in situ perfusion in rodents to explore intestinal drug absorption: challenges and opportunities
Jef Stappaerts, Joachim Brouwers, Pieter Annaert and Patrick Augustijns
Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, Leuven, Belgium
Corresponding author:
Patrick Augustijns
Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences
Gasthuisberg O&N 2 - Herestraat 49 box 921 - 3000 Leuven - Belgium
tel: +32-16-330301 - fax: +32-16-330305
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Keywords: transporter-metabolism interplay; site dependent absorption; knockout animals; solubility-permeability interplay; supersaturation; intestinal perfusion
Abstract
The in situ intestinal perfusion technique in rodents is avery 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 vitroabsorption 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, thein situ intestinal perfusion modelhas exhibited an exceptional ability to adapt to the latest challenges in drug absorption profiling. For instance, the introduction of the mesenteric vein cannulation allows determining the appearance of compounds in the blood and is of great use, especially when evaluating the absorption of compounds undergoing intestinal metabolism. Moreover, the use of the closed loop intestinal perfusion set-up is interesting when compounds or perfusion media are scarce. Compatibility with emerging trends in pharmaceutical profiling, such as the use of knockout or transgenic animals, generates unparalleled possibilities to gain mechanistic insight into specific absorption processes.
Notwithstanding the fact that the in situ experiments are technically challenging and relatively time-consuming, the model offers great opportunities to gain insight into the processes determining intestinal drug absorption.
Contents
Abstract
1.Introduction
2.Permeability assessment – disappearance (Peff) versus appearance (Papp)
2.1 Measuring disappearance from the perfusion solution - Effective permeability
2.2 Measuring appearance in the blood - Apparent permeability
2.3 Vascular perfusion
2.4 Open loop and closed loop intestinal perfusions
3.Exploring the biochemical barrier function of the small intestine using in situ perfusion
3.1 Use of effective permeability in transporter – metabolism studies
3.2 Use of apparent permeability in transporter – metabolism studies
3.3 Intestinal absorption of ester prodrugs
3.4 Evaluating the specific contribution of drug transporters and metabolizing enzymes: use of knockout animals
3.5 The effect of induction on the biochemical barrier function of the small intestine
3.6 Regional absorption studies – site dependent expression of transporters and metabolizing enzymes
3.6.1 Regional in situ intestinal absorption studies– transporter substrates
3.6.2 Regional in situ intestinal absorption studies– dual substrates
3.7 In situ intestinal excretion upon intravenous administration
4.Towards the use of more complex media
4.1 In situ intestinal perfusions using biorelevant media – solubility-permeability interplay
4.2 Beyond solubility: supersaturation
5.Future perspectives
5.1.Evaluation of barrier functions: specific inhibitors versus knockout animals
5.2.Predictive and mechanistic studies in rodents
5.3Selection of appropriate perfusion media and drug concentrations
5.4 Towards a more dynamic absorption model
5.5 Formulation evaluation
6.Concluding Remarks
1.Introduction
Since oral intake remains the preferred route of drug administration, the need to develop and validate suitable models to evaluate intestinal absorption is self-evident. In the pharmaceutical industry, there is a strong tendency towards the use of in vitro tools to study intestinal permeability because of their suitability to be implemented in high-throughput programs(Bohets et al., 2001). The Caco-2 model is nowadays considered the gold standard in intestinal permeability screening. This cell line expresses most of the transporters that are relevant for drug absorption in humans, rendering it useful to study absorption mechanisms. Moreover, for compounds that are passively absorbed and exhibit low intestinal metabolism, permeability values observed in the Caco-2 model allow good predictions of the fraction of the administered dose of a drug that will be absorbed in humans(Artursson et al., 2001). Nevertheless, despite its wide applicability in permeability profiling, this in vitro model sometimes fails to address the complexity of intestinal processes which eventually determinein 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 situintestinal 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.
