and certain class II drugs, some
correlation of r2 = 0.6 was achieved. However, high permeability 1669
1605 absorption may also occur in the more distal intestine for IR formu-
1606 lations. However, many drugs have too low and highly variable
1607 absorption in the colon to be suitable for extended release delivery
1608 (Wilding and Prior, 2003). In the worst case, poor colonic drug
1609 absorption may terminate the development of the MR product.
1610 Hence, a regional drug absorption assessed, preferable in humans,
1611 should be conducted prior to starting the MR formulation develop-
1612 ment. However, for new chemical entities it is desirable to evaluate
1613 regional absorption properties already in pre-clinical screening.
1614 There has been a clear progress in recent years to use in vitro
1615 tools for regional absorption assessment including permeability
1616 aspects (Sjöberg et al., 2013; Tannergren et al., 2009), solubility as-
1617 pects (Vertzoni et al., 2010), and drug degradation in colonic lumen
1618 by bacteria (Sousa et al., 2008). However, the advantage of in vivo
1619 models compared to in vitro testing is that they capture the com-
1620 plexity and dynamics of critical factors in the GI tract influencing
1621 drug release and absorption. The main limitation of the usage of
1622 animal models is that no single species resembles all physiological Fig. 8. Relationship between relative bioavailability in dogs and humans following
1623 properties of man, even if the rat model seems to be predictive oral and colon administration (Sutton et al., 2006).
Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
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1670 compounds had consistently higher relative bioavailability and low 9. Excipients effects 1734
1671 permeability compounds had lower values in agreement with the
1672 work by Sutton (Sutton et al., 2006). A couple of low permeability 9.1. Introduction 1735
1673 compounds, cimetidine and ranitidine, had significantly lower val-
1674 ues in man compared to the dog as was noticed for atenolol (Sutton Excipients encompass a wide range of properties that are of 1736
1675 et al., 2006). This supports the suggestion by Sutton that for smal- importance for the resulting drug product ensuring stability, con- 1737
1676 ler hydrophilic molecules, the dog colonic mucosa is somewhat tent uniformity and bioavailability of the incorporated APIs among 1738
1677 more ‘‘leaky’’ than in humans. Another interesting deviation be- many other factors. The total absorption of an API can be affected 1739
1678 tween dog and man in the AstraZeneca data set was for an internal by the combination of excipients included in the formulation. This 1740
1679 developmental compound where relative colonic bioavailability may be due to changes in the solid state of the API itself or the 1741
1680 was 48% and 100% in dog and man, respectively. This compound formulation characteristics of physical form (particle size, tablet 1742
1681 was identified to have intestinal Phase II metabolism indicating hardness, porosity, hydrophilic properties of the total matrix, 1743
1682 that the regional difference with less metabolism in colon in man etc.) or the interactions between the components of the product 1744
1683 is not reflected by the dog model. Tajiri and co-workers studied (Panakanti and Narang, 2012). 1745
1684 diclofenac, dilitiazem, cevimeline, felodipine, morphine, metfor- Excipients may chemically react with the API, and thereby neg- 1746
1685 min and felodipine in a dog colonic absorption model (Tajiri atively influencing its availability for absorption. Such unintended 1747
1686 et al., 2010a,b). Again a good correspondence was obtained with incompatibilities have been reviewed lately by Bharate et al. 1748
1687 human data where good absorption was obtained in man for all (2010). For example, lactose led to degradation of acyclovir (Mona- 1749
1688 compounds except metformin. The main deviation was obtained jjemzadeh et al., 2009), amlodipine (Abdoh et al., 2004), metformin 1750
1689 for felodipine which is almost completely absorbed from long act- (Santos et al., 2008) and other amine-compounds in compatibility 1751
1690 ing extended release formulations in man while colonic relative studies, whereas PVP led to degradation of oxprenolol (Botha and 1752
1691 bioavailability was only 30%. This may be due to differences in Lötter, 1990) and sulfathiazole (Voigt et al., 1984). Inclusion of 1753
1692 in vivo dissolution between dog and man since felodipine is a magnesium stearate has shown to decrease the stability of moex- 1754
1693 low solubility compound (water solubility 1 lg/mL). However, an ipril hydrochloride and beta-lapachone, potentially by increasing 1755
1694 alternative or complimentary explanation is the difference be- the humidity in the formulation (Cunha-Filho et al., 2007; Stanisz 1756
1695 tween the species of regional intestinal CYP3A4 metabolism. The et al., 2013). Conventional and modern QbD formulation develop- 1757
1696 latter explanation is also supported by other data from Tajiri for ment strategies will generally avoid such incompatibilities to occur 1758
1697 diltiazem, where the relative colonic bioavailability in dog was in drug products. 1759
1698 lower than for man at relevant doses (Tajiri et al., 2010b). Also data From a biopharmaceutics point of view, functional excipients 1760
1699 from Sutton for nifedipine, another drug with intestinal CYP3A4 may exert a number of well-known effects including enhancement 1761
1700 metabolism, showed lower relative colonic bioavailability in the of wettability, dissolution rates and even solubility of the incorpo- 1762
1701 dog compared to man, further supporting the possibility of species rated active ingredients. The solubility of an API is determined by 1763
1702 differences in regional intestinal metabolism. Recent work on gene its physical–chemical characteristics including its polymorphic 1764
1703 expression of enzymes and transporters in beagle dogs also con- form. This polymorphic form may be stabilized by addition of spe- 1765
1704 cluded that there are significant differences in distribution of CYP cific excipients (Singhal and Curatolo, 2004; Telang et al., 2009). 1766
1705 enzymes between dog and man (Haller et al., 2012). Particle size is a determinant for the dissolution rate and the 1767
1706 In conclusion, the dog model seems relevant with respect to effective surface area for dissolution may also be influenced by ex- 1768
1707 identifying drugs with permeability-limited colonic drug absorp- cipients (Vialpando et al., 2011). Chemical or physical derivation of 1769
1708 tion even though absolute levels for some drugs in this class seem the API itself may be obtained during production of the active 1770
1709 to be somewhat higher in the dog compared to man. However, this substance and/or of the finished product. See the section on models 1771
1710 is possible to predict by use of simpler in vitro methods for API-formulation approaches later in this review for more exam- 1772
1711 (Tannergren et al., 2009). Regarding solubility limitations, data ples in this context. 1773
1712 are complex, due to the influence of formulations and aspect of There are also less well-documented effects that may affect the 1774
1713 regional intestinal metabolism, which makes clear conclusions bioperformance of a drug product such as modulation of intestinal 1775
1714 hard to be drawn. Regarding regional variation of intestinal metab- transit times, interference with drug metabolizing enzymes and 1776
1715 olism, it seems that there is a clear species difference but this other constituents of the GI-tract, e.g. bile salts, which may in a un- 1777
1716 needs to be further elucidated. Overall, the dog has shown a ique way affect the absorption of a drug. Effects on physiological 1778
1717 reasonable good resemblance with humans regarding regional conditions and processes involved in drug absorption may lead 1779
1718 bioavailability data but if it offers any clear advantage with regard to changes in the absorption profile. 1780
1719 to predictive value on top of simpler in vitro/in silico approaches Excipient effects on bioavailability are best shown by in vivo 1781
1720 remains to be verified. bioavailability studies in humans. Knowledge of these excipient 1782
effects is especially relevant in the context of comparative studies: 1783
1721 8.4.3. Rat model formulation A versus formulation B containing different excipients. 1784
1722 Data on regional absorption comparison versus man is less well In many situations, comparative bioavailability studies are needed 1785
1723 studied for the rat despite the fact that it is a simpler model. The for new formulations to demonstrate equivalence. Such relative 1786
1724 work by Ungell et al. using rat tissue from different regions of the bioavailability/BE studies are not only relevant in the context of 1787
1725 GIT showed consistently a reduction in permeability in colon for generic oral drug products, but also in the cases of a change in 1788
1726 low permeability compounds whereas high permeability composition of an existing product. Understanding of intended or 1789
1727 compounds, if anything, had a more similar permeation across the unintended effects of excipients on the pharmacokinetics of a drug 1790
1728 entire GIT (Ungell et al., 1998). Thus, the rat model can be substance is particularly interesting from the perspective of waiv- 1791
1729 expected to be a better discriminating system for drugs with ing in vivo BE studies for IR dosage forms. 1792
1730 passive permeability limitations. However, as metabolism differs Current EU Guidance on the investigation of BE states that BE 1793
1731 between rat and man, both in regard of enzyme type and distribu- studies for oral solutions of multisource drug products may be 1794
1732 tion, it is not likely that the rat model will correctly capture such waived (EMA, 2010). However, if the excipients in the dosage 1795
1733 effects in a manner relevant for man (Komura and Iwaki, 2011). forms involved affect GI transit (e.g. sorbitol, mannitol), absorption 1796

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
 PHASCI 2973 No. of Pages 53, Model 5G
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1797 (e.g. surfactants or excipients that may affect transport proteins), Complex formation may reduce the availability of the API for 1861
1798 in vivo solubility (e.g. co-solvents) or in vivo stability of the active absorption and consequently reduce its bioavailability. For in- 1862
1799 substance, a BE study should be conducted, unless the differences stance, PEG 4000 formed insoluble complexes with phenobarbital, 1863
1800 in the amounts of these excipients can be adequately justified by leading to decreased permeation across excised rat intestine (Singh 1864
1801 reference to other data (EMA, 2010). et al., 1966). A reduction of bioavailability of the API was also ob- 1865
1802 A BCS-based biowaiver may be considered for BCS class I and III served when Tween 80 and sodium lauryl sulfate were combined 1866
1803 (in the EU) drug compounds. As a general rule, for both BCS class I with chlorpromazine and calcium salts and for magnesium carbon- 1867
1804 and III drug substances, well established excipients in usual ate with tetracycline (Chin and Lach, 1975; Nakano, 1971; Zak 1868
1805 amounts should be employed and possible interactions affecting et al., 1978). This might have been caused by a complex formed 1869
1806 drug bioavailability and/or solubility characteristics should be between the API and the excipient(s). Complex formation was also 1870
1807 considered and discussed. A description of the function of the ex- postulated as mechanism for the interaction of calcium sulfate 1871
1808 cipients is required with a justification whether the amount of with phenytoin. When calcium sulfate was replaced by lactose 1872
1809 each excipient is within the normal range. the bioavailability of phenytoin increased so that the maximal safe 1873
1810 The following overview will go through the physiological pro- plasma concentration was exceeded. However, additional studies 1874
1811 cesses that a solid oral dosage form encounters after oral adminis- did not confirm this mechanism and other authors suggested that 1875
1812 tration and give examples on how excipients may modulate these the higher hydrophilicity of lactose as compared to that of calcium 1876
1813 processes. It will discuss how the knowledge of these excipients is sulfate promoted increased dissolution of phenytoin. Nevertheless, 1877
1814 applied from a regulatory point of view and when mathematical other authors reported interactions of phenytoin with antacids and 1878
1815 models may be applied, especially in the context of biowaivers. summaries of product characteristics of phenytoin include a warn- 1879
ing that concomitant use of antacids or calcium salts may reduce 1880
1816 9.2. Modulation of drug release from dosage form: disintegration, the absorption of phenytoin (Bochner et al., 1972; Cacek, 1986; 1881
1817 dissolution and solubility Chapron et al., 1979; Garnett et al., 1980). 1882
The calcium sulfate-phenytoin data were correlated to result in 1883
1818 Drug release from the dosage form is essential for drug absorp- bioinequivalence. However, in other cases there is often a lack of 1884
1819 tion as this will make the API available for absorption. The use of information on the magnitude of the excipient effect on the 1885
1820 specific disintegrants to facilitate drug release is well-established in vivo bioavailability of a specific API. The FDA Inactive Ingredient 1886
1821 for IR oral dosage forms. For example, the inclusion of sodium Database includes levels of excipients used in combination with 1887
1822 bicarbonate as disintegrant in the formulation led to a more rapid the administration route (FDA, 2013). However, as the API included 1888
1823 absorption of ibuprofen in humans compared to a formulations in the concerned dosage form is not listed together with these 1889
1824 including aluminium hydroxide (Hannula et al., 1991). An amounts, it is not possible to correlate the study data directly to 1890
1825 enhancement in carbon dioxide production led to enhanced levels actually used or to deduce safe levels from it. Additional 1891
1826 in vivo disintegration of the capsule, enhanced in vivo dissolution information on the quantitative composition of bioequivalent 1892
1827 of the drug and enhanced gastric emptying rate. Examples of excip- products would be necessary to conclude on safe levels in combi- 1893
1828 ients that have been shown to improve in vitro dissolution rates nation with specific APIs. 1894
1829 are microcrystalline cellulose, D-glucosamine hydrochloride and
1830 PEG 6000 (Al-Hamidi et al., 2010; Alsaidan et al., 1998; Vijaya 9.3. Modulation of stomach physiology 1895
1831 Kumar and Mishra, 2006). However, some super disintegrants have
1832 been described to interact with drugs, leading to decreased in vitro The characteristics and volume of the pharmaceutical product 1896
1833 dissolution, although these interactions do not seem to affect the as present in the stomach will determine the subsequent process 1897
1834 in vivo performance of the formulation (Fransén et al., 2008; Na- steps. The authors are not aware of excipients that have been 1898
1835 rang et al., 2012). shown to change the function of the stomach physiology i.e. having 1899
1836 Excipients can also improve the solubility and dissolution of APIs an effect on the volume of the stomach itself or of the mechanistic 1900
1837 via different mechanisms, e.g., solubilization, precipitation and aspects of stomach motility. Excipients affecting the luminal condi- 1901
1838 supersaturation, as addressed elsewhere in this review. Cyclodex- tions including the stomach content are discussed below. The vol- 1902
1839 trins have for example been studied with the aim to increase the sol- ume of the excipients, their liquid or solid state and perhaps also 1903
1840 ubility and thereby the bioavailability of in vivo in different animal the effects on the viscosity of the stomach content may affect the 1904
1841 models by complexation of the drugs, e.g., griseofulvin, cinnarizin, gastric emptying time. See description of the stomach physiology 1905
1842 glibenclamide, ibuprofen and nifedipine (Dhanaraju et al., 1998; Jar- in this review for information on relevant mechanisms in this 1906
1843 vinen et al., 1995) (Emara et al., 2002; Nambu et al., 1978; Savolai- context. 1907
1844 nen et al., 1998). In regulatory practice however, cyclodextrins are Strategies to increase residence time of an API in the stomach 1908
1845 not widely applied. The FDA Inactive Ingredients Database lists only include the use of bioadhesive microspheres that slow intestinal 1909
1846 one oral preparation in which hydroxypropyl-b-cyclodextrin is transit and gastroretentive dosage systems (Ahmed and Ayres, 1910
1847 applied and seven parenteral products including cyclodextrines 2007; Davidovich-Pinhas and Bianco-Peled, 2010; Davis, 2005; 1911
1848 (FDA, 2013). Having said that, a number of cyclodextrin-containing Prajapati et al., 2013). Small particles with bioadhesive properties 1912
1849 oral dosage forms are available in worldwide formularies (Davis and and sometimes also floating capacity (on stomach content) are 1913
1850 Brewster, 2004). used to physiologically and physically reduce the gastric emptying. 1914
1851 On the other side, several studies have shown that excipients Positively charged molecules are thought to adhere to the nega- 1915
1852 may also negatively affect the solubility of an API. In a recent study tively charged sialic acid groups on the gastric mucus. Strategies 1916
1853 an interaction between SLS with intrinsic intestinal mixed micelles to enhance gastroretention may also make use of an enlarged ob- 1917
1854 composed of bile salts and lecithin has been shown (Buch et al., ject size and include swelling and floating agents. An example of 1918
1855 2010, 2009). The interaction led to a destruction of the micellar such a formulation are superporous hydrogels formed in situ from 1919
1856 structure and a decrease in solubility of the low water soluble polyethylene oxide with swelling capacity in combination with 1920
1857 fenofibrate by SLS. Thus, instead of increasing the solubility of hydroxypropyl methylcellulose (HPMC) as sustained release 1921
1858 the drug by the addition of surfactant, the opposite effect was pro- matrix have been investigated (Kousar et al., 2013). 1922
1859 voked with a concomitant decrease in the bioavailability of the SLS Formulations including gastroretentive mechanism are usually 1923
1860 containing fenofibrate formulations (Buch et al., 2010, 2009). tested based on their specific physical characteristics using 1924

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
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1925 in vitro techniques which may include in vitro release testing as 9.6. Modulation of GI transit and motility 1964
1926 well as floating/buoyancy tests, the latter demonstrating the
1927 capacity of the dosage form to float on the gastric contents. How- The residence time and transit speed of an API are determined 1965
1928 ever, an in vivo performance test in human or animal models is by the GI motility, which can be influenced by e.g., food intake or 1966
1929 usually needed. Such investigations are generally carried out using composition of the formulation. 1967
1930 radiology or scintigraphy visualization, endoscopic techniques or Examples of excipients affecting gastric emptying time are de- 1968
1931 magnetic monitoring to show the location of the formulation or scribed above. Another commonly discussed phenomenon is the 1969
1932 alternatively 13C octanoic acid breath testing to determine the gas- reduction of the SITT and the consequences for absorption of 1970
1933 tric residence time (Prajapati et al., 2013). See also the section drugs (Yuen, 2010). Well-known excipients that reduce the intes- 1971
1934 above on imaging techniques. tinal transit time in humans include the alcohol monosaccharides 1972
mannitol and xylitol as well as lactulose (Adkin et al., 1995a,b; 1973
1935 9.4. Modulation of intestinal surface area Read et al., 1982; Salminen et al., 1989; Staniforth, 1989). Other 1974
examples are sodium acid pyrophosphate, polyethylene glycol 1975
1936 Morphological changes to the structure of the intestinal surface and sorbitol (Adkin et al., 1995a; Basit et al., 2001, 2002; Chen 1976
1937 of the GI tract may change the absorption of drugs by changing the et al., 2007; Chusid and Chusid, 1981; Islam and Sakaguchi, 1977
1938 effective surface for absorption. Surfactants have been shown to 2006; Koch et al., 1993; Payne et al., 1997; Schulze et al., 1978
1939 cause (reversible) mucosal damage resulting in enhanced absorp- 2003). The EMA pointed out that, since there is no information 1979
1940 tion of co-administered APIs. Rat intestinal perfusion studies also on the actual threshold of an effect of sorbitol on the PK of highly 1980
1941 showed that co-administration of a mucolytic agent and a non-io- permeable drugs, strict compliance with the BE guideline is rec- 1981
1942 nic surfactant improved the intestinal absorption of poorly ommended, i.e., quantitative differences are not accepted in the 1982
1943 absorbed hydrophilic (Swenson et al., 1994; Takatsuka et al., context of biowaivers (EMA, 2010, 2013). Other excipients that 1983
1944 2006, 2008) compounds. Histological evaluation also showed are known to enhance GI motility are mentioned in Table 5. 1984
1945 mucosal damage which is therefore thought to play a role in the There are also excipients that increase the intestinal transit time 1985
1946 observed absorption enhancement. See also below for more exam- such as oleic acid and lipids in general (Dobson et al., 1999; 1986
1947 ples of permeability enhancers. Martinez et al., 1995; Pilichiewicz et al., 2006). Formulations 1987
containing large amounts of lipids, like self-emulsifying systems, 1988

1948 9.5. Modulation of pH of the GIT liposomes or micelles have the potential to modulate of 1989
intestinal transit times, especially for digestible lipids (Porter 1990

1949 Excipients may interact with the luminal components and et al., 2004). 1991

1950 thereby indirectly influence the dissolution and absorption of an

1951 API. For example, sodium bicarbonate increased the bioavailability 9.7. Modulation of GI fluids 1992
1952 of the acid-labile drug erythromycin acistrate in humans (Marvola
1953 et al., 1991), probably due to an increase in the gastric pH. The Excipients may also affect the composition of the matrix in 1993
1954 effects of excipients on the gastric pH could be simulated by which the drug is transported through the GI tract. Lipid excip- 1994
1955 in vitro tests, for example by simulation based on known com- ients in conjunction with the role of the animal model and its 1995
1956 pounds pH-stability profiles and the assumed residence times in gastrointestinal fluid composition in characterizing the perfor- 1996
1957 the stomach. There also exist artificial stomach-duodenum models mance of lipid excipients containing dosage forms has been 1997
1958 which incorporate the stomach to duodenum transit step with a reviewed elsewhere (Hauss, 2007). Mucus formation may also 1998
1959 concomitant shift in pH (Carino et al., 2010). However, so far be influenced by the presence of excipients; mucolytic agents 1999
1960 animal and human studies provide the best measures of these are discussed above in the context of the intestinal surface area. 2000
1961 potential effects. In the extrapolation from animal to humans it Osmotic effects are discussed in the section on transit time, but 2001
1962 is however important to account for differences in animal GI phys- could also be considered as an effect of modulation of the GI 2002
1963 iology compared to man, as described in the sections above. fluid content. 2003

Table 5
Examples of modulators of residence time and/ or transit speed.

