Absorption characterization: the in situ intestinal perfusion technique in mice

To investigate the mechanisms by which a drug is absorbed from the intestinal lumen into the blood, many techniques have been developed over the years. In early drug discovery, the absorption properties of new chemical entities are usually assessed by high-throughput techniques, e.g., automated Caco-2 screening. In the later drug discovery stages, when drug properties are being optimized and/or when detailed insight into the mechanisms underlying oral drug disposition isrequired, Caco-2 cells or more advanced absorption models (Ussing Chambers or intestinal perfusion systems) are being used. The Caco-2 model has many advantages, including the fact that (1) it is from human origin, (2) it is suitable for high-throughput screening, (3) it allows retrieving absorption mechanisms as it expresses a wide variety of uptake and efflux transporters, and (4) it provides a good prediction ofthe fraction absorbed (Fa) for passively transported drugs in humans. Unfortunately, this model has also some drawbacks: (1) the expression of CYP3A4 (which is the most abundant phase I drug-metabolizing enzyme present in the human small intestine) is very low to non-existent, whichmay result in overprediction of drug transport, (2) there is no mucus layer which protects the cells and forms an extra barrier for compounds to reach the cells, (3) pregnane X receptor (PXR)-mediated drug-drug interactions cannot be studied since this nuclear receptor is not expressed, (4) there are interlaboratory differences in enzyme and drug transporter expression levels. Another absorption model which can be considered in late discovery and early development stages is the in situintestinal perfusion with mesenteric blood collection. Of all presentlyavailable models, it is the closest one to the in vivo situation becauseof (1) an intact intestinal mucosa, nerve system and blood flow; (2) thepresence of sink conditions; and (3) the expression of all relevant enzymes and transporters. The rat is the standard animal used in this technique. Recently, the in situ intestinal perfusion technique with mesenteric blood sampling was successfully downsized from the rat to themouse in our laboratory, which opens new opportunities in pharmaceuticalresearch. First, the use of knockout mice allows studying the involvement of one specific drug transporter or enzyme in the absorptionof a drug. This may not be possible with the use of chemical inhibitorsdue to a lack of specificity. Second, transgenic (humanized) mice, which carry human genes in their genome, may be invaluable in order to eliminate species differences between rodents and humans.The aim of this dissertation research was to evaluate the use of the mouse in the in situ intestinal perfusion technique with mesenteric blood sampling. We investigated the use of wild-type, knockout and humanized mice in different studies, of which the specific objectives were to:1),,Validate the use of P-gp knockout mice in the in situ intestinal perfusion technique, and explore the role of P-gp in the intestinal permeability for darunavir.2),,Investigate the use of the mouse in situ intestinal perfusion technique to elucidate the role of both efflux transporters (P-gp) and metabolism (P450) towards the intestinal absorption of HIV protease inhibitors.3),,Evaluate the use of PXR/CYP3A4-humanized mice for studying drug-druginteractions involving intestinal P-glycoprotein. The effect of rifampicin (PXR activator) was investigated on the intestinal permeability for darunavir.4),,Explore a novel combination of human intestinal fluids (HIF) with the mouse in situ intestinal perfusion technique to assess food effects. We used indinavir as model compound.HIV protease inhibitors (PIs) were selected as model compounds in our studies because of their interesting pharmacokinetic properties. The drugs of this class are characterized by an extensive first-pass elimination as they are metabolized by CYP3A4 present in the liver and intestine. In addition, these drugs have been shown to be substrates ofefflux transporters present in the intestine, which potentially limit their absorption. The most important and best characterized intestinal efflux transporter is P-glycoprotein (P-gp), present in the apical membrane of enterocytes. Although all PIs were shown to be substrates of P-gp, the exact role of P-gp in limiting their intestinal absorption in vivo remains to be elucidated. As a consequence of their unfavorablepharmacokinetics, PIs are always co-administered with the pharmacokinetic booster ritonavir, which is known to inhibit their CYP3A4-mediated metabolism. In addition, ritonavir is a known P-gp inhibitor, thereby potentially inhibiting their intestinal efflux.In the first part of this doctoral research, we evaluated the use of P-gp knockout mice in the in situ intestinal perfusion technique. We explored the contribution of P-gp to the transport characteristics of darunavir (up to 100 µM) using Caco-2 monolayers and the in situ intestinal perfusion technique using wild-type and mdr1a/1b(#/#) mice. We observed that, in vitro, P-gp has a modulatory effect on the absorption of darunavir, even at a concentration of 100 µM (efflux ratio= 25). Fasted state simulated intestinal fluids (FaSSIF) partially inhibited P-gp functionality, which was further inhibited by including the P-gp inhibitors verapamil, PSC833 (valspodar), GF120918 (elacridar),or ritonavir. Using the in situ intestinal perfusion technique, we demonstrated that co-perfusion with ritonavir resulted in a similar apparent permeability coefficient to that observed using P-gp knockout mice, which