In situ perfusion in rodents to explore intestinal drug absorption: challenges and opportunities.

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Contents lists available at ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

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Review

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In situ perfusion in rodents to explore intestinal drug absorption: Challenges and opportunities

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Jef Stappaerts, Joachim Brouwers, Pieter Annaert, Patrick Augustijns *

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

A R T I C L E I N F O

A B S T R A C T

Article history: Received 2 October 2014 Received in revised form 13 November 2014 Accepted 14 November 2014 Available online xxx

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 timeconsuming, the model offers great opportunities to gain insight into the processes determining intestinal drug absorption. ã 2014 Published by Elsevier B.V.

Keywords: Transporter–metabolism interplay Site dependent absorption Knockout animals Solubility–permeability interplay Supersaturation Intestinal perfusion

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Permeability assessment–disappearance (Peff) versus appearance (Papp) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Measuring disappearance from the perfusion solution–effective permeability . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Measuring appearance in the blood–apparent permeability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Vascular perfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Open loop and closed loop intestinal perfusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Exploring the biochemical barrier function of the small intestine using in situ perfusion . . . . . . . . . . . . . . . . . . . . . . . Use of effective permeability in transporter–metabolism studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Use of apparent permeability in transporter–metabolism studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Intestinal absorption of ester prodrugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Evaluating the specific contribution of drug transporters and metabolizing enzymes: use of knockout animals 3.4. The effect of induction on the biochemical barrier function of the small intestine . . . . . . . . . . . . . . . . . . . . . . . 3.5. Regional absorption studies–site dependent expression of transporters and metabolizing enzymes . . . . . . . . . 3.6. Regional in situ intestinal absorption studies–transporter substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.1.

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* Corresponding author. Tel.: +32 16 330301; fax: +32 16 330305. E-mail address: [email protected] (P. Augustijns). http://dx.doi.org/10.1016/j.ijpharm.2014.11.035 0378-5173/ ã 2014 Published by Elsevier B.V.

Please cite this article in press as: Stappaerts, J., et al., In situ perfusion in rodents to explore intestinal drug absorption: Challenges and opportunities. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.11.035

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3.6.2. Regional in situ intestinal absorption studies–dual substrates . . . . . . . . . . . . . In situ intestinal excretion upon intravenous administration . . . . . . . . . . . . . . . . . . . . . . 3.7. The effect of age on biochemical barrier function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8. Towards the use of more complex media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In situ intestinal perfusions using biorelevant media–solubility–permeability interplay 4.1. Beyond solubility: supersaturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evaluation of barrier functions: specific inhibitors versus knockout animals . . . . . . . . . 5.1. 5.2. Predictive and mechanistic studies in rodents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Selection of appropriate perfusion media and drug concentrations . . . . . . . . . . . . . . . . 5.3. Towards a more dynamic absorption model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. 5.5. Formulation evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

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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 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.

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

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2.1. Measuring disappearance from the perfusion solution–effective permeability

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In the original set-up of the in situ intestinal perfusion, a segment of the small intestine of an anesthetized 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 concentration and the absorptive area of the intestinal segment, the effective permeability value can be calculated using Eq. (1):

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Peff ¼ F

ð1 C out =C in Þ 2pRL

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(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 transportermediated absorption Cao et al. (2006). Human intestinal permeability values used in this study were obtained from jejunal perfusion studies using the Loc-I-gut1 technique (Lennernäs et al., 1992).

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2.2. Measuring appearance in the blood–apparent permeability

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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

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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. 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 (Fig. 1). Donor blood is supplied via the vena jugularis to maintain the hemodynamic balance. This technique allows calculating the apparent permeability (Eq. (2) and Fig. 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. Papp

dQ 1 ¼ dt 2pRL C donor

(2)

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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 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).

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Fig. 2. The cumulative amount of drug appearing in the mesenteric blood as a function of time.

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.

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2.4. Open loop and closed loop intestinal perfusions

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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). Fig. 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

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Fig. 1. Schematic representation of the in situ intestinal perfusion set-up with mesenteric blood sampling.


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permeability calculations in the closed-loop modus, require frequent sampling of the perfusion solution, while for open-loop experiments, 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. Caco2 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 Caco2 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 1a,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 Pgp (Mudra et al., 2011). Interestingly, there have been reports both on cooperative and counteracting functioning of these detoxifying mechanisms. Consequently, 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. (2000b) demonstrated low esterase activity in Caco-2 cells, which may lead to overestimation of the intestinal transport of ester prodrugs.

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).

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3.1. Use of effective permeability in transporter–metabolism studies

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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 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. (2002) 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. (2012). By integrating the effective permeability obtained in situ and several additional disposition parameters from in vitro experiments in a physiologically based pharmacokinetic (PBPK)

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model, Abuasal et al. (2012) managed to predict the bioavailability of the dual P-gp/CYP3A4 substrate UK343,664 and explain its nonlinear absorption behavior. Km and Vmax values for CYP3A4 and Pgp were determined in vitro using supersomes and the Caco2 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). 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. (2012) 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.