Excipient/modulator Drug/marker Model Observed effect Reference

compound
Sodium alginate/karay gum gel Barium sulfate Rat/mice Gastric retention Foster et al. (2012)
Gellan and sodium alginate Paracetamol Rabbit and Rat Gastric retention Kubo et al. (2003)
Unfolding multilayer polymeric films Riboflavin Beagle dogs Gastric retention Klausner et al. (2002)
Swellable polymer films Riboflavin Dogs and Gastric retention Ahmed and Ayres (2007)
Human
Polycarbophil with albumin Chlorothazid Human Gastric retention Longer et al. (1985)
Poly(acrylic acid), in gelatin Oxprenolol Rat Gastric retention Preda and Leucuta (2003)
microspheres hydrochloride
Sodium bicarbonate Aluminium hydroxide Human Reduced gastric emptying time Hannula et al. (1991)
Sodium acid pyrophosphatee Ranitidine Human Decreased small intestinal transit Adkin et al. (1995a), Koch et al. (1993)
time
Alcohol monosaccharides mannitol, – Human Decreased small intestinal transit Adkin et al. (1995a,b), Read et al. (1982),
xylitol and lactulose time Salminen et al. (1989), Staniforth (1989)
PEG 400 Ranitidine Human Basit et al. (2001, 2002), Schulze et al. (2003)
Sorbitol Ranitidine and Human Chen et al. (2007), Chusid and Chusid (1981),
metoprolol Islam and Sakaguchi (2006), Payne et al. (1997)
Oleic acid – Human Increased intestinal transit time Dobson et al. (1999)
Lipids – Human Enhanced gastrointestinal Martinez et al. (1995), Pilichiewicz et al. (2006)
motility

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E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx 23

2004 9.8. Modulation of metabolism and intestinal degradation processes 9.9.1. Paracellular route: tight junction modulation 2066
Several authors reviewed possibilities for absorption enhance- 2067
2005 Pharmaceutical excipients may interact with metabolic ment by the paracellular pathway (Cano-Cebrian et al., 2005) and 2068
2006 enzymes in the GI tract. Particularly nonionic surfactants and the implication of tight junction modulation for drug delivery 2069
2007 polymers have been shown to inhibit CYP activity. Examples of ex- (Salama et al., 2006). Numerous potential modulators have been 2070
2008 cipients with documented in vitro effect on CYP3A include different investigated with the purpose of increasing permeability by open- 2071
2009 types of polyethylene glycol, Tween, Cremophor, Triton x, SLS, sol- ing tight junctions. For many of these compounds, the dose needed 2072
2010 utol, Lecithin and Vit C (Bittner et al., 2002; Bravo Gonzalez et al., to reach an effect was so high that cytotoxicity limited the applica- 2073
2011 2002; Christiansen et al., 2011; Rao et al., 2010; Ren et al., 2009, tion and further explorations were stopped. This applied for exam- 2074
2012 2008; Tompkins et al., 2010; Wandel et al., 2003). PEG 400 also ple for calcium chelator EDTA, sodium dodecyl sulfate, 2075
2013 inhibited CYP3A metabolism of Digoxin and Verapamil in excised cytochalasins, saponines and acylcarnitines. These compounds 2076
2014 rat jejunum (Johnson et al., 2002). Another type of excipient, were tested mainly in cell culture models like Caco-2 and/or on 2077
2015 b-cyclodextrin inhibited CYP3A4 and CYP2C19 in vitro on cDNA ex- intestinal mucosa (Maher et al., 2008), but some also in animal 2078
2016 pressed human cytochrome P-450 (Ishikawa et al., 2005). As studies (see above at modulation of the intestinal surface). 2079
2017 shown by the above examples, the excipients vary in their inducing Some permeability enhancers, such as medium-chain amphi- 2080
2018 or inhibiting effects on CYPs. It is also conceivable that excipients pathic fatty acid like sodium caprate and polymeric enhancers like 2081
2019 affect other metabolising enzymes than CYPs, e.g., UGTs and SULT. chitosan and its derivatives, carbomers and thiolated polymers, 2082
2020 In vitro digestion models are available that simulate the digestion reached the phase of in vivo studies in pre-clinical species, e.g., rats, 2083
2021 of formulations in (parts of) the GI tract (Dahan and Hoffman, pigs and dogs, as well as in the clinic (Cano-Cebrian et al., 2005; 2084
2022 2008; Mohsin, 2012; Versantvoort et al., 2004), but publications Maher et al., 2008; Thanou et al., 2001b). 2085
2023 in the context of excipient effects are limited. In vitro tests to study Permeability can also be enhanced by making use of the struc- 2086
2024 specific enzyme interactions also exist, but are mainly used in the tural similarity of the active substance and the enhancers. The 2087
2025 context of drug–drug interactions (EMA, 2012). Generally there is a absorption process of peptide drugs may be improved by 2088
2026 lack of in vivo data on excipients inhibition effects, in particularly peptide-based permeability enhancers, which being proteins 2089
2027 in humans, so extrapolations of in vitro effects to the in vivo situa- themselves, share physicochemical properties, diffusion character- 2090
2028 tion are difficult. istics and stability issues with the therapeutic proteins being 2091
2029 A combination of carboxymethyl-starch excipients and enzyme delivered (Maher et al., 2008). However, oral formulations are 2092
2030 inhibitors may enhance the gastroresistance and stability against not available as far as known to the authors of this paper. Clinical 2093
2031 gastrointestinal enzymes of an active substance in the GIT (De Kon- studies suggest that there is no toxicity of concern and the absorp- 2094
2032 inck et al., 2010; Nassar et al., 2008). In addition to their potential tion-promoting effects were transient and complete in <1 h. Other 2095
2033 role in increasing the solubility of the API as discussed above, cy- examples of compounds which have been studied for their effects 2096
2034 clodextrines may form a non-degradable complex with the API on the paracellular transport are listed in Table 6. 2097
2035 thereby protecting it from degradation and increasing its bioavail-
2036 ability (Challa et al., 2005). 9.9.2. Transporter-mediated absorption 2098
The influence of excipients on transporter-mediated absorption 2099
(Table 7) has lately been reviewed by Grube and Langguth and by 2100
2037 9.9. Modulation of membrane transport Goole et al. (Goole et al., 2010; Grube and Langguth, 2007). Maher 2101
described cell-penetrating peptides (CPP’s) that have been studied 2102
2038 Polarity of the molecule and characteristics like particle size to improve the delivery of protein drugs in the target cells (Maher 2103
2039 determine the intrinsic permeability of the API. These aspects et al., 2008). These cationic amphipathic peptides include poly-L- 2104
2040 could also be modulated in presence of specific excipients. lysines, poly-L-arginine. These two peptides were mainly studied 2105
2041 Micronisation to microparticles or even nanoscale particle size for delivery in the nasal or tracheal epithelial cells. Transportan 2106
2042 increases the contact surface of the active substance with the and penetratin are two other CPP’s having cell-penetrating effects 2107
2043 intestinal membrane. Ross and Toth reviewed the use of micro- of which analogs are thought to be potentially interesting perme- 2108
2044 particles and nanoparticles containing heparins that enlarged ability enhancers. 2109
2045 the contact surface of the heparin (Ross and Toth, 2005). Biode-
2046 gradable poly-caprolactone and poly(lactic-co-glycolic acid) and 9.9.3. Passive diffusion and endocytosis 2110
2047 nonbiodegradable positively charged polymers such as Eudragit Passive diffusion could be affected by changes in either the 2111
2048 RS and RL were reported to enhance the absorption of heparin. apical or the basolateral membrane. Guan et al. describe an inves- 2112
2049 The polymers were used alone or in 1:1 combination and both mi- tigation of the mechanisms of improved oral bioavailability of 2113
2050 cro- and nanoparticles were shown to increase the bioavailability bergenin by complexation of bergenin with phospholipid and con- 2114
2051 of heparin. The mechanism of the nanoparticles absorption clude that the complex could transport across enterocytes by both 2115
2052 enhancement was however not determined. Ross and Toth also passive diffusion and active transport by receptor-mediated endo- 2116
2053 described how polyanionic low molecular weight heparins cytosis (Guan et al., 2013). The authors used experimental models 2117
2054 (LMWH) were paired with polycationic lipophilic-core dendrons such as the ex vivo everted rat gut sac model and in vitro Caco-2 cell 2118
2055 (PLCDs) (Ross and Toth, 2005). Lipoamino acids were designed monolayers and the effect was limited (Guan et al., 2013). 2119
2056 to increase the lipophilicity of the complex. These PLCDs may also
2057 act by perturbing the cell membrane. Several successful studies in 9.10. Models for testing excipient effects and their application for 2120
2058 which the concept of lipophilic ion-pairing with diamines, triam- biowaivers 2121
2059 ines and lipoamino acids was shown were summarized (Ross and
2060 Toth, 2005). However, according to the authors, the variety of To study the effects of excipients on the bioavailability the 2122
2061 models (in vivo models like rat oral gavage, rat intraduodenal, pharmaceutical industry and academia apply different techniques, 2123
2062 mouse oral gavage, pig intraduodenal, rabbit oral gavage, monkey each with their own limitations. 2124
2063 oral gavage, dog oral gavage and also Caco-2 cell monolayers and The current regulatory biowaiver guidance limits itself to the first 2125
2064 Ussing chamber) made it difficult to compare the results of step in the GI absorption of the API from an immediate release solid 2126
2065 different studies. oral dosage form: drug release (EMA, 2010). As the review above 2127

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in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
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24 E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx

Table 6
Examples of paracellular transport modulators and marker compounds.

Excipient/modulator Drug/marker Model Observed effect Reference

compound
EDTA PEG4000 Caco-2 Enhanced permeability Tomita et al. (1994)
EDTA Iron Caco-2 Increased absorptoin Kibangou et al. (2008)
Sodium caprate Macromolecules Intestinal cells Increased permeation Krug et al. (2013)
Sodium decanoate Antisense Pigs Enhanced bioavailability Raoof et al. (2002)
oligonucleotides
Sodium decanoate Cefmetazole In situ loop study Increased jejunal Tomita et al. (1992)
absorption
Sodium decanoate Ampicillin Human Absorption after rectal Lindmark et al. (1997)
administration increased
Sodium decanoate in GIPET Acycline Human Oral Amory et al. (2009)
Palmitoyl carnitine Mannitol and PEG Caco-2 and IEC-18 Enhanced permeability Duizer et al. (1998)
4000
Palmitoyl carnitine Cefoxitin Dogs and rats Enahanced bioavailability Sutton et al. (1993)
Chitosan-coated nanoparticles Insulin Rats Lin et al. (2007)
Chitosan Oxaprozin Caco-2 Enhanced permeability Maestrelli et al. (2011)
Chitosan hydrochloride and glutamate salts Peptides In vitro Enhanced absorption Kotzé et al. (1997)
Chitosan hydrochloride Rats Enhanced absorption Luessen et al. (1996)
Trimethyl chitosan Octreotid acetate Pigs Intraduodenal application Thanou et al. (2001c)
increased bioavailablity
N-sulfonato-N,O-carboxymethylchitosan Macromolecules Caco-2 Enhanced permeation Thanou et al. (2007)
Mono-N-carboxymethyl chitosan Low molecular In vitro and in vivo rat Increased intestinal Thanou et al., 2001a
weight heparin model absorption
N-trimethyl chitosan (TMC) Insulin loaded Caco-2 and excised rat Increased permeation Sandri et al. (2010)
nanoparticles jejunum
N-trimethyl chitosan (TMC) Buserelin and Rat and pig Enhanced bioavailability Thanou et al. (2007, 2001c)
octreotid acetate
Sodium lauryl sufate Cefradoxil Rat duodenum Enhanced absorption Sancho-Chust et al. (1995)
Tetradecylmaltoside Enoxaparin C2BBe1-cells and Rats Enhanced absorption Yang et al. (2005)
Octylglucoside Insulin Caco-2 and T84 Enhanced permeation Tirumalasetty and Eley (2006)
monolayers
Chenodeoxycholates Oligonucleotides Rat jejunum and ilem Enhanced paracellular Tsutsumi et al. (2008)
diffusion
Sodium taurocholate Insulin Caco-2 Increased permeation Degim et al. (2004)
Thiolated polycarbophil/glutathione FITC dextran and Excised rat jejunum Perera et al. (2011)
sodium fluorescein
Other thiolated polymers (e.g. chitosan-cysteine Hydrophilic In vitro Enhanced permeability Bernkop-Schnurch et al. (2004),
and chitosan-4-thio-butylamidine) compounds Bernkop-Schnürch et al. (1999)
Ò
Butylated methacrylate copolymer (Eudragit E ) Mannitol, Talinolol, Caco-2 Enhanced permeability Grube et al. (2008)
and Trospium

2128 shows, animal models are commonly applied by pharmaceutical disintegration or dissolution could be tested by the pharmacopoe- 2155
2129 industry to test differences in drug release from pharmaceutical ial models; however, no specific excipient information or other de- 2156
2130 formulations. However, in the context of BE questions, these models tails are given on the evaluation of any test outcome. 2157
2131 do play a limited role: animal models are not approved as models for
2132 biowaivers. From a regulatory perspective, a human volunteer is the 9.11. Gap analysis and consequences for biowaivers 2158
2133 only acceptable ‘model’ for comparative bioavailability testing
2134 when biowaiver conditions are not met and in absence of an Excipients may theoretically affect the GI absorption of API in 2159
2135 in vivo in vitro correlation of the dissolution versus plasma data. many ways. However, the in vivo relevance of data obtained by 2160
2136 Cell culture models for testing permeability effects are models is not always clear. 2161
2137 well-known and in vitro digestion models and enzyme interaction In addition, the current usability of the available models to 2162
2138 models also exist. However, these are not approved to confirm BE detect excipient effects in the context of regulatory applications 2163
2139 of formulations either. The GI transit of APIs can be studied using in the EU is limited. Only one in vitro model is sufficiently validated 2164
2140 different in vivo techniques, but comparative in vitro models vali- and approved from a regulatory point of view: the dissolution test. 2165
2141 dated for their biorelevance of this parameter are not known to However, many other models exist and many relevant data on 2166
2142 the authors. In conclusion, apart from the dissolution test, there excipient effects are available at pharmaceutical industry. Little 2167
2143 is no comparative in vitro test model validated and approved for of this knowledge obtained in drug development studies is 2168
2144 comparative testing of disposition effects. currently translated into regulatory guidance on excipients. 2169
2145 The regulatory guidance is limited as to allowed (difference in) Authorities may, therefore, seem unnecessary restrictive in the 2170
2146 levels of excipients: as a general rule for BCS class I and III drug acceptance of differences in excipients. However, it should also 2171
2147 substances well-established excipients in usual amounts should be noted that authorities do not dispose of these, mostly confiden- 2172
2148 be employed and possible interactions affecting drug bioavailabil- tial, company data and development of regulatory acceptable mod- 2173
2149 ity and/or solubility characteristics should be considered and dis- els depends on the availability of public data and shared 2174
2150 cussed. Even in the case of Class I drugs it is advisable to use knowledge. 2175
2151 similar amounts of the same excipients in the composition of test The available guidance on biowaivers shows that EU regulators 2176
2152 like in the reference product. If a biowaiver is applied for a BCS are in principle open to submissions including adequate justifica- 2177
2153 class III drug substance, excipients have to be qualitatively the tion of full BCS based biowaivers or waivers in the context of a 2178
2154 same and quantitatively very similar (EMA, 2010). Effects on change in composition of the product. For BCS class I biowaivers 2179

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
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Table 7
Examples of modulators of transporter mediated absorption.