2.Permeability assessment – disappearance (Peff) versus appearance (Papp)
2.1 Measuring disappearance from the perfusion solution - Effective permeability
In the original set-up of the in situ intestinal perfusion, a segment of the small intestine of an anaesthetized animal is cannulated and perfused with a solution containing a predefined concentration of a drug of interest. During the experiment, the animal is kept unconscious and its body temperature is maintained by the use of a heating pad or an overhead lamp. Upon perfusion of the intestinal segment, drug will be absorbed to some extent, depending on its physicochemical and biopharmaceutical properties, and the drug concentration in the perfusion solution will decrease. Through comparison of the donor concentration and the concentration of the solution that exits the isolated segment, the amount of drug that has permeated the apical membrane of the small intestinal barrier (transcellular transport) or has passed through the intercellular space (paracellular transport) can be calculated. By correcting the amount of drug that disappeared from the perfusion solution over time for the donor concentration and the absorptive area of the intestinal segment, theeffective permeability value can be calculated using equation (1):
(1)
withF the flow rate of the perfusion solution, Cout and Cin the outlet and inlet concentration, respectively, and R the radius and Llength 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 transportermediatedabsorption (Cao et al., 2006). Human intestinal permeability values used in this study were obtained from jejunal perfusion studies using the Loc-I-gut® technique(Lennernäs et al., 1992).
2.2 Measuring appearance in the blood - Apparent permeability
It is essential, however, to be aware of the fact that the effective permeability does not necessarily give a reliable prediction of the amount of drug that will appear in the blood. Non-specific binding to perfusion tubing or the isolated intestinal segment can result in a decrease in Coutwhich may be erroneously interpreted asdrug absorption. Moreover, for compounds that undergo a high intestinal metabolic extraction, a lower fraction will generally reach the blood circulation than would be predictedbased 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(Figure 1). Donor blood is supplied via the vena jugularis to maintain the hemodynamic balance.
This technique allows calculating the apparent permeability (Equation (2) and Figure 2),where dQ/dt is the slope of the cumulative amount of drug appearing into the mesenteric blood over time,R the radius and L length of the perfused intestinal segment. Cdonor is the donor concentration of the perfusion solution.
Obviously, by taking samples from the perfusion solution at the inlet and outlet of the cannulated intestinal segment, the effective permeability can still be determined.
2.3 Vascular perfusion
It is clear that mesenteric vein cannulation in combination with intestinal perfusion experiments improves insight into intestinal drug absorption mechanisms. Additional cannulation of the mesenteric artery enables perfusion of the mesenteric capillary bed, creating the possibility to control both intestinal and vascular perfusion of the cannulated small intestinal segment.Vascular perfusion solutions mostly consist of oxygenated buffer solutions containing albumin, circumventing the need for donor blood. An additional advantage of the vascular perfusion set-up, is the ability to vary the blood supply to the intestinal segment. For example, in postprandial conditions, the blood flow to the small intestine is higher than in the fasted state and this may consequently influence the absorption rate. For instance, Tamura et al. demonstrated that the absorbed amount of tacrolimus at a vascular perfusion rate of 2.5 ml/min was significantly higher than the absorption at a flow rate of 1 ml/min (Tamura et al., 2003).
A downside of vascular perfusion with oxygenated buffers is the increased interference with physiological processesin this set-up. For instance, the distribution of blood to the small intestine via the mesenteric arteries follows a pulsatile pattern, whereas a constant flow is generated upon vascular perfusion. Moreover, care should be taken not to disrupt the fragile capillaries when imposing a certain flow rate through the vascular bed.
2.4 Open loop and closed loop intestinal perfusions
A small intestinal segment can be perfused in the open loop or the closed loop set-up. In the open loop set-up, the perfusion solution that exits the cannulated segment goes directly to waste. However, when perfusion media are scarce (e.g. when using intestinal fluids) or when only small amounts of compound are available (e.g. early development stages), the closed loop set-up can be applied; in this configuration, the perfusion solution is continuously recirculated through the intestinal segment, dramatically decreasing the volume of perfusion medium needed to perform the experiment(Doluisio et al., 1969). Figure 3 gives a schematic representation of the open and closed loop set-up. Depending on the specifications of the materials used, including the internal diameter and the length of the tubing, 5 ml of medium can be sufficient to perform a closed-loop perfusion. It is clear that, upon absorption in the closed-loop set-up, Cdonorwill decrease during the experiment, whereas in the open-loop set-up, the donor concentration will generally be constant if the compound of interest is stable in the perfusion medium. Therefore, apparent permeability calculations in the closed-loop modus, require frequent sampling of the perfusion solution, while for open-loop experiments, 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 usingin situ perfusion
The rapidly growing body of literature on intestinal drug disposition evidences the complex nature of the processes underlying intestinal absorption. The small intestine is equipped with a number of efficient detoxifying mechanisms, hampering the uptake of xenobiotics. Membrane transporters and metabolizing enzymes have been shown to affect both rate and extent of intestinal drug absorption(FDA, 2011). The use of in vitro models allows investigators to study isolated processes such as the involvement of transporters in intestinal drug absorption. Caco-2 cells express most of the transporters that are relevant for intestinal drug transport in human and therefore, they have proven to be very convenient in transporter studies. For the assessment of intestinal P450 mediated metabolism, however, investigators have to rely on otherin vitro tools, including intestinal microsomes or homogenates. Indeed, one of the major drawbacks of the Caco-2 model is the very low to non-existent expression of cytochrome P450 enzymes. Therefore, application ofthis in vitro model to assess intestinal permeability for compounds exhibiting a high metabolic extraction in the gutmay generate an overestimation of the intestinal transport. Despite efforts to induce the expression of CYP3A4 in selected clones of Caco-2 cells using 1α,25-dihydroxyvitamin D3, the metabolic activity was still low compared to human intestinal tissue homogenates (Schmiedlin-Ren et al., 1997).
