In vitro and in vivo evaluation of effects of sodium caprate on enteral peptide absorption and on mucosal morphology

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Abstract

Sodium salts of medium-chain fatty acids, sodium caprate (C10) in particular, have been used as absorption-enhancing agents to promote transmucosal drug absorption. In this study, we conducted both in vitro and in vivo experiments to investigate the effects of C10 on intestinal permeabilities and mucosal morphology. Mucosal addition of C10 (13–25 mM) reduced the transepithelial electric resistance (TEER) of cultured monolayers of the human intestinal cell line Caco-2 by 40–65% and, upon removal of C10, a marked tendency of TEER recovery was recorded. C10 added mucosally at 13–50 mM increased the transports of mannitol and polyethylene glycol (PEG) 900 across Caco-2 in a dose-dependent manner. In contrast, the transport of a model d-decapeptide was maximally enhanced with 20–25 mM C10. No noticeable morphological alteration of the Caco-2 monolayers was observed after a 1-h mucosal pretreatment with C10. Co-delivery with C10 (0.05–0.5 mmol/kg) into the rat terminal ileum increased the d-decapeptide bioavailability (BA) dose-dependently. With 0.5 mmol/kg C10 co-administered, d-decapeptide percent BA was elevated from 2 to 11%. Following a 1-h incubation with 0.5 mmol/kg C10 (in liquid or powder form) non-invasively delivered into the rectal lumen, no signs of histological change in the rectal mucosa were detected. These results demonstrate that C10 can promote intestinal absorption of a small peptide without causing detrimental alterations of the intestinal mucosa. C10 thus seems to be a good candidate as an enhancing agent for improving the oral BA of small therapeutic peptides.

Introduction

Application of absorption-enhancing agents as formulation excipients to improve the oral bioavailability (BA) of biopharmaceuticals has remained an active area of research in drug delivery technology (Lee et al., 1991, Aungst, 1996). In recent years, particular attention has been given to enhancing agents that are endogenous and/or metabolizable by mucosal tissues (Fix, 1987, Muranishi, 1990, Chao et al., 1998a). As a good example, sodium salts of medium-chain fatty acids, sodium caprate (C10) in particular, have been shown to promote the transmucosal absorption of drugs and marker compounds (Tomita et al., 1992, Lindmark et al., 1998, Shimazaki et al., 1998), apparently mainly by enhancing the permeabilities of the paracellular pathway (Sawada et al., 1991, Anderberg et al., 1993). C10 is a dairy product constituent that has been approved in a few countries for use in humans as an absorption-enhancing agent (Watts and Illum, 1997).

It seems that so far most of the published studies on effects of C10 on intestinal permeabilities have involved in vitro transport analysis of small-molecule, non-peptide marker compounds. Also, data are sparse regarding the in vitro and in vivo effect of C10 on morphology/viability of the mucosal epithelial tissues. To complement the information available in the literature, in the present study we conducted both in vitro and in vivo investigations of effects of C10 on intestinal absorption of model compounds including a peptidase-resistant model decapeptide, and on mucosal morphology. Our results show that C10, when co-administered as a formulation excipient, can effectively enhance the intestinal absorption of small peptide and other small-molecule compounds without causing significant morphological alterations of the gastrointestinal (GI) mucosa.

Section snippets

Materials

Caco-2 cells were purchased from American Type Culture Collection (Rockville, MD). Male Sprague–Dawley rats were from Charles River (Wilmington, MD). Cell culture media and reagents were from Life Technologies (Grand Island, NY). Fetal bovine serum was from HyClone Laboratories (Logan, UT). Synthetic d-decapeptide (constituent amino acid sequence: d-Glu-d-Tyr-d-Leu-d-Lys-d-Ala-d-Leu-d-Leu-d-Ser-d-Lys-d-Leu-NH2) and d-[3H]decapeptide (35 Ci/mmol) were from Cambridge Research Biochemicals

Effect of C10 on the TEER of Caco-2 cell monolayers

The effect of C10 on the in vitro permeability of intestinal epithelium and the reversibility of this effect were first examined electrophysiologically by monitoring the TEER values of cultures of Caco-2 cells. The baseline TEER of the Caco-2 monolayers was 244±8 (S.E.) Ω-cm2 (n=28). As shown in Fig. 1, the mucosal addition of C10 in the concentration range of 13–25 mM quickly reduced the Caco-2 TEER by 40–65%, with a T1/2 (the time required to show the half-maximal effect of C10) of ∼5 min.

Discussion

Mucosal addition of C10 rapidly reduced the Caco-2 TEER (T1/2, ∼5 min) and, upon its washout, a marked propensity for recovery of TEER was observed. This result is in concert with the recent finding by Söderholm et al. (1998) that apical C10 causes a reversible drop in VE and increases in vitro EDTA permeation across rat ileum, and suggests that C10 does not cause long-term damage to the Caco-2 monolayer and that its effect on Caco-2 permeability is reversible.

To study the effect of C10 on

Acknowledgements

We wish to thank Dr Patrick Wong and Dr Liang Dong (Oral Products R&D, ALZA Corporation, USA) for their helpful discussions.

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1

Present address: Eli Lilly and Company, Lilly Corporate Center, Drop Code 6414, Indianapolis, Indiana 46285, USA.

2

Present address: Stanford Research Center, Yamanouchi-Shaklee Pharma, Palo Alto, CA 94304, USA.

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