Drug absorption studies of prodrug esters using the Caco-2 model: evaluation of ester hydrolysis and transepithelial transport

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Abstract

The design of lipophilic ester prodrugs is a widely used approach to obtain enhanced oral delivery of poorly membrane permeable compounds. The present study was conducted in order to assess the influence of intestinal metabolism on transepithelial flux of prednisolone prodrugs using the Caco-2 system. In addition, distribution of esterase activity along the GI tract was evaluated using homogenates of scraped intestinal mucosa from various parts of small intestine and colon of rat and pig. Prednisolone acetate (lipophilic prodrug) and prednisolone hemisuccinate (hydrophilic prodrug) were selected as model compounds for transport studies. In transport studies using prednisolone acetate (100 μM), almost complete ester hydrolysis and an increased transepithelial flux of prednisolone were observed. Virtually no transport nor metabolism was observed when the hemisuccinate ester was used, illustrating its poor ability to cross membranes. Incubation studies with purified carboxylesterase showed that prednisolone acetate was rapidly degraded (t1/2=2.94 min), while prednisolone hemisuccinate degradation was very low. Studies on site dependency of esterase activity using p-nitrophenyl acetate as a substrate showed an important interspecies difference, rat intestine possessing much higher activity than pig intestine, and a gradual decrease of esterase activity along the GI tract for the two species tested. Esterase activity in Caco-2 monolayers was twice as high as observed in colon of rat and pig, but much lower than activities measured in the small intestine. It can be concluded that the rat may not be a suitable choice for oral bioavailability studies of ester prodrugs; it may also be advantageous to target ester prodrugs of hydrophilic compounds to the colon, thus preventing significant accumulation of the parent compound inside the mucosal cells.

Introduction

In oral drug delivery programs, ester prodrugs are commonly used to enhance membrane permeation and transepithelial transport of hydrophilic drugs by increasing the lipophilicity of the parent compound, resulting in enhanced transmembrane transport by passive diffusion (Balant et al., 1990, Taylor, 1996). Acyloxyalkyl ester derivatives have, for instance, been introduced to increase oral bioavailability of phosphate or phosphonate compounds (Friis and Bundgaard, 1996). After absorption, the parent compound is expected to be released in the plasma through either enzymatic or chemical cleavage of the prodrug. This interesting approach can however be jeopardized if the prodrug is poorly resistant to esterase activity: rapid intracellular conversion of the prodrug by esterases inside the mucosal cells will result in the entrapment of the hydrophilic parent compound in the intestinal mucosa. Previous studies with the Caco-2 model conducted in our laboratory have shown that esterase mediated degradation of bis(pivaloyloxymethyl)-PMEA, a bis-ester prodrug of the antiviral compound phosphonylmethoxyethyladenine (PMEA), inside the cells was responsible for very high intracellular entrapment of negatively charged metabolites of bis(POM)-PMEA, thus diminishing the efficiency of the prodrug used (Annaert et al., 1997).

The objective of this report was to study the influence of metabolism on the transepithelial transport of ester prodrugs of a neutral compound in the Caco-2 system. Caco-2 monolayers are generally accepted as an in vitro model for drug transport studies as these cells have been shown to express most of the enzymatic, functional and morphological characteristics of the intestinal mucosa (Hidalgo et al., 1989, Audus et al., 1990, Hilgers et al., 1990, Gan et al., 1994). The Caco-2 system has already been used in several studies to characterise the biomembrane permeation of ester prodrugs (Hovgaard et al., 1995).

In this study, prednisolone acetate (lipophilic prodrug) and hemisuccinate (hydrophilic prodrug) were chosen as model prodrugs of a neutral compound, prednisolone. In addition to transport studies, esterase mediated degradation in homogenates of Caco-2 cells was compared to degradation in tissue homogenates from various sites in the GI tract of rat and pig using p-nitrophenyl acetate as a model substrate. These species were chosen as rats are commonly being used in drug bioavailability studies, while pigs are considered as the most suitable animal model for oral drug delivery since it resembles the human situation with respect to anatomy and physiology (Kararli, 1995, Gardner et al., 1996).

Section snippets

Reagents

Hanks' balanced salt solution, Dulbecco's Modified Eagle Medium (DMEM) containing glutaMAX™, N-[2-hydroxyethyl]piperazine-N-ethanesulfonic acid (HEPES), 0.05% trypsin and 0.02% EDTA in PBS, non-essential amino acids (NEAA), penicillin-streptomycin (10 000 IU/ml—10 000 μg/ml) and fetal bovine serum were from Gibco BRL (N.V. Life Technologies, Paisley, UK). MeOH and CH3CN were from BDH, Poole, UK. NaH2PO4, acetic acid, and sodium fluorescein were purchased from UCB, Leuven, Belgium; p-nitrophenyl

Results and discussion

The design of ester prodrugs with increased lipophilicity is one approach to enhance transport across membranes and to increase the oral bioavailability of charged compounds. One of the prerequisites of an efficient prodrug ester is that the drug is eventually released, be it enzymatically or chemically. For a charged parent drug, however, it may be possible that, if resistance to esterase mediated cleavage of the prodrug moiety is too low, metabolism in the intestinal cells by intracellular

Acknowledgements

This study was supported by a grant from the Fonds voor Wetenschappelijk Onderzoek – Vlaanderen (FWO, Belgium). P. Annaert acknowledges the receipt of a fellowship from the Flemish Institute for the promotion of scientific-technological research in the industry (IWT). The technical assistance of Ing. H. Herbots is greatly acknowledged.

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