IN SITU PERFUSION IN RODENTS TO EXPLORE INTESTINAL DRUG

ABSORPTION: CHALLENGES AND OPPORTUNITIES

Jef Stappaerts, Joachim Brouwers, Pieter Annaert and Patrick Augustijns

Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, Leuven, Belgium

Corresponding author:

Patrick Augustijns

Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences

Gasthuisberg O&N 2 - Herestraat 49 box 921 - 3000 Leuven - Belgium

tel: +32-16-330301 - fax: +32-16-330305

e-mail: patrick.augustijns@pharm.kuleuven.be

Keywords: transporter-metabolism interplay; site dependent absorption; knockout animals;

solubility-permeability interplay; supersaturation; intestinal perfusion

1

ABSTRACT

The in situ intestinal perfusion technique in rodents is a very important absorption model, not

only because of its predictive value, but it is also very suitable to unravel the mechanisms

underlying intestinal drug absorption. This literature overview covers a number of specific

applications for which the in situ intestinal perfusion set-up can be applied in favor of

established in vitro absorption tools, such as the Caco-2 cell model. Qualities including the

expression of drug transporters and metabolizing enzymes relevant for human intestinal

absorption and compatibility with complex solvent systems render the in situ technique the

most designated absorption model to perform transporter-metabolism studies or to evaluate

the intestinal absorption from biorelevant media.

Over the years, the in situ intestinal perfusion model has exhibited an exceptional ability to

adapt to the latest challenges in drug absorption profiling. For instance, the introduction of the

mesenteric vein cannulation allows determining the appearance of compounds in the blood

and is of great use, especially when evaluating the absorption of compounds undergoing

intestinal metabolism. Moreover, the use of the closed loop intestinal perfusion set-up is

interesting when compounds or perfusion media are scarce. Compatibility with emerging

trends in pharmaceutical profiling, such as the use of knockout or transgenic animals,

generates unparalleled possibilities to gain mechanistic insight into specific absorption

processes.

Notwithstanding the fact that the in situ experiments are technically challenging and relatively

time-consuming, the model offers great opportunities to gain insight into the processes

determining intestinal drug absorption.

2

CONTENTS

Abstract.................................................................................................................................................................................... 2

1. Introduction.................................................................................................................................................................... 4

2. Permeability assessment – disappearance (Peff) versus appearance (Papp).................................................5

2.1 Measuring disappearance from the perfusion solution - Effective permeability..................................5

2.2 Measuring appearance in the blood - Apparent permeability.....................................................................6

2.3 Vascular perfusion.....................................................................................................................................................8

2.4 Open loop and closed loop intestinal perfusions.............................................................................................8

3. Exploring the biochemical barrier function of the small intestine using in situ perfusion..................9

3.1 Use of effective permeability in transporter – metabolism studies................................................11

3.2 Use of apparent permeability in transporter – metabolism studies........................................................13

3.3 Intestinal absorption of ester prodrugs.............................................................................................................15

3.4 Evaluating the specific contribution of drug transporters and metabolizing enzymes: use of knockout animals............................................................................................................................................................17

3.5 The effect of induction on the biochemical barrier function of the small intestine..........................20

3.6 Regional absorption studies – site dependent expression of transporters and metabolizing enzymes............................................................................................................................................................................. 22

3.6.1 Regional in situ intestinal absorption studies– transporter substrates...........................................25

3.6.2 Regional in situ intestinal absorption studies– dual substrates.......................................................26

3.7 In situ intestinal excretion upon intravenous administration....................................................................29

4. Towards the use of more complex media..........................................................................................................31

4.1 In situ intestinal perfusions using biorelevant media – solubility-permeability interplay..............32

4.2 Beyond solubility: supersaturation....................................................................................................................35

5. Future perspectives....................................................................................................................................................38

5.1. Evaluation of barrier functions: specific inhibitors versus knockout animals............................38

5.2. Predictive and mechanistic studies in rodents........................................................................................39

5.3 Selection of appropriate perfusion media and drug concentrations................................................40

5.4 Towards a more dynamic absorption model...........................................................................................41

5.5 Formulation evaluation......................................................................................................................................... 42

6. Concluding Remarks................................................................................................................................................43

3

1. INTRODUCTION

Since oral intake remains the preferred route of drug administration, the need to develop and

validate suitable models to evaluate intestinal absorption is self-evident. In the pharmaceutical

industry, there is a strong tendency towards the use of in vitro tools to study intestinal

permeability because of their suitability to be implemented in high-throughput programs

(Bohets et al., 2001). The Caco-2 model is nowadays considered the gold standard in

intestinal permeability screening. This cell line expresses most of the transporters that are

relevant for drug absorption in humans, rendering it useful to study absorption mechanisms.

Moreover, for compounds that are passively absorbed and exhibit low intestinal metabolism,

permeability values observed in the Caco-2 model allow good predictions of the fraction of

the administered dose of a drug that will be absorbed in humans (Artursson et al., 2001).

Nevertheless, despite its wide applicability in permeability profiling, this in vitro model

sometimes fails to address the complexity of intestinal processes which eventually determine

in vivo intestinal absorption. Two major downsides of using Caco-2 cells include (i) the very

low expression levels of P450 enzymes, important for compounds undergoing significant

intestinal metabolic extraction and (ii) the absence of a protective mucus layer, causing the

cells to be vulnerable upon direct contact with more complex media, including human and

simulated intestinal fluids of the fed state. Moreover, the lack of a mucus layer renders the

Caco-2 cells more sensitive to pH changes of the apical media, as compared to mammalian

intestinal tissue (Lee et al., 2005). Additionally, the Caco-2 model cannot be used for regional

absorption studies, for obvious reasons.

Therefore, the use of more robust, biorelevant and versatile models is crucial to understand

and predict key mechanisms defining drug transport across the small intestinal barrier. The in

situ intestinal perfusion technique in rodents has been around for decades and since its

4

introduction by Schanker in 1958, this model has exhibited the ability to adapt to

contemporary challenges (Schanker et al., 1958). This versatility has rendered the in situ

intestinal perfusion model indispensible in the field of intestinal absorption research.

This review aims to provide a critical overview of the use and applications of the in situ

intestinal perfusion technique in rodents. More specifically, some unique assets of this model

will be discussed, such as its applicability in evaluating the transporter-metabolism interplay,

regional absorption processes and its compatibility with complex media, which is of utmost

importance in the study of food effects and absorption enhancing strategies.

2. PERMEABILITY ASSESSMENT – DISAPPEARANCE (PEFF) VERSUS

APPEARANCE (PAPP)

2.1 MEASURING DISAPPEARANCE FROM THE PERFUSION SOLUTION - EFFECTIVE PERMEABILITY

In the original set-up of the in situ intestinal perfusion, a segment of the small intestine of an

anaesthetized animal is cannulated and perfused with a solution containing a predefined

concentration of a drug of interest. During the experiment, the animal is kept unconscious and

its body temperature is maintained by the use of a heating pad or an overhead lamp. Upon

perfusion of the intestinal segment, drug will be absorbed to some extent, depending on its

physicochemical and biopharmaceutical properties, and the drug concentration in the

perfusion solution will decrease. Through comparison of the donor concentration and the

concentration of the solution that exits the isolated segment, the amount of drug that has

permeated the apical membrane of the small intestinal barrier (transcellular transport) or has

passed through the intercellular space (paracellular transport) can be calculated. By correcting

the amount of drug that disappeared from the perfusion solution over time for the donor

5

concentration and the absorptive area of the intestinal segment, the effective permeability

value can be calculated using equation (1):

(1)

with F the flow rate of the perfusion solution, Cout and Cin the outlet and inlet concentration,

respectively, and R the radius and L length of the perfused intestinal segment. Due to the fact

that water absorption or secretion upon intestinal perfusion may influence the measured

concentrations, correction methods for this water flux have been introduced, including the use

of non-absorbable markers in the perfusion solution or gravimetric methods (Sutton et al.,

2001).

Cao et al. demonstrated a good correlation between the effective permeability of rat intestine

and human intestine for a series of 17 compounds, exhibiting both passive and

transporter-mediated absorption (Cao et al., 2006). Human intestinal permeability values used

in this study were obtained from jejunal perfusion studies using the Loc-I-gut® technique

(Lennernäs et al., 1992).

2.2 MEASURING APPEARANCE IN THE BLOOD - APPARENT PERMEABILITY

It is essential, however, to be aware of the fact that the effective permeability does not

necessarily give a reliable prediction of the amount of drug that will appear in the blood. Non-

specific binding to perfusion tubing or the isolated intestinal segment can result in a decrease

in Cout which may be erroneously interpreted as drug absorption. Moreover, for compounds

that undergo a high intestinal metabolic extraction, a lower fraction will generally reach the

blood circulation than would be predicted based on the disappearance from the perfusion

solution.

6

(1 )

2

out

ineff

CCP FRL

These concerns can be addressed by using the in situ intestinal perfusion technique with

mesenteric blood sampling. In this adaptation of the classical set-up, the mesenteric vein,

draining the blood from the perfused intestinal segment, is cannulated and blood samples are

collected over predefined intervals to determine the actual amount of drug that is present in

the blood (Figure 1). Donor blood is supplied via the vena jugularis to maintain the

hemodynamic balance.

This technique allows calculating the apparent permeability (Equation (2) and Figure 2),

where dQ/dt is the slope of the cumulative amount of drug appearing into the mesenteric

blood over time, R the radius and L length of the perfused intestinal segment. Cdonor is the

donor concentration of the perfusion solution.

Obviously, by taking samples from the perfusion solution at the inlet and outlet of the

cannulated intestinal segment, the effective permeability can still be determined.

2.3 VASCULAR PERFUSION

It is clear that mesenteric vein cannulation in combination with intestinal perfusion

experiments improves insight into intestinal drug absorption mechanisms. Additional

cannulation of the mesenteric artery enables perfusion of the mesenteric capillary bed,

creating the possibility to control both intestinal and vascular perfusion of the cannulated

small intestinal segment. Vascular perfusion solutions mostly consist of oxygenated buffer

solutions containing albumin, circumventing the need for donor blood. An additional

advantage of the vascular perfusion set-up, is the ability to vary the blood supply to the

7

Papp=dQdt

× 12 πRL×Cdonor

(2)

intestinal segment. For example, in postprandial conditions, the blood flow to the small

intestine is higher than in the fasted state and this may consequently influence the absorption

rate. For instance, Tamura et al. demonstrated that the absorbed amount of tacrolimus at a

vascular perfusion rate of 2.5 ml/min was significantly higher than the absorption at a flow

rate of 1 ml/min (Tamura et al., 2003).

A downside of vascular perfusion with oxygenated buffers is the increased interference with

physiological processes in this set-up. For instance, the distribution of blood to the small

intestine via the mesenteric arteries follows a pulsatile pattern, whereas a constant flow is

generated upon vascular perfusion. Moreover, care should be taken not to disrupt the fragile

capillaries when imposing a certain flow rate through the vascular bed.

2.4 OPEN LOOP AND CLOSED LOOP INTESTINAL PERFUSIONS

A small intestinal segment can be perfused in the open loop or the closed loop set-up. In the

open loop set-up, the perfusion solution that exits the cannulated segment goes directly to

waste. However, when perfusion media are scarce (e.g. when using intestinal fluids) or when

only small amounts of compound are available (e.g. early development stages), the closed

loop set-up can be applied; in this configuration, the perfusion solution is continuously

recirculated through the intestinal segment, dramatically decreasing the volume of perfusion

medium needed to perform the experiment (Doluisio et al., 1969). Figure 3 gives a schematic

representation of the open and closed loop set-up. Depending on the specifications of the

materials used, including the internal diameter and the length of the tubing, 5 ml of medium

can be sufficient to perform a closed-loop perfusion. It is clear that, upon absorption in the

closed-loop set-up, Cdonor will decrease during the experiment, whereas in the open-loop set-

up, the donor concentration will generally be constant if the compound of interest is stable in

the perfusion medium. Therefore, apparent permeability calculations in the closed-loop

modus, require frequent sampling of the perfusion solution, while for open-loop experiments,

8

determining the concentration of the donor solution at the beginning and the end of the

experiment is usually sufficient.

3. EXPLORING THE BIOCHEMICAL BARRIER FUNCTION OF THE SMALL

INTESTINE USING IN SITU PERFUSION

The rapidly growing body of literature on intestinal drug disposition evidences the complex

nature of the processes underlying intestinal absorption. The small intestine is equipped with a

number of efficient detoxifying mechanisms, hampering the uptake of xenobiotics. Membrane

transporters and metabolizing enzymes have been shown to affect both rate and extent of

intestinal drug absorption (FDA, 2011). The use of in vitro models allows investigators to

study isolated processes such as the involvement of transporters in intestinal drug absorption.

Caco-2 cells express most of the transporters that are relevant for intestinal drug transport in

human and therefore, they have proven to be very convenient in transporter studies. For the

assessment of intestinal P450 mediated metabolism, however, investigators have to rely on

other in vitro tools, including intestinal microsomes or homogenates. Indeed, one of the major

drawbacks of the Caco-2 model is the very low to non-existent expression of cytochrome

P450 enzymes. Therefore, application of this in vitro model to assess intestinal permeability

for compounds exhibiting a high metabolic extraction in the gut may generate an

overestimation of the intestinal transport. Despite efforts to induce the expression of CYP3A4

in selected clones of Caco-2 cells using 1α,25-dihydroxyvitamin D3, the metabolic activity

was still low compared to human intestinal tissue homogenates (Schmiedlin-Ren et al., 1997).

For some compounds, a complex interplay may exist between transporters and metabolizing

enzymes upon intestinal transport, as has been observed for dual substrates of CYP3A

enzymes and P-gp (Mudra et al., 2011). Interestingly, there have been reports both on

cooperative and counteracting functioning of these detoxifying mechanisms. Consequently,

9

incubations of dual P-gp/CYP3A substrates in intestinal microsomes or homogenates,

combined with permeability data from Caco-2 will not necessarily create a reliable picture of

the key mechanisms dictating the intestinal absorption. Therefore, simultaneous assessment of

transporter and metabolism functioning is advisable for these compounds.

In addition to the lack of P450 enzyme expression, Van Gelder et al. demonstrated low

esterase activity in Caco-2 cells, which may lead to overestimation of the intestinal transport

of ester prodrugs (Van Gelder et al., 2000b).

As mice and rats express both intestinal transporters and P450 enzymes, the in situ intestinal

perfusion technique in rodents has been used to study the intestinal absorption of drugs that

are affected by intestinal metabolism and efflux transporters. Obviously, species differences

exist with reference to substrate specificities and kinetic parameters. For example, CYP3A9 is

the rat ortholog for human CYP3A4 with a sequence identity of 76.5% (Wang et al., 1996).