Excipient/modulator Drug/marker compound Model Observed effect Reference

Acconon E Digoxin Everted rat gut sac Increased uptake Cornaire et al. (2004)
Cholesterol cytotoxic agents ? ATPase activity of P-gp Shu and Liu (2007)
Cremophor EL Digoxin Everted rat gut sac Increased uptake Cornaire et al. (2004)
Cremophor EL Rhodamine 123 Caco-2 and rat intestinal Increased transport Rege et al. (2002)
membranes
Cremophor EL Taxol Caco2 Increased transport Hugger et al. (2002b)
Cremophor RH 40 Digoxin Human Reduced bioavailability Tayrouz et al. (2003)
Imwitor 742 Digoxin Everted rat gut sac Increased uptake Cornaire et al. (2004)
Labrasol Celiprolol Everted rat gut sac Increased uptake Cornaire et al. (2004)
Miglyol Digoxin Everted rat gut sac Increased uptake Cornaire et al. (2004)
MS-310, MO-310 and MS-500 Ceftibuten BBM Vesicles Increased uptake Koga et al. (2000)
N-dodecyl-b-D-maltopyranoside Rhodamine 123 Rat intestinal membrane Increased transport Shono et al. (2004)
PEG 300 Taxol Caco-2 and MDR1-MDCK Increased transport Hugger et al. (2002a)
cells
PEG 400 Digoxin Rat jejunal tissue Inhibition of efflux Johnson et al. (2002)
Pluronic L61 Vinblastine LLC-MDR1 cells Increased transport Evers et al. (2000)
Different types of Pluronics Rhodamine 123 KBv cells Increased uptake Batrakova et al. (1999a)
Pluronic P85 Different substances Caco-2- and BBMEC-cells Increased transport Batrakova et al. (1999b)
Polysorbate 20 Digoxin Everted rat gut sac Increased uptake Cornaire et al. (2004)
Polysorbate 20 Ceftibuten BBMV vesicles Increased uptake Cornaire et al. (2004)
Polysorbate 40 Ceftibuten BBMV vesicles Increased uptake Koga et al. (2000)
Polysorbate 80 Rhodamine 123 and other Caco2-cell-monolayers and Increased transport Rege et al. (2001)
substances rat intestinal membrane
Sodium lauryl sulfate Different substances Caco-2 Increased transport Cornaire et al. (2004)
Softigen 767 and SS-500 Digoxin Everted rat gut sac Increased uptake Cornaire et al. (2004)
TGPS 1000 Talinolol Caco-2 and human Increased trasnport and Rege et al. (2002)
increased AUC
TGPS Colchicine and doxorubicin G185 cells Decreased permeation Dintaman and Silverman (1999)
TGPS 800 and TS-500 Ceftibuten BBMV vesicles Increased uptake Koga et al. (2000)

2180 ‘qualitative differences’ are acceptable, if appropriately justified Mills et al., 2005; Rodriguez-Fragoso et al., 2011; Schmidt and 2213
2181 and provided that excipients that might affect the bioavailability Dalhoff, 2002; Singh, 1999; Welling, 1977; Won et al., 2010, 2012). 2214
2182 are qualitatively and quantitatively the same. In case of a change Food effects are derived from two basic principles of mecha- 2215
2183 in composition, ‘minor changes’ in excipient content may be ac- nisms: the impact of the meal’s content itself and the postprandial 2216
2184 cepted based on dissolution data only i.e. without addressing other changes of GI physiology. From a physiological perspective, food 2217
2185 steps in the absorption process. In such a case, the classification of intake, compared to the fasted state, can particularly provoke 2218
2186 the change as ‘minor’ is to be assessed on a case-by-case basis. (Fleisher et al., 1999; Welling, 1977): 2219
2187 To avoid the need for comparative testing of each difference in
2188 composition and clarify the classification of changes as ‘minor’ or  Changes of visceral blood and lymph flow. 2220
2189 ‘major’, publication of information of the effects of specific  Intraluminal composition in qualitative and quantitative terms. 2221
2190 excipients in combination with specific APIs seems useful. Formu-  Time- and region-specific alterations of hydrodynamics and 2222
2191 lation development handbooks advise on usual concentrations of mechanical forces within the GI tract. 2223
2192 well-known excipients but do not indicate ‘no effect levels’ of  Modulation of drug metabolizing enzymes and transporters 2224
2193 excipients or ‘safe windows’ in the context of their effect on the resulting in distinct absorption, distribution and elimination 2225
2194 bioavailability of active substances. And, information on the quan- characteristics. 2226
2195 titative composition of pharmaceutical products is confidential 2227
2196 while actual levels of excipients are not clear in the EU. So far, Despite these physiological and physicochemical variations, 2228
2197 the FDA Inactive Ingredients Database is used by some authors to based on the BCS, several reviews stated the absence of food effects 2229
2198 refer to actually approved levels of excipients, e.g. in the biowaiver on the oral drug bioavailability for BCS class I compounds (Custo- 2230
2199 monographs published by the FIP on http://www.fip.org/bcs. A dio et al., 2008; Fleisher et al., 1999). Likewise, the positive effect 2231
2200 public database on excipient effects could lead to cut-off values of high-fat meals on the absorption of non-ionizable and weak 2232
2201 for specific excipient effects and may allow building up ‘safe acidic BCS class II drugs in immediate-release formulations seems 2233
2202 excipient ranges’ or a ‘safe space’ per excipient. In addition, such to be predictable (Custodio et al., 2008; Fleisher et al., 1999). 2234
2203 database could clarify which excipients are known to physiologi- Including transporter-related aspects according to the BDDCS, Cus- 2235
2204 cally affect the bioavailability of APIs and identify ‘suspect excipi- todio et al. (2008) hypothesized distinct trends to be expected for 2236
2205 ents’ or ‘bioavailability modulators’ in the context of biowaivers. the oral bioavailability of BDDCS class I and II compounds with 2237
concomitant food intake (Custodio et al., 2008). For modified-re- 2238
2206 10. Food effects lease formulations, BCS class III and IV drugs, however, the inter- 2239
play of various parameters in the fed state can yield more 2240
2207 10.1. Introduction complex scenarios – in vivo models could be of great value in these 2241
cases, presuming that the species characteristics regarding certain 2242
2208 Altered human drug PK in the presence of food (including dietary physiological parameters are kept in mind. 2243
2209 supplements and nutraceuticals) has been extensively examined Categorizing different effects of food based on the biopharma- 2244
2210 from a scientific and regulatory perspective (Abdel-Rahman et al., ceutical processes that are affected, the next paragraphs of this re- 2245
2211 2011; Boullata and Hudson, 2012; Chan, 2002; Custodio et al., view focus on the use of common laboratory animals to evaluate 2246
2212 2008; Fleisher et al., 1999; Genser, 2008; Joshi and Medhi, 2008; food effects in vivo. 2247

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
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26 E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx

2248 10.2. In vivo assessment of food effects on the pharmacokinetics of needed to return to basal acidity after food digestion is significantly 2313
2249 orally administered drugs longer in monkey than in human (Chen et al., 2008; Kondo et al., 2314
2003a). This was so even after replacing the standard biscuit-type 2315
2250 10.2.1. Disintegration, dissolution, diffusivity and intra-luminal meal by fruits or jelly type food, respectively (Kondo et al., 2003b). 2316
2251 reactions Summarizing the available reports, the effect of food on drug sol- 2317
2252 Modified dissolution and precipitation characteristics via solu- ubility and stability has been vastly investigated, mostly using dogs, 2318
2253 bilization (Charman et al., 1993; Rolan et al., 1994), pH alteration GI aspirates and perfusion studies, whereas the dietary impact on 2319
2254 (Carver et al., 1999; Zimmermann et al., 1994) and reactions with formulation integrity and diffusivity of the released drug to the GI 2320
2255 meal components (Huupponen et al., 1984; Jung et al., 1997; mucosa is scarcely described for any species. There is evidence that 2321
2256 Neuvonen et al., 1991) have been proposed to explain changes of the poor correlation of food-induced enhancement of solubility 2322
2257 human drug pharmacokinetics in the presence of food. To elucidate with dissolution rates in vivo might be ascribed to reduced diffusiv- 2323
2258 these mechanisms, intestinal perfusion techniques can be of great ity of the forming mixed micelles (Charman et al., 1997; Lennernäs, 2324
2259 value. Fuse et al. (1989), for example, investigated the influence of 2007c). Moreover, studies in dogs and humans implicated that post- 2325
2260 pectin as dietary fiber by means of intestinal perfusion in rats and prandial increased viscosity in the upper GI tract modulated 2326
2261 humans, concluding that not binding of bile acids, but expansion of absorption profiles by decreasing gastric emptying times, dissolu- 2327
2262 the unstirred water layer is the major factor leading to decreased tion rates and diffusivity of drugs with pronounced region-specific 2328
2263 absorption of linoleic acid and glucose (Fuse et al., 1989). With a absorption (Pao et al., 1998; Reppas et al., 1998). Hence, combining 2329
2264 porcine intestinal perfusion model, Persson et al. (2008) showed methods to assess mechanical forces (Marciani et al., 2001b; McIn- 2330
2265 that solubilization rather than P-gp inhibition is responsible for nes et al., 2008), media conditions e.g. viscosity (Marciani et al., 2331
2266 the positive food effect observed for danazol as a model compound 2000, 2001b, 1998) and fluid volumes (Schiller et al., 2005) as well 2332
2267 for low solubility drugs (Persson et al., 2008). as in vivo dosage form performance (Sutton, 2004) should be con- 2333
2268 Among whole animal models, the dog model seems most sidered in future in vivo food effect studies. 2334
2269 appropriate to investigate food effects on the disintegration and
2270 dissolution of oral solid formulations (Abrahamsson et al., 2004; 10.2.2. Permeability and region-specific absorption 2335
2271 Wu et al., 2004), due to physiological and dosage form related With respect to para- and transcellular transport, food may 2336
2272 advantages as well as the suitability to receive infrequent large modulate intestinal permeability by direct, bile salts-induced or 2337
2273 meals on command (Lentz, 2008; Sutton, 2004). In contrast, over- microflora-mediated changes in membrane integrity and fluidity 2338
2274 prediction of the solubility enhancing impact of food (Campbell (Bagchi et al., 1998; Kvietys et al., 1991; Ten Bruggencate et al., 2339
2275 and Rosin, 1998; Humberstone et al., 1996; Paulson et al., 2001; 2006), altering the pH dependent extent of ionization (Charman 2340
2276 Xu et al., 2012) and insufficient differentiation between fasted et al., 1997; Marasanapalle et al., 2009), forming mixed bile salt 2341
2277 and fed state with respect to dose dumping behavior of a matrix micelles thus reducing the free fraction of lipophilic drugs 2342
2278 tablet formulation have been reported (McInnes et al., 2008). Con- (Charman et al., 1997; Poelma et al., 1991) and increasing fluid flux 2343
2279 sequently, higher bile salts level (Carlsson et al., 2002; Dressman, facilitated passive diffusion (Kitazawa et al., 1978; Lane et al., 2344
2280 1986; Kalantzi et al., 2006), lower gastric acidity in fasted state 2006; Lu et al., 1992; See and Bass, 1993). The latter has been sub- 2345
2281 opposing lower postprandial pH (Dressman, 1986; Lui et al., ject of controversial discussion since contrary to data generated in 2346
2282 1986; Mahar et al., 2012; Sagawa et al., 2009) (Table 1) and higher rodent models, the nutrient-induced solvent drag effect on 2347
2283 mechanical forces in the in the stomach (Kamba et al., 2001, 2002) intestinal permeability has not always been observed in human 2348
2284 are some physiological factors that need to be considered when (Fagerholm et al., 1995; Fine et al., 1993; Lennernäs, 1995; Nilsson 2349
2285 extrapolating from canine disintegration and dissolution data to et al., 1994). This apparently is due to the complexity of factors in- 2350
2286 human. Looking at the controversial comparative solubility data volved such as molecular weight, ionization, segmental difference, 2351
2287 in fed intestinal aspirates (Kalantzi et al., 2006; Persson et al., ion partitioning effects, bulk flow transport towards the intestinal 2352
2288 2005) and the variety of applied test meals reviewed by Lentz wall, tight junction contribution, motility and relative magnitude 2353
2289 et al. (Lentz, 2008), meal volume and composition should be stan- of transcellular versus paracellular water movement (Fagerholm 2354
2290 dardized to improve correlations. Simulating human stomach pH et al., 1999; Johno and Kitazawa, 1985; Lennernäs, 1995, 1998; 2355
2291 by stimulating gastric acid secretion, e.g. with pentagastrin, is an Pappenheimer and Reiss, 1987; Soergel, 1993). 2356
2292 additional approach that requires further validation for fasted ver- There are evident physiological and methodological discrepan- 2357
2293 sus fed state comparisons (Ajayi et al., 1999; Akimoto et al., 2000; cies for estimating intestinal permeability between rats and hu- 2358
2294 Fancher et al., 2011; Lentz et al., 2007; Polentarutti et al., 2010). mans, including about 4-fold higher thickness of the unstirred 2359
2295 Other species, e.g. rats (Morita et al., 2006), rabbits (Dongowski water layer in the rat (DeSesso and Jacobson, 2001; Hurst et al., 2360
2296 et al., 2005) and pigs (Grove et al., 2007), are less represented 2007; Kararli, 1995), dynamic changes of the functional absorptive 2361
2297 throughout literature concerning the impact of food on GI absorp- area as a function of the species, interspecies differences in villus 2362
2298 tion. The pig might be an alternative to the canine model for solu- tip osmolality and unknown transporter contribution, anesthesia 2363
2299 bility related food effects (Grove et al., 2007) as the biliary system effects and segmental distensions in situ (Bijlsma et al., 1995; 2364
2300 and pancreatic duct of minipigs, for example, are more consistent Lennernäs, 2007a). Nevertheless reviews confirmed reasonable 2365
2301 with human data in size and function (Kararli, 1995; Swindle and correlation between the jejunal permeability of rat and human 2366
2302 Smith, 1998). In principle, the monkey could also serve as an for passively transported drugs, regardless of the permeability 2367
2303 animal model to study the effect of food on drug absorption, albeit classification of the compounds (Lennernäs, 1998, 2007c), and 2368
2304 reported human-primate discrepancies with respect to intestinal the suitability of the rat model as a predictor of the fraction of drug 2369
2305 metabolism which is addressed in another paragraph of this review absorbed in human (Chiou and Barve, 1998). For this reason, the rat 2370
2306 (Ikegami et al., 2003). Still, apart from ethical aspects as well as is the most used model for determining permeability-related nutri- 2371
2307 difficulties in animal supply, handling and high costs, defining a ent–drug interactions, either by using in vivo perfusion techniques 2372
2308 suitable standard meal yielding postprandial pH profiles compara- or noninvasive differential urinary excretion approach (Lane et al., 2373
2309 ble to human appears to be one of the major obstacles for the use of 2006; Lu et al., 1992; Schepens et al., 2008; See and Bass, 1993; 2374
2310 the monkey model for predicting food effects on absorption. In spite Song et al., 2011; Suzuki and Hara, 2010). 2375
2311 of quantitative discrepancies probably attributable to the different Compared to the rat, the canine small intestine exposes a higher 2376
2312 measuring techniques, two studies indicated that the overall time permeability to hydrophilic substances which is presumably 2377

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
 PHASCI 2973 No. of Pages 53, Model 5G
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E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx 27