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3.2. Use of apparent permeability in transporter–metabolism studies

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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. (2011), 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. (2003) observed an increase in apparent permeability of verapamil when using PSC833 (valspodar, typical P-gp inhibitor) and midazolam (typical CYP3A substrate), whereas Mudra et al. (2011) 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 knockout 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

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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, 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).

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3.3. Intestinal absorption of ester prodrugs

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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 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

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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 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 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 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. Pgp, 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 4methylumbelliferone (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,


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G Model

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but no statistical difference was observed in effective permeability of the parent 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 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). 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-16a-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

7

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 increased expression of P-gp, whereas intestinal metabolism remained negligible (Salphati and Benet, 1999). Sandström and Lennernäs 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. (2013a,b) also determined expression levels of P-gp and Q2 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.

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

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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

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G Model

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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 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 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.

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 Pgp 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; Raju 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 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 Pept1 in the small intestine is the main causative factor for the higher permeability as compared to the colon.

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

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


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G Model

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of intracellular metabolism. Within the same research group, the site dependent permeability of another dual substrate, UK343,664, 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 to 50 mM. At a UK343,664 concentration of 50 mM, 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 increase in permeability for UK343,664 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

9

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 is stronger at intestinal sites where their relative expression is lower. Fig. 4 represents the concentrations of cyclosporine A and its main metabolite M17 in portal venous blood.

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3.7. In situ intestinal excretion upon intravenous administration

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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.

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Fig. 4. The concentrations of cyclosporin A (a) and M17 (b) in portal venous blood 45 min after in situ administration of cyclosporin A (40 nmol) into an upper or lower intestinal loop of wild-type or mdr1a/1b knockout mice with or without dexamethasone treatment (75 mg/kg, daily, 7 times), at 1.5 h after the last administration. White bars: wild-type without dexamethasone treatment; dotted bars: wild-type with dexamethasone treatment; striped bars: mdr1a/1b knockout without dexamethasone treatment; gray bars: mdr1a/1b knockout with dexamethasone treatment. Each column and bar represent the mean S.D. of four mice. Significantly different at *P < 0.05 and **P < 0.01, respectively. (NS) no significant difference.


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G Model

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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. Fig. 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 Q4 (Dautrey et al., 1999; Stappaerts et al., 2014a) (Fig. 6). 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).

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3.8. The effect of age on biochemical barrier function

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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 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.

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Enterocytes (ii) Lipid absorption

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D D D D Multilamellar vesicles

Bile Blood

Intesnal perfusate Fig. 5. Schematic overview of the experimental set-up of the in situ excretion model: drug is intravenously administered via the vena jugularis and samples are collected from systemic blood (arteria carotis), bile and intestinal perfusate.

(ii) Lipid absorption

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Intravenous administraon

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Weak bases > neutral > weak acids)

Fig. 6. Generation of supersaturation upon processing of colloidal phases (adopted from Yeap et al.).

Q14

Lindahl et al. demonstrated similar permeability values for compounds undergoing passive paracellular (atenolol) or transcellular (metoprolol) transport and carrier-mediated transport (Dglucose) in rats in the age interval between 5 and 30 weeks (Lindahl et al., 1997). Similarly, Yuasa et al. (1997) 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. (1997) 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.

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4. Towards the use of more complex media

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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 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).

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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 mg/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 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 followup 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 environment, in both solubility and intestinal absorption models. Simulated

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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 Q6 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). 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 b-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-b-cyclodextrins, the disappearance of dexamethasone from the perfusion solution was twofold lower than the 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).

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4.2. Beyond solubility: supersaturation

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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,b,c). 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

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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 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 mesoporous silica suspension also outperformed the marketed amorphous solid dispersion formulation of itraconazole, Sporanox1. 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 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 Hawksworth, 2012; Zamek-Gliszczynski et al., 2012).

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5.2. Predictive and mechanistic studies in rodents

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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 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.

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5.3. Selection of appropriate perfusion media and drug concentrations

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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

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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 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,b)

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5.4. Towards a more dynamic absorption model

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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 b-blocker carvedilol, Stappaerts et al. (2014a,b) observed a very strong decrease in the absorptive flux in postprandial as compared to fasted state conditions. This reduced intestinal uptake was 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,c). 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

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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 vivo experimental conditions. Moreover, it is sufficiently robust and versatile to implement modifications, further increasing biorelevance.

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5.5. Formulation evaluation

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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). 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.

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6. Concluding Remarks

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The in situ intestinal perfusion technique with mesenteric blood sampling in rats exhibits unique qualities, which allow

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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.

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Acknowledgments

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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 Q8 from ‘Onderzoeksfonds’ of the KU Leuven in Belgium. References

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