Moreover, CYP3A9 expression in rat small intestine was shown to be much higher than

CYP3A4 in human intestine, which could result in different metabolic extraction (Cao et al.,

2006). As a result, inter species metabolism rates may significantly differ. By any means,

from a qualitative point of view, the in situ intestinal perfusion model in rodents remains very

useful in mechanistic studies. Recent advances in the field of transgenic animals (e.g. mice

expressing human CYP3A4) may further increase the relevance of using rodents in the

evaluation of the intestinal absorption of compounds that are subject to significant intestinal

metabolic extraction (Ma et al., 2008; van Waterschoot and Schinkel, 2011).

3.1 USE OF EFFECTIVE PERMEABILITY IN TRANSPORTER – METABOLISM STUDIES

As mentioned in section 2, determining the effective intestinal permeability for a compound

that undergoes significant metabolic extraction may result in an overestimation of the fraction

that will reach the blood. For some compounds, however, it is possible to follow the

appearance of metabolites, originating from intracellular metabolism, in the perfusion

10

medium. These metabolites can reach the apical side of the enterocytes via active or passive

transport processes and serve as a measure of the intracellular metabolism. Li et al. monitored

the concentration of metabolite ‘M6’ in the perfusion solution upon perfusion of the rat small

intestine with the dual P-gp/CYP3A substrate indinavir and used this metabolite to estimate

intestinal metabolism. Extensive metabolism of indinavir in the jejunum was demonstrated,

generating a larger concentration difference for indinavir over the apical membrane, thereby

facilitating the transport of indinavir across the apical membrane of the enterocytes. The fact

that M6 is also a P-gp substrate and may consequently compete with indinavir efflux was also

postulated as a possible mechanism by which the intestinal metabolism increases the effective

permeability of indinavir (Li et al., 2002).

A more indirect approach to gain insight into the interplay between P-gp and CYP3A

metabolism was presented by Abuasal et al. By integrating the effective permeability obtained

in situ and several additional disposition parameters from in vitro experiments in a

physiologically based pharmacokinetic (PBPK) model, Abuasal et al. managed to predict the

bioavailability of the dual P-gp/CYP3A4 substrate UK343,664 and explain its non-linear

absorption behavior. Km and Vmax values for CYP3A4 and P-gp were determined in vitro

using supersomes and the Caco-2 model, respectively. Using the PBPK model, it was clearly

demonstrated that the relative involvement of P-gp and CYP3A4 metabolism is largely

dependent upon the concentration of the compound. At lower concentrations, P-gp efficiently

effluxes the compound out of the enterocytes, leading to low unbound intracellular

concentrations of UK343,664. This way, P-gp renders the compound unavailable to the

metabolizing enzymes. At higher concentrations, saturation of P-gp will occur and the

extraction ratio will increase up to the point where (at the highest concentrations tested) also

intestinal CYP3A4 gets saturated. Obviously, saturation of intestinal metabolism will in turn

reduce the extraction ratio (Abuasal et al., 2012).

11

As is evidenced by these studies, sampling from the perfusion medium may generate indirect

information with reference to the extent and the rate of intestinal absorption as well as

intestinal metabolism. Nevertheless, no unambiguous information on the actual appearance of

parent compound or metabolite into the blood is gathered. The study performed by Abuasal et

al. demonstrates that PBPK modeling is highly promising as a predictive and descriptive tool

for intestinal absorption. It is important to note, however, that, in order to obtain reliable

predictions of drug absorption from PBPK modeling, several kinetic and physiological

parameters need to be assessed first.

3.2 USE OF APPARENT PERMEABILITY IN TRANSPORTER – METABOLISM STUDIES

In view of the drawbacks associated with using the effective permeability to study the

transporter-metabolism interplay, the ability to determine the absorption of both parent

compound and metabolites in the mesenteric blood, is a huge step forward. This way, a more

accurate image of the contribution of metabolism and drug transporters to intestinal

absorption is generated. Moreover, compared to systemic sampling, mesenteric blood

collection excludes the confounding interference of non-intestinal pharmacokinetic

phenomena.

The importance of determining concentrations of a dual substrate in the mesenteric blood has

for instance been demonstrated for verapamil. Upon measuring disappearance from the

perfusion solution, Johnson et al. observed similar effective permeability values for verapamil

in absence or presence of P-gp and/or CYP3A inhibitors (Johnson et al., 2003). Similarly,

based on effective permeability, Mudra et al. could not demonstrate non-linear behavior in the

intestinal absorption of verapamil (Mudra and Borchardt, 2010). Nevertheless, in both studies,

the appearance of verapamil in the mesenteric blood was found to be significantly increased

in presence of dual inhibitors of both P-gp and CYP3A. Interestingly, Johnson et al. observed

12

an increase in apparent permeability of verapamil when using PSC833 (valspodar, typical P-

gp inhibitor) and midazolam (typical CYP3A substrate), whereas Mudra et al. could only

demonstrate significant increases when using dual substrates. This inconsistency is probably

due to differences in inhibitor concentrations applied, as PSC833 and midazolam, when being

used at higher concentrations, also inhibit CYP3A metabolism and P-gp, respectively.

Comparison of these studies advocates the necessity to use specific inhibitors or knock-out

animals, lacking the expression of a specific transporter or metabolizing enzyme.

In a study reported by Cummins et al, P-gp functionality was found to enhance the

metabolism of cysteine protease inhibitor K77, as inhibition studies using the P-gp inhibitor

GF120918 (elacridar) resulted in a decreased extraction ratio (Cummins et al., 2003). This

finding supports the concept of P-gp increasing the mean residence time of a compound inside

the cell, increasing its exposure to metabolizing enzymes (Benet et al., 2004). In the same

study, appearance of K77 in the blood was much lower than expected judging from the

disappearance from the perfusion solution. Moreover, the authors reported the difficulty to

accurately quantify the loss of compound from the perfusion solution, exposing an additional,

analytical limitation for the determination of permeability based on drug disappearance from

the perfusion solution. Indeed, especially for low permeability compounds, the relative

decrease in concentration from the perfusion solution is mostly very small compared to the

appearance of compound in the blood.

Holmstock et al. revealed that even within the same class of compounds, the relative

contribution of P450 mediated metabolism and P-gp may differ significantly. By making use

of the in situ intestinal perfusion technique in mice, the authors unraveled the mechanism by

which ritonavir increases the intestinal transport of the HIV protease inhibitors darunavir,

indinavir and lopinavir (Holmstock et al., 2012). Using the diagnostic inhibitors

1-aminobenzotriazole and GF120918, inhibiting P450 mediated metabolism and P-gp,

13

respectively, it was shown that ritonavir enhances the intestinal permeability for darunavir and

indinavir, mostly by inhibiting P-gp, whereas for lopinavir, the increase in permeability is due

to inhibition of P450 metabolizing enzymes. An additional study on the absorption of the HIV

protease inhibitor saquinavir was performed by Usansky et al., who applied the rat in situ

intestinal perfusion to demonstrate that, as was observed for darunavir and indinavir, P-gp

mediated efflux is the main mechanism responsible for the low apparent permeability for

saquinavir (Usansky et al., 2008).

3.3 INTESTINAL ABSORPTION OF ESTER PRODRUGS

Generally, ester prodrugs are designed to overcome poor permeability, which is often caused

by the presence of polar, hydrophilic groups. Most commonly, an ester bond is added to the

active compound with the aim of increasing lipophilicity and thereby improving the passive

diffusion over the cell membrane of the enterocytes (Beaumont et al., 2003). Other rationales

for using ester prodrugs include targeting of active uptake transporters, such as the PEPT1

transporter, to increase poor intestinal permeability (Cao et al., 2012; Eriksson et al., 2010;

Gupta et al., 2011; Han et al., 1998).

The small intestine exhibits significant esterase activity, resulting in intracellular hydrolysis of

ester prodrugs. The active compound is subsequently transported to the apical or basolateral

side of the enterocyte, by passive diffusion or by active transport. Carboxylesterases (CES)

have been reported to be the major family of enzymes involved in the intestinal hydrolysis of

exogenous and endogenous esters (Imai and Ohura, 2010). Despite the fact that esterase

acitivity has been observed in Caco-2 cells, some issues have been reported with reference to

the use of this cell monolayer in studying the intestinal absorption of ester prodrugs. For

instance, Van Gelder et al. reported low esterase activity in Caco-2 cells as compared to

human intestinal tissues (Van Gelder et al., 2000b). Moreover, Caco-2 cells mostly express

14

CES1, whereas in humans, CES2 is the predominant carboxylesterase isoenzyme in the small

intestine (Imai and Ohura, 2010).

Notwithstanding possible differences in substrate specificity, CES2 isoenzymes have also

been observed to be the most abundantly expressed carboxylesterase in rats. Moreover,

degradation rates of tenofovir disoproxil in rat and human ileal tissues were found to be

similar (Van Gelder et al., 2000b).

Tenofovir and adefovir are antiviral agents for which a prodrug approach has been applied

because of their hydrophilic nature, resulting in a low intestinal permeability. In view of the

observed intestinal degradation of tenofovir disoproxil, Van Gelder et al. hypothesized that in

situ intestinal perfusion in presence of ester containing fruit extracts would increase the

intestinal absorption of tenofovir disoproxil in rats (Van Gelder et al., 2000a). Indeed, the

amount of tenofovir equivalents in the mesenteric blood increased by 7-fold in presence of a

strawberry extract, containing a multitude of small esters, competitively inhibiting the

hydrolysis of tenofovir disoproxil. Notwithstanding the success of this approach, Masaki et al.

demonstrated that inhibition of intestinal CES, could lead to increased intracellular

concentration of the prodrug, thereby decreasing the driving force across the apical membrane

of the enterocytes, resulting in a decreased effective permeability (Masaki et al., 2007).

Annaert et al. evaluated the intestinal absorption of ester prodrug adefovir dipivoxil in the in

situ intestinal perfusion technique with mesenteric sampling in rats. Results were compared

with data obtained from Caco-2 and diffusion chambers experiments (Annaert et al., 2000). In

agreement with the aforementioned tenofovir study performed by Van Gelder et al., ester

hydrolysis of adefovir dipivoxil was demonstrated to be relatively low in Caco-2 cells.

Adefovir dipivoxil was found to be the major species appearing on the basolateral side of the

cell monolayer, whereas in situ, no prodrug could be measured in the mesenteric blood.

Interestingly, addition of the P-gp inhibitor verapamil increased the apparent permeability for

15

total adefovir in situ and in vitro, but not ex vivo in the diffusion chambers. Moreover,

diffusion chambers were less discriminative than the in situ and in vitro models to

demonstrate a prodrug effect on the absorption of adefovir. These data suggest that the in situ

intestinal perfusion technique is the most suitable model to study the intestinal absorption of

ester prodrugs, especially when the hydrolysis product is a substrate for intestinal transporters.

Consistent with these findings, other authors have confirmed the necessity to take both

esterase activity and transporter mechanisms into account, when studying the intestinal

absorption of prodrugs. Significant disappearance from the perfusion medium was observed

for ethyl-fexofenadine and M3229, ester prodrugs of the antihistaminic agent fexofenadine

and the glycoprotein IIb/IIIa antagonist M3277, respectively (Ohura et al., 2012; Okudaira et

al., 2000). Nevertheless, for both drugs, the appearance of active compound in the mesenteric

blood was low compared to what would be expected, based on disappearance of prodrug and

hydrolytic activity in the enterocytes. This apparent disparity could be explained by the fact

that the intracellularly formed active compounds underwent significant efflux towards the

intraluminal environment. Clearly, as could be concluded for drugs undergoing P450

mediated metabolism, mesenteric vein cannulation offers important additional information on

the overall absorption process of ester prodrugs.

It is important to note that, for a prodrug approach to be successful, intraluminal stability is

mostly required. Several reports have described the degradation of ester prodrugs in aspirated

human intestinal fluids, advocating the need for stability assessment of ester prodrugs in

biorelevant media (Borde et al., 2012; Granero and Amidon, 2006; Stoeckel et al., 1998).

3.4 EVALUATING THE SPECIFIC CONTRIBUTION OF DRUG TRANSPORTERS AND METABOLIZING ENZYMES: USE OF KNOCKOUT ANIMALS

Numerous membrane transporters have been identified along the human small intestine and,

for a lot these proteins, rodents express isoenzymes that are similar with reference to amino

16

acid sequence and functionality. However, for most of these transporters, there is no (or not

enough) evidence that they are clinically relevant for drug disposition in vivo (International

Transporter Consortium et al., 2010). Despite the introduction of specific and potent

diagnostic inhibitors of drug transporters and metabolizing enzymes, it often remains very

difficult to estimate their relative importance in the intestinal absorption of a compound. A lot

of inhibitors that are frequently used in mechanistic research exhibit cross specificity, even at

low concentrations (Choo et al., 2000). Therefore, the use of animal models in which a

specific gene encoding a drug transporter or metabolizing enzyme has been inactivated, so

called knockout animals, is of great benefit to pharmacokinetic research. In situ intestinal

perfusion studies using knockout animals have been performed to evaluate the specific role of

transporters in intestinal drug absorption.

For instance, the significance of PEPT1 in the intestinal absorption of drugs is difficult to

evaluate. PEPT1 is an oligopeptide transporter present at the apical membrane of the small

intestine and exhibits a heterogeneous expression along the small intestine and broad substrate

specificity which overlaps with other peptide transporters (e.g. peptide/histidine transporters).

These elements impede estimating the specific contribution of the PEPT1 transporter to the

overall intestinal absorption (Jappar et al., 2010). The use of Pept1 knockout mice offers the

possibility to discard these confounding factors. The applicability of this absorption model in

pharmacokinetic research was evaluated through the use of the dipeptide glycylsarcosine. Hu

et al. demonstrated that deletion of Pept1 resulted in a 20-fold reduction in the effective

permeability upon intestinal perfusion of glycylsarcosine in knockout mice compared to the

wild-type mice, whereas upon intravenous administration, the plasma profiles of the dipeptide

were very similar between the two groups (Hu et al., 2008). Jappar et al. confirmed the very

low intestinal uptake of glycylsarcosine in knockout mice along the entire length of the small

intestine (Jappar et al., 2010). These studies evidenced the reliable use of the Pept1 knockout

17

mice and the absorption tool has been adopted by other authors to confirm the role of PEPT1

in the intestinal transport of commonly used drugs such as valacyclovir, a peptide prodrug of

the antiviral drug acyclovir (Yang and Smith, 2013).

