Absorption of angiotensin II antagonists in Ussing chambers, Caco-2, perfused jejunum loop and in vivo:: Importance of drug ionisation in the in vitro prediction of in vivo absorption

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Abstract

The aims of this study were (i) to compare the absorption of three closely related inhibitors of angiotensin II, RU60018, RU60079 and HR720, in various in vitro and in vivo models, and (ii) to explain the differences in the results and to assess the importance of drug ionisation to predict absorption. Drug absorption was investigated in Ussing chambers, Caco-2 cell monolayers, perfused rat jejunum loops and in vivo after oral, intraduodenal or intravenous administration. In Ussing chambers, the analogues showed the same site-related absorption profile and a common mechanism involving the paracellular pathway. At pH 7.4 in Ussing chambers, perfused jejunum loop or Caco-2 transport studies, the three compounds exhibited low and comparable permeability values suggesting that a similar level of oral absorption may be expected for all three compounds. However, after oral or intraduodenal administration, only HR720 was significantly absorbed. The in vivo results can be explained by the ionic distribution profile which indicated that only HR720 possessed a significant amount of uncharged species at pH values close to that found in the upper part of intestinal tract. Hence, it is expected that in this part of the intestine, only HR720 absorption is favoured. This is supported by Caco-2 transport studies performed when the pH of the apical medium was lowered from 7.4 to 6.0, in which a dramatic increase in permeability was observed for HR720 compared to those of the other analogues. This study highlights the usefulness of different absorption models for drug screening and demonstrates that ionisation profiles must be carefully considered to avoid rejection of promising compounds.

Introduction

Various physicochemical parameters, such as molecular size and shape, lipophilicity, hydrogen bonding capacity and molecular surface properties, appear to play a crucial role in absorption of drugs which cross the intestinal barrier by passive diffusion, either through the transcellular or paracellular pathway (Karls et al., 1991, Conradi et al., 1992, Stewart et al., 1995, Palm et al., 1996). In recent years, many studies have been devoted to the assessment of relationships between such parameters and drug absorption in various model systems (Caro et al., 1995, Hillgren et al., 1995, Stewart et al., 1995, Rubas et al., 1996, Ungell, 1997). This approach appears to be promising in both drug design and rank ordering of compounds in order to select new drug candidates (Kim et al., 1993, Stewart et al., 1995, Artursson et al., 1996, Bayley et al., 1996, Levet-Trafit et al., 1996, Gan and Thakker, 1997). However, few studies have examined the importance of ionisation in drug absorption screening and candidate selection.

In this work, three non-tetrazole compounds with high selective affinity for the angiotensin II receptor AT1 have been selected: RU60079, RU60018 and HR720. These compounds exhibit anti-hypertensive effects in vivo, when intravenously administered to spontaneously hypertensive (SHR) rats. However, only HR720 provides the expected effect on blood pressure when given orally.

The aim of the present study was to investigate the permeability and transepithelial transport mechanisms of the analogues in three different models: (a) Caco-2 cell monolayer, (b) intestinal segments mounted in Ussing chambers, and (c) isolated-perfused jejunum loops in situ. Permeability results were then compared and correlated with both physicochemical parameters and pharmacokinetic data obtained following intravenous, intraduodenal or oral administration to the rat.

Section snippets

Chemicals

HR720 (2-butyl-4-(methylthio)-1-((2′-(((propylamino)carbonyl)amino)sulfonyl) (1,1′-biphenyl)-4yl)methyl)-1H-imidazole-5-carboxylic acid), its sulfoxide RU60018 and its sulfone RU60079 were used throughout the study. The compounds were 99, 94 and 98% pure, respectively. 14C-labelled HR720, RU60018 and RU60079, all at a specific activity of 2.22 GBq/mmol and greater than 95% purity were synthesised by Hoechst-Marion-Roussel, Romainville, France (Fig. 1). d-[1-14C]mannitol (spec. act. 1.11

Physicochemical properties

Solubility of RU60018, RU60079 and HR720 in Ringer medium at 37°C exceeded 10−2 M. Each compound exhibited two macroscopic pKa values referred to as pKa1 and pKa2 which reflected ionisation of the carboxylic and sulfonylurea groups, respectively. For RU60018, pKa1 and pKa2 were 3.0 and 6.2, for RU60079 2.5 and 6.1, and HR720 5.1 and 6.2. Respective proportions of ionised species at pH 2.5, 6.0 and 7.4 are given in Table 1. The results showed that at pH 7.4 greater than 90% of each drug was

Discussion

The Papp values obtained in perfused jejunum loops, were remarkably similar to those obtained from transport studies where the jejunum was mounted in Ussing chambers.

Permeability data obtained with the Ussing chamber model showed that HR720, RU60018 and RU60079 may be preferentially absorbed in the jejunum, ileum and colon. Indeed, the Papp values determined for the three compounds at these intestinal sites were comparable, except at the ileum where the mean Papp value for HR720 was

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