2378 related to differences in the paracellular pathway and villi tively to the interdigestive migrating motility complex (Collins 2443
2379 morphology (He et al., 1998; Lennernäs, 2007a; Martinez et al., et al., 1996; Davis et al., 1986; Fleisher et al., 1999; Kaniwa et al., 2444
2380 2002; Sutton, 2004) resulting in a poor correlation of fraction of 1988b; Lin et al., 1993; Meyer et al., 1985; Welling, 1977). Thus, 2445
2381 dose absorbed in man (Chiou et al., 2000). Similarly, studies con- postprandial conditions are often associated with altered absorp- 2446
2382 ducted at whole-animal level indicated that paracellular absorp- tion profiles of substrates and drug formulations with pronounced 2447
2383 tion is higher in pigs than in rats, while the underlying absorption window due to site-specific dissolution, instability, 2448
2384 mechanism is still unknown (Delahunty and Hollander, 1987; targeting concept et cetera (Gouda et al., 1987; Ishibashi et al., 2449
2385 Lavin et al., 2007). Therefore, the use of pigs and dogs for 1999a; Marathe et al., 1998; Pao et al., 1998; Sunesen et al., 2450
2386 nutrient-induced modulation of drug permeability is limited. Still, 2005; Yuen, 2010). For BCS Class I and III compounds which are 2451
2387 dietary effects need to be accounted for during breeding as certain rapidly absorbed in proximal parts, for example, gastric emptying 2452
2388 food constituents may alter mucosal integrity and function in these is critical for the absorption rate, but not necessarily for the overall 2453
2389 mammals (Watson et al., 2006; Zhang and Guo, 2009). extent of absorption (Fleisher et al., 1999; Pao et al., 1998). 2454
2390 Although the upper GI segments are considered to be the main Although the SI transit times (SITT) is hardly affected by con- 2455
2391 site of absorption, distal regions including ileum and colon can sig- comitant food consumption (Billa et al., 2000; Davis et al., 1986; 2456
2392 nificantly contribute to the overall absorption as well. Segmental Fleisher et al., 1999; Kararli, 1995; Kenyon et al., 1995; Yuen 2457
2393 single-pass perfusion (Fagerholm et al., 1997) and site-specific et al., 1993), some nutrient-induced feedback mechanisms on gas- 2458
2394 administration (Lindahl et al., 2004) have been applied to the rat tric emptying (Lin et al., 1993, 1992), species difference in the 2459
2395 intestine to investigate regional differences in absorption. Sakuma fasted state (Kararli, 1995) and the gastro-ileocaecal reflex (Fadda 2460
2396 et al. (2007) demonstrated by using a rat model in vivo combined et al., 2009; Kerlin et al., 1982; Schiller et al., 2005) can have 2461
2397 with surgical assessment of GI transit of food components that consequences on fasted versus fed state comparisons. The in vivo 2462
2398 varying the administration site could help decreasing the negative relevance of these alterations has to be evaluated in dependence 2463
2399 food effect for several model drugs (Sakuma et al., 2007). Yet, un- of the dosage form – monolithic formulations, for example, have 2464
2400 like human, the absorptive surface area is more evenly distributed been reported to be more affected than multiparticulate prepara- 2465
2401 in the rat (DeSesso and Jacobson, 2002) and some inconclusive re- tions (Fadda et al., 2009; Mundy et al., 1989; Yuen et al., 1993). 2466
2402 sults e.g. regarding colonic permeability (Fagerholm et al., 1997; Overall, substantial interspecies differences in GITT in fasted 2467
2403 Hollander et al., 1989; Krugliak et al., 1994) along with solid dos- and fed state mitigate the accuracy in predicting food effects, as 2468
2404 age form related restrictions as mentioned elsewhere limit the summarized in several reviews (Martinez et al., 2002; Martinez 2469
2405 use of the rodent model for the prediction of more complex and Papich, 2009; Sutton, 2009). Moreover, uncertainties in captur- 2470
2406 absorption profiles. Moreover, despite the development of sophis- ing the known influence of meal viscosity, lipid digestion products 2471
2407 ticated intubation techniques to study regional absorption in and breed size, etc. on GITT (Bourreau et al., 2004; Ehrlein and 2472
2408 human such as triple-lumen tubing and rectal perfusion (Gramatte, Prove, 1982; Fix et al., 1993; Meyer et al., 1994) can impair the 2473
2409 1994; Lennernäs et al., 1995), directly in vivo obtained permeabil- prediction quality of in vivo models. 2474
2410 ity values for human ileum and colon are lacking, most likely due Nutrient-induced alterations of GITT seem to be similar in qual- 2475
2411 to experimental difficulties (Lennernäs, 2007a). Hence, extrapola- itative terms, but highly variable in quantitative evaluations. Again, 2476
2412 tion from preclinical data to human is a delicate challenge for the dog is the most studied preclinical model in this aspect, show- 2477
2413 low permeability drugs and formulations for which the distal ing rather poor correlation up to over-prediction of food effects due 2478
2414 region is the preferable site of drug release and absorption. to more pronounced postprandial delay of gastric emptying and 2479
2415 However, efforts are being made to develop more precise preclin- faster GITT in the fasted state as compared to human (Campbell 2480
2416 ical models for this purpose, e.g. by using GI transit time controlled and Rosin, 1998; Kaniwa et al., 1988a; Paulson et al., 2001) (Table 2481
2417 beagle dogs for the successful estimation of the bioavailability of 1). For the assessment of modified-release formulation behavior 2482
2418 paracetamol from a sustained-release formulation (Yamada et al., in both fasted and fed state, human-canine dissimilarities in bio- 2483
2419 1995) or by introducing a canine colonoscopy model as a surrogate availability have also been partly attributed to distinct residence 2484
2420 for human intubation studies exploring controlled-release formu- times in the targeted region, especially for monolithic dosage forms 2485
2421 lation behavior (Sutton et al., 2006). All the same, the suitability (Fix et al., 1993; Ishibashi et al., 1999a; Kabanda et al., 1994). The 2486
2422 of the dog for investigating modified-release formulation and porcine model, too, has to be evaluated with caution, since its stom- 2487
2423 site-specific absorption – with or without food – has been contro- ach residence time is remarkably longer than in human (Aoyagi 2488
2424 versially evaluated (Akimoto et al., 1995; Cook et al., 1990; Ishib- et al., 1992), although the overall GITT time seems to be more com- 2489
2425 ashi et al., 1999a,b; Kulkarni et al., 2012; Li et al., 2001; Pao parable to human than that obtained from the canine model (Karar- 2490
2426 et al., 1998; Sutton, 2004, 2009, 2006; Wu et al., 2004; Yamada li, 1995; Kulkarni et al., 2012). Considering the rat which may be 2491
2427 et al., 1995). For extended release formulations, the porcine model used for oral disperse formulations, there are indeed some similar- 2492
2428 might be preferred which can partly be explained by more similar ities e.g. regarding the intestinal transit time (Hurst et al., 2007), but 2493
2429 GI surface area and transit times to human (Kulkarni et al., 2012; obviously, rodent models are inappropriate for large infrequent 2494
2430 Lennernäs, 2007a). Despite some shortcomings in terms of quanti- meals which is needed to simulate human dietary behavior. With 2495
2431 tative estimation, the dog remains a useful model to explore respect to GITT in fasted and postprandial conditions, the monkey 2496
2432 underlying mechanisms of food effects on oral dosage form behav- appears most suitable albeit some dissimilarities in SITT, according 2497
2433 ior, irrespective of dissolution (Wu et al., 2004) or permeation to Ikegami et al. (2003). Its application remains limited for reasons 2498
2434 dependent regional differences in absorption (Li et al., 2001; Pao mentioned above, though. 2499
2435 et al., 1998; Sutton, 2004). Nonetheless, especially for modified-re- Regardless of which in vivo model is employed, if food-induced 2500
2436 lease formulations and compounds with site-specific absorption, alteration of the GI residence times of a dosage form occurs, the 2501
2437 food-induced changes in GI transit times have to be taken into ac- risk of subsequent changed stability, unexpected absorption path- 2502
2438 count which is addressed in the next paragraph. ways as well as modified metabolism and transport pattern should 2503
be taken into consideration which, in turn, varies across species. 2504
2439 10.2.3. Transit times
2440 Food intake is known to influence GI transit times (GITT), e.g. by 10.2.4. Lymphatic uptake 2505
2441 delaying gastric emptying time in dependence of volume, caloric Intestinal lymphatic transport is considered a relevant pathway 2506
2442 content, stomach pH, viscosity, lipid digestion and timing rela- to circumvent hepatic first pass extraction and for the delivery of 2507

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
 PHASCI 2973 No. of Pages 53, Model 5G
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28 E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx

2508 certain antiviral, immuno-modulatory and anticancer drugs dous interest, as reflected in myriad reports and reviews on this to- 2573
2509 (Porter and Charman, 2001; Trevaskis et al., 2008; Yáñez et al., pic which often refer to results obtained from rodent and human 2574
2510 2011). The contribution of lymphatic uptake to oral bioavailability studies (Farkas and Greenblatt, 2008; Fuhr, 1998; Mandlekar 2575
2511 can be estimated from physicochemical characteristics of the mol- et al., 2006; Rodriguez-Fragoso et al., 2011; Won et al., 2010). 2576
2512 ecules, still, it is important to take account of the very low rate of However, clinical relevance is drug-dependent, and the metabo- 2577
2513 lymphatic fluid transport – approx. 0.2% (v/v) – relative to portal lism model used is critical for extrapolation to human, since the 2578
2514 blood (Charman et al., 1997). In general, fat-soluble vitamins, amount, activity, substrate specificity and tissue distribution of 2579
2515 dietary or synthetic lipids and lipophilic peptide-like compounds metabolic enzymes considerably vary across species (Hurst et al., 2580
2516 are prone to absorption into the lymphatic system (Charman 2007; Komura and Iwaki, 2011; Martignoni et al., 2006; Tang and 2581
2517 et al., 1997; Porter and Charman, 2001; Trevaskis et al., 2008; Prueksaritanont, 2010). For instance, intestinal metabolism has 2582
2518 Yáñez et al., 2011). been reported to be remarkably more extensive in cynomolgus 2583
2519 The postprandial increase of luminal lipid concentration monkey than in human (Komura and Iwaki, 2011; Takahashi 2584
2520 evidently favoured lymphatic uptake of the lipophilic prodrug tes- et al., 2009, 2010). Appreciable interspecies dissimilarities in met- 2585
2521 tosterone undecanoat relative to testosterone resulting in higher abolic fate have been shown for indinavir and atomoxetine, among 2586
2522 systemic exposure of the former in the fed state (Bagchus et al., others, contributing to up to 10-fold difference in oral bioavailabil- 2587
2523 2003; Frey et al., 1979). The absorption of halofantrine, a com- ity (Lin et al., 1996; Mattiuz et al., 2003). In addition, typically 2588
2524 pound with remarkable positive food effect (Milton et al., 1989), diminished metabolic reactions have been outlined for distinct 2589
2525 has also been proven to be mediated by lymphatic transport (Por- species such as acetylation in dogs or sulfate conjugation in pigs 2590
2526 ter et al., 1996). All the same, the interplay of bile salts and pH on (Martinez et al., 2002). 2591
2527 solubility and extent of ionization should be considered when eval- According to a recent review by Tang et al. (2010), multiple ani- 2592
2528 uating the potential of a drug to be associated to lipid digestion mal models can be used for CYP3A inhibition studies, whereas 2593
2529 products for the subsequent transport into the lymph (Charman induction data may be better gained with rhesus monkeys, cyno- 2594
2530 et al., 1997; Yáñez et al., 2011). The role of lymphatic transport is molgus monkeys and beagle dogs, under the tacit assumption that 2595
2531 hardly sufficiently assessed by plasma concentration–time profiles standard inhibitors and inducers are involved (Tang and Prueksa- 2596
2532 only, since the increased lymphatic uptake after a high-fat meal ritanont, 2010). The porcine model has been excluded from most 2597
2533 may result in lower plasma levels of the drug in question (Charman of the reviews evaluating animal models for predicting drug 2598
2534 et al., 1997; Porter and Charman, 2001). Therefore, animal models metabolism (Martignoni et al., 2006; Tang and Prueksaritanont, 2599
2535 allowing sampling or exhaustive collection of lymph are indispens- 2010). Porcine CYP3A29 is known to exhibit comparably high pro- 2600
2536 able to assess the contribution of lymphatic transport to enhanced tein similarity to human CYP3A4 (Suenderhauf and Parrott, 2013) 2601
2537 postprandial bioavailability. Theoretical and practical aspects of and the pig has been sporadically used for assessing drug–nutrient 2602
2538 the sophisticated techniques including advantages and limitations interactions at the metabolic level (Wein et al., 2012). There is a 2603
2539 of the respective models have been described profoundly in need for more detailed investigations on porcine drug-metaboliz- 2604
2540 reviews on this topic (Edwards et al., 2001; Trevaskis et al., ing enzymes, though (Puccinelli et al., 2011). The shortcomings 2605
2541 2008; Yáñez et al., 2011). Briefly, the conscious and unconscious, of in vivo models to assess intestinal microflora metabolism as 2606
2542 restrained or unrestrained rat models have been widely used to implicated by the development of a rat model with associated 2607
2543 gain mechanistic understanding of lymphatic contribution to drug human colonic bacteria (Hurst et al., 2007) may be faced with 2608
2544 absorption (Charman et al., 1986; Jandacek et al., 2009; Porter the porcine model since the pig’s colon is populated with bacterial 2609
2545 et al., 1996; Turner and Barrowman, 1977). Larger animal models microflora resembling human conditions (Martinez et al., 2002). 2610
2546 like dogs, pigs, sheep and rabbits as well as an indirect pharmaco- However, the (intermediate) metabolites from fermented sub- 2611
2547 logical approach avoiding lymph-duct canulation have also been strates produced by pig colon microbiota can be very different 2612
2548 developed (Dahan and Hoffman, 2005; Khoo et al., 1998, 2002, from that of human colon microbiota, indicating that the microbial 2613
2549 2003; Shackleford et al., 2003; White et al., 1991; Yáñez et al., composition on species/strain level is different between pigs and 2614
2550 2011). In summary, reports on lymphatic transport and veterinary humans. 2615
2551 drug absorption stated the more representative fasted and fed Especially for the pig which, being fed ad libitum, is sometimes 2616
2552 states of the porcine and canine model, but also outlined the gen- used to model obesity, but for any in vivo metabolism model in 2617
2553 eral restriction in comparability of the models among each other general, diet restrictions and standardization are recommendable, 2618
2554 and relative to human. This is mainly because of experimentally since dietary habits can confound metabolic activities (Martinez 2619
2555 caused bias related to the variable surgical and anaesthetic et al., 2002; Suenderhauf and Parrott, 2013). In general, the rodent 2620
2556 methodologies and the overall insufficient knowledge of species model appears to be less appropriate for compounds that undergo 2621
2557 difference in drug lymphatic uptake (Cook et al., 1998; Hurst extensive intestinal metabolism, as indicated by qualitative and 2622
2558 et al., 2007; Martinez et al., 2002; Trevaskis et al., 2008; Yáñez quantitative human-rodent discrepancies in the expression of 2623
2559 et al., 2011). Hence, investigations on the impact of food with re- major intestinal drug metabolizing enzymes (Cao et al., 2006; 2624
2560 spect to lymphatic transport remain rather case-specific, and the Komura and Iwaki, 2008; Tang and Prueksaritanont, 2010), even 2625
2561 relevance with respect to the drug systemic exposure in vivo can in transgenic rodents expressing human CYP3A4 (Lin, 2008). 2626
2562 be considerably affected by interspecies differences regarding lym- Irrespective of the animal model, knowledge about the absorption, 2627
2563 phatic flow and mechanism of lymphatic absorption. disposition and metabolism profile of both drug and nutrient is 2628
essential for in vivo–in vivo extrapolation of food–drug interactions 2629
2564 10.2.5. Metabolism (Benet, 2009; Wu and Benet, 2005). 2630
2565 Food may interfere with drug metabolism or enterohepatic
2566 recirculation, e.g. by inhibiting hydrolytic enzymes produced by 10.2.6. Transporter-mediated processes 2631
2567 intestinal bacteria (Schmidt and Dalhoff, 2002). Transporter-related nutrient–drug interactions can occur at any 2632
2568 Metabolism-related food–drug interactions are highly site with absorptive and extractive characteristics. Intestinal, 2633
2569 dependent on the composition of the food, namely, they are mostly hepatobiliary and renal carrier-mediated transport as well as 2634
2570 associated with fruits, vegetables, alcoholic beverages, teas and transporter modulation at the blood–brain barrier have been of 2635
2571 herbs (Rodriguez-Fragoso et al., 2011; Won et al., 2012). Above predominant concern throughout literature (Chandra and Brou- 2636
2572 all, fruit juices and food-derived flavonoids have evoked tremen- wer, 2004; ITC, 2010; Lai, 2009; Tang and Prueksaritanont, 2010; 2637

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
 PHASCI 2973 No. of Pages 53, Model 5G
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2638 Won et al., 2012; Xia et al., 2007). Food-induced modulation of rodent models are preferred as in case of permeability, lymphatic 2702
2639 transporter processes can therefore affect absorption, distribution, uptake or transporter-related interaction studies. Pigs and monkeys 2703
2640 metabolism and excretion to different extents in dependence of the are alternative preclinical surrogates which require more validated 2704
2641 exposure to the causative ingredients at the various sites. Similar approaches in assessing drug bioavailability in the fed state. The 2705
2642 to metabolism-related interactions, fruits, vegetables, herbs and monkey seems to bear potential for overcoming physiological lim- 2706
2643 their secondary metabolites have drawn increasing attention with itations of the canine model. Irrespective of the animal model, in- 2707
2644 respect to modulation of transporters during the last two decades, tra-species differences in the postprandial state related to sex, 2708
2645 starting with the intensively described inhibition of P-gp and CYP age, strain, diet and housing conditions need to be assessed system- 2709
2646 3A4 by grapefruit juice and gradually widening out to specific phy- atically in future work for estimating the robustness of each model 2710
2647 tochemical-based alteration of both influx and efflux processes and establishing adequate ranges of study parameters. 2711
2648 (Bailey, 2010; Deferme and Augustijns, 2003; Rodriguez-Fragoso Adaptation of the study performance might be considered to 2712
2649 et al., 2011; Won et al., 2012; Zhang et al., 2009). Interestingly, yield GI responses which best resemble human postprandial condi- 2713
2650 lipids and bile salts can also contribute to a decrease of transporter tions. For example by pharmacological stimulation of gastric acid 2714
2651 function in the postprandial intestine, implicating that not only secretion and gastric emptying time controlling in the dog or diet 2715
2652 specific food products and phytochemicals derived therefrom can restriction in the pig (Lentz et al., 2007; Suenderhauf and Parrott, 2716
2653 affect transporter-mediated drug absorption, but also the ingestion 2013; Yamada et al., 1995). Of course, each additional modification 2717
2654 of a fat-containing meal in general (Custodio et al., 2008). needs to be validated carefully and progressive use of standardized 2718
2655 Focusing on intestinal absorption, perfusion studies in rodents techniques is necessary to confirm the benefits and uncover sys- 2719
2656 appear to be advantageous for elucidating mechanisms and regio- tematic limitations, respectively. With regard to the standard 2720
2657 nal characteristics of interactions, allowing simultaneous and meals recommended by the regulators for human studies, there 2721
2658 localized assessment of intestinal permeability, metabolism and is an urgent need to define specific meal compositions to be 2722
2659 transport (Deferme et al., 2002; Lennernäs, 2007a; Shirasaka applied in fed animal studies, as the common practice ranges 2723
2660 et al., 2010). Excluding the poor correlation with respect to intesti- between liquid diet mixtures, standard or enriched animal and hu- 2724
2661 nal metabolism and oral bioavailability, Cao et al. (2006) depicted man diet of various quantities (Kondo et al., 2003b; Lentz, 2008). 2725
2662 reasonable similarities between rat and human regarding drug Looking beyond acute responses to meal digestion, well defined 2726
2663 intestinal absorption profiles and expression pattern of PepT1, feeding experiments can provide interesting insights into the effect 2727
2664 SGLT-1, GLUT5 and MRP2 in the small intestine (Cao et al., 2006). of dietary habits on drug absorption, distribution and metabolism. 2728
2665 Detailed reviews of in vitro and in vivo models used for the evalu- This aspect has been accounted for in the draft guidance on drug 2729
2666 ation of transporter-mediated interactions have been published interaction studies released by the FDA in 2012, recommending 2730
2667 (ITC, 2010; Xia et al., 2007). In summary, comprehensive investiga- that uncontrolled consumption of dietary/nutritional supplements 2731
2668 tions of transporter expression and activities in large animal and distinct food products or beverages containing alcohol, grape- 2732
2669 models such as pig, dog and monkey are requisite to establish bet- fruit, apple, orange, vegetable from the mustard green family, 2733
2670 ter in vivo surrogates of transporter-related interaction studies in chargrilled meat and tobacco should be avoided for 1 week prior 2734
2671 human. Moreover, selective inhibitors or antibodies have not yet to the start of the interaction study until its conclusion (FDA, 2735
2672 been identified for most transporters, especially with respect to 2012). Focusing further on possible effects of distinct food compo- 2736
2673 drug uptake pathways (Xia et al., 2007). nents on drug bioavailability, determining food-derived causative 2737
ingredients and exploring their pharmacokinetic properties as well 2738
2674 10.3. Conclusions on food effects and in vivo model selection as (multiple) interaction mechanisms across species are challenges 2739
yet to be faced. Eventually, the evaluation of the BCS and BDDCS in 2740
2675 It is evident that well defined procedures are required to common laboratory species revealing interspecies classification 2741
2676 encompass the variety of potential drug–nutrient interactions to differences and similarities might be a reasonable approach bene- 2742
2677 ensure therapeutic efficacy in clinical practice. For this purpose, ficial to both veterinary and human pharmaceutical research. 2743
2678 in vivo models are indispensable tools since the impact of food
2679 digestion is best assessed in intact animals expressing dynamic
11. In vitro in vivo correlations 2744
2680 responses in drug absorption, distribution, metabolism and
2681 elimination rates in accordance to the complexity of physiological
It is necessary to understand how changes during development 2745
2682 reactions to food intake in human.
to formulation and/or manufacturing process affect in vivo perfor- 2746
2683 Animal models usually require highly sophisticated approaches
mance, and thereby safety and efficacy. This is also a central aspect 2747
2684 comprising varied types of formulation, administration sites and
to ensure that batches produced during routine manufacture will 2748
2685 probe substrates with defined sampling schemes and surgical
continue to give consistent local and/or systemic exposures to 2749
2686 interventions. These efforts allow the estimation of the extent to
those evaluated in the pivotal clinical studies. As it is not practical 2750
2687 which food and food-derived components affect drug exposure in
to measure the PK of every batch of drug product in man, some of 2751
2688 plasma and specific tissues. Combination with in vivo imaging
this understanding and verification must be based on in vitro test- 2752
2689 techniques, ex vivo and post mortem investigations is recommend-
ing. The development of IVIVC or in vivo in vitro relationship 2753
2690 able for an improved understanding of the relevance of food effect
(IVIVR) is a key topic in all drug development programs and 2754
2691 in oral drug bioavailability. Since the mechanisms of drug absorp-
submission for marketing approvals, as this is the basis for under- 2755
2692 tion and distribution often differ across species, comprehensive
standing how product performance measured in vitro is likely to 2756
2693 knowledge about model-specific pathways is necessary. Especially
relate to performance in vivo. The gaps and issues in developing 2757
2694 for large laboratory animals such as pigs, dogs and monkeys, qual-
IVIVC or IVIVR for orally administered drug products are discussed 2758
2695 itative and quantitative information about transporter and enzyme
from an industrial and regulatory perspective. 2759
2696 expression and function is poor.
2697 Overall, the dog is the most studied in vivo model to evaluate
2698 common food effects on drug absorption due to physiological and 11.1. Definitions 2760
2699 dosage form related advantages as well as the ability to consume
2700 large infrequent meals on command. For mechanistic studies and An IVIVC is traditionally defined as a predictive mathematical 2761
2701 investigations requiring extensive surgical or genetic interventions, relationship between in vitro dissolution and some aspect of 2762