As is the case for uptake transporters, also the clinical importance of efflux transporters is a

topic of much debate. P-gp, BCRP and MRP2 are abundantly expressed at the apical

membrane of enterocytes and have been shown to interfere with the absorption of a high

number of drugs. To specifically explore the contribution of these transporters, genetically

modified animals lacking the expression of specific proteins can be extremely useful. Eisai

hyperbilirubinemic rats (EHBRs) and TR- rats are defective for the efllux transporter Mrp2

(Adachi et al., 2005; Sesink et al., 2005). The Mrp2 deficient rats and Bcrp knockout mice

have been used to evaluate the fate of compounds that undergo significant phase-II

metabolism in the enterocytes, generating sulfates and glucuronides. These conjugates are

mostly good substrates for MRP2 and BCRP. Intestinal perfusion with naturally occurring

products such as genistein (a flavonoid) and 4-methylumbelliferone (a coumarin) in Bcrp

knockout mice revealed an important role for BCRP in effluxing sulfate and glucuronide

conjugates from the intracellular environment to the apical side of the enterocytes (Adachi et

al., 2005; Yang et al., 2012). The appearance of sulfates and glucuronides of genistein in the

perfusion medium was significantly lower in the knockout animals than in the wild type mice,

indicating that BCRP may cause alterations in the distribution of conjugates to the systemic

circulation. Only minor involvement of MRP2 could be demonstrated in these studies.

Despite the large difference in efflux of conjugates between the knockout and the wild type

mice, this was not reflected in the disappearance of parent compound from the perfusion

solution. Similarly, significant differences in efflux rates of glucuronide and sulfate

conjugates of 4-methylumbelliferone were demonstrated between Bcrp knockout and wild

type mice, but no statistical difference was observed in effective permeability of the parent

18

compound. The appearance of parent compound and metabolites in the mesenteric blood was

not assessed in these studies.

In a study of Rong et al., the appearance of tolmetin and its metabolites in the plasma upon

intestinal perfusion with prodrug amtolmetin guacyl was shown to be significantly increased

in Bcrp knockout mice compared to wild type mice (Rong et al., 2013). However, since blood

samples were taken from the systemic circulation (vena jugularis), and the expression of Bcrp

is not limited to the small intestine, the exact role of Bcrp at the level of the small intestine

cannot be unambiguously evaluated in this study. Indeed, BCRP may also be involved in the

hepatobiliary elimination of drugs.

In an effort to combine the benefits of using knockout mice, which remain more readily

available than knockout rats, and preserving the ability of evaluating the apparent

permeability, Mols et al. downscaled the in situ intestinal perfusion with mesenteric blood

sampling to mice (Mols et al., 2009). The same research group reported a study in which P-gp

knockout mice were used to assess the importance of P-gp in the intestinal absorption of the

HIV protease inhibitor darunavir and pointed out the ability of ritonavir to exert its function

as a booster, not only at the hepatic level, but also at the level of the small intestine

(Holmstock et al., 2010).

The in situ intestinal perfusion with mesenteric blood sampling in mice is obviously

technically challenging and, as a result, the success rate is low compared to using rats.

Therefore, further construction and validation of knockout rats may prove to be extremely

useful in the evaluation of intestinal drug absorption pharmacokinetic studies in general

(Farooq and M. Hawksworth, 2012). Recently, P-gp, Mrp2 and Bcrp knockout rats were

demonstrated to be a good alternative for knockout mice(Zamek-Gliszczynski et al., 2012).

19

3.5 THE EFFECT OF INDUCTION ON THE BIOCHEMICAL BARRIER FUNCTION OF THE SMALL INTESTINE

The effective treatment of chronic diseases mostly requires adherence to a prolonged, often

lifelong drug regimen. Long-term use of drugs has been shown to upregulate the expression

of transporters and metabolizing enzymes involved in the disposition of these compounds.

This adaptive process of induction is mediated through nuclear receptors (e.g. pregnane X

receptor (PXR) and constitutive androstane receptor (CAR)), which ‘sense’ the presence of

xenobiotics and upregulate detoxifying proteins such as metabolizing enzymes and efflux

transporters (Willson and Kliewer, 2002). These phenomena can not be adequately studied in

the Caco-2 model as this cell model does not express the PXR nuclear receptor (Thummel et

al., 2001). Therefore, several authors have applied rodent models to study the effects of

induction on intestinal absorption of drugs.

Ho et al. demonstrated that 15 days of consecutive administration of a St. John’s wort extract

to rats significantly reduced the concentrations of indinavir in the portal venous blood. Both

intestinal and hepatic CYP3A mediated metabolism was identified to be at the origin of this

induction phenomenon. P-gp induction was not evaluated in this study despite the fact that P-

gp and CYP3A induction pathways involve the same nuclear receptors (PXR and CAR). This

has been recognized by several authors who evaluated the effect of typical inducers such as

dexamethasone and pregnenolone-16α-carbonitrile (PCN) on the expression and functionality

of P-gp and CYP3A metabolism (Liu et al., 2006; Sandström and Lennernäs, 1999). A

general observation is that the repeated administration of these compounds decreased the

permeability for dual CYP3A/P-gp substrates such as verapamil and digoxin across the small

intestine of rats and mice. Liu et al. observed increased expression not only of P-gp but also

of CYP3A upon PCN pretreatment. Despite the reported extensive metabolism of digoxin by

CYP3A in rats, the decrease in effective permeability for digoxin was found to be due to

20

increased expression of P-gp, whereas intestinal metabolism remained negligible (Salphati

and Benet, 1999). Sandström et al. demonstrated that the effective permeability for verapamil

was already significantly decreased at day one of the oral dosing regimen of dexamethasone.

The induction of CYP3A mediated metabolism, determined by measuring the extent of

norverapamil formation in the perfusion solution, was slower and only significant upon 14

days of once daily oral dexamethasone administration (Sandström and Lennernäs, 1999).

Based on these studies, the rat seems to be a suitable model to evaluate the mechanism

underlying altered intestinal permeability as a result of induction phenomena.

Important to note, however, is that compounds which are potent inducers in humans do not

necessarily induce strong upregulation of detoxifying proteins in rodents. Rifampicin for

example induces strong activation of human PXR, whereas it appears to be a less effective

activator of rodent PXR. In an effort to further increase the biorelevance of the mouse models,

mice carrying functional human genes (e.g. mice expressing human PXR and CYP3A4) have

been developed and validated (Ma et al., 2008). Holmstock et al. made use of these

PXR/CYP3A4 humanized mice to evaluate the induction effect of rifampicin (Holmstock et

al., 2013b). In the humanized mice, a decreased permeability for dual P-gp/CYP3A substrate

darunavir was observed after pretreatment with rifampicin for three days. An increased efflux

by P-gp was found to be at the origin of this drop in permeability. Holmstock et al. also

determined expression levels of P-gp and CYP3A4 enzymes and found that only the

expression of P-gp was increased upon pretreatment with rifampicin. It was hypothesized in

this study that the baseline expression of CYP3A4 is already relatively high in these

PXR/CYP3A4 humanized mice and, as a result, an additional induction of CYP3A4 is less

likely to occur.

21

3.6 REGIONAL ABSORPTION STUDIES – SITE DEPENDENT EXPRESSION OF TRANSPORTERS AND METABOLIZING ENZYMES

The expression of membrane transporters and metabolizing enzymes along the small intestine

is far from homogenous. Tissue samples obtained from different regions of the human small

intestine reveal a significant site dependency for a number of transporters and enzymes that

are known to affect drug absorption. Nevertheless, limited availability of healthy human

tissue and high interindividual differences in expression levels result in a low number of

studies clearly demonstrating regional expression patterns along the human small intestine.

Therefore, it remains highly difficult to draw general conclusions with regard to the site

dependent expression of drug transporters and metabolizing enzymes.

From the scarce data that is present, however, it appears that the expression of CYP3A4,

which is the most abundantly expressed isoenzyme of the cytochrome P450 superfamily in

the small intestine, is higher in proximal parts of the small intestine than in distal regions

(Berggren et al., 2007; Canaparo et al., 2007). In contrast, efflux transporters P-gp and BCRP

have been shown to exhibit a higher expression at distal segments of the small intestine

(Englund et al., 2006).

Studying site-dependent absorption is of great relevance for drugs that exhibit poor

dissolution, solubility or permeability characteristics, as these drugs are likely to be exposed

to the entire length of the small intestine. Moreover, knowledge on the regional absorption

profile of a compound may aid in the development and evaluation of controlled release

formulations (Tannergren et al., 2009; Thombre, 2005). For instance, a modified-release

formulation of hydrocortisone could be developed based on permeability data in both human

small and large intestine (Johannsson et al., 2009; Lennernäs, 2014)

Despite the limited data from human intestinal tissues, it appears that for a number of human

transporters and metabolizing enzymes, the rodent isoenzymes exhibit similar expression

22

patterns along the intestinal tract. As is the case in humans, the expression of CYP3A

enzymes is highest in proximal parts of the small intestine of mice and rats and the expression

of P-gp increases from duodenum to ileum, making these animal models very useful for

mechanistic studies concerning regional absorption of substrates of transporters or

metabolizing enzymes (Jin et al., 2006; MacLean et al., 2008; Mitschke et al., 2008; Stephens

et al., 2001; Takara et al., 2003). Similarities in expression profiles between human and

rodent models have also been observed for uptake transporters. The expression of OATP2B1,

the most predominant OATP isoenzyme in human small intestine, has been observed to be

higher in the ileum than in the duodenum, although this difference was not statistically

significant (Meier et al., 2007). Similarly, the rat isoenzymes Oatp2b1 and Oatp1a5 exhibit

higher expression levels at distal sites of the small intestine (MacLean et al., 2010). For the

oligopeptide transporter PEPT1, a dissimilarity is observed between human and rat, as

hPEPT1 appears to be more abundantly expressed in the proximal small intestine, whereas in

rats, no significant regional differences have been observed (Herrera-Ruiz et al., 2001;

Ingersoll et al., 2012).

Obviously, site dependent absorption mechanisms cannot be studied using Caco-2 cells. In

contrast, diffusion chambers are very suitable for this application as mounting of tissues from

different intestinal regions allows reliable determination of site dependent intestinal transport.

Moreover, use of human intestinal tissue strongly enhances the biorelevance of this

absorption model. Sjöberg et al. demonstrated a good correlation between the apparent

permeability across human intestinal tissue in the diffusion chambers and the fraction

absorbed in humans (Sjöberg et al., 2013). A major drawback related to this model is the poor

availability of viable human intestinal tissue. Moreover, careful manipulation of the excised

tissue is required prior to mounting. Serosa and muscularis mucosae present a barrier to

compound permeation, which is not relevant in vivo as blood vessels in the submucosa

23

guarantee suitable sink conditions. Therefore, stripping of serosa and the longitudinal muscle

layer of the intestinal tissue is generally performed ahead of mounting. Nevertheless, despite

removal of this longitudinal muscle layer, the circular muscle layer underlying the submucosa

cannot be removed.

3.6.1 REGIONAL IN SITU INTESTINAL ABSORPTION STUDIES– TRANSPORTER SUBSTRATES

P-gp is the most extensively documented intestinal drug transporter and there is compelling

evidence on the impact of its site dependent expression on the regional absorption of P-gp

substrates. Upon perfusion of model compounds such as talinolol, tacrolimus and digoxin, P-

gp was demonstrated to limit the absorption rate to a higher extent at distal sites of the small

intestine than at proximal sites (Sababi et al., 2001; Tamura et al., 2002; Wagner et al., 2001).

Valenzuela et al. examined the site dependent permeability for P-gp substrate salbutamol and

evaluated these findings in view of mRNA and protein expression levels. An inverse

relationship between expression of P-gp and absorption rate of salbutamol was observed

(Valenzuela et al., 2004). Notwithstanding the fact that the role of P-gp in the absorption of

numerous compounds was becoming increasingly evident, Cao et al. pointed out the

importance to keep taking passive permeability into account (Cao et al., 2005). The P-gp

substrate verapamil was shown to be unaffected by the 6-fold difference in expression level of

P-gp in rat, due to its high passive permeability. Nevertheless, the impact of regional

expression of P-gp on site dependent permeability has been confirmed for several other

compounds, amongst which the HIV protease inhibitor darunavir, the antimalarial compound

lumefantrine and the fluoroquinolone CNV97100 (González-Alvarez et al., 2007; Stappaerts

et al., 2013; Wahajuddin et al., 2014).

The general acceptance of the impact of the site dependent expression of P-gp on the

increasing number of identified substrates, prompted Shirasaka et al. to develop a prediction

24

model for the intestinal absorption of P-gp substrates in humans (Shirasaka et al., 2008).

Based on Km and Vmax values obtained from cell monolayers exhibiting different levels of P-

gp expression, regional absorption profiles were predicted for rats and validated by in situ

intestinal perfusions. Although, theoretically, the implementation of regional P-gp expression

levels obtained from biopsies in human could lead to adequate predictions, the reality of the

strong interindividual variation in humans makes it very difficult to obtain reliable predictions

(Berggren et al., 2007; Canaparo et al., 2007). Moreover, for several compounds, involvement

of multiple transporters in the intestinal absorption has been observed, rendering site

dependent predictions extremely challenging.

Dahan et al. showed that multiple efflux transporters affected the absorption of colchicine (P-

gp and Mrp2) and sulfasalazine (Mrp2 and Bcrp) in rat (Dahan and Amidon, 2009; Dahan et

al., 2009). For other compounds, such as ciprofloxacin, atazanavir and pitavastatin, intestinal

permeability has been demonstrated to be influenced by both uptake (Oatp) and efflux

transporters (P-gp) (Arakawa et al., 2012; Kis et al., 2013; Shirasaka et al., 2011). It is clear

that the combination of involvement of multiple transporters in intestinal absorption and their

heterogeneous expression along the gastrointestinal tract adds complexity to the interpretation

of their relative roles in the absorption of substrates. As mentioned in section 3.4, the use of

knockout animals may resolve this intricacy.

Knockout mice have been used to estimate the regional differences of Pept1 involvement in

the absorption of model compound glycylsarcosine and the antiviral drug valacyclovir (Jappar

et al., 2010; Yang and Smith, 2013). In wild type mice, in situ permeability for these

compounds was observed to be significantly lower in the colon compared to permeability in

the small intestine. However, perfusion experiments in knockout mice revealed a complete

loss of site-dependent differences in permeability, demonstrating that the higher expression of

25

Pept1 in the small intestine is the main causative factor for the higher permeability as

compared to the colon.

3.6.2 REGIONAL IN SITU INTESTINAL ABSORPTION STUDIES– DUAL SUBSTRATES

Section 3.1 and 3.2 illustrated the often complex nature of transporter-metabolism interplay

and, more specifically, the interaction between P-gp and CYP3A mediated metabolism. It is

self-evident that the intricacy of the regional expression profiles of these proteins further

complicates the intestinal absorption studies of dual substrates. In situ perfusion experiments

have been used to gain mechanistic insight into the site dependent P-gp/CYP3A interplay

affecting the permeability for dual substrates.