was 2.7-fold higher than in control mice. From these data, we conclude that in mice, even at a relevant intraluminal concentration of darunavir, P-gp has a modulatory effect on the absorption of darunavir. However, this P-gp-mediated darunavir transport is inhibitedwhen it is combined with ritonavir.In the next study, we subsequently expanded our focus from darunavir to a broad series of PIs. In addition, we investigated the role of both P-gp and intestinal metabolism on their absorption. We explored the impact of ritonavir on the intestinal absorption of PIs in two models: the Caco-2 system and the in situ intestinal perfusion model using wild-type mice. In the Caco-2 model, the effect of ritonavir on the permeability of the other PIs was significant for saquinavir (2-fold increase) and indinavir (3-fold increase), negligible for darunavir and amprenavir, and nonexistent for nelfinavir, lopinavir, tipranavir, and atazanavir. However, performing the in situ intestinal perfusion technique in mice for three selected PIs showed a significant increase in the intestinal permeability for all: indinavir (3.2-fold), lopinavir (2.3-fold), and darunavir (3-fold). The effect of aminobenzotriazole (anonspecific P450 inhibitor) on lopinavir permeability was comparable with using ritonavir, whereas there was no effect for indinavir and darunavir. We conclude that ritonavir can boost drug absorption by inhibiting P-glycoprotein and/or metabolism, in a compound-specific manner. Because the dual effect of ritonavir on metabolism and transporter inhibition could only be observed in a system co-expressing both, the Caco-2 model might be insufficient when studying drug-drug interactions at the level of the intestinal mucosa.In the third study, we evaluated the use of humanized mice in the in situ intestinal perfusion technique. Humanized mice hold great promise to overcome species differences between rodents and humans. Conventional rodent models are less suitable for predicting drug-drug interactions at the level of the human intestinal mucosa, especially when nuclear receptors like pregnane X receptor (PXR) are involved. Recently, a transgenic mouse model, expressing both human PXR and CYP3A4, was developed and shown to be a better predictor of CYP3A4 induction by xenobiotics in humans compared to wild-type mice. In the present study, we tested the hypothesis that this mouse model can also predict PXR-mediated induction of intestinal P-gp in humans. By use of the in situ intestinal perfusion technique with mesenteric blood sampling, the effect of oral rifampicin treatment on the intestinal permeability for darunavir was investigated. Rifampicin treatment lowered the intestinal permeability for darunavir by 50 % compared to non-treated mice. The P-gp inhibitor GF120918 increased the permeability for darunavir by 400 % in rifampicin-treated mice, while this was only 56 % in mice that were not treated, thus indicating P-gp induction by rifampicin. The non-specific P450 inhibitor aminobenzotriazole (100 µM) did not affect the permeability for darunavir. Quantitative Western blot analysis of the intestinal tissue showed that rifampicin treatment induced intestinal P-gp levels four-fold, while CYP3A4 levels remained unchanged. Oral co-administration of rifampicin with the phytochemical sulforaphane (present in many cruciferous vegetables including broccoli) for three days increased the permeability for darunavir by 50 % compared to rifampicin treatment alone. These data show that PXR/CYP3A4-humanized mice can be used to study the inducing effects of xenobiotics on intestinal P-gp. In addition, food-drug interactions playing at the level of the intestine can be investigated.In the last study, we explore a novel combination of human intestinal fluids (HIF) with the mouse in situ intestinal perfusion technique to assess food effects. Fed state conditions cannot be easily implemented in the presently available permeability tools, including the frequently used Caco-2 system. Therefore, exploring food effects during drug development can be quite challenging. In this study, we investigated the effect of fasted and fed state conditions on the intestinal absorption of the PI indinavir using simulated and human intestinal fluids in the in situ intestinal perfusion technique in mice. Indinavirwas selected as model compound because it is very susceptible to a negative food effect in humans. Although the solubility of indinavir was 6-fold higher in fed state human intestinal fluid (FeHIF) as compared to fasted state HIF (FaHIF), the intestinal permeation of indinavir was 22-fold lower in FeHIF as compared to FaHIF. Dialysis experiments showed that only a small fraction of indinavir is accessiblefor absorption in FeHIF due to micellar entrapment, possibly explaining its low intestinal permeation. These findings could not be confirmed using simulated intestinal fluids. From these data, we conclude that the use of HIF in the in situ intestinal perfusion model is very promising for biorelevant absorption evaluation as it allows to directlyexplore the complex solubility/permeability interplay on drug absorption.In conclusion, the results obtained in this dissertation research using the mouse in situ intestinal perfusion technique with mesenteric blood sampling clearly show that this powerful tool holds great promise in elucidating drug absorption mechanisms, drug-drug interactions and food effects in the most biorelevant way. The possibility to use knockout and knockin (humanized) mice emphasizes the benefit of using the mouse instead of the rat in this model.

Publication year:2013

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