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
 PHASCI 2973 No. of Pages 53, Model 5G
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30 E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx

2763 in vivo exposure, covering either the entire absorption curve (Level ter stages of development (i.e., during and after the pivotal clinical 2826
2764 A) or an individual parameter associated with the rate or extent of safety and efficacy studies), IVIVC/R is used to provide regulatory 2827
2765 absorption (as in Level C correlations). However, in literature and evidence of relative bioavailability or BE to support formulation 2828
2766 practice the term IVIVC is often used in a more holistic sense, to and process changes, definition of manufacturing ranges or design 2829
2767 describe a wide range of approaches linking some aspect of space. Here, the evidence to support IVIVC/R is more likely to be 2830
2768 in vitro formulation behavior to the measured or predicted clinical drawn from a single appropriately powered human bioavailability 2831
2769 performance of dosage forms. Indeed, the BCS system represents study performed specifically for this purpose. At the time of 2832
2770 an example of a different kind of IVIVR, as it defines when changes product registration, the understanding gained from IVIVC/R is also 2833
2771 in in vitro dissolution will have no impact on BE, for specific useful to link the proposed in vitro quality control release methods 2834
2772 compound types and test conditions. This thinking is effectively and acceptance criteria to in vivo performance registration, to give 2835
2773 the basis of biowaivers for BCS Class I (and BCS class III within assurance that batches produced during routine commercial man- 2836
2774 EU) compounds. Stimulated by the advent of QbD, this thinking ufacture will be of appropriate clinical quality (i.e. be bioequivalent 2837
2775 has recently evolved further into the ‘safe space’ concept, in which to batches used in the pivotal safety and efficacy studies). From a 2838
2776 a risk-based approach is applied to determine the range over which regulatory perspective, it is of course desirable to have a dissolu- 2839
2777 dissolution may vary without altering bioavailability, for a specific tion test and acceptance criterion which are not only discriminat- 2840
2778 drug product and API. This concept has been proposed as a basis for ing between batches of different quality, but are also relevant for 2841
2779 setting in vivo relevant in vitro specifications in the same manner in vivo performance. However, while the guidances from FDA and 2842
2780 as a traditional IVIVC. EMA allows for biowaivers based on IVIVC or BCS, there is cur- 2843
2781 There are three possible relationships between in vitro dissolu- rently no regulatory guidance which mentions the use of other 2844
2782 tion and in vivo performance, as described by Dickinson et al. forms of IVIVR in this context. 2845
2783 (2008).
11.3. Learning from previous reviews of IVIVC/IVIVR 2846
2784 1. A mathematical correlation between in vitro dissolution and
2785 in vivo performance, such that a given change in in vitro disso- The gaps and limitations in the practice of IVIVC and IVIVR have 2847
2786 lution can be used to predict the corresponding change in an previously been discussed by other authors. These reviews have 2848
2787 in vivo exposure parameter (e.g. area under the curve (AUC) mainly focused on modified release MR formulations, which repre- 2849
2788 or maximum concentration), i.e. a classical IVIVC. sent a high proportion of IVIVC studies described in the literature. 2850
2789 2. Changes in in vitro dissolution can be tolerated without any This is unsurprising, as this formulation type is a priori more likely 2851
2790 impact on in vivo performance, resulting in a dissolution ‘safe to produce IVIVC, having been purposefully designed so that disso- 2852
2791 space’. lution will become rate limiting for the overall absorption. The 2853
2792 3. Small changes in in vitro dissolution performance have no main points from each of these reviews which are pertinent to 2854
2793 impact on in vivo exposures, but larger changes do. the topic under discussion are recapped below. 2855
2794 Dokoumetzidis and Macheras (2008) ascribe failure of IVIVC 2856
2795 For the purposes of this article, the term ‘IVIVR’ will be used to for MR products to an inability to adequately reproduce or 2857
2796 refer to scenarios 2 and 3 above, i.e., relationships other than a simulate the complexity of the holistic in vivo environment using 2858
2797 classical IVIVC, developed using both in vivo and in vitro data, that current in vitro or in silico techniques (Dokoumetzidis and 2859
2798 enables the impact of a given in vitro dissolution profile on in vivo Macheras, 2008). Specific examples of this cited by the authors 2860
2799 performance to be understood. This is sometimes described by include: 2861
2800 other authors as a nonlinear IVIVC (e.g., Polli, 2000) (this is not
2801 the same as a mathematical nonlinear correlation e.g., a nonlinear  Inability to adequately replicate the composition of the luminal 2862
2802 Level A) (Polli, 2000). It should be noted that these three scenarios media in which in vivo dissolution takes place, including 2863
2803 are part of a continuum – ultimately when dissolution is slowed changes in composition along the intestine (which can poten- 2864
2804 beyond a certain rate it will begin to impact in vivo exposures. tially bring about an interplay between hydrodynamics, luminal 2865
2805 The relationship detected in an in vivo study depends on where fluid composition and dissolution behavior). 2866
2806 in the dissolution space the profiles to be tested lie. All three sce-  Under-stirring and heterogeneity of mixing meaning that what 2867
2807 narios can be used to understand the relevance of a particular appears to be a well-controlled and reproducible dissolution 2868
2808 in vitro test result for in vivo performance. Scenarios 1 and 3, i.e., behavior in vitro is not the case in vivo. 2869
2809 where at least one of the profiles tested produces a change in expo-  Inability to adequately simulate intestinal hydrodynamics and 2870
2810 sures, are perceived to offer a greater degree of control, as the flow, which may lead to a discrepancy between in vitro and 2871
2811 detectability of an in vivo ‘failure’ provides assurance of the rele- in vivo results, or induce such a high degree of variability for 2872
2812 vance of the dissolution test. However, for routine manufacture drug products which are sensitive to these factors that IVIVC 2873
2813 Scenario 2 offers a greater degree of assurance, as it demonstrates is not shown. 2874
2814 that the product lies within a ‘safe space’ where changes in in vitro  Complex dynamic interplay between several characteristics of 2875
2815 performance will not be reflected in vivo. the in vivo environment which are important for dissolution, 2876
which cannot be adequately captured by reproducing these 2877
2816 11.2. Purposes of IVIVC/IVIVR in drug development individual factors in vitro or in silico. 2878
2879
2817 The purpose and application of IVIVC/R will evolve as a drug Cardot and Davit (2012) described some of the limitations of 2880
2818 progresses through development. In the early phases, the focus is performing mathematical IVIVC for MR and IR formulations (Car- 2881
2819 likely to be on establishing understanding of the potential clinical dot and Davit, 2012). The authors describe several aspects of the 2882
2820 impact of formulation switches and changes made during manu- data manipulation step which must be carefully considered to 2883
2821 facturing process development. At this stage, IVIVC/R tends to be maximize the likelihood of successful IVIVC: 2884
2822 drawn from across several data sources (e.g., performance of sim-
2823 ple formulations in SAD/MAD studies vs. early solid dosage form  The use of mean vs. individual in vivo curves. 2885
2824 prototypes, data form preclinical studies, TNO-TIM1) to create a  Whether it is appropriate to correct for a lag time or apply time 2886
2825 holistic picture of the likely impact of a given change in man. At la- scaling. 2887

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
 PHASCI 2973 No. of Pages 53, Model 5G
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E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx 31

2888  The potential confounding effects of ‘flip-flop’ kinetics (where marketed gemfibrozil formulations, using dissolution tests in 2952
2889 absorption rate is significantly slower than elimination rate), simple pharmacopoeial apparatus (Rouini et al., 2008). Kovacevic 2953
2890 or using data from formulations with different bioavailabilities. et al. (2009) report Level A IVIVC for carbamazepine developed 2954
2891  Differences between subjects used to build the original IVIVC across both IR and MR formulations, again relating to a 2955
2892 and those used to validate its predictive ability. simple pharmacopoieal dissolution test and media (Kovacevic 2956
2893 et al., 2009). 2957
2894 The authors describe a potential error in correcting for lag time For other compounds, use of more complex in vitro systems has 2958
2895 when this is caused by gastric emptying, stating that this is not enabled an IVIVC to be developed. Buch et al. (2009) describe the 2959
2896 reproducible between subjects and so should not be corrected use of a combined dissolution and permeation system, to predict 2960
2897 for. They also raise the issue that an IVIVC for highly variable drugs the in vivo performance of fenofibrate (Buch et al., 2009). Six differ- 2961
2898 is problematic, as within-subject variability may mask formulation ent fenofibrate formulations were tested in this system using bio- 2962
2899 differences. Taken together, these two points highlight an interest- relevant dissolution media, and the in vitro data obtained was 2963
2900 ing assumption inherent in the in vivo component of IVIVC/R stud- shown to correlate with exposure in the rat. In a later study, it 2964
2901 ies, namely that the in vivo factors which govern dissolution of the was necessary to modify this system to predict the performance 2965
2902 dosage form are so reproducible between individuals that they do of formulations in man, to focus mainly on the effect of micellar 2966
2903 not affect the outcome of the study. In addition, variability in the entrapment by surfactant in the formulations on the permeation 2967
2904 in vivo factors governing dissolution can have an impact on step (Buch et al., 2011). Dissolution testing in USP2/Paddle appara- 2968
2905 whether an IVIVC is likely to be successful. This emphasizes a tus was not able to predict the in vivo performance of the formula- 2969
2906 gap in current practices of clinical IVIVC studies – when perform- tions. Okumu et al. (2008) studied the dissolution behavior of 2970
2907 ing in vitro dissolution measurements we need to routinely moni- montelukast sodium tablets in USP4/Flow-through cell apparatus, 2971
2908 tor and control aspects of the system to ensure that differences in using biorelevant dissolution media and a dynamic pH change pro- 2972
2909 the testing apparatus do not confound our ability to measure the tocol (Okumu et al., 2008). By using the dissolution profile from 2973
2910 performance of the dosage form. However, no such characteriza- this system in combination with GastroplusÒ modeling, they were 2974
2911 tion of the in vivo system can be performed, despite the fact that able to obtain a good fit for the in vivo plasma profile obtained from 2975
2912 the scope for variation between test systems here is far greater. this formulation. Dissolution profiles from USP2/Paddle apparatus 2976
2913 Characterization of parameters such as gastric emptying time, local under various testing conditions were not able to model the clini- 2977
2914 pH or even pressure forces prevalent in the GI environment could cal data as closely. 2978
2915 be performed in a relatively simple and non-invasive manner. The The examples above demonstrate that the use of more complex 2979
2916 collection of such data on each individual dosing occasion may en- in vitro dissolution systems and protocols may increase the likeli- 2980
2917 hance our interpretation of the PK and increase the likelihood of hood of a successful IVIVC, as they mimic the in vivo dissolution/ 2981
2918 building a quality IVIVR. absorption environment more closely than simple USP1/Basket or 2982
2919 Jiang et al. (2011) discussed the use of physiologically based USP2/Paddle apparatus. However, while they are very useful to 2983
2920 biopharmaceutical modeling in drug development (establishing guide formulation and process development, these systems are 2984
2921 IVIVC) from a regulatory perspective (Jiang et al., 2011). This mod- not suitable for routine batch release testing. Simpler release tests 2985
2922 eling is a valuable tool in the development and regulatory environ- more suitable for routine use would therefore need to be devel- 2986
2923 ments, in particular providing opportunities for exploring BE of oped to utilize the full benefits of IVIVC during and after product 2987
2924 complex drug products and in the QbD environment, and registration. However, the mechanistic understanding gleaned 2988
2925 encourage drug companies to explore its application. However, from more complex in vitro systems may enable the rate 2989
2926 they express concerns over the ‘black box’ nature of many in silico controlling mechanisms for dissolution to be determined, so that 2990
2927 models, and suggest that the same scientific question should be as- a simpler test which reflects these can subsequently be 2991
2928 sessed using more than one modeling software package to ensure developed. 2992
2929 that consistent results are obtained. Additionally, a need for stan- The majority of reports in the literature where IVIVC has been 2993
2930 dard modeling study designs and acceptance criteria is identified. attempted utilize BCS Class II compounds. Due to the lack of 2994
2931 Polli (2000) states that a Level A IVIVC is the least likely reporting of failed IVIVCs, it cannot be stated with certainty 2995
2932 outcome for IR products, as they tend not to have dissolution-rate whether this is due to a lack of effort for drugs from other BCS clas- 2996
2933 limited absorption (Polli, 2000). However, nonlinear forms of ses, or whether a large number of examples of failed attempts for 2997
2934 IVIVC, (i.e., where the plot of fraction dose absorbed vs. fraction these classes exists which have not been reported. The former sit- 2998
2935 dissolved is non-linear, with dissolution occurring faster than uation seems the more likely. The boundaries for ‘high’ solubility 2999
2936 absorption) are applicable and useful. Polli argues that the term and permeability in the BCS system are set very conservatively, 3000
2937 ‘IVIVR’ is preferable to ‘IVIVC’, to remove the implication that a which is appropriate to their current use in defining an area of very 3001
2938 study has failed if a linear mathematical relationship is not devel- low risk for biowaivers which encompasses any given formulation, 3002
2939 oped. To maximize the benefits of IVIVR in drug development and drug substance properties, manufacturing process and manufac- 3003
2940 in the regulatory context, a better understanding of both in vivo turing site. 3004
2941 dissolution and the in vitro dissolution test is needed. However, in practicality this also means that, with the excep- 3005
tion of BCS Class I, each BCS class will contain compounds with a 3006
2942 11.4. IVIVC for IR products broad spectrum of properties, and therefore potentially different 3007
rate limiting steps for absorption. For example, a low solubility 3008
2943 In general, the topic of IVIVC and IVIVR for IR products has not compound which narrowly misses the 90% fabs boundary (or 3009
2944 received the same level of attention in the literature as IVIVC for 85% for EMA) would fall into BCS Class IV, and yet is very 3010
2945 MR products, but there exist a number of reports where IVIVC unlikely to have permeability-limited absorption – for such a 3011
2946 has been achieved. For instance, a Level C IVIVC was developed compound, IVIVC is still likely to be possible irrespective of its 3012
2947 for four marketed carbamazepine IR tablets (Lake et al., 1999). BCS IV classification. Similarly, not all compounds in Class II 3013
2948 The in vitro data from two simple pharmacopoeial-type dissolution should be expected to show IVIVR, an example of which is 3014
2949 tests is related to their relative bioavailability in man, and used described below. The BCS as it currently stands is therefore an 3015
2950 to calculate a release specification which will assure formulation inadequate system to assess the likelihood of IVIVC for a partic- 3016
2951 BE. Rouini et al. (2008) reported a similar example for five ular compound. 3017

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
 PHASCI 2973 No. of Pages 53, Model 5G
19 March 2014