Li et al. observed a high extent of intestinal metabolism of the HIV protease inhibitor

indinavir in the jejunum as compared to the ileum (Li et al., 2002). In the latter intestinal

region, metabolism was found to be low to non-existent. Nevertheless, in the ileum, the

permeability for indinavir was significantly lower than in the jejunum due to the higher

expression of P-gp. The CYP3A inhibitor ketoconazole strongly decreased the effective

permeability in jejunum, most likely because it decreases the indinavir concentration gradient

over the apical membrane, through the inhibition of intracellular metabolism. Within the same

research group, the site dependent permeability of another dual substrate, UK-343,664, was

evaluated (Kaddoumi et al., 2006). Permeability for this compound was found to be

influenced mostly by P-gp and only poor intestinal metabolism of UK-343,644 was observed

both in jejunum and in ileum. Consistent with the increasing P-gp expression from proximal

to distal parts of the small intestine, intestinal permeability for this compound was higher in

jejunum than in ileum in the concentration range from 5-50 µM. At a UK-343,664

concentration of 50 µM, P-gp inhibition caused an increase in both permeability and fraction

metabolized at the level of the jejunum, indicating that for this compound at this substrate

concentration, P-gp decreases the fraction metabolized in the jejunum. Nevertheless, the

26

increase in permeability for UK-343,664 upon P-gp inhibition was higher in the ileum, which

is in good agreement with the higher expression of P-gp at this intestinal site. Due to the

lower expression of CYP3A metabolizing enzymes at the level of the ileum, the increase in

fraction metabolized upon P-gp inhibition was lower than that observed in the jejunum.

Tamura et al. emphasized the benefit of determining the apparent permeability for a

compound that is metabolized (Tamura et al., 2003). The disappearance of tacrolimus from

the perfusion solution was found to be twofold higher in the jejunum than in the ileum. This is

in accordance with the higher expression of P-gp at distal sites of the small intestine. The

apparent permeability, measured upon vascular perfusion, however, was similar in the

intestinal segments. Using midazolam as a CYP3A inhibitor, higher metabolic extraction of

tacrolimus was demonstrated in jejunum, as compared to ileum.

An excellent study, underscoring the value of the in situ model to gain mechanistic insight,

was performed by Jin et al. who studied the site dependent absorption of the dual substrate

cyclosporine A as well as the induction effects mediated by dexamethasone on the absorption

profile (Jin et al., 2006). Both wild-type mice and mdr1a/1b knockout mice were used to

discriminate between effects exerted by P-gp and CYP3A mediated metabolism. Taking

blood samples from both portal and jugular vein, absorption of cyclosporine A from proximal

sites of the small intestine was shown to be higher than absorption from distal sites in wild-

type mice. Using the knockout mice, P-gp was shown to limit the absorption of cyclosporine

A in the distal small intestine, but not in the upper part, whereas the formation of metabolites

was demonstrated to be highest in the upper part. Interestingly, upon administration of

dexamethasone for 7 days, absorption of cyclosporine A from the proximal small intestine

was decreased due to increased P-gp expression, whereas in distal loops, mostly CYP3A

mediated metabolism was induced, resulting in a higher proportion of metabolites appearing

in the blood. This finding indicates that induction of P-gp and CYP3A metabolizing enzymes

27

is stronger at intestinal sites where their relative expression is lower. Figure 4 represents the

concentrations of cyclosporine A and its main metabolite M17 in portal venous blood.

3.7 IN SITU INTESTINAL EXCRETION UPON INTRAVENOUS ADMINISTRATION

Whereas the number of applications of most in vitro techniques is limited, the more

sophisticated nature of the in situ intestinal perfusion model allows optimizing the technique

for a specific purpose. Several authors have exploited the versatility of the model and

implemented an alternative set-up to study transporter involvement in intestinal drug

elimination: upon intravenous administration of a drug of interest, its appearance in blank

perfusion medium can be determined. Moreover, the contribution of efflux transporters to this

intestinal excretion can be studied using diagnostic inhibitors.

The intestinal excretion upon intravenous administration of talinolol and digoxin was shown

to be influenced by P-gp (Hanafy et al., 2001; Sababi et al., 2001). As discussed in section

3.6, regional differences in the expression of transporters may affect the intestinal excretion of

substrates. For instance, the percentage of an intravenously administered darunavir dose,

excreted in distal segments was significantly higher than in proximal segments, which is in

good agreement with the regional expression of P-gp along the small intestine. Upon

intravenous coadministration of P-gp inhibitor zosuquidar, similar intestinal excretion values

were observed for the two intestinal regions (Stappaerts et al., 2014a).

It is clear, however, that in contrast to the conventional in situ intestinal absorption set-up,

extra-intestinal factors influence the outcome of these excretion studies. Gao et al.

demonstrated the involvement of both P-gp and CYP3A mediated metabolism in the

interference of indinavir with the intestinal excretion of other HIV protease inhibitors

including amprenavir, nelfinavir and saquinavir (Gao et al., 2003). As CYP3A enzymes are

abundantly present in both hepatocytes and enterocytes, the involvement of intestinal

metabolism is difficult to estimate in this set-up.

28

Interestingly, the in situ intestinal excretion set-up can be complemented with additional bile

duct cannulation, which enables the simultaneous assessment of intestinal and biliary

excretion as well as the estimation of the relative impact of these processes on the overall

systemic drug exposure. Figure 5 illustrates this in situ excretion set-up. For instance, biliary

and intestinal excretion were demonstrated to be major excretion routes for the macrolide

antibiotics clarithromycin and roxythromycin, respectively (Arimori et al., 1998). Moreover,

bile cannulation allows assessing the involvement of transporters in hepatobiliary drug

disposition. For instance, P-gp substrates ciprofloxacin and darunavir exhibited a decreased

intestinal and biliary excretion upon intravenous coadministration of P-gp inhibitors (Dautrey

et al., 1999; Stappaerts et al., 2014a).

As metabolism often impedes the unambiguous assessment of transporter involvement in

intestinal and hepatobiliary excretion, again, genetically modified animals may provide a

solution. Using Mrp2 deficient GY/TR- rats, Mallants et al. demonstrated the involvement of

Mrp2 in the biliary excretion but not in the intestinal excretion of total tenofovir upon

intravenous administration of tenofovir disoproxil fumarate (Mallants et al., 2005).

3.8 THE EFFECT OF AGE ON BIOCHEMICAL BARRIER FUNCTION

As age-dependent changes in the expression of metabolizing enzymes and transporters have

been described in man, the efficiency of the biochemical barrier function of the small intestine

may be age related. For instance, an age-dependent increase in the expression and

functionality of CYP3A4 has been observed in duodenal sections from a pediatric population

(Johnson and Thomson, 2008). Similarly, the expression of MDR1 mRNA was found to

strongly vary among different age groups. Intestinal perfusions using very young or very old

animals might yield important information on intestinal absorption of drugs in young or

elderly populations. Since the in situ intestinal perfusion technique with mesenteric blood

29

sampling was validated in mice, this technique should also be feasible in very young rats

(Mols et al., 2009). Moreover, mesenteric blood sampling could again provide additional

information on the metabolic capacity of young versus old animals.

Lindahl et al. demonstrated similar permeability values for compounds undergoing passive

paracellular (atenolol) or transcellular (metoprolol) transport and carrier-mediated transport

(D-glucose) in rats in the age interval between 5 and 30 weeks (Lindahl et al., 1997).

Similarly, Yuasa et al. observed comparable permeability values for passively absorbed

compounds as well as for the carrier-mediated uptake of cephradine. In contrast with the

results of Lindahl et al., the intestinal permeability for D-glucose was shown to be 50% lower

in older rats (54 weeks) than in young rats (8 weeks) (Yuasa et al., 1997). Oguri et al. reported

high intestinal permeability for the amino acids alanine, arginine and aspartic acid in very

young rats (up to 8 weeks) as compared to older rats (8-104 weeks) (Oguri et al., 1999). In

general, based on the limited amount of studies performed, it appears that age-dependent

changes in the permeability of compounds are mild to moderate with some exceptions in very

young or very old animals, especially when absorption is carrier-mediated.

4. TOWARDS THE USE OF MORE COMPLEX MEDIA

In pharmaceutical industry, high-throughput screening programs to identify new drug

candidates are generally directed towards rapid identification of compounds with a high

potency against the biological target. These high-affinity compounds tend to exhibit a

relatively high lipophilicity, which is usually associated with a low aqueous solubility (Varma

et al., 2010). As a result, the proportion of compounds with poor aqueous dissolution and

solubility characteristics is increasing, both in drug development and on the market

(Stegemann et al., 2007). Consequently, a major challenge for the pharmaceutical industry

30

today is to get sufficiently high concentrations of an orally administered drug at the site of

absorption, i.e. the small intestine. Several formulation strategies have proven to be successful

in overcoming this solubility issue. These so-called ‘enabling formulations’ rely on different

principles and include the use of surfactants, particle size reduction, solid dispersions and

lipid based formulations (Buckley et al., 2013; Williams et al., 2013a).

It is becoming increasingly clear, however, that the raise in solubility that can be achieved

using these formulations is not always accompanied by a proportional increase in the

intestinal absorption. Therefore, it is of utmost importance to evaluate solubility as well as

permeability when studying enabling formulations.

Caco-2 has been shown to be compatible with a number of commonly used pharmaceutical

excipients within specific concentration ranges (Ingels and Augustijns, 2003). Nevertheless,

the protective mucus layer that is naturally present on enterocytes is not produced by this cell

monolayer, rendering the cells more vulnerable than naturally occurring enterocytes

(Cepinskas et al., 1993; Meaney and O’Driscoll, 1999). The more robust in situ intestinal

perfusion technique provides a tool to overcome this hurdle and study intestinal absorption

from more complex media. For example, in a study performed by Schipper et al., a 10 to 15-

fold increase in the permeability for atenolol, a paracellular marker, was seen in Caco-2 cells

in the presence of the polysaccharide chitosan (50 µg/ml), whereas the effect of chitosans on

permeability in situ was only modest (Schipper et al., 1999).

4.1 IN SITU INTESTINAL PERFUSIONS USING BIORELEVANT MEDIA – SOLUBILITY-PERMEABILITY INTERPLAY

Several authors have demonstrated the solubility-permeability trade-off that is present upon

micellar solubilization (Fischer et al., 2011; Katneni et al., 2006; Miller et al., 2011; Yano et

al., 2010). When using surfactants at a concentration above their critical micellar

concentration (CMC), micellar solubilization tends to positively influence the solubility of

31

lipophilic compounds. Nevertheless, due to this micellar entrapment, the free, bioaccessible

fraction also decreases, thereby offsetting the gain in apparent solubility. The importance of

studying the behavior of drugs in micellar solutions cannot be overestimated, as colloidal

structures, including micelles and vesicles, are omnipresent in the small intestine. Both

exogenous substances, such as food- or formulation derived lipid digestion products and

endogenous compounds, such as bile salts, may contribute to the formation of these micellar

and vesicular structures.

Using the intestinal perfusion technique in rats, Poelma et al. demonstrated a reduction in the

absorption rate of lipophilic compounds griseofulvin (log P = 2.18) and ketoconazole (log P =

4.35) upon addition of the bile salt taurocholate at concentrations above the CMC. In contrast,

the absorption rate of hydrophilic compounds paracetamol (log P = 0.46) and theophylline

(log P = -0.02) remained unaltered by taurocholate, indicating that the effect of micellar

entrapment increases with increasing lipophilicity (Poelma et al., 1990). Moreover, the fact

that the absorption rate of the hydrophilic compounds remained unaltered in presence of high

concentrations of the bile salt (up to 20 mM), suggests that the barrier function of the

intestinal wall was intact throughout the experiment. These concentrations of taurocholate

would be toxic to Caco-2 cells, illustrating the superior robustness of the in situ technique

(Ingels and Augustijns, 2003). In a follow-up study, earlier findings were confirmed in

perfusion media containing lysophosphatidylcholine and oleic acid, apart from taurocholate

(Poelma et al., 1991). These substances, creating mixed micelles, were included in an attempt

to generate biorelevant experimental conditions for mimicking the postprandial intraluminal

environment. In presence of the mixed micelles, the more lipophilic compounds were again

shown to exhibit the strongest decrease in absorption rate.

The effect of bile salt containing micelles on solubility and permeability has prompted

investigators to explore the use of media that are more relevant for the intraluminal

32

environment, in both solubility and intestinal absorption models. Simulated intestinal fluids

were developed to evaluate the intestinal disposition in a more biorelevant manner. These

media were optimized to mimic the intraluminal fluids in the fasted or the fed state and are

very useful to study food effects (Vertzoni et al., 2004).

Whereas simulated intestinal fluids of the fasted state (FaSSIF) are commonly applied in

absorption models such as Caco-2 cells, simulated fluids of the fed state are detrimental to

this cell monolayer (Fossati et al., 2008; Ingels et al., 2002). Despite attempts to generate

simulated media that mimic the fed state and retain compatibility with Caco-2 cells, it remains

challenging to avoid the trade-off between compatibility and biorelevance. For instance,

Markopoulos et al. generated simulated intestinal media of the fasted and fed state that are

compatible with Caco-2 cells. Nevertheless, the concentration of taurocholate that was used

for the fed state (6.8 mM) is low compared to conventional fed state simulated fluids (FeSSIF

v1: 15 mM and FeSSIF v2:10 mM) (Markopoulos et al., 2013).

Holmstock et al. used the in situ intestinal perfusion technique in mice to explore the negative

food effect that is clinically observed for the HIV protease inhibitor indinavir (Holmstock et

al., 2013a). In addition to simulated intestinal fluids, aspirated human intestinal fluids of

fasted and fed state conditions were used as solvent systems to evaluate intestinal solubility

and permeability of indinavir upon food intake. As compared to the fasted state, a higher

solubility accompanied by a lower absorptive flux was observed in postprandial conditions.

Stappaerts et al. confirmed this solubility-permeability trade-off for lipophilic compounds in

human aspirated fluids and demonstrated an increase in micellar entrapment upon increasing

lipophilicity for a series of structurally related β-blockers (Stappaerts et al., 2014b).

The solubility-permeability reciprocity is not limited to micellar media and was also

observed for cyclodextrin-based formulations. In presence of hydroxypropyl-β-cyclodextrins,

the disappearance of dexamethasone from the perfusion solution was twofold lower than the

33

permeability of the free drug (Beig et al., 2013). Therefore, it is generally accepted that a

formulation can only be successful if the increase in apparent solubility is associated with an

increase in the free, bioaccessible fraction or when the dissociation from the entrapped

fraction is fast as compared to permeation (Frank et al., 2012; Miller et al., 2012).