For some compounds, a complex interplay may exist between transporters and metabolizing enzymes upon intestinal transport, as has been observed for dual substrates of CYP3A enzymes and P-gp(Mudra et al., 2011). Interestingly, there have been reports both on cooperative and counteracting functioning of these detoxifying mechanisms.Consequently, incubations of dual P-gp/CYP3A substratesin intestinal microsomes or homogenates, combined with permeability data from Caco-2 will not necessarily create a reliable picture of the key mechanisms dictatingthe intestinal absorption. Therefore, simultaneous assessment of transporter and metabolism functioning is advisable for these compounds.
In addition to the lack of P450 enzyme expression, Van Gelder et al. demonstrated low esterase activity in Caco-2 cells, which may lead to overestimation of the intestinal transport of ester prodrugs(Van Gelder et al., 2000b).
As mice and rats express both intestinal transporters and P450 enzymes, the in situ intestinal perfusion technique in rodents has been used to study theintestinal 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 remainsvery useful in mechanistic studies. Recent advances in the field of transgenic animals (e.g. mice expressing human CYP3A4) may further increase the relevance of using rodents in the evaluation of the intestinal absorption of compounds that are subject to significant intestinal metabolic extraction(Ma et al., 2008; van Waterschoot and Schinkel, 2011).
3.1 Use of effective permeability in transporter – metabolism studies
As mentioned in section 2, determining the effective intestinal permeability for a compound that undergoes significant metabolic extraction may result in an overestimation of the fraction that will reach the blood. For some compounds, however, it is possible to follow the appearance of metabolites, originating from intracellular metabolism, in the perfusion medium. These metabolites can reach the apical side of the enterocytes via active or passive transport processes and serve as a measure of the intracellular metabolism.Li et al. monitored the concentration of metabolite ‘M6’ in the perfusion solution upon perfusion of the rat small intestine with the dual P-gp/CYP3A substrate indinavir and used this metabolite to estimate intestinal metabolism. Extensive metabolism of indinavir in the jejunum was demonstrated, generating a larger concentration difference for indinavir over the apical membrane, thereby facilitating the transport of indinavir across the apical membrane of the enterocytes. The fact that M6 is also a P-gp substrate and may consequently compete with indinavir efflux was also postulated as a possible mechanism by which the intestinal metabolism increases the effective permeability of indinavir(Li et al., 2002).
A more indirect approach to gain insight into the interplay between P-gp and CYP3A metabolism was presented by Abuasal et al. By integrating the effective permeability obtained in situ and several additional disposition parameters fromin vitro experiments inaphysiologically based pharmacokinetic (PBPK) model,Abuasal et al. managed to predict the bioavailability of the dual P-gp/CYP3A4 substrate UK343,664 and explain its non-linear absorption behavior. Km and Vmax values for CYP3A4 and P-gp were determined in vitro using supersomes and the Caco-2 model, respectively. Using the PBPK model, it was clearlydemonstrated 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 unambiguousinformation on the actual appearance of parent compound or metabolite into the blood is gathered. The study performed by Abuasal et al. demonstrates that PBPK modeling is highly promising as a predictive and descriptive tool for intestinal absorption. It is important to note, however, that, in order to obtain reliable predictions of drug absorption from PBPK modeling, severalkinetic and physiological parameters need to be assessed first.