32 E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx

3018 11.5. IVIVR for IR products this would help set expectation regarding whether an IVIVC could 3083
truly be developed, or whether other outcomes such as ‘safe space‘ 3084
3019 The implementation of QbD has lead to a renewed focus on the are more likely, and may thus give additional confidence to regula- 3085
3020 relationship between formulation and manufacturing process vari- tors when these outcomes are achieved. 3086
3021 ables and in vivo performance during product development. This An interesting gap in the reported data in the literature is the 3087
3022 has led to the establishment of dissolution tests and release spec- absence of IVIVC or IVIVR studies in patient groups. Naturally, 3088
3023 ifications based on clinical bioavailability data, which are used in healthy volunteers are usually the dosing group of choice for 3089
3024 defining the Design Space and are a key component of the Control relative bioavailability and BE studies, as the absence of the con- 3090
3025 Strategy. Dickinson et al. (2008) present a case study applying this founding effects of disease and co-medications on the absorptive 3091
3026 approach to a BCS Class II compound (Dickinson et al., 2008). environment should reduce the ‘noise’ and make them more dis- 3092
3027 Tablet variants were manufactured based on the commercial criminatory for detecting differences in formulation performance. 3093
3028 formulation but incorporating the highest risk process and formu- However, for certain classes of compound (e.g., cytotoxics used in 3094
3029 lation dissolution failure modes, as determined by a product-spe- the oncology setting), studies in healthy volunteers are not permit- 3095
3030 cific Quality Risk Assessment. These tablet variants were then ted from a safety perspective. Additionally, it would be unethical to 3096
3031 dosed in a clinical relative bioavailability study, and exposures dose formulation variants which may have suboptimal perfor- 3097
3032 compared to the standard tablet and an oral solution. The study mance to patients who are expecting to receive therapeutic benefit 3098
3033 showed that all of the tablet variants gave equivalent exposures from the treatment. This raises the question of how best to link 3099
3034 to the standard tablet, despite having slower dissolution in vitro. in vitro and in vivo performance for these compound types. During 3100
3035 Additionally, even the slowest dissolving tablet variant gave development, increased reliance is likely to be placed on pre-clin- 3101
3036 equivalent exposures to the oral solution. Thus, a ‘safe space’ for ical studies, complex in vitro dissolution (e.g.TNO-TIM-1) and in 3102
3037 dissolution performance had been established, i.e. a range of disso- silico modeling to support formulation bridges and design the dis- 3103
3038 lution profiles which would result in BE in vivo. This indicates that solution release test. However, the absence of a robust way to 3104
3039 dissolution was not rate-limiting over the range of profiles tested. prove that these models to adequately describe drug absorption 3105
3040 The authors hypothesized that this was due to sufficiently high sol- in man for the compound in question limits the degree of reliance 3106
3041 ubility so that the dissolution was faster than other physiological which can be placed on them. More work is needed to develop 3107
3042 processes (such as permeability and/or gastric emptying), despite innovative ways of defining IVIVR for these compound types, for 3108
3043 that it is classified as a BCS Class II compound. The authors subse- use both during development and in the regulatory BE 3109
3044 quently used the results of the clinical relative bioavailability study environment. 3110
3045 to select an appropriate dissolution test, and set a release specifica-
3046 tion which would assure BE was maintained. Similarly, Buggins 11.6. Gaps in the current state of the art in IVIVC/R 3111
3047 et al. (2011) described a case study for a BCS Class IV compound,
3048 where three tablet variants with slowed in vitro dissolution profiles While there are some interesting and innovative examples of 3112
3049 encompassing the highest risk failure modes for dissolution gave the development and application of IVIVC and IVIVR in product 3113
3050 equivalent exposures to a standard tablet (Buggins et al., 2011). development, further work is needed to increase the likelihood of 3114
3051 This was attributed to the fact that the compound had good developing a successful IVIVC/IVIVR, and enable them to be fully 3115
3052 permeability and high intestinal solubility, despite its BCS IV utilized in drug development and regulatory practice. Current gaps 3116
3053 classification. in the state of the art are summarized below. 3117
3054 This approach is starting to gain acceptance from a regulatory
3055 perspective. In a recent FDA presentation, the ‘safe space’ concept
11.6.1. General gaps 3118
3056 was included as a method of setting dissolution specifications
3119
3057 which are linked to clinical performance, which may lead to a
 Better understanding and mapping of the compound space over 3120
3058 wider dissolution specification being granted than if no data
which dissolution is likely to be rate determining for in vivo 3121
3059 linking dissolution to in vivo performance had been generated
exposures is needed. This would form the basis for intelligent 3122
3060 (Pope Miksinski, 2011). However, the IVIVR/‘safe space’ concept
design of IVIVC/R studies, and provide a platform of common 3123
3061 is not currently included in any regulatory guidance documents,
understanding on which to base discussions with health 3124
3062 meaning it is not as well established from a regulatory perspective
authorities. The current BCS system does not adequately fulfil 3125
3063 as traditional IVIVC. This is somewhat paradoxical, as it is more
this, and can create false expectation as to the likelihood and 3126
3064 difficult to gain regulatory flexibility for a product where in vivo
risk associated with IVIVR in the minds of scientists and health 3127
3065 performance has been proven to be insensitive to dissolution
authority reviewers. 3128
3066 changes, than for a product where any change in dissolution re-
 More examples of IVIVR are needed in healthy volunteers and 3129
3067 lease rate results in measurable changes in the rate or extent of
patients, to enable success factors and limitations to be better 3130
3068 absorption. This may be because for IVIVC, the presence of a math-
understood. 3131
3069 ematical correlation can be perceived to give a greater assurance of
 More examples of the use of in silico simulations as part of 3132
3070 control. Incorporation of in silico modeling approaches into the de-
IVIVC/R, and an understanding of how to use these in a regula- 3133
3071 sign and interpretation of ’safe space’ studies may help to dispel
tory context. 3134
3072 this perception, for example by demonstrating that in vivo perfor-
 Better knowledge is needed about the acceptable use of IVIVR 3135
3073 mance would not be expected to show sensitivity to dissolution
and the ‘safe space’ concept in the regulatory environment, as 3136
3074 over the range of profiles tested in the clinical study.
this is currently not described in any of the BE guidelines. The 3137
3075 The examples above indicate that, for a well designed IR tablet,
current guidances allow for biowaivers based on IVIVC or BCS 3138
3076 ‘safe space’ may be a more likely outcome than IVIVC. They also
Class I and III ‘safe spaces’, but do not allow for compound-spe- 3139
3077 demonstrate that the use of BCS class alone can be misleading
cific ‘safe spaces’. 3140
3078 regarding the likelihood of IVIVC development. A more detailed
 More examples of the use of IVIVR as part of an overall QbD 3141
3079 understanding of the space over which formulation dissolution is
strategy are needed, including examples of selection of tablet 3142
3080 truly rate limiting for a particular API from a particular formulation
variants on the basis of highest product specific risk mecha- 3143
3146
3081 is needed. As well as enabling more efficient design of IVIVC/IVIVR
nisms. Also, better understanding of the use of such IVIVRs in 3144
3147
3082 studies and development programs from an industrial perspective,
the regulatory context is required. 3145
Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
 PHASCI 2973 No. of Pages 53, Model 5G
19 March 2014

E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx 33

3148 11.6.2. In vitro systems interactions as well as early approaches to frame formulation 3210
3149 appropriateness as a function of two important drivers including 3211
3150  Difficulty in mimicking the complex and dynamic environment the needs of the compound and the ability of a particular organiza- 3212
3151 for in vivo dissolution in vitro. tion to address these needs into which category items such as 3213
3152  Difficulty in translating the understanding gained form complex downstream capabilities and capacity, cost of goods and related 3214
3153 in vitro apparatus into a simple pharmacopoeial-type test and factors fall. Ultimately, deciding whether a simple formulation 3215
3154 acceptance criterion. platform could be considered or if enabled strategies are required 3216
3155 is the goal of these evaluation methodologies. 3217
3156 11.6.3. Difficult compound types For discovery-based projects, the opportunity exists for an early 3218
3157 assessment of drug characteristics and contribute to the compound 3219
3158  Compounds where human studies cannot be performed in selection and development (Ding et al., 2012; Ku, 2008; Li and 3220
3159 healthy volunteers (e.g. oncology) – how do we generate under- Zhao, 2007; Maas et al., 2007; Saxena et al., 2009). These working 3221
3160 standing for these compounds to support relevant dissolution models have been evolving over time in many organizations. This 3222
3161 tests and specifications? evolution relies on interconnectivity with upstream medicinal 3223
3162  Highly variable drugs – how do we demonstrate IVIVR for these chemistry and biological assessments components. To this end, 3224
3163 compounds without dissolution changes being swamped by problematic compounds and projects are often identified at early 3225
3164 in vivo variability? stages allowing for drug-ability, formulate-ability and formulation 3226
3165  Failed attempts at IVIVC/R are not generally reported, making it process-ability assessments to be leveraged early in the develop- 3227
3166 difficult to fully define which other types of compound are ment cycle. These evaluations which are part and parcel of the 3228
3167 problematic. internal formulation decision tree will be outlines as a function 3229
3168 of this section. 3230
The general formalism of a formulation decision tree is rooted 3231
3169 11.6.4. Use of innovative clinical study designs
3170
in a number of science-based, data-driven factors incorporated into 3232
pharmaceutical company practices that are refined by experience 3233
3171  In vivo factors impacting dissolution are not routinely
as well as the information from the external scientific community. 3234
3172 characterized in IVIVR studies, however this would enrich the
The general strategy for selecting a formulation often follows the 3235
3173 information gained and aid data interpretation.
suggestion of Branchu et al. (2007) which divides the problems 3236
3174  Adaptive study designs appear to be under-utilized in IVIVR.
into three parts (Fig. 9) (Branchu et al., 2007): 3237
3175 This approach is likely to increase the efficiency of IVIVR stud-
3176 ies, reducing the number of testing arms by allowing the study
1. What is the nature of the formulation challenge and are conven- 3238
3177 either to stop once the ‘safe space’ region is reached, or allowing
tional or enabled strategies most appropriate for the selected 3239
3178 specific mechanisms to be probed based on feedback from the
API. 3240
3179 initial study arms.
2. If enablement is needed which enabled system is most 3241
3180
appropriate. 3242
3181 11.7. Conclusion regarding IVIVR/IVIVC 3. For a selected enabled formulation strategy, what are the most 3243
important design space considerations that can be suggested 3244
3182 Developing an understanding of the link between in vitro based on the nature of the data available. 3245
3183 performance and clinical exposures is of critical importance for 3246
3184 all drug products. This understanding is needed in order to stream- These factors, by definition, heavily target ‘‘compound need’’ 3247
3185 line the development process, to understand the risk of a change in elements and how these are balanced with capacity, capability 3248
3186 formulation or process significantly affecting bioavailability, and and costs represent important downstream considerations in 3249
3187 ultimately to provide assurance of consistent clinical quality of bringing the successful product to the market. 3250
3188 batches produced during routine manufacture. However, several A variety of factors may contribute to deciding whether formu- 3251
3189 gaps in the current state of the art impede the full and efficient uti- lation enablement will be likely needed. API properties are crucial 3252
3190 lization of IVIVC and IVIVR approaches. Working to address these to understanding how a compound should be formulated and these 3253
3191 will be of benefit to both drug developers and regulators, and ulti- are derived from several important assessments associated with 3254
3192 mately to the patient. drug absorption. While there are many factors that impact the 3255
PK and PD aspects of a drug candidate, the most important feature 3256
3193 12. Models for predicting API and API-formulation approaches include the exposure of the body to the drug candidates both in 3257
3194 and their correlation with in vitro and human terms of its rate and extent of absorption. Thus for an IR solid oral 3258
dosage form (representing about 80% of current formulation devel- 3259
3195 Processes for appropriately and accurately selecting formulation opment), it must disintegrate and dissolve releasing the API in a 3260
3196 strategies to progress important drug candidates have a number of solubilized form. Solubility and permeability therefore form the 3261
3197 benefits. These include directing the right resources to the right prob- basis for BCS which attempts to describe and categorize drugs 3262
3198 lems at the right time with the right level of effort. Aligning formula- based on their biopharmaceutical properties. The scale-up and post 3263
3199 tion strategies that provide for adequate pharmaceutical and approval change (SUPAC) apply BCS in regulatory decision. These 3264
3200 biopharmaceutical performance as well as clinical outcomes without values and boundary conditions incorporated in the BCS are 3265
3201 using systems that exaggerate needs and costs are crucial to effective intended as guidelines for granting biowaivers for clinical studies 3266
3202 drug development. The earlier that these concerns can be addressed, intended to validate formulation changes post-approval meaning 3267
3203 the lower will be the need for rework with ensuring shorter cycle that the definitions are designed to be strict and specific to that 3268
3204 times, lower costs and potentially higher overall quality. purpose. While solubility and permeability are essential to 3269
3205 The purpose of this section is to present formulation selection understand when and how much support is needed to validating 3270
3206 philosophies focussing on preclinical in vivo tools. This section will a formulation change, the underpinning scientific framework is 3271
3207 also include a discussion on important drug candidate properties also useful in assessing drug candidates in terms of their formu- 3272
3208 impinging on formulation selection such as API physicochemical late-ability (Wei et al., 2008). This is done using a variant of the 3273
3209 properties, pharmaceutical manifestations and biopharmaceutical BCS, i.e. the developability classification system or DCS (Butler 3274

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
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34 E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx

Fig. 9. Selection strategy for enabled (non-conventional) formulations.

3275 and Dressman, 2010). This paradigm differs from BCS in that it 12.1. Physicochemical API properties as indicators of oral delivery 3314
3276 focuses on the formulation rather than on the API and attempts challenges 3315
3277 to assess factors useful in judging the formulation difficulty which
3278 may be encountered. The DCS separates Class II candidates into For poorly water-soluble compounds, the causes of their poor 3316
3279 two types: namely Class IIa, compounds which are dissolution-rate solubility can also aid in improving oral bioavailability through 3317
3280 limited in terms of their oral bioavailability and Class IIb, appropriate formulation concepts. This can be appreciated by 3318
3281 compounds which are solubility limited. This difference can be assessing the empirical relationships described by Ran and 3319
3282 appreciated by considering the Noyes–Whitney equation: Yalkowsky such as the relationship presented below: 3320
3321
3283 
log Sw ¼ 0:5  log P  0:01ðMP ð CÞ  25Þ 3323
dC DA
¼
3285 dt VhðC s  C t Þ where the log of the water solubility is given as a function of the 3324
log P and the melting point (MP) (Ran and Yalkowsky, 2001). In 3325
3286 where dC/dt is the dissolution rate, A is the surface area of the other words, compounds can be limited in their aqueous solubility 3326
3287 drug, D is the diffusion coefficient of the drug, V is the system by wettability factors or by crystal lattice forces. Knowing which 3327
3288 volume, h is the thickness of the unstirred water layer separating component is more influential in limiting the water-solubility can 3328
3289 the drug and bulk media, Cs is the saturation solubility of the be instructive in selecting a solubilization strategy. In addition, 3329
3290 drug and Ct is the concentration of drug at time, t. In a formula- the complexity of finding a solution is suggested by the two limiting 3330
3291 tion context, if reducing the particle size or increasing wettability conditions wherein log P-limited water solubility is more easily ad- 3331
3292 (i.e., increasing A or decreasing h) leads to an increased oral bio- dressed than melting point-limited solubility. When crystal lattice 3332
3293 availability, then such API’s could be considered as dissolution- energy limits solubility, the drug is not only insoluble in water 3333
3294 rate limited. If these manipulations do not impact dissolution rate but in other solvents and carriers as well. Based on these two 3334
3295 or oral bioavailability, the system could be considered solubility- factors, ‘‘grease-balls’’ (high log P materials) may be more easily for- 3335
3296 limited. This latter situation suggests that either the drug form mulated using solubilizing strategies while ‘‘brick-dust’’ (high melt 3336
3297 should be changed to increase drug absorption (i.e., the chemical point compounds) might lend themselves better to particle size 3337
3298 potential of the API should be increased) or that the additives reduction. 3338
3299 should be included to decrease the chemical potential of the drug Historically, many attempts have been made to use physico- 3339
3300 in the dissolved state. Thus, both in the context of DCS chemical properties to scout out formulation trajectories based 3340
3301 assessments and more broadly, knowledge of whether the drug on the discussions outlined in the last few paragraphs. These 3341
3302 is dissolution-rate or solubility-limited in terms of its oral bio- considerations together with permeability features were used to 3342
3303 availability sets the stage for assessing formulation complexity. suggest not only if an enabling formulation approach is needed 3343
3304 That is, this simple system provides an interesting insight as to but also to give insight as to which systems might best add value. 3344
3305 the how the oral bioavailability of a drug candidate is limited This has taken the form of the following ‘‘play ground’’ diagram 3345
3306 which, by inference, can suggest how the limitation is best ad- (Fig. 10). 3346
3307 dressed. Type I compounds are soluble and permeable meaning Assessing a large group of formulated drugs, Branchu et al. 3347
3308 that only factors that impact their ability to reach absorption assessed which were formulated conventionally and which were 3348
3309 sites are of consequence (e.g., gastric emptying) making these formulated using enabled technologies and then deconvoluted 3349
3310 systems relatively formulation independent. Class II compounds the physicochemical properties of the API (Branchu et al., 2007). 3350
3311 by contrast are limited in their absorption by their dissolution This retrospective analysis found that the two groups of com- 3351
3312 rate or solubility meaning that factors which enhance these sys- pounds had different properties which could be generally de- 3352
3313 tem properties could be bioavailability-promoting. scribed such that enabled formulation were needed if the log Do 3353

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
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E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx 35

Fig. 10. Schematic view of solubility and derivative factors, including permeability, falling within conventional formulation space.