4.2 BEYOND SOLUBILITY: SUPERSATURATION

The thermodynamically metastable state of supersaturation in the intraluminal environment

increases the apparent solubility of a compound without a simultaneous decrease of the free

fraction. Therefore, inducing supersaturation at the level of the small intestine is very

beneficial to drug absorption. Both endogenous, physiological pathways such as

gastrointestinal transfer and digestive processes, and formulation strategies have been

described to generate intraluminal supersaturation (Bevernage et al., 2013; Williams et al.,

2013b).

Yeap et al. recognized the disparity between the rich body of data describing the trade-off

between solubility and permeability and, on the other hand, the irrefutable clinical

observations describing the positive influence of food and lipid based formulations on the oral

bioavailability of drugs. The rat in situ intestinal perfusion was very elegantly used as a

preclinical tool to evaluate possible endogenous mechanisms triggering supersaturation of

drugs from colloidal phases originating from dietary or formulation lipids. Physiologically

relevant mechanisms such as dilution by bile secretion and lipid absorption are proposed as

possible inducers of supersaturation (Yeap et al., 2013a, 2013b, 2013c). Bile mediated

dilution of colloidal phases containing weak bases, weak acids or neutral compounds was

evaluated as a causative trigger inducing supersaturation. Rat bile, which was collected

beforehand, was added to the perfusion solution directly prior to entering the small intestinal

segment. This is crucial, as the presence of an absorptive compartment has been shown to

34

sustain the supersaturated state of compounds (Bevernage et al., 2012). Upon increasing the

bile salt : lipid ratio in the colloidal phases, increased solubilization was observed for the

acidic and neutral compounds, whereas the apparent solubility of the bases cinnarizine and

halofantrine, decreased. As a result, periods of supersaturation followed by increased flux

towards the mesenteric vein could be induced upon addition of bile to the perfusion solution

for the weak base cinnarizine, whereas the effect on neutral compound danazol was less

pronounced. Apart from the dilution effect, the absorption of post-digestion lipids such as

oleic acid, present in the perfusion solution, was also suggested as a factor possibly

contributing to supersaturation. This hypothesis could be confirmed in a follow-up study

revealing the mechanism of lipid absorption as a trigger for supersaturation of cinnarizine

from oleic acid containing mixed micelles. Moreover, the acidic microclimate of the unstirred

water layer was demonstrated to play a role in converting the fatty acids to their unionized

state, hereby facilitating their absorption and, consequently, increasing the bile salt : lipid

ratio. Yeap et al. evidenced that, upon inclusion of amiloride, a competitive inhibitor of the

plasma membrane Na+/H+ exchanger, the flux of cinnarizine towards the mesenteric vein was

significantly compromised.

This series of reports points out the invaluable role of the in situ intestinal perfusion technique

as a biorelevant model to gain insight into the interplay between intraluminal concentrations

and drug permeability during supersaturation events.

Other authors have used the in situ intestinal perfusion to evaluate the performance of

supersaturation inducing formulations. Mellaerts et al. assessed the use of ordered

mesoporous silica loaded with itraconazole to generate supersaturation in the intraluminal

environment (Mellaerts et al., 2008). Upon suspension of the formulation in fasted state

simulated intestinal fluid, transport of itraconazole from the ordered mesoporous silica was

found to be more than 20-fold higher than from a saturated solution. Interestingly, the ordered

35

mesoporous silica suspension also outperformed the marketed amorphous solid dispersion

formulation of itraconazole, Sporanox®.

Nevertheless, the amorphous solid dispersions remain of great interest in overcoming

solubility and dissolution related issues associated with poorly soluble drugs. Recently, this

formulation approach was demonstrated to increase the apparent solubility of progesterone

and nifedipine without decreasing permeability, thus escaping the solubility-permeability

trade-off (Dahan et al., 2013; Miller et al., 2012).

5. FUTURE PERSPECTIVES

5.1. EVALUATION OF BARRIER FUNCTIONS: SPECIFIC INHIBITORS VERSUS KNOCKOUT ANIMALS

The in situ intestinal perfusion technique is frequently applied in mechanistic studies

evaluating the role of transporters and metabolizing enzymes in the intestinal uptake of drugs.

As the relative contribution of these mechanisms to the overall absorption is often difficult to

determine, the need for potent and specific inhibitors is self-evident. Nevertheless, numerous,

commonly used inhibitors have been shown to interfere with multiple transporters or

metabolic processes. For instance, inhibitors of P-gp often inhibit CYP3A enzymes as well

(Choo et al., 2000). Quinidine, cyclosporine A and ketoconazole are examples of frequently

used dual P-gp/CYP3A inhibitors. Therefore, in order to discriminate between the relative

involvement of transporters or metabolizing enzymes in intestinal absorption, it is important

to use diagnostic inhibitors at concentrations causing specific inhibition of the mechanism of

interest.

The introduction of knockout animals is a great step forward towards resolving the issue

encountered when using non-specific inhibitors. Comparison of intestinal permeability in

knockout and wild-type animals enables estimating the contribution of a specific absorption

36

process, without the need for diagnostic inhibitors. Nowadays, knockout mice for numerous

intestinal transporters are readily available (Tang et al., 2013). Moreover, the arrival of

knockout mice lacking specific metabolizing enzymes, combination knockout mice for both

transporters and metabolizing enzymes, tissue specific knockouts and humanized mice, has

created great opportunities for pharmacologic studies, including absorption profiling.

(Holmstock et al., 2013b, 2010; van Waterschoot and Schinkel, 2011). Despite the fact that

the use of genetically modified animals is very promising, thorough validation of the models

is required, as upregulation of compensatory mechanisms has been observed in knockout

animals (Lagas et al., 2012; Schuetz et al., 2000).

The small size and relatively fragile nature are some drawbacks of using mice. Moreover, as

compared to rats, the mouse model is less suitable to use in experiments involving multiple

manipulations. These factors negatively affect the success rate of the in situ intestinal

perfusion experiments in mice, especially when the cannulation of the mesenteric vein is

performed. Unfortunately, development of knockout rat models is lagging behind, rendering

mice still the most designated option when considering the use of genetically modified

animals. Nevertheless, recent advances in the field of construction of knockout rat models,

may bring the rat back center stage in future research (Farooq and M. Hawksworth, 2012;

Zamek-Gliszczynski et al., 2012).

5.2. PREDICTIVE AND MECHANISTIC STUDIES IN RODENTS

The fraction of a drug that is absorbed upon oral intake is a crucial factor determining the oral

bioavailability. Assessment of this pharmacokinetic parameter is costly as it requires

performing expensive clinical studies. Therefore, alternative methods to reliably predict the

fraction absorbed in humans are extremely valuable in the evaluation of drug candidates.

Strong correlations have been established for the fraction absorbed and the effective

37

permeability between rats and humans for a series of structurally diverse compounds,

including transporter substrates (Cao et al., 2006; Chiou and Barve, 1998). Consequently, the

in situ model in rats remains the most reliable model to predict the fraction absorbed in

humans (Lennernäs, 2014).

When intestinal metabolism is taken into account, however, species differences in isoforms of

metabolizing enzymes, substrate specificity and expression levels may impede quantitative

predictions of the fraction escaping gut metabolism in humans (Cao et al., 2006; Martignoni

et al., 2006). Nevertheless, despite the reduction in predictive power when using the in situ

intestinal perfusion model for compounds that undergo intestinal metabolic extraction, still,

important mechanistic insight into the involvement of metabolizing enzymes during intestinal

absorption can be gathered when performing the in situ intestinal perfusion in rodents.

5.3 SELECTION OF APPROPRIATE PERFUSION MEDIA AND DRUG CONCENTRATIONS

The majority of the in situ experiments, reported in the literature, involves intestinal perfusion

with a solution containing the drug of interest at a predetermined concentration. Often, simple

aqueous buffer solutions are selected as solvent systems. It is becoming abundantly clear,

however, that, as compared to aqueous media, the use of biorelevant fluids may significantly

alter dissolution, solubility and permeability characteristics. As already discussed in section 4,

the solubility of most lipophilic drugs is positively influenced by the presence of bile salts and

phospholipids in biorelevant fluids. Nevertheless, absorptive flux may be compromised due to

micellar inclusion of lipophilic compounds. Moreover, components specific to intestinal

fluids may affect the intestinal permeability of drugs. Taurocholate, for instance, has been

shown to inhibit P-gp functionality (Ingels et al., 2004). In addition, for some ester prodrugs,

poor stability has been reported in aspirated human intestinal fluids, which may compromise

38

the usefulness of the prodrug approach (Borde et al., 2012; Brouwers et al., 2007; Granero

and Amidon, 2006; Stoeckel et al., 1998).

Therefore, in order to address the complexity of the intraluminal environment, the use of

biorelevant fluids is advised. In this respect, simulated intestinal fluids of the fasted and fed

state are very suitable and readily available. Moreover, solubility values in simulated

intestinal fluids and human intestinal fluids of both fasted and fed state conditions are well

correlated (Augustijns et al., 2014). Solubility and dissolution studies in biorelevant media are

crucial to reliably estimate the concentrations to use in the absorption models.

Ideally, in vivo intraluminal concentrations profiling upon oral administration of a dosage

form to healthy volunteers, offers direct information of relevant concentrations to use in the

absorption models (Brouwers and Augustijns, 2014). These in vivo concentrations are

resulting from a myriad of physiological and physicochemical factors that are usually poorly

addressed in most absorption studies.

In addition to using biorelevant fluids, it is often suitable to use perfusion media with varying

pH values. Indeed, the intraluminal pH seems to increase from proximal (pH 6) to distal sites

(pH 7.4) of the small intestine (Fallingborg, 1999). As a result, compounds with a pKa value in

this pH range, may exhibit site dependent absorption related to their ionized fractions. This

has been reported for several compounds in aqueous buffer media (Dahan et al., 2010; Zur et

al., 2014a, 2014b)

5.4 TOWARDS A MORE DYNAMIC ABSORPTION MODEL

To the best of our knowledge, two reports have been published in which intestinal perfusions

were performed using human intestinal fluids of the fasted and fed state as perfusion media

(Holmstock et al., 2013a; Stappaerts et al., 2014b). Interestingly, for the lipophilic β-blocker

carvedilol, Stappaerts et al. observed a very strong decrease in the absorptive flux in

postprandial as compared to fasted state conditions. This reduced intestinal uptake was

39

strongly correlated with a decrease in the free, bioaccessible fraction of carvedilol and could

not be compensated for by the increase in solubility in fed state conditions, resulting in an

overall negative food effect on the intestinal absorption of carvedilol from a saturated

suspension. Nevertheless, since no clinical effect of food has been reported for carvedilol, it

appears likely that other important mechanisms, such as further dispersion and digestion of

the human intestinal fluids or gastrointestinal transfer may significantly affect the intestinal

absorption of this lipophilic compound.

Indeed, Yeap et al. demonstrated that dispersion of colloidal media by bile and absorption of

fatty acids can induce periods of supersaturation (Yeap et al., 2013b, 2013c). The colloidal

media used in this study represent different phases that form in the small intestine during the

digestion of triglycerides. Moreover, the same research group demonstrated in vitro

generation of supersaturation upon digestion of lipid based formulations (Anby et al., 2012).

It is therefore very plausible that further processing of human intestinal fluids collected in fed

state conditions will also affect the permeation of lipophilic compounds. The same processes

of dispersion and digestion may increase, the free, bioaccessible fraction of micellarly

entrapped drugs again, resulting in increased absorption rates. Therefore, performing

intestinal perfusion experiments with human intestinal fluids of the fed state complemented

with pancreatic extract, may be very interesting to evaluate the effect of digestion on the

absorptive flux of compounds. Thorough characterization of the digestion process will be of

great benefit to the use of human intestinal fluids in absorption models (Williams et al., 2012).

It is clear that the intraluminal environment is a very complex and highly dynamic climate in

which interaction between endogenous mechanisms and the dosage form may significantly

influence the concentrations available for absorption. The in situ intestinal perfusion

technique can already be considered a highly biorelevant technique as it exhibits close to in

40

vivo experimental conditions. Moreover, it is sufficiently robust and versatile to implement

modifications, further increasing biorelevance.

5.5 FORMULATION EVALUATION

Over the years, a strong increase has been observed in the proportion of poorly water soluble

drug candidates in drug development programs (Stegemann et al., 2007). In response to this

trend, formulation scientists have come upon ways to address this issue and the use of

enabling formulations, generating suitable intraluminal drug concentrations, has rapidly

gained interest (Buckley et al., 2013; Williams et al., 2013a).

Notwithstanding the beneficial effect of these formulations on drug solubility and dissolution,

it has become clear that the rise in apparent solubility is not always a reliable measure for the

expected gain in drug absorption, as was discussed in section 4. Therefore, it is advisable to

combine data from dissolution experiments with data obtained using intestinal absorption

models. In order to study the effect of formulations on the intestinal absorption, it is clear that

an absorption model should be selected, which is compatible with the components of the

formulation. The in situ intestinal perfusion exhibits suitable robustness to serve as an

absorption tool for the evaluation of formulations.

Despite the fact that most intestinal perfusion studies are performed using solutions of a drug,

some authors have described the evaluation of the intestinal permeability of drugs from

enabling formulations in an in situ set-up. The assessment of drug absorption from lipid based

formulations, cyclodextrin-containing media and ordered mesoporous silica have been

reported and this type of studies may prove very valuable to gain insight into the intestinal

disposition of a drug upon oral intake of a dosage form (Beig et al., 2013; Mellaerts et al.,

2008; Yeap et al., 2013a).

41

As it is often difficult to unambiguously define a donor concentration when working with

drug formulations, calculation of permeability values remains problematic. In most cases, the

permeability of the small intestine for a compound remains unaltered upon perfusion with

different formulations. In contrast, drug concentrations, more specifically the free

concentrations that are generated when using different formulations, may strongly differ and

lead to changes in the overall absorption of a compound. Therefore, these formulation

evaluation studies usually have a comparative character and mostly report the resulting

absorptive flux upon perfusion with a formulation. As mentioned in section 4.2, formulations

that increase the free, bioaccessible fraction of a compound are of great interest to overcome

poor drug absorption caused by solubility or dissolution issues.

6. CONCLUDING REMARKS

The in situ intestinal perfusion technique with mesenteric blood sampling in rats exhibits

unique qualities, which allow investigators to overcome the hurdles encountered when using

in vitro tools. In combination with the expression of the most important drug transporters,

P450 enzyme expression in rat enterocytes enables the evaluation of transporter-metabolism

interactions. During the in situ procedure, blood flow and innervation remain intact, creating

experimental conditions that are very close to the in vivo situation. Moreover, the robustness

of the system permits using more biorelevant but complex perfusion media, which are often

detrimental to Caco-2 cells.

