Frog intestinal sac as an in vitro method for the assessment of intestinal permeability in humans: Application to carrier transported drugs

This work is dedicated to the memory of our colleague Prof. Gaetano Liso, recently deceased.
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Abstract

The aim of this study was to investigate the presence of pharmaceutically relevant drug transporters in frog intestine which has been proposed as model for intestinal permeability screening assays of passively absorbed drugs in humans [Trapani, G., Franco, M., Trapani, A., Lopedota, A., Latrofa, A., Gallucci, E., Micelli, S., Liso, G., 2004. Frog intestinal sac: a new in vitro method for the assessment of intestinal permeability. J. Pharm. Sci. 93, 2909–2919]. The expression of transporters in frog intestine was supported by the following observations: (i) the involvement of purine nucleobase transport system was deduced by inhibition of acyclovir transport in the presence of adenine; (ii) baclofen or l-dopa transport was inhibited by the digitalis steroid ouabain and it may be related to the Na+ electrochemical potential difference, presumably involving amino acid transporters; (iii) the presence of proton-dependent peptide transporters was argued evaluating the effect of the pH change (from pH 5.9 to pH 7.4) on the transport of glutathione; (iv) the possible expression in the frog intestine of an efflux system distinct from P-glycoprotein (Pgp) in the benzylpenicillin transport was deduced using a glucose enriched frog Ringer with or without the known Pgp inhibitor verapamil; (v) the contribution of Pgp-mediated efflux system in determining the frog intestinal absorption of drugs was supported by the specific inhibition of cimetidine or nadolol transport in the presence of verapamil. These results indicate that pharmaceutically relevant drug transporters should be also expressed in frog intestine.

In this work, an attempt was also made to compare the measured Papp values in the frog intestinal model for the aforementioned series of actively/effluxed transported drugs in humans to the corresponding literature values for the fraction absorbed. The Papp values used in these comparisons were obtained at high concentrations of drugs at which probably saturation of the carrier occurs. Interestingly, it was found that drugs that are completely absorbed had Papp values >3 × 10−6 cm/s, while drugs absorbed <90% had Papp values lower than 1 × 10−6 cm/s. In these cases, indeed, a borderline region characterized by the apparent permeability coefficient Papp value between 1 × 10−6  and 3 × 10−6 cm/s should be considered for which the prediction of the absorbed fraction after oral administration in humans become more uncertain by the frog intestinal sac system.

Introduction

The rate-limiting barriers for the absorption of orally administered drugs are aqueous solubility and intestinal permeability (Lipinski et al., 1997). Thus, assessment of intestinal permeability represents one essential part in the prediction of oral bioavailability of new drug candidates. For the assessment of intestinal drug permeability, different in vitro methods are available such as the use of excised tissues, epithelial cell culture models, artificial membranes and these techniques have been recently reviewed (Tukker, 2000). Among these different approaches, the methods based on drug transport across intestinal epithelial monolayers, such as Caco-2 cells, are at present most utilized. In fact, they show features similar to the human small intestine giving rise to good correlations with the fraction absorbed in humans for a variety of compounds. However, some important drawbacks, such as time consuming, high cost per assay, possibility of microbial contamination, and wide inter-laboratory variations, are considered the weakness of the Caco-2 cell culture model as screening tool. Moreover, Caco-2 cells as other cell lines are generally lacking mucus layer which covers the intestinal epithelium. Therefore, alternative procedures to the use of cultured cells are currently pursued (Castella et al., 2006).

Recently, we reported a new experimental protocol utilizing isolated frog intestinal sac as a new in vitro screening tool for assessing human intestinal permeability (Trapani et al., 2004). We showed that the in vitro permeability coefficient deduced from isolated frog intestinal experiments is a reasonable predictor of in vivo peroral absorption in humans for compounds that are passively absorbed. We also demonstrated that the frog intestinal permeability model could be used to classify these drugs into the Biopharmaceutics Classification System (BCS) (Trapani et al., 2004). Following our interest in this field, we decided to investigate the applicability of the frog intestinal sac to carrier transported drugs. It should be noted that while for active transport, the majority of studies reported in the literature are aimed at determining the expression of drug transporter systems in the examined experimental model for assessing human intestinal permeability, for passive transport attention has been focused mainly to prediction of the fraction absorbed (FA) in humans. In this last case, in silico models that predict drug permeability from molecular descriptors has also been developed. About the prediction of FA for actively transported drugs in humans, the existing methods for the assessment of in vitro intestinal permeability showed some limitations and generally unsatisfactory results were obtained. Thus, it has been demonstrated that both the Caco-2 and the small-intestine like 2/4/A1 cell models are not able to predict FA for compounds exhibiting significant active transport mechanisms in vivo (Chong et al., 1996, Matsson et al., 2005).

To account for these limitations it should be also considered that for actively absorbed drugs the transport is concentration dependent and hence it could change considerably with different donor concentrations.

In this work, we explored the possible presence of pharmaceutically relevant drug transporters in frog intestine. In this regard, although there have been only a few studies reporting the expression of glucose (GLUT) and amino acid (AAT) transporters as well as metabolising enzymes (CYP1A) in frog intestine (Csàky and Gallucci, 1977, Huang et al., 2001), to the best of our knowledge, a deeper study on the expression of pharmaceutically important transporter systems was not reported in literature. In addition, an attempt was made to compare the measured Papp values in the frog intestinal model for a series of actively/effluxed transported drugs in humans to the corresponding literature values of FA.

Section snippets

Chemicals

Baclofen, benzylpenicillin, cimetidine, l-dopa, enalapril maleate, phloridzin dihydrate (1-[2-(β-d-glucopyranosyloxy)-4,6-dihydroxyphenyl]-3-(4-hydroxyphenyl)-1-propanone, glutathione, nadolol, (±)-verapamil hydrochloride and (−)ouabain hydrate were purchased from Sigma-Aldrich Chemical Co. (USA). Acyclovir was extracted from tablets of Acyclovir EG® purchased from a local drugstore. Identity and purity of the latter compound was checked by means of spectroscopic methods (IR, 1H NMR, and mass

Carrier-mediated transport systems in frog intestine

To evaluate whether important transporters are expressed in frog intestine, a series of actively transported drugs in humans was selected. For these drugs both the absorbed fraction after oral administration in humans (FA) and the active transport mechanism were deduced from literature sources (Table 4), (Matsson et al., 2005, Steffansen et al., 2004, Varma et al., 2005). In particular, acyclovir is substrate for the purine nucleobase transporter (ENT) proteins, baclofen and l-dopa are

Conclusions

In this work, evidence for the expression of specific transporter systems in frog intestine was obtained on the basis of the effect of selected inhibitors. Although further studies are necessary to fully demonstrate that the compounds examined in this study (exhibiting significant active transport mechanisms in humans) are also carrier transported in frog intestine, results indicate that pharmaceutically relevant drug transporters should be also expressed in this last intestine. Moreover, it

Acknowledgment

Thanks are due to Prof. E. Gallucci (Dipartimento Farmaco-Biologico, Facoltà di Farmacia, Università degli Studi di Bari) for helpful discussion.

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