3354 (Dose Number) < 1.7, log D > 1.2, hydrogen-binding donors ple dosing design then generates a 2  2 matrix which can suggest 3383
3355 (HBD) = 0, hydrogen-binding acceptors (HBA) < 7 and molecule a formulation trajectory along with other experimental and theo- 3384
3356 surface area >406 Å2. The usefulness of this and related approaches retical findings. 3385
3357 has been limited when used prospectively with a number of root If a solution and suspension give equivalent exposure, this sug- 3386
3358 causes associated with the poor predictivity in the guise of both gests that the in vivo dissolution rate of the suspension in no way 3387
3359 Type I and II errors. One of the most glaring is related to the biorel- limits oral bioavailability. This suggests that the impact of a formu- 3388
3360 evance of these measurements. Thermodynamic solubilities, for lation on the ability of the API to be absorbed is relatively low. For 3389
3361 example, of drug candidates in compendial buffers under carefully cases where solution and suspension give similar exposure and the 3390
3362 controlled conditions have the potential of dramatically over- or absolute oral bioavailability is high, a BCS I or I-like system is sug- 3391
3363 underestimating the solubility of that drug material in the body gested. If on the other hand, a mismatch of expected maximum 3392
3364 not only as a function of the dissolving media but also by inappro- bioavailability (based on intravenous clearance) and observed 3393
3365 priately considering supersaturation and absorption. bioavailability (lower than expected) occurs then this indicates 3394
permeability issues or precipitation within the GI tract suggesting 3395
3366 12.2. Solution versus suspension dosing comparison as in vivo a BCS III or III-like system or that the API is subject of high 3396
3367 formulation finding strategy first-pass effects. Again for both of these systems, conventional 3397
formulation strategies would seem to be appropriate. For the 3398
3368 Intravenous drug testing is often important for deriving param- situation where a solution gives significantly higher exposure than 3399
3369 eters such as intrinsic clearance and other factors associated with the suspension, the data suggests that in vivo dissolution rate or 3400
3370 the interaction of the drug with the biological systems (Balani solubility is limiting, pointing to a BCS II material and one likely 3401
3371 et al., 2005; Neervannan, 2006), however these screening vectors in need of enablement (i.e., a BCS II-complex compound). The 3402
3372 do not provide insight as to whether a solid dosage form might fourth possibility is one in which a suspension provides for better 3403
3373 be useful. To this latter end, Mackie et al., described a simple oral bioavailability than a solution. Several root causes might be 3404
3374 solution–suspension–intravenous comparison (Fig. 11) of drug ascribed to this behavior including poor chemical stability of the 3405
3375 candidates in test animals, most usually the rat and dog (Mackie API when in solution within the stomach environment, saturation 3406
3376 et al., 2008). In this companion assay to API property and pharma- of an uptake transport mechanism or initial supersaturation fol- 3407
3377 ceutical assessments, a simple drug solution (generally as aqueous lowed by precipitation of the API from the solution into a more 3408
3378 20% w/v 2-hydroxypropyl-b-cyclodextrin (HPbCD)) is compared poorly dissolving form than that associated with the suspension. 3409
3379 with a drug suspension (usually as 0.5% MethocelÒ, processed If acid instability is assigned as the root cause, a number of formu- 3410
3380 using a CovarisÒ homogenizer). The dose for both formulations is lation design elements can be considered such as enteric coating or 3411
3381 10 mg/kg in the rat and 5 mg/kg in the dog. Blood levels are then co-administration of the drug with an antacid or proton pump 3412
3382 determined using appropriate LC/MS-based techniques. This sim- inhibitor, the latter under the assumption that no interactions 3413
occur. Thus, the rat and dog are used as bioreactors to assess 3414
solubility, dissolution rate and permeability. 3415
This approach has now been applied to more than 100 com- 3416
pounds (Mackie et al., 2012). Based on this perspective, a number 3417
of points have been made: A suitable suspension could be prepared 3418
for the vast majority of compounds using 0.5% w/v Methocel. In 3419
two situations, a slight modification was needed such that Metho- 3420
cel with Tween 20 was optimal for one compound and, in a second 3421
case, where HPbCD and Tween 20 were the best suspending 3422
agents. Drug solutions were possible using 20% HPbCD in 87% of 3423
cases. Other systems that were assessed included water for injec- 3424
Fig. 11. Solution–suspension–intravenous outcomes in a test species and their tion (1%), citrate–phosphate buffer (1%), TPGS in oleic acid (1%), 3425
alignment with BCS. SBEbCD (5%) and PEG400 (6%). Use of PEG 400 or others however 3426

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
 PHASCI 2973 No. of Pages 53, Model 5G
19 March 2014

36 E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx

3427 presents a difficult situation as PEG has influences on motility and clearance less than the hepatic blood flow and a bioavailability from 3493
3428 water flux in animals and humans to varying extents (Schulze the selected formulation of >30%. Li and Zhao (2007) suggested a 3494
3429 et al., 2003). It should be used cautiously or in low concentrations. similar approach with a comparison of solutions and suspensions 3495
3430 The 2  2 matrix could be bucketed using several classification to suggest whether simple or complex formulation would likely 3496
3431 cut-offs with AUC ratios of solution–suspension dosing <0.8 (sus- be of benefit (Li and Zhao, 2007). Zheng et al. (2012) suggested 3497
3432 pension more bioavailable than solution), 0.8–1.2 (solution and assigning formulation risk based on in vitro solubility as well as 3498
3433 suspension gave equivalent bioavailabilities), 1.2–2 (solutions in vivo drug levels in test species after oral dosing of solutions 3499
3434 were modestly better than suspensions) and >2 (solutions were (Zheng et al., 2012). Low risk compounds were those generating 3500
3435 significantly better than suspensions). The data suggested that: useful blood levels after dosing with vehicles containing <30% of 3501
3436 18% of compounds demonstrated better bioavailability from a sus- an organic modifier and high risk systems were those requiring 3502
3437 pension than a solution, 45% of compounds demonstrated equiva- higher levels of an organic modifier. The decision tree outlined sug- 3503
3438 lent exposure from a solution and a suspension, 26% of compounds gested that low risk compounds could be formulated using salts or 3504
3439 were modestly more bioavailable from a solution than a suspen- particle size reduction while high risk compounds would likely re- 3505
3440 sion and 10% of compounds were significantly more bioavailable quire enabled systems including amorphous solid dispersions, 3506
3441 from a solution than a suspension. Application of traditional tools nanosuspensions or lipid-based strategies. Based on the possible 3507
3442 based on thermodynamic solubility, log P, pKa, intrinsic dissolution complexity of high risk systems, additional work in parallel was 3508
3443 rate, PAMPA (parallel artificial membrane permeability assay) and suggested to optimize formulation finding. 3509
3444 related assays suggested that approximately 65% of the
3445 compounds would require some type of enablement to generate 12.3. Selection of the most appropriate formulation technology 3510
3446 a useful form preclinically or clinically. That suggests that using
3447 only physicochemical properties may overdiscriminate the need The next step, after an assessment of formulation complexity is 3511
3448 for enabled systems with the application of technologies of higher derived, is then to suggest which enabled technology is best placed 3512
3449 complexities and costs than needed as well as a higher risk of to solve the specific issues associated with the drug candidates 3513
3450 rework. This combination of API, pharmaceutical and biopharma- being considered (Kawakami, 2009). One approach is to extract 3514
3451 ceutical data then suggest whether formulation enablement is value from various theoretical as well as down-scaled, automated 3515
3452 likely needed. filters to align a formulation type with the compound of interest 3516
3453 A similar suspension vs. solution approach has been performed (Fig. 12). 3517
3454 by Muenster et al. using a physicochemical diverse Bayer pipeline The philosophy associated with the formulation filters is based 3518
3455 compound set (Muenster et al., 2011). Here, a correlation of in vivo on a deconstruction of important formulation elements and then 3519
3456 dissolution in the rat at respective predicted therapeutic doses in the development of tests to assess these aspects. Four possible en- 3520
3457 humans vs. actual in vivo dissolution data in humans (tablet or sus- abled strategies are included in this process approach including 3521
3458 pension vs. solution) of the same compounds was established. Data nano-crystalline suspensions, amorphous solid dispersion, liquid- 3522
3459 suggest that if AUCnorm suspension vs. AUCnorm solution in the rat is filled capsules and ‘‘others’’. 3523
3460 >50%, in vivo dissolution in humans is sufficient for the develop-
3461 ment of a standard IR tablet, without enabling technologies 12.4. Inclusion of computational assessments as additional tools 3524
3462 needed. Furthermore, correlation of suspension vs. solution in rat
3463 vs. dose/solubility ratios at various pH revealed a good correlation Theoretical assessments are based on PBPK models with the two 3525
3464 at pH 4.5 and 7, indicating that at dose/solubility ratios of main tools including GastroPlus™, an advanced compartmental and 3526
3465 <100 mL/kg (pH = 4.5) and <500 mL/kg (pH = 7) no enabling formu- transit (ACAT) model (SimulationsPlus, Lancaster, CA) and SimCyp, 3527
3466 lation technology is needed for the development of an oral market an advanced dissolution, absorption and metabolism (ADAM) mod- 3528
3467 formulation for humans. However, a poor correlation between el (Simcyp, Sheffield, UK). GastroPlus™ can be used both to predict 3529
3468 in vivo dissolution in the rat and dose/solubility ratios at pH = 1 API, pharmaceutical and biopharmaceutical properties based only 3530
3469 was observed, suggesting that the rat is good predictor for neutral on the chemical structure or with additional experimentally 3531
3470 and acidic API dissolution, but may underestimate in vivo dissolu- determined data with an increasing predictivity as a function of 3532
3471 tion of weak bases in humans. more and higher quality data (Hosea and Jones, 2013; Kuentz 3533
3472 Early formulation finding strategies have also been described by et al., 2006; Sjögren et al., 2013; Tsume et al., 2012). Thus, even with 3534
3473 Maas et al. who also assessed API, formulation and biopharmaceuti- only the chemical structure, initial suggestions of the compound 3535
3474 cal factors (Maas et al., 2007). In their approach, a number of solubility and absorb-ability can be estimated and reported in terms 3536
3475 formulation platforms are identified to address both simple-to-for- of a dose, dissolution and absorption number (Do, Dn and An, respec- 3537
3476 mulation API’s as well as those in need of enablement. Intravenous tively) as well as the maximum absorbable dose (MAD). The terms 3538
3477 dosing is first completed to verify that PK properties are appropriate are defined as follows: 3539
3478 and then is an oral suspension dosed, first in rat and subsequently in
3479 dog. If exposure is satisfactory, formulation trajectories are selected  Dose number (Do) – the dose divided by the product of 3540
3480 from the conventional toolbox. If suspension dosing provides for delivered volume (250 mL) and solubility of the drug: 3541
3481 poor exposure, other manipulations of the API are considered Do = Dose/(V  Cs) (the lower the better). 3542
3482 including milling. These studies then chart a way forward for the  Dissolution number (Dn) – the ratio of mean residence time and 3543
3483 possible formulation possibilities. Saxena et al. used a similar strat- mean dissolution time (the higher the better). 3544
3484 egy with a comparison of drug solution and suspensions (Saxena  Absorption number (An) – the ratio of the mean residence time 3545
3485 et al., 2009). This decision tree thus suggested that bioavailability and mean absorption time (the higher the better). 3546
3486 from a solution should be reasonable. If exposure of the suspension  Maximum Absorbable Dose (MAD) – the product of the drug 3547
3487 was more than 2-fold lower, a number of alternative processing solubility, absorption rate constant, fluid volume and transit 3548
3488 technologies were suggested including salt screening, solid disper- time (i.e., solubility and permeability are compensatory): 3549
3489 sions, lipid based methods and suspension/nanosuspensions. The MAD = S  Ka  V  T. 3550
3490 approach suggested that the preclinical formulations should result 3551
3491 in certain minimal pharmacokinetic properties to justify progress- An assessment of itraconazole (including API, pharmaceutical 3552
3492 ing the compound including an acceptable terminal half-life, a total and biopharmaceutical properties) was completed using only the 3553

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
 PHASCI 2973 No. of Pages 53, Model 5G
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E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx 37

Fig. 12. Decision approach for the selection of an enabled technology.

3554 chemical structure of the compound. This information-risk read- and increasing the drug solubility from 0.0006 mg/mL to 0.06 mg/ 3591
3555 out suggests that itraconazole is a BCS Type II/DCS type IIb drug mL would increase the drug fabs to >75%. Clearly, these are gross 3592
3556 with a low predicted water-solubility (0.0006 mg/mL) and a estimates that do not take into account a number of important 3593
3557 MAD of 48 mg. This is in contrast to doses that are needed to processes and factors but these early assessments can give some 3594
3558 generate a useful anti-fungal effect (200 mg loading dose and insight within a specified error as to whether a particular formula- 3595
3559 100 mg maintained dose). A parameter sensitivity plot can also tion direction is possible or less interesting. 3596
3560 be rendered that suggests what the effect of drug solubilization In addition to solubilizing strategies, PBPK modeling can also 3597
3561 might be on the fabs (in this case the amount of drug reaching shed light on the possible application of particle size reduction 3598
3562 the portal blood) (Brewster et al., 2007). with regard to improved oral bioavailability. This is again 3599
3563 These calculations suggest that the low fabs is strongly influ- completed using a parameter sensitivity analysis but in this case 3600
3564 enced by the degree of drug solubilization and that even modest the variable is API particle size. That is, the percent drug absorbed 3601
3565 increases in this factor can strongly influence the amount of drug (into the portal circulation) is assessed as a function of reducing 3602
3566 taken up into the presystemic circulation based on high intrinsic the particle size into the nano-domain. Data thus derived suggest 3603
3567 permeability. This analysis suggests that increasing the drug that, in this case, reducing the particle size even to 1 nm would 3604
3568 solubility from 0.0006 mg/mL to 0.1 mg/mL would make drug not influence the oral bioavailability. This finding has since been 3605
3569 absorption almost quantitative. This information may then initiate corroborated in animal studies suggesting that the solubility of 3606
3570 an assessment of solubilizing technologies to generate an appropri- the compound is so low that particle size reduction techniques 3607
3571 ate liquid or solid dosage forms. In the case of itraconazole, two are not useful or that the saturation solubility is reached faster 3608
3572 useful solubilizing vehicles were identified including PEG400 (sol- than disappearance of dissolved API is happening by diffusion/ 3609
3573 ubility of itraconazole = 2 mg/mL) and 40% w/v HPbCD (solubility permeability. 3610
3574 of itraconazole = 10 mg/mL). GastroPlus™ suggested that both of Theoretical assessments (which are or are not bolstered by 3611
3575 these systems would increase the fabs and this was verified in both other data) are useful inputs but cannot fully position or align a 3612
3576 animal and clinical studies. The HPbCD vehicle served as the basis formulation approach and compound. The formulation process is 3613
3577 for a marketed oral solution and intravenous formulation. Another based on several factors including the need for processing informa- 3614
3578 use of the parameter sensitivity (spider plot) is to assess the possi- tion as well as data on the excipient design space, etc. To fill these 3615
3579 ble application of an amorphous dosage form concept. In assessing knowledge gaps, experimental work is suggested. In order to 3616
3580 this trajectory, an estimate is made of the solubility enhancing complete these experiments in a time- and compound-efficient 3617
3581 effect of converting the drug from its crystalline form to the manner, down-scaled, automated tests have been designed and 3618
3582 amorphous phase. A variety of approaches can be assessed. The evolved to assess not only the most appropriate technology for 3619
3583 formalism of Hancock and Parks (2000) can be applied wherein the particular API but also to give insight into processing aspects 3620
3584 the increased solubility is inferred using thermo-analytical data of formulation. These miniaturized, automated workflows are de- 3621
3585 obtained by Differential Scanning Calorimetry (DSC) including signed to address key formulation questions to help identify the 3622
3586 the melting point and heat of fusion of the crystalline phase, the most appropriate dosage form platform, enabled strategy or direc- 3623
3587 glass transition temperature and change in heat capacity at the tion as well as to eliminate possibilities that are not likely to add 3624
3588 glass transition temperature (Tg) for the amorphous phase value. Implicit in all of these simplified models is that their output 3625
3589 (Hancock and Parks, 2000). In the case of itraconazole, the solubil- needs to be continuously confirmed and checked as a function of 3626
3590 ity ratio of amorphous/crystalline drug is 100. Assessing these data compound development. 3627

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
 PHASCI 2973 No. of Pages 53, Model 5G
19 March 2014

38 E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx

3628 Key questions are therefore collected as a function of the four 12.6. Solid dispersion feasibility 3692
3629 formulation directions. For a nanosuspension to be useful, the abil-
3630 ity to mill an API to a desired size is important (mill-ability) as is Important elements in the construction of a useful amorphous 3693
3631 the physical stability of the milled dispersion. For an amorphous solid dispersion include the ability to dissolve or disperse a drug 3694
3632 solid dispersion to be useful, glassy carriers that adequately dis- in a glassy or semi-crystalline polymer as well as the stability of 3695
3633 perse the amorphous drug are needed and the resulting systems the formed dispersion. As a consequence, amorphous solid disper- 3696
3634 need to be stable to phase separation and crystallization. Finally, sions should act as supersaturating drug delivery systems. That is, 3697
3635 liquid-filled capsule strategies need to contain excipients that ade- the release of the amorphous API, the rate of which is ideally 3698
3636 quately solubilize the drug dose in an appropriate volume and the governed by the dissolution of the glassy carrier, is such that nucle- 3699
3637 said dispersion needs to be stable both chemically and physically. ation and crystal growth is delayed and the formed supersaturated 3700
3638 Thus once enablement is tangibly decided upon, all three enabled system is produced in a way to allow for sufficient drug absorption. 3701
3639 possibilities are assessed using both fast and compound-sparing Thus, excipients that act as nucleation or crystal growth inhibitors 3702
3640 experimental approaches with a selection based on the assigned can increase the efficacy of the formulation. In the best case, the 3703
3641 inclusion and exclusion criteria. precipitation inhibitor is the glassy carrier. Alternatively, precipita- 3704
tion inhibitors can be added to the glassy carrier. Finding compo- 3705
3642 12.5. Nanosuspensions feasibility: mill-ability and dispersion stability nents that may impact supersaturation stability is therefore 3706
integral to building up a useful solid dispersion (Bevernage et al., 3707
3643 The workflow identified to assess the likelihood that a nanosus- 2012; Brouwers et al., 2009; Takano et al., 2010). Specialized 3708
3644 pension might serve as the enabled formulation strategy, are based workflows have been developed to screen for these and other prop- 3709
3645 on several purpose-built and generic automation platforms erties. Specific tools to assess precipitation inhibition, film casting 3710
3646 including a multiplexed nanomill. This attrition mill consists of and down-scaled processing are available and these will be 3711
3647 10 independent milling heads that can be independently con- discussed in turn. Assessing whether excipients are available to 3712
3648 trolled both in terms of milling speed and time. The systems makes impact precipitation rate is conducting using a solvent shift/ 3713
3649 use of 2.8 mL disposable milling chambers which can be filled quench approach (Vandecruys et al., 2007; Warren et al., 2010; 3714
3650 using automated workflows and each milling station is actively Yamashita et al., 2011). A supersaturated system is generated by 3715
3651 cooled. Milling media are generally 0.5 mm highly reticulated adding the drug dissolved in a water-miscible organic solvent to 3716
3652 polystyrene beads and particle size analysis is completed by har- an aqueous solution of the excipient of interest present at a con- 3717
3653 vesting the nanosuspension using an insulin syringe (U-100, centration of 2.5% (w/v) at an appropriate pH. The concentration 3718
3654 0.5 mL, 0.33 mm  12.7 mm) followed by the particle size distribu- achieved in this system as well as the rate of precipitation is 3719
3655 tion assessment measured using a Malvern Mastersizer coupled assessed analytically using either nephelometry or filtration fol- 3720
3656 with a Hydro lP dispersant unit and a Wyatt DLS plate reader. lowed by UPLC (Ultra performance liquid chromatography) as a 3721
3657 The multiplexed mill is designed to assess multiple formulation function of time. Excipients usually used in this assay include 3722
3658 aspects and design space elements in a concerted fashion to rapidly rheological and other polymers including cellulosic systems, sur- 3723
3659 find fit-for-purpose formulations. In addition, the mill is configured factants, cyclodextrins and related materials. Both the extent and 3724
3660 to scale to larger processing situations (i.e., to the nanomill, stability of the formed supersaturated systems is important in 3725
3661 dynomill and Netzsch mill). The experimental workflows generally assessing the possible use of the excipients as components in the 3726
3662 begin with an assessment of excipient space (including primary amorphous solid dispersion-based formulation. Once this excipient 3727
3663 and secondary stabilizers) and processing space (including milling space has been assessed, these materials are used to form films 3728
3664 speed and time). The important endpoint derived from these with the API. This exercise is intended to interrogate API-excipient 3729
3665 experiments includes mill-ability (the ability to reduce the particle miscibility in the solid state as well as the tendency for the 3730
3666 size to a proscribed average and distribution) and dispersion stabil- dispersed API to remain as such over time (that is to maintain 3731
3667 ity over time and under various conditions. the dispersion without phase separation or recrystallization) 3732
3668 A practical example may be useful in outlining the utility of this (Janssens et al., 2010; Weuts et al., 2011). Films are case out of a 3733
3669 approach. Itraconazole was evaluated as an injectable nanosuspen- common solvent (a solvent providing for good solubility of both 3734
3670 sion in several clinical studies (Mouton et al., 2006). In developing the API and polymer/excipient) and the formed film is analytically 3735
3671 this formulation, two key factors were screened as a function of assessed (usually using white light and birefringence microscopy, 3736
3672 development including which surfactant manifested the best mill- XRD and DSC). In addition, the dissolution properties of the films 3737
3673 ing and stabilizer properties and what was the optimal milling are assessed to see whether supersaturation occurs and whether 3738
3674 time. Both of these could be assessed in the downscaled the API remains in solution. These tasks are performed in an auto- 3739
3675 multiplexed mill. Itraconazole was milled in the presence of sev- mated manner using specially designed 96-well plates to allow for 3740
3676 eral potential ionic and non-ionic stabilizers at ratios of 1:4 relative the analytical assessments as well as the dissolution evaluation 3741
3677 to the API. Standard protocols included milling the material for (Brewster et al., 2011). The microscopy can suggest changes in 3742
3678 60 min at 4000 rpm in the presence of 0.5 mm highly reticulated the degree of miscibility including phase separation and crystalli- 3743
3679 polystyrene milling media. Particle sizes were then assessed at zation while XRD and DSC are confirmatory method to assess 3744
3680 the end of milling as well as at one and two weeks after milling any increase in crystalline content over time. 3745
3681 with the samples stored at various conditions (5, 25 and 40 °C). The application of these approaches to a poorly water-soluble 3746
3682 Of the 25 unitary or binary stabilizer systems assessed, Poloxamer drug candidate can be illustrative. A drug candidate has a melting 3747
3683 388 proved to be the most useful under these conditions. Based on point of 270 °C and a measured Tg of 113 °C with a molecular 3748
3684 this excipient, milling curves were generated wherein itraconazole weight of 433 g/mol. The compound has a pKa of 3.5 and a solubil- 3749
3685 was milled for times varying between 15 and 300 min with phys- ity in simulated intestinal fluid of 0.6 lg/mL. Excipient screening 3750
3686 ical stability follow-up. Based on d90 and d99 measurements, a studies were completed using the automated 96-well plate meth- 3751
3687 suspension of a useful size could be generated after 120 min of od. In this screen a total of 41 excipients were assessed alone or in 3752
3688 milling, less time generated undermilled systems. At long milling combination at four different concentrations. The study suggested 3753
3689 times (i.e. 300 min), particle growth on storage was noted sug- that the following materials provided for significant effects on the 3754
3690 gested that these systems were overmilled. Conditions from the extent and duration of supersaturation: Solutol, Poloxamer 407 3755
3691 downscaled multiplexed mill could be scaled to the Netzsch mill. and TPGS. Films then were cast using the automated protocol in 3756