ACKNOWLEDGMENTS

We would like to thank Yan Yan Yeap for providing us with the overview figure illustrating

the generation of supersaturation upon processing of colloidal phases. This research was

funded by a grant from ‘Onderzoeksfonds’ of the KU Leuven in Belgium.

42

REFERENCESAbuasal, B.S., Bolger, M.B., Walker, D.K., Kaddoumi, A., 2012. In silico modeling for the nonlinear

absorption kinetics of UK-343,664: a P-gp and CYP3A4 substrate. Mol. Pharm. 9, 492–504. doi:10.1021/mp200275j

Adachi, Y., Suzuki, H., Schinkel, A.H., Sugiyama, Y., 2005. Role of breast cancer resistance protein (Bcrp1/Abcg2) in the extrusion of glucuronide and sulfate conjugates from enterocytes to intestinal lumen. Mol. Pharmacol. 67, 923–928. doi:10.1124/mol.104.007393

Anby, M.U., Williams, H.D., McIntosh, M., Benameur, H., Edwards, G.A., Pouton, C.W., Porter, C.J.H., 2012. Lipid digestion as a trigger for supersaturation: evaluation of the impact of supersaturation stabilization on the in vitro and in vivo performance of self-emulsifying drug delivery systems. Mol. Pharm. 9, 2063–2079. doi:10.1021/mp300164u

Annaert, P., Tukker, J.J., van Gelder, J., Naesens, L., de Clercq, E., van Den Mooter, G., Kinget, R., Augustijns, P., 2000. In vitro, ex vivo, and in situ intestinal absorption characteristics of the antiviral ester prodrug adefovir dipivoxil. J. Pharm. Sci. 89, 1054–1062.

Arakawa, H., Shirasaka, Y., Haga, M., Nakanishi, T., Tamai, I., 2012. Active intestinal absorption of fluoroquinolone antibacterial agent ciprofloxacin by organic anion transporting polypeptide, Oatp1a5. Biopharm. Drug Dispos. 33, 332–341. doi:10.1002/bdd.1809

Arimori, K., Miyamoto, S., Fukuda, K., Nakamura, C., Nakano, M., 1998. Characteristic difference in gastrointestinal excretion of clarithromycin and roxithromycin. Biopharm. Drug Dispos. 19, 433–438.

Artursson, P., Palm, K., Luthman, K., 2001. Caco-2 monolayers in experimental and theoretical predictions of drug transport 27–43.

Augustijns, P., Wuyts, B., Hens, B., Annaert, P., Butler, J., Brouwers, J., 2014. A review of drug solubility in human intestinal fluids: implications for the prediction of oral absorption. Eur. J. Pharm. Sci. Off. J. Eur. Fed. Pharm. Sci. 57, 322–332. doi:10.1016/j.ejps.2013.08.027

Beaumont, K., Webster, R., Gardner, I., Dack, K., 2003. Design of ester prodrugs to enhance oral absorption of poorly permeable compounds: challenges to the discovery scientist. Curr. Drug Metab. 4, 461–485.

Beig, A., Agbaria, R., Dahan, A., 2013. Oral delivery of lipophilic drugs: the tradeoff between solubility increase and permeability decrease when using cyclodextrin-based formulations. PloS One 8, e68237. doi:10.1371/journal.pone.0068237

Benet, L.Z., Cummins, C.L., Wu, C.Y., 2004. Unmasking the dynamic interplay between efflux transporters and metabolic enzymes. Int. J. Pharm. 277, 3–9. doi:10.1016/j.ijpharm.2002.12.002

Berggren, S., Gall, C., Wollnitz, N., Ekelund, M., Karlbom, U., Hoogstraate, J., Schrenk, D., Lennernäs, H., 2007. Gene and protein expression of P-glycoprotein, MRP1, MRP2, and CYP3A4 in the small and large human intestine 252–257.

Bevernage, J., Brouwers, J., Annaert, P., Augustijns, P., 2012. Drug precipitation-permeation interplay: supersaturation in an absorptive environment. Eur. J. Pharm. Biopharm. Off. J. Arbeitsgemeinschaft Für Pharm. Verfahrenstechnik EV 82, 424–428. doi:10.1016/j.ejpb.2012.07.009

Bevernage, J., Brouwers, J., Brewster, M.E., Augustijns, P., 2013. Evaluation of gastrointestinal drug supersaturation and precipitation: strategies and issues. Int. J. Pharm. 453, 25–35. doi:10.1016/j.ijpharm.2012.11.026

Bohets, H., Annaert, P., Mannens, G., Van Beijsterveldt, L., Anciaux, K., Verboven, P., Meuldermans, W., Lavrijsen, K., 2001. Strategies for absorption screening in drug discovery and development. Curr. Top. Med. Chem. 1, 367–383.

Borde, A.S., Karlsson, E.M., Andersson, K., Björhall, K., Lennernäs, H., Abrahamsson, B., 2012. Assessment of enzymatic prodrug stability in human, dog and simulated intestinal fluids. Eur. J. Pharm. Biopharm. Off. J. Arbeitsgemeinschaft Für Pharm. Verfahrenstechnik EV 80, 630–637. doi:10.1016/j.ejpb.2011.11.011

43

Brouwers, J., Augustijns, P., 2014. Resolving intraluminal drug and formulation behavior: Gastrointestinal concentration profiling in humans. Eur. J. Pharm. Sci. Off. J. Eur. Fed. Pharm. Sci. 61, 2–10. doi:10.1016/j.ejps.2014.01.010

Brouwers, J., Tack, J., Augustijns, P., 2007. In vitro behavior of a phosphate ester prodrug of amprenavir in human intestinal fluids and in the Caco-2 system: illustration of intraluminal supersaturation 302–309.

Buckley, S.T., Frank, K.J., Fricker, G., Brandl, M., 2013. Biopharmaceutical classification of poorly soluble drugs with respect to “enabling formulations.” Eur. J. Pharm. Sci. Off. J. Eur. Fed. Pharm. Sci. 50, 8–16. doi:10.1016/j.ejps.2013.04.002

Canaparo, R., Finnström, N., Serpe, L., Nordmark, A., Muntoni, E., Eandi, M., Rane, A., Zara, G.P., 2007. Expression of CYP3A isoforms and P-glycoprotein in human stomach, jejunum and ileum 1138–1144.

Cao, F., Jia, J., Yin, Z., Gao, Y., Sha, L., Lai, Y., Ping, Q., Zhang, Y., 2012. Ethylene glycol-linked amino acid diester prodrugs of oleanolic acid for PepT1-mediated transport: synthesis, intestinal permeability and pharmacokinetics. Mol. Pharm. 9, 2127–2135. doi:10.1021/mp200447r

Cao, X., Gibbs, S.T., Fang, L., Miller, H.A., Landowski, C.P., Shin, H.-C., Lennernas, H., Zhong, Y., Amidon, G.L., Yu, L.X., Sun, D., 2006. Why is it challenging to predict intestinal drug absorption and oral bioavailability in human using rat model 1675–1686.

Cao, X., Yu, L.X., Barbaciru, C., Landowski, C.P., Shin, H.-C., Gibbs, S., Miller, H.A., Amidon, G.L., Sun, D., 2005. Permeability dominates in vivo intestinal absorption of P-gp substrate with high solubility and high permeability. Mol. Pharm. 2, 329–340. doi:10.1021/mp0499104

Cepinskas, G., Specian, R.D., Kvietys, P.R., 1993. Adaptive cytoprotection in the small intestine: role of mucus. Am. J. Physiol. 264, G921–927.

Chiou, W.L., Barve, A., 1998. Linear correlation of the fraction of oral dose absorbed of 64 drugs between humans and rats. Pharm. Res. 15, 1792–1795.

Choo, E.F., Leake, B., Wandel, C., Imamura, H., Wood, A.J., Wilkinson, G.R., Kim, R.B., 2000. Pharmacological inhibition of P-glycoprotein transport enhances the distribution of HIV-1 protease inhibitors into brain and testes. Drug Metab. Dispos. Biol. Fate Chem. 28, 655–660.

Cummins, C.L., Salphati, L., Reid, M.J., Benet, L.Z., 2003. In vivo modulation of intestinal CYP3A metabolism by P-glycoprotein: studies using the rat single-pass intestinal perfusion model 306.

Dahan, A., Amidon, G.L., 2009. Small intestinal efflux mediated by MRP2 and BCRP shifts sulfasalazine intestinal permeability from high to low, enabling its colonic targeting G371–377.

Dahan, A., Beig, A., Ioffe-Dahan, V., Agbaria, R., Miller, J.M., 2013. The twofold advantage of the amorphous form as an oral drug delivery practice for lipophilic compounds: increased apparent solubility and drug flux through the intestinal membrane. AAPS J. 15, 347–353. doi:10.1208/s12248-012-9445-3

Dahan, A., Miller, J.M., Hilfinger, J.M., Yamashita, S., Yu, L.X., Lennernäs, H., Amidon, G.L., 2010. High-Permeability Criterion for BCS Classification: Segmental/pH Dependent Permeability Considerations. Mol. Pharm. 7, 1827–1834. doi:10.1021/mp100175a

Dahan, A., Sabit, H., Amidon, G.L., 2009. Multiple efflux pumps are involved in the transepithelial transport of colchicine: combined effect of p-glycoprotein and multidrug resistance-associated protein 2 leads to decreased intestinal absorption throughout the entire small intestine. Drug Metab. Dispos. Biol. Fate Chem. 37, 2028–2036. doi:10.1124/dmd.109.028282

Dautrey, S., Felice, K., Petiet, A., Lacour, B., Carbon, C., Farinotti, R., 1999. Active intestinal elimination of ciprofloxacin in rats: modulation by different substrates. Br. J. Pharmacol. 127, 1728–1734. doi:10.1038/sj.bjp.0702703

Doluisio, J.T., Billups, N.F., Dittert, L.W., Sugita, E.T., Swintosky, J.V., 1969. Drug absorption. I. An in situ rat gut technique yielding realistic absorption rates. J. Pharm. Sci. 58, 1196–1200.

44

Englund, G., Rorsman, F., Rönnblom, A., Karlbom, U., Lazorova, L., Gråsjö, J., Kindmark, A., Artursson, P., 2006. Regional levels of drug transporters along the human intestinal tract: co-expression of ABC and SLC transporters and comparison with Caco-2 cells 269–277.

Eriksson, A.H., Varma, M.V.S., Perkins, E.J., Zimmerman, C.L., 2010. The intestinal absorption of a prodrug of the mGlu2/3 receptor agonist LY354740 is mediated by PEPT1: in situ rat intestinal perfusion studies. J. Pharm. Sci. 99, 1574–1581. doi:10.1002/jps.21917

Fallingborg, J., 1999. Intraluminal pH of the human gastrointestinal tract. Dan. Med. Bull. 46, 183–196.

Farooq, M., M. Hawksworth, G., 2012. The Arrival of “knockout” Rats. J. Mol. Cloning Genet. Recomb. 01. doi:10.4172/2325-9787.1000e101

FDA, 2011. Drug Interactions & Labeling - Drug Development and Drug Interactions: Table of Substrates, Inhibitors and Inducers. [WWW Document]. URL http://www.fda.gov/drugs/developmentapprovalprocess/developmentresources/druginteractionslabeling/ucm093664.htm (accessed 9.10.14).

Fischer, S.M., Brandl, M., Fricker, G., 2011. Effect of the non-ionic surfactant Poloxamer 188 on passive permeability of poorly soluble drugs across Caco-2 cell monolayers. Eur. J. Pharm. Biopharm. Off. J. Arbeitsgemeinschaft Für Pharm. Verfahrenstechnik EV 79, 416–422. doi:10.1016/j.ejpb.2011.04.010

Fossati, L., Dechaume, R., Hardillier, E., Chevillon, D., Prevost, C., Bolze, S., Maubon, N., 2008. Use of simulated intestinal fluid for Caco-2 permeability assay of lipophilic drugs. Int. J. Pharm. 360, 148–155. doi:10.1016/j.ijpharm.2008.04.034

Frank, K.J., Rosenblatt, K.M., Westedt, U., Hölig, P., Rosenberg, J., Mägerlein, M., Fricker, G., Brandl, M., 2012. Amorphous solid dispersion enhances permeation of poorly soluble ABT-102: true supersaturation vs. apparent solubility enhancement. Int. J. Pharm. 437, 288–293. doi:10.1016/j.ijpharm.2012.08.014

Gao, W., Kageyama, M., Inoue, Y., Tadano, J., Fukumoto, K., Fukushima, K., Yamasaki, D., Nishimura, A., Yoshikawa, Y., Shibata, N., Takada, K., 2003. Effect of indinavir on the intestinal exsorption of amprenavir, saquinavir and nelfinavir after intravenous administration in rats. Biol. Pharm. Bull. 26, 199–204.

González-Alvarez, I., Fernández-Teruel, C., Casabó-Alós, V.G., Garrigues, T.M., Polli, J.E., Ruiz-García, A., Bermejo, M., 2007. In situ kinetic modelling of intestinal efflux in rats: functional characterization of segmental differences and correlation with in vitro results. Biopharm. Drug Dispos. 28, 229–239. doi:10.1002/bdd.548

Granero, G.E., Amidon, G.L., 2006. Stability of valacyclovir: implications for its oral bioavailability. Int. J. Pharm. 317, 14–18. doi:10.1016/j.ijpharm.2006.01.050

Gupta, S.V., Gupta, D., Sun, J., Dahan, A., Tsume, Y., Hilfinger, J., Lee, K.-D., Amidon, G.L., 2011. Enhancing the intestinal membrane permeability of zanamivir: a carrier mediated prodrug approach. Mol. Pharm. 8, 2358–2367. doi:10.1021/mp200291x

Hanafy, A., Langguth, P., Spahn-Langguth, H., 2001. Pretreatment with potent P-glycoprotein ligands may increase intestinal secretion in rats. Eur. J. Pharm. Sci. Off. J. Eur. Fed. Pharm. Sci. 12, 405–415.

Han, H., de Vrueh, R.L., Rhie, J.K., Covitz, K.M., Smith, P.L., Lee, C.P., Oh, D.M., Sadée, W., Amidon, G.L., 1998. 5’-Amino acid esters of antiviral nucleosides, acyclovir, and AZT are absorbed by the intestinal PEPT1 peptide transporter. Pharm. Res. 15, 1154–1159.

