Prodrugs of purine and pyrimidine analogues for the intestinal di/tri-peptide transporter PepT1: affinity for hPepT1 in Caco-2 cells, drug release in aqueous media and in vitro metabolism

Abstract

A general drug delivery approach for increasing oral bioavailability of purine and pyrimidine analogues such as acyclovir may be to link