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
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E. Sjögren et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx 39

3757 which 95 excipients or excipient combinations were assessed systems showing good solubility can then be added to water to as- 3821
3758 based on unitary, binary or ternary systems at two API to sess emulsifying or self-emulsifying properties based on a two- or 3822
3759 excipient(s) ratios. Hits from the precipitation inhibition screen three-phase diagram. Isotopically clear systems indicative of 3823
3760 were included in the film experiments. Identified dispersions micro/nano-emulsification can be assessed using white light and 3824
3761 which were amorphous and stable over time included: birefringence microscopy. 3825
3762 API:HPbCD:TPGS, API:HPbCD:HPMC, API:HPMC:Poloxamer 407, A case study optimizing a poorly water-soluble drug candidate 3826
3763 API:HPMC-P:Poloxamer 407 and API:HPbCD:Solutol. Dissolution as a self-emulsifying system was addressed. The compound gave 3827
3764 profiles were completed for films that performed well in stability poor exposure when dosed as a suspension compared to a solution 3828
3765 and physicochemical property assessment using a 96-well two- (a relative bioavailability of 1.7%). Precipitation inhibition screen- 3829
3766 stage dissolution approach in which simulated gastric fluid is ing using a 96-well plate assay suggested that the best materials 3830
3767 added at time 0 followed by FaSSIF, which was added at 60 min. were surfactants with the three most important hits being TPGS, 3831
3768 This protocol assessed supersaturation under conditions which Cremophor RH40 and Tween 20. These materials were then in- 3832
3769 might represent the stomach to intestine transition. Dissolution cluded in a larger SEDDS (self-emulsifying drug delivery system) 3833
3770 profile for the API as well as for cast films suggested an increasing screen which made used of a list of 50 lipids and surfactants in 3834
3771 supersaturation tendency in the order API < API:HPMC various ratios. In this assay, solubility was optimized with 18 hits 3835
3772 < API:HPMC;TPGS < API:HPbCD:HPMC. In aggregate, the data sug- giving solubilities >50 mg/g. These hits were then screened for bio- 3836
3773 gested that the most robust system was the API:HPbCD:HPMC in pharmaceutical properties in the rat and compared with a standard 3837
3774 terms of (1) manifesting good supersaturation, (2) showing mini- solution (drug dissolved in TPGS/NMP). Based on the drug solution, 3838
3775 mal precipitation upon SGF-FaSSIF transfer and (3) demonstrating 5 formulations gave significant increases in exposure with the best 3839
3776 insensitivity to composition. These systems were then used to formulation containing Capmul PG8, Cremophor RH40 and lauric 3840
3777 configure test formulations to assess biopharmaceutics in the rat. acid. This formulation gave a relative bioavailability of 165% versus 3841
3778 Formulations involved spray drying the ingredients and adminis- the drug solution and increased the absolute bioavailability to 3842
3779 tering the spray dried powders to rats by gavage in a 0.5% methocel almost 50%. 3843
3780 suspension. The test formulations administered in this way in-
3781 cluded: API:HPbCD:TPGS (1:3:1), API:HPMC:TPGS (1:3:1), 12.8. Predictability of animal models for humans 3844
3782 API:HPMC (1:1), drug milled into the nano-domain and micronized
3783 drug. Using the micronized suspension as a reference, all of the While these factors target API and pharmaceutical aspects in 3845
3784 enabled systems provided for varying degrees of benefit. The formulation finding, an assessment of biopharmaceutical proper- 3846
3785 nano-sized suspension was almost 3-fold more bioavailable than ties are likewise of interest. The selection of an appropriate animal 3847
3786 the micronized suspension while the HPMC dispersion gave almost model to assess these formulation concepts is complex (see 3848
3787 4-fold higher exposure. Consistent with the dissolution profiles, sections above) and is impacted by an amalgam of API, pharmaceu- 3849
3788 the best formulation in the rat was the API:HPbCD:TPGS which tical and biopharmaceutical features. Wu et al. (2004) suggested 3850
3789 gave an oral bioavailability almost 9-fold greater than that of the that the dog was a useful model for assessing nanosuspension 3851
3790 simple micronized drug. These rough designs were then converted based on both translate-ability to man as well as the ability of 3852
3791 to formulations that might be tested clinically. These more refined the dog model to predict food effects attenuated by the nanosus- 3853
3792 systems included: (A) a spray-dried solid dispersion of pension approach (Wu et al., 2004). The model compounds as- 3854
3793 API:HPbCD:TPGS filled into a gelatin capsule, (B) a dispersion of sessed in these studies were MK-0869 (aprepitant). By contrast, 3855
3794 API:HPbCD:HPMC coated onto an inert Mono-N-carboxymethyl Mackie et al. (2009) and Ouwerkerk-Mahadevan et al. (2011) sug- 3856
3795 chitosan sphere using a closed Wurster process and filled in a cap- gested that the rat was a better model to assess nanosuspension 3857
3796 sule, (C) a solid dispersion of API:HPMC pressed into a tablet, (D) a based on comparison of rat, dog and human clinical data (Mackie 3858
3797 nanosuspension-based tablet and (E) API in capsule. These systems et al., 2009; Ouwerkerk-Mahadevan et al., 2011). In these assess- 3859
3798 were then assessed in a traditional USP II dissolution apparatus ments, two compounds were evaluated and the alignment of rat 3860
3799 using a two-phase transfer model approach. Not only did the opti- and human data were thought to be related to pH difference in 3861
3800 mized formulations retain the dissolution profiles of the simple the rat and dog GI tract as well as the more human-like transit 3862
3801 dispersions from where they were derived, but the USP II data were time. The differences in outcome of these two sets of studies 3863
3802 well correlated with the 96-well two-phase dissolution method. may be related to the API chemotype assessed in that aprepitant 3864
3803 Importantly, the oral bioavailability assessment seen in rats of (pKa = 9.7, MP = 254 °C, log P = 4.8 and water solubility of 3–7 lg/ 3865
3804 the simple systems was maintained in other animal models with mL) and the compounds from the second set of studies (Compound 3866
3805 the optimized dosage forms. A, pKa = 3.45, MP = 216 °C, log P = 3.1, water solubility <0.1 lg/mL; 3867
Compound B, pKa < 2, MP = 156 °C, log P = 4.6, water solubility 3868
3806 12.7. Liquid-filled capsules (lipids/surfactants/S(M/N)EDDS/solvent) <0.1 lg/mL) differ in several important respects. These include 3869
3807 feasibility the pKa and water solubility potentially biasing either the rat or 3870
dog to be the better model of translation. 3871
3808 A third possible enabling formulation trajectory included sol- Newman et al. (2012) discussed the application of various 3872
3809 vent-, lipid-, surfactant- or lipid/surfactant-based systems. These in vivo models in the assessment of solid dispersions (Newman 3873
3810 are important in the industry with an estimated 2–4% of marketed et al., 2012). In their retrospective analysis of 40 studies, the fol- 3874
3811 oral dosage forms using these concepts. Key questions that need to lowing animal models were described including the dog (41%), 3875
3812 be answered regarding the possible use of these technologies rat (24%), rabbit (15%) and monkey (2%). In some cases, the dog 3876
3813 include whether the intended dose can be solubilized in an appro- model was altered using agent to modify the GI pH. In 3877
3814 priate volume of vehicle (capsules usually limit this to 1 mL) and at vitro–in vivo relationships could be constructed using animal and 3878
3815 an appropriate pill burden and whether the API so solubilized is dissolution data in all but 1 rat study, 3 rabbit studies and 2 dog 3879
3816 physically and chemically stable over time. In addition, if a S(M/ studies. Generally, selection criteria of the animal model for a par- 3880
3817 N)EDDS (Self microemulsifying/nanoemulsifying drug delivery ticular formulation is most often aligned with biopharmaceutics 3881
3818 system) is intended, does the system perform in vivo as designed. (including physiological and metabolic similarities to man, trans- 3882
3819 The pharmaceutical questions are assessed using an automated, late-ability to man) and less on pharmaceutical and API properties. 3883
3820 down-scaled workflow based on viscous liquid handling. Solvent Nonetheless, the appropriate selection of an animal model will 3884

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
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3885 require all three axes to be incorporated into a decision tree includ- 13. Overall gap analysis 3950
3886 ing comparisons of fluid volumes as a function of GI location (as
3887 well as when comparing the fed and fasted model) as well as GI This review has made clear that there are important gaps in our 3951
3888 regional pH, composition and ability to support supersaturation. understanding of the human GIT as well as the physiology of ani- 3952
3889 In a line of reasoning similar to the nanosuspension discussion mals that are currently used for in vivo drug and formulation 3953
3890 the selection of an animal model for amorphous solid dispersion characterization. 3954
3891 is best completed on a case-by-case basis – factors to be taken into Typical gaps in knowledge about human GI physiology affecting 3955
3892 account include also the relevant API and its pharmaceutic and bio- dosage form performance have been mentioned in this review 3956
3893 pharmaceutic properties. Other species such as the pig and minipig including e.g. intraluminal water availability in particular in seg- 3957
3894 may also be of value in these assessments in addition to the more ments that are low in water content but important for dosage form 3958
3895 generally applied models (see Section 4). performance and absorption such as the small and large intestines. 3959
3896 Lipid-based formulations have a number of factors in common Likewise little information is available on the magnitude and fre- 3960
3897 with the other formulation types described but also a number of sig- quency of intraluminal pressures and hydrodynamics that act as 3961
3898 nificant differences. In common with other approaches, formulation mechanical stress factors and thus affect the integrity of dosage 3962
3899 processing can generate supersaturation which may be an impor- forms (disintegration, erosion). This information is however needed 3963
3900 tant system aspect for enablement (Brouwers et al., 2009; Williams in order to design more meaningful in vitro test systems that are 3964
3901 et al., 2013). By contrast, lipid formulations may also be processed reflective of the in vivo situation. Relevant for the active ingredient 3965
3902 by digestion and the API-lipid or digestion products may be useful itself is the question about its mechanism of intestinal permeation 3966
3903 modalities for targeting lymphatic absorption. The importance of and possibly metabolism. In recent years some progress has been 3967
3904 these factors means that more animal model development and opti- made but not sufficient for a clear understanding about carrier- 3968
3905 mization have to be completed as a function of the pharmaceutical mediated and passive components of drug absorption in the intes- 3969
3906 axis for these systems. In addition since a number of chemotype tine. Identification of carriers, their expression along the GIT and 3970
3907 rules have been postulated related to log P, molecular weight and li- relationships between expression and drug affinity are just some 3971
3908 pid solubility requirements, more API insight is also applied to the examples where progress needs to be made. This holds true for hu- 3972
3909 selection of an animal model. Generally, rat and dog studies are mans but also for most of the animal models that are used for drug 3973
3910 used for all three assessments. The dog is more commonly used to and dosage form characterization. Important differences e.g. in gas- 3974
3911 assess the effect of lipid on absorption consistent with its use in tric and intestinal pH, gastric emptying and intestinal transit times 3975
3912 evaluating food effects however models for lymph duct cannulation and motility as well as intestinal permeability may be taken as the 3976
3913 are available for both species (Porter and Charman, 2007). Dogs also underlying cause for differences between animal species and insuf- 3977
3914 have the advantage that human-scaled dosage forms can be admin- ficient predictability of the effects in humans. An analysis of dat- 3978
3915 istered (Charman et al., 1997; O’Driscoll, 2002). abases covering different formulations and drugs in different 3979
3916 As the use of enabling formulations increases and as API’s species is needed in order to better define the relationships between 3980
3917 becoming increasingly difficult to formulate, appropriate animal chemical space of the compound, formulation space and usefulness 3981
3918 models to study these systems are essential. While historically, as screening tool and predictability for humans. Imaging tools for 3982
3919 the focus associated with selecting an animal model has been bio- animals and for humans represent important tools to investigate 3983
3920 pharmaceutical in nature, more and more, factors associated with some of these phenomena and need to be established further. 3984
3921 model alignment as a function of API properties and formulation Progress needs also to be made in particular for understanding 3985
3922 processing will likely grow in importance. This is based on both and better predicting the activity of compounds exerting low water 3986
3923 the need for increased translation from the preclinical species to solubility in order to design formulations that will predictively de- 3987
3924 man but also in an effort to generally reduce the number of liver their active ingredients in various patient groups. Among 3988
3925 animals used in formulation optimization and drug development. these factors, variability in composition and relevance of GI fluids 3989
3926 In vivo factors associated with supersaturation, excipient for drug solubilization should be mentioned. On the same token lit- 3990
3927 processing and API uptake at the intestinal mucosa will also have tle is known about the intraluminal behavior of formulations, i.e. 3991
3928 increasing impact on the choice of a useful animal model (Bever- the concentrations of drugs following oral administration of their 3992
3929 nage et al., 2012; Brouwers et al., 2009; Takano et al., 2010). formulations in the lumen of the stomach and the small intestine, 3993
3930 In contrast to the various animal models being available to their solubilization and precipitation also as a function of chemical 3994
3931 make predictions of in vivo dissolution and permeability for IR for- structure (acids versus bases versus neutral compounds) and sites 3995
3932 mulations in humans, to date, there is no established animal model (stomach versus small intestine) and dose. This knowledge is 3996
3933 published that would reliably predict colonic API stability needed to understand the performance of enabling formulations 3997
3934 (microbiota), permeability, and dissolution. A biopharmaceutical for low soluble compounds as well as to better reflect the solubil- 3998
3935 colon model would be of high interest for the developability ity/permeability relationships that result from the dissolution and 3999
3936 assessment of slow release formulations. Human microflora con- solubilization of the API and its permeation across the intestinal 4000
3937 sisting of 10–100 trillion cells, from 160 species is a complex epithelium. Ignorance of these items leads to a trial and error ap- 4001
3938 mixture which requires certain technical know-how to culture. proach in the design of in vitro experimental conditions that may 4002
3939 Also, it is important to keep the in vitro culture media to the most or may not reflect the in vivo situation appropriately. 4003
3940 physiological relevant composition to allow the microbiota to In particular for controlled release dosage forms the knowledge 4004
3941 exhibit their natural enzymatic activity (Qin et al., 2010). Experi- about their in vivo transit and processing as well as the absorption 4005
3942 mentally, in vitro API stability and dissolution experiments have of the released drug in different intestinal segments is crucial but 4006
3943 been performed (www.TNO.nl.pharma; www.prodigest.eu), frequently not available. Thus sufficient resources may be spent 4007
3944 however, the number of compounds of which colonic stability in vain trying to develop formulations for compounds that 4008
3945 and dissolution data have been validated against human PK data intrinsically are not prone to be delivered in oral sustained release 4009
3946 are very limited, and respective in vivo animal and human colonic systems simply as a consequence of their cumbersome absorption 4010
3947 absorption and/or dissolution data are not publically available to and metabolism properties. 4011
3948 an extent that would allow the generation of a predictive biophar- Another wide open field is the personalized medicine approach 4012
3949 maceutical colon model. and the need to predict drug product performance not just in 4013

Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
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potential carrier in solid dispersion formulations. Colloids Surf. B, Biointerfaces 4086
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in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
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in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010
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Please cite this article in press as: Sjögren, E., et al. In vivo methods for drug absorption – Comparative physiologies, model selection, correlations with
in vitro methods (IVIVC), and applications for formulation/API/excipient characterization including food effects. Eur. J. Pharm. Sci. (2014), http://
dx.doi.org/10.1016/j.ejps.2014.02.010