Herrera-Ruiz, D., Wang, Q., Gudmundsson, O.S., Cook, T.J., Smith, R.L., Faria, T.N., Knipp, G.T., 2001. Spatial expression patterns of peptide transporters in the human and rat gastrointestinal tracts, Caco-2 in vitro cell culture model, and multiple human tissues. AAPS PharmSci 3, E9.

Holmstock, N., Bruyn, T.D., Bevernage, J., Annaert, P., Mols, R., Tack, J., Augustijns, P., 2013a. Exploring food effects on indinavir absorption with human intestinal fluids in the mouse intestine. Eur. J. Pharm. Sci. Off. J. Eur. Fed. Pharm. Sci. 49, 27–32. doi:10.1016/j.ejps.2013.01.012

45

Holmstock, N.F., Annaert, P.P., Augustijns, P., 2012. Boosting of HIV Protease Inhibitors by Ritonavir in the Intestine: The Relative Role of Cyp and P-gp Inhibition Based on Caco-2 monolayers Versus In Situ Intestinal Perfusion in Mice.

Holmstock, N., Gonzalez, F.J., Baes, M., Annaert, P., Augustijns, P., 2013b. PXR/CYP3A4-humanized mice for studying drug-drug interactions involving intestinal P-glycoprotein. Mol. Pharm. 10, 1056–1062. doi:10.1021/mp300512r

Holmstock, N., Mols, R., Annaert, P., Augustijns, P., 2010. In situ intestinal perfusion in knockout mice demonstrates inhibition of intestinal p-glycoprotein by ritonavir causing increased darunavir absorption 1407–1410.

Hu, Y., Smith, D.E., Ma, K., Jappar, D., Thomas, W., Hillgren, K.M., 2008. Targeted disruption of peptide transporter Pept1 gene in mice significantly reduces dipeptide absorption in intestine. Mol. Pharm. 5, 1122–1130.

Imai, T., Ohura, K., 2010. The role of intestinal carboxylesterase in the oral absorption of prodrugs. Curr. Drug Metab. 11, 793–805.

Ingels, F., Beck, B., Oth, M., Augustijns, P., 2004. Effect of simulated intestinal fluid on drug permeability estimation across Caco-2 monolayers 221–232.

Ingels, F., Deferme, S., Destexhe, E., Oth, M., Van den Mooter, G., Augustijns, P., 2002. Simulated intestinal fluid as transport medium in the Caco-2 cell culture model. Int. J. Pharm. 232, 183–192.

Ingels, F.M., Augustijns, P.F., 2003. Biological, pharmaceutical, and analytical considerations with respect to the transport media used in the absorption screening system, Caco-2. J. Pharm. Sci. 92, 1545–1558. doi:10.1002/jps.10408

Ingersoll, S.A., Ayyadurai, S., Charania, M.A., Laroui, H., Yan, Y., Merlin, D., 2012. The role and pathophysiological relevance of membrane transporter PepT1 in intestinal inflammation and inflammatory bowel disease. Am. J. Physiol. Gastrointest. Liver Physiol. 302, G484–492. doi:10.1152/ajpgi.00477.2011

International Transporter Consortium, Giacomini, K.M., Huang, S.-M., Tweedie, D.J., Benet, L.Z., Brouwer, K.L.R., Chu, X., Dahlin, A., Evers, R., Fischer, V., Hillgren, K.M., Hoffmaster, K.A., Ishikawa, T., Keppler, D., Kim, R.B., Lee, C.A., Niemi, M., Polli, J.W., Sugiyama, Y., Swaan, P.W., Ware, J.A., Wright, S.H., Yee, S.W., Zamek-Gliszczynski, M.J., Zhang, L., 2010. Membrane transporters in drug development. Nat. Rev. Drug Discov. 9, 215–236. doi:10.1038/nrd3028

Jappar, D., Wu, S.-P., Hu, Y., Smith, D.E., 2010. Significance and regional dependency of peptide transporter (PEPT) 1 in the intestinal permeability of glycylsarcosine: in situ single-pass perfusion studies in wild-type and Pept1 knockout mice. Drug Metab. Dispos. Biol. Fate Chem. 38, 1740–1746. doi:10.1124/dmd.110.034025

Jin, M., Shimada, T., Yokogawa, K., Nomura, M., Ishizaki, J., Piao, Y., Kato, Y., Tsuji, A., Miyamoto, K.-I., 2006. Site-dependent contributions of P-glycoprotein and CYP3A to cyclosporin A absorption, and effect of dexamethasone in small intestine of mice 1042–1050.

Johannsson, G., Bergthorsdottir, R., Nilsson, A.G., Lennernas, H., Hedner, T., Skrtic, S., 2009. Improving glucocorticoid replacement therapy using a novel modified-release hydrocortisone tablet: a pharmacokinetic study. Eur. J. Endocrinol. Eur. Fed. Endocr. Soc. 161, 119–130. doi:10.1530/EJE-09-0170

Johnson, B.M., Chen, W., Borchardt, R.T., Charman, W.N., Porter, C.J.H., 2003. A kinetic evaluation of the absorption, efflux, and metabolism of verapamil in the autoperfused rat jejunum. J. Pharmacol. Exp. Ther. 305, 151–158. doi:10.1124/jpet.102.045328

Johnson, T.N., Thomson, M., 2008. Intestinal metabolism and transport of drugs in children: the effects of age and disease. J. Pediatr. Gastroenterol. Nutr. 47, 3–10. doi:10.1097/MPG.0b013e31816a8cca

Kaddoumi, A., Fleisher, D., Heimbach, T., Li, L.Y., Cole, S., 2006. Factors influencing regional differences in intestinal absorption of UK-343,664 in rat: possible role in dose-dependent pharmacokinetics. J. Pharm. Sci. 95, 435–445. doi:10.1002/jps.20527

46

Katneni, K., Charman, S.A., Porter, C.J.H., 2006. Permeability assessment of poorly water-soluble compounds under solubilizing conditions: the reciprocal permeability approach. J. Pharm. Sci. 95, 2170–2185. doi:10.1002/jps.20687

Kis, O., Zastre, J.A., Hoque, M.T., Walmsley, S.L., Bendayan, R., 2013. Role of drug efflux and uptake transporters in atazanavir intestinal permeability and drug-drug interactions. Pharm. Res. 30, 1050–1064. doi:10.1007/s11095-012-0942-y

Lagas, J.S., Damen, C.W.N., van Waterschoot, R.A.B., Iusuf, D., Beijnen, J.H., Schinkel, A.H., 2012. P-glycoprotein, multidrug-resistance associated protein 2, Cyp3a, and carboxylesterase affect the oral availability and metabolism of vinorelbine. Mol. Pharmacol. 82, 636–644. doi:10.1124/mol.111.077099

Lee, K.-J., Johnson, N., Castelo, J., Sinko, P.J., Grass, G., Holme, K., Lee, Y.-H., 2005. Effect of experimental pH on the in vitro permeability in intact rabbit intestines and Caco-2 monolayer. Eur. J. Pharm. Sci. Off. J. Eur. Fed. Pharm. Sci. 25, 193–200. doi:10.1016/j.ejps.2005.02.012

Lennernäs, H., 2014. Regional intestinal drug permeation: biopharmaceutics and drug development. Eur. J. Pharm. Sci. Off. J. Eur. Fed. Pharm. Sci. 57, 333–341. doi:10.1016/j.ejps.2013.08.025

Lennernäs, H., Ahrenstedt, O., Hällgren, R., Knutson, L., Ryde, M., Paalzow, L.K., 1992. Regional jejunal perfusion, a new in vivo approach to study oral drug absorption in man. Pharm. Res. 9, 1243–1251.

Li, L.Y., Amidon, G.L., Kim, J.S., Heimbach, T., Kesisoglou, F., Topliss, J.T., Fleisher, D., 2002. Intestinal metabolism promotes regional differences in apical uptake of indinavir: coupled effect of P-glycoprotein and cytochrome P450 3A on indinavir membrane permeability in rat 586–593.

Lindahl, A., Krondahl, E., Grudén, A.C., Ungell, A.L., Lennernäs, H., 1997. Is the jejunal permeability in rats age-dependent? Pharm. Res. 14, 1278–1281.

Liu, S., Tam, D., Chen, X., Pang, K.S., 2006. P-glycoprotein and an unstirred water layer barring digoxin absorption in the vascularly perfused rat small intestine preparation: induction studies with pregnenolone-16alpha-carbonitrile. Drug Metab. Dispos. Biol. Fate Chem. 34, 1468–1479. doi:10.1124/dmd.105.008227

MacLean, C., Moenning, U., Reichel, A., Fricker, G., 2008. Closing the gaps: a full scan of the intestinal expression of p-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated protein 2 in male and female rats 1249–1254.

MacLean, C., Moenning, U., Reichel, A., Fricker, G., 2010. Regional absorption of fexofenadine in rat intestine 670–674.

Mallants, R., Van Oosterwyck, K., Van Vaeck, L., Mols, R., De Clercq, E., Augustijns, P., 2005. Multidrug resistance-associated protein 2 (MRP2) affects hepatobiliary elimination but not the intestinal disposition of tenofovir disoproxil fumarate and its metabolites 1055–1066.

Markopoulos, C., Thoenen, F., Preisig, D., Symillides, M., Vertzoni, M., Parrott, N., Reppas, C., Imanidis, G., 2013. Biorelevant media for transport experiments in the Caco-2 model to evaluate drug absorption in the fasted and the fed state and their usefulness. Eur. J. Pharm. Biopharm. Off. J. Arbeitsgemeinschaft Pharm. Verfahrenstechnik EV. doi:10.1016/j.ejpb.2013.10.017

Martignoni, M., Groothuis, G.M.M., de Kanter, R., 2006. Species differences between mouse, rat, dog, monkey and human CYP-mediated drug metabolism, inhibition and induction. Expert Opin. Drug Metab. Toxicol. 2, 875–894. doi:10.1517/17425255.2.6.875

Masaki, K., Hashimoto, M., Imai, T., 2007. Intestinal first-pass metabolism via carboxylesterase in rat jejunum and ileum. Drug Metab. Dispos. Biol. Fate Chem. 35, 1089–1095. doi:10.1124/dmd.106.013862

Ma, X., Cheung, C., Krausz, K.W., Shah, Y.M., Wang, T., Idle, J.R., Gonzalez, F.J., 2008. A double transgenic mouse model expressing human pregnane X receptor and cytochrome P450 3A4 2506–2512.

47

Meaney, C., O’Driscoll, C., 1999. Mucus as a barrier to the permeability of hydrophilic and lipophilic compounds in the absence and presence of sodium taurocholate micellar systems using cell culture models. Eur. J. Pharm. Sci. 8, 167–175. doi:10.1016/S0928-0987(99)00007-X

Meier, Y., Eloranta, J.J., Darimont, J., Ismair, M.G., Hiller, C., Fried, M., Kullak-Ublick, G.A., Vavricka, S.R., 2007. Regional distribution of solute carrier mRNA expression along the human intestinal tract 590–594.

Mellaerts, R., Mols, R., Kayaert, P., Annaert, P., Van Humbeeck, J., Van den Mooter, G., Martens, J.A., Augustijns, P., 2008. Ordered mesoporous silica induces pH-independent supersaturation of the basic low solubility compound itraconazole resulting in enhanced transepithelial transport. Int. J. Pharm. 357, 169–179. doi:10.1016/j.ijpharm.2008.01.049

Miller, J.M., Beig, A., Carr, R.A., Spence, J.K., Dahan, A., 2012. A win-win solution in oral delivery of lipophilic drugs: supersaturation via amorphous solid dispersions increases apparent solubility without sacrifice of intestinal membrane permeability. Mol. Pharm. 9, 2009–2016. doi:10.1021/mp300104s

Miller, J.M., Beig, A., Krieg, B.J., Carr, R.A., Borchardt, T.B., Amidon, G.E., Amidon, G.L., Dahan, A., 2011. The solubility-permeability interplay: mechanistic modeling and predictive application of the impact of micellar solubilization on intestinal permeation. Mol. Pharm. 8, 1848–1856. doi:10.1021/mp200181v

Mitschke, D., Reichel, A., Fricker, G., Moenning, U., 2008. Characterization of cytochrome P450 protein expression along the entire length of the intestine of male and female rats. Drug Metab. Dispos. Biol. Fate Chem. 36, 1039–1045. doi:10.1124/dmd.107.019687

Mols, R., Brouwers, J., Schinkel, A.H., Annaert, P., Augustijns, P., 2009. Intestinal perfusion with mesenteric blood sampling in wild-type and knockout mice: evaluation of a novel tool in biopharmaceutical drug profiling 1334–1337.

Mudra, D.R., Borchardt, R.T., 2010. Absorption barriers in the rat intestinal mucosa: 1. Application of an in situ perfusion model to simultaneously assess drug permeation and metabolism. J. Pharm. Sci. 99, 982–998. doi:10.1002/jps.21912

Mudra, D.R., Desino, K.E., Desai, P.V., 2011. In silico, in vitro and in situ models to assess interplay between CYP3A and P-gp 750–773.

Oguri, S., Kumazaki, M., Kitou, R., Nonoyama, H., Tooda, N., 1999. Elucidation of intestinal absorption of D,L-amino acid enantiomers and aging in rats. Biochim. Biophys. Acta 1472, 107–114.

Ohura, K., Soejima, T., Nogata, R., Adachi, Y., Ninomiya, S., Imai, T., 2012. Effect of intestinal first-pass hydrolysis on the oral bioavailability of an ester prodrug of fexofenadine. J. Pharm. Sci. 101, 3264–3274. doi:10.1002/jps.23182

Okudaira, N., Tatebayashi, T., Speirs, G.C., Komiya, I., Sugiyama, Y., 2000. A study of the intestinal absorption of an ester-type prodrug, ME3229, in rats: active efflux transport as a cause of poor bioavailability of the active drug. J. Pharmacol. Exp. Ther. 294, 580–587.

Poelma, F.G., Breäs, R., Tukker, J.J., 1990. Intestinal absorption of drugs. III. The influence of taurocholate on the disappearance kinetics of hydrophilic and lipophilic drugs from the small intestine of the rat. Pharm. Res. 7, 392–397.

Poelma, F.G., Breäs, R., Tukker, J.J., Crommelin, D.J., 1991. Intestinal absorption of drugs. The influence of mixed micelles on on the disappearance kinetics of drugs from the small intestine of the rat. J. Pharm. Pharmacol. 43, 317–324.

Rong, Z., Xu, Y., Zhang, C., Xiang, D., Li, X., Liu, D., 2013. Evaluation of intestinal absorption of amtolmetin guacyl in rats: breast cancer resistant protein as a primary barrier of oral bioavailability. Life Sci. 92, 245–251. doi:10.1016/j.lfs.2012.12.010

Sababi, M., Borgå, O., Hultkvist-Bengtsson, U., 2001. The role of P-glycoprotein in limiting intestinal regional absorption of digoxin in rats. Eur. J. Pharm. Sci. Off. J. Eur. Fed. Pharm. Sci. 14, 21–27.

48

Salphati, L., Benet, L.Z., 1999. Metabolism of digoxin and digoxigenin digitoxosides in rat liver microsomes: involvement of cytochrome P4503A. Xenobiotica Fate Foreign Compd. Biol. Syst. 29, 171–185. doi:10.1080/004982599238722

Sandström, R., Lennernäs, H., 1999. Repeated oral rifampicin decreases the jejunal permeability of R/S-verapamil in rats. Drug Metab. Dispos. Biol. Fate Chem. 27, 951–955.

Schanker, L.S., Tocco, D.J., Brodie, B.B., Hogben, C.A., 1958. Absorption of drugs from the rat small intestine. J. Pharmacol. Exp. Ther. 123, 81–88.

Schipper, N.G., Vârum, K.M., Stenberg, P., Ocklind, G., Lennernäs, H., Artursson, P., 1999. Chitosans as absorption enhancers of poorly absorbable drugs. 3: Influence of mucus on absorption enhancement. Eur. J. Pharm. Sci. Off. J. Eur. Fed. Pharm. Sci. 8, 335–343.

Schmiedlin-Ren, P., Thummel, K.E., Fisher, J.M., Paine, M.F., Lown, K.S., Watkins, P.B., 1997. Expression of enzymatically active CYP3A4 by Caco-2 cells grown on extracellular matrix-coated permeable supports in the presence of 1alpha,25-dihydroxyvitamin D3. Mol. Pharmacol. 51, 741–754.

Schuetz, E.G., Umbenhauer, D.R., Yasuda, K., Brimer, C., Nguyen, L., Relling, M.V., Schuetz, J.D., Schinkel, A.H., 2000. Altered expression of hepatic cytochromes P-450 in mice deficient in one or more mdr1 genes. Mol. Pharmacol. 57, 188–197.

Sesink, A.L.A., Arts, I.C.W., de Boer, V.C.J., Breedveld, P., Schellens, J.H.M., Hollman, P.C.H., Russel, F.G.M., 2005. Breast cancer resistance protein (Bcrp1/Abcg2) limits net intestinal uptake of quercetin in rats by facilitating apical efflux of glucuronides. Mol. Pharmacol. 67, 1999–2006. doi:10.1124/mol.104.009753

Shirasaka, Y., Sakane, T., Yamashita, S., 2008. Effect of P-glycoprotein expression levels on the concentration-dependent permeability of drugs to the cell membrane. J. Pharm. Sci. 97, 553–565. doi:10.1002/jps.21114

Shirasaka, Y., Suzuki, K., Shichiri, M., Nakanishi, T., Tamai, I., 2011. Intestinal absorption of HMG-CoA reductase inhibitor pitavastatin mediated by organic anion transporting polypeptide and P-glycoprotein/multidrug resistance 1. Drug Metab. Pharmacokinet. 26, 171–179.

Sjöberg, Å., Lutz, M., Tannergren, C., Wingolf, C., Borde, A., Ungell, A.-L., 2013. Comprehensive study on regional human intestinal permeability and prediction of fraction absorbed of drugs using the Ussing chamber technique. Eur. J. Pharm. Sci. Off. J. Eur. Fed. Pharm. Sci. 48, 166–180. doi:10.1016/j.ejps.2012.10.007

Stappaerts, J., Annaert, P., Augustijns, P., 2013. Site dependent intestinal absorption of darunavir and its interaction with ketoconazole. Eur. J. Pharm. Sci. Off. J. Eur. Fed. Pharm. Sci. 49, 51–56. doi:10.1016/j.ejps.2013.01.015

Stappaerts, J., Fattah, S., Annaert, P., Augustijns, P., 2014a. Hepatobiliary and intestinal elimination of darunavir in an integrated preclinical rat model. Xenobiotica Fate Foreign Compd. Biol. Syst. 44, 489–497. doi:10.3109/00498254.2013.861541

Stappaerts, J., Wuyts, B., Tack, J., Annaert, P., Augustijns, P., 2014b. Human and simulated intestinal fluids as solvent systems to explore food effects on intestinal solubility and permeability. Eur. J. Pharm. Sci. Off. J. Eur. Fed. Pharm. Sci. 63C, 178–186. doi:10.1016/j.ejps.2014.07.009

Stegemann, S., Leveiller, F., Franchi, D., de Jong, H., Lindén, H., 2007. When poor solubility becomes an issue: from early stage to proof of concept. Eur. J. Pharm. Sci. Off. J. Eur. Fed. Pharm. Sci. 31, 249–261. doi:10.1016/j.ejps.2007.05.110

Stephens, R.H., O’Neill, C.A., Warhurst, A., Carlson, G.L., Rowland, M., Warhurst, G., 2001. Kinetic profiling of P-glycoprotein-mediated drug efflux in rat and human intestinal epithelia 584–591.

Stoeckel, K., Hofheinz, W., Laneury, J.P., Duchene, P., Shedlofsky, S., Blouin, R.A., 1998. Stability of cephalosporin prodrug esters in human intestinal juice: implications for oral bioavailability. Antimicrob. Agents Chemother. 42, 2602–2606.

Sutton, S.C., Rinaldi, M.T., Vukovinsky, K.E., 2001. Comparison of the gravimetric, phenol red, and 14C-PEG-3350 methods to determine water absorption in the rat single-pass intestinal perfusion model. AAPS PharmSci 3, E25.

49

Takara, K., Ohnishi, N., Horibe, S., Yokoyama, T., 2003. Expression profiles of drug-metabolizing enzyme CYP3A and drug efflux transporter multidrug resistance 1 subfamily mRNAS in small intestine 1235–1239.

Tamura, S., Ohike, A., Ibuki, R., Amidon, G.L., Yamashita, S., 2002. Tacrolimus is a class II low-solubility high-permeability drug: the effect of P-glycoprotein efflux on regional permeability of tacrolimus in rats. J. Pharm. Sci. 91, 719–729.

Tamura, S., Tokunaga, Y., Ibuki, R., Amidon, G.L., Sezaki, H., Yamashita, S., 2003. The site-specific transport and metabolism of tacrolimus in rat small intestine. J. Pharmacol. Exp. Ther. 306, 310–316. doi:10.1124/jpet.103.050716

Tang, S.C., Hendrikx, J.J.M.A., Beijnen, J.H., Schinkel, A.H., 2013. Genetically modified mouse models for oral drug absorption and disposition. Curr. Opin. Pharmacol. 13, 853–858. doi:10.1016/j.coph.2013.08.011

Tannergren, C., Bergendal, A., Lennernäs, H., Abrahamsson, B., 2009. Toward an increased understanding of the barriers to colonic drug absorption in humans: implications for early controlled release candidate assessment. Mol. Pharm. 6, 60–73. doi:10.1021/mp800261a

Thombre, A.G., 2005. Assessment of the feasibility of oral controlled release in an exploratory development setting. Drug Discov. Today 10, 1159–1166. doi:10.1016/S1359-6446(05)03551-8

Thummel, K.E., Brimer, C., Yasuda, K., Thottassery, J., Senn, T., Lin, Y., Ishizuka, H., Kharasch, E., Schuetz, J., Schuetz, E., 2001. Transcriptional Control of Intestinal Cytochrome P-4503A by 1α,25-Dihydroxy Vitamin D3. Mol. Pharmacol. 60, 1399–1406. doi:10.1124/mol.60.6.1399

Usansky, H.H., Hu, P., Sinko, P.J., 2008. Differential roles of P-glycoprotein, multidrug resistance-associated protein 2, and CYP3A on saquinavir oral absorption in Sprague-Dawley rats 863.

Valenzuela, B., Nácher, A., Ruiz-Carretero, P., Martín-Villodre, A., López-Carballo, G., Barettino, D., 2004. Profile of P-glycoprotein distribution in the rat and its possible influence on the salbutamol intestinal absorption process. J. Pharm. Sci. 93, 1641–1648. doi:10.1002/jps.20071

Van Gelder, J., Deferme, S., Annaert, P., Naesens, L., De Clercq, E., Van den Mooter, G., Kinget, R., Augustijns, P., 2000a. Increased absorption of the antiviral ester prodrug tenofovir disoproxil in rat ileum by inhibiting its intestinal metabolism. Drug Metab. Dispos. Biol. Fate Chem. 28, 1394–1396.

Van Gelder, J., Shafiee, M., De Clercq, E., Penninckx, F., Van den Mooter, G., Kinget, R., Augustijns, P., 2000b. Species-dependent and site-specific intestinal metabolism of ester prodrugs. Int. J. Pharm. 205, 93–100.

Van Waterschoot, R.A.B., Schinkel, A.H., 2011. A critical analysis of the interplay between cytochrome P450 3A and P-glycoprotein: recent insights from knockout and transgenic mice. Pharmacol. Rev. 63, 390–410. doi:10.1124/pr.110.002584

Varma, M.V., Ambler, C.M., Ullah, M., Rotter, C.J., Sun, H., Litchfield, J., Fenner, K.S., El-Kattan, A.F., 2010. Targeting intestinal transporters for optimizing oral drug absorption. Curr. Drug Metab. 11, 730–742.

Vertzoni, M., Fotaki, N., Kostewicz, E., Stippler, E., Leuner, C., Nicolaides, E., Dressman, J., Reppas, C., 2004. Dissolution media simulating the intralumenal composition of the small intestine: physiological issues and practical aspects. J. Pharm. Pharmacol. 56, 453–462. doi:10.1211/0022357022935

Wagner, D., Spahn-Langguth, H., Hanafy, A., Koggel, A., Langguth, P., 2001. Intestinal drug efflux: formulation and food effects S13–31.

Wahajuddin, null, Raju, K.S.R., Singh, S.P., Taneja, I., 2014. Investigation of the functional role of P-glycoprotein in limiting the oral bioavailability of lumefantrine. Antimicrob. Agents Chemother. 58, 489–494. doi:10.1128/AAC.01382-13

Wang, H., Kawashima, H., Strobel, H.W., 1996. cDNA cloning of a novel CYP3A from rat brain. Biochem. Biophys. Res. Commun. 221, 157–162. doi:10.1006/bbrc.1996.0562

50

Williams, H.D., Sassene, P., Kleberg, K., Bakala-N’Goma, J.-C., Calderone, M., Jannin, V., Igonin, A., Partheil, A., Marchaud, D., Jule, E., Vertommen, J., Maio, M., Blundell, R., Benameur, H., Carrière, F., Müllertz, A., Porter, C.J.H., Pouton, C.W., 2012. Toward the establishment of standardized in vitro tests for lipid-based formulations, part 1: method parameterization and comparison of in vitro digestion profiles across a range of representative formulations. J. Pharm. Sci. 101, 3360–3380. doi:10.1002/jps.23205

Williams, H.D., Trevaskis, N.L., Charman, S.A., Shanker, R.M., Charman, W.N., Pouton, C.W., Porter, C.J.H., 2013a. Strategies to address low drug solubility in discovery and development. Pharmacol. Rev. 65, 315–499.

Williams, H.D., Trevaskis, N.L., Yeap, Y.Y., Anby, M.U., Pouton, C.W., Porter, C.J.H., 2013b. Lipid-Based Formulations and Drug Supersaturation: Harnessing the Unique Benefits of the Lipid Digestion/Absorption Pathway. Pharm. Res. 30, 2976–2992. doi:10.1007/s11095-013-1126-0

Willson, T.M., Kliewer, S.A., 2002. PXR, CAR and drug metabolism. Nat. Rev. Drug Discov. 1, 259–266. doi:10.1038/nrd753

Yang, B., Smith, D.E., 2013. Significance of peptide transporter 1 in the intestinal permeability of valacyclovir in wild-type and PepT1 knockout mice. Drug Metab. Dispos. Biol. Fate Chem. 41, 608–614. doi:10.1124/dmd.112.049239

Yang, Z., Zhu, W., Gao, S., Yin, T., Jiang, W., Hu, M., 2012. Breast cancer resistance protein (ABCG2) determines distribution of genistein phase II metabolites: reevaluation of the roles of ABCG2 in the disposition of genistein. Drug Metab. Dispos. Biol. Fate Chem. 40, 1883–1893. doi:10.1124/dmd.111.043901

Yano, K., Masaoka, Y., Kataoka, M., Sakuma, S., Yamashita, S., 2010. Mechanisms of membrane transport of poorly soluble drugs: role of micelles in oral absorption processes. J. Pharm. Sci. 99, 1336–1345. doi:10.1002/jps.21919

Yeap, Y.Y., Trevaskis, N.L., Porter, C.J.H., 2013a. The potential for drug supersaturation during intestinal processing of lipid-based formulations may be enhanced for basic drugs. Mol. Pharm. 10, 2601–2615. doi:10.1021/mp400035z

Yeap, Y.Y., Trevaskis, N.L., Porter, C.J.H., 2013b. Lipid absorption triggers drug supersaturation at the intestinal unstirred water layer and promotes drug absorption from mixed micelles. Pharm. Res. 30, 3045–3058. doi:10.1007/s11095-013-1104-6

Yeap, Y.Y., Trevaskis, N.L., Quach, T., Tso, P., Charman, W.N., Porter, C.J.H., 2013c. Intestinal bile secretion promotes drug absorption from lipid colloidal phases via induction of supersaturation. Mol. Pharm. 10, 1874–1889. doi:10.1021/mp3006566

Yuasa, H., Soga, N., Kimura, Y., Watanabe, J., 1997. Effect of aging on the intestinal transport of hydrophilic drugs in the rat small intestine. Biol. Pharm. Bull. 20, 1188–1192.

Zamek-Gliszczynski, M.J., Bedwell, D.W., Bao, J.Q., Higgins, J.W., 2012. Characterization of SAGE Mdr1a (P-gp), Bcrp, and Mrp2 knockout rats using loperamide, paclitaxel, sulfasalazine, and carboxydichlorofluorescein pharmacokinetics. Drug Metab. Dispos. Biol. Fate Chem. 40, 1825–1833. doi:10.1124/dmd.112.046508

Zur, M., Gasparini, M., Wolk, O., Amidon, G.L., Dahan, A., 2014a. The low/high BCS permeability class boundary: physicochemical comparison of metoprolol and labetalol. Mol. Pharm. 11, 1707–1714. doi:10.1021/mp500152y

Zur, M., Hanson, A.S., Dahan, A., 2014b. The complexity of intestinal permeability: Assigning the correct BCS classification through careful data interpretation. Eur. J. Pharm. Sci. Off. J. Eur. Fed. Pharm. Sci. 61, 11–17. doi:10.1016/j.ejps.2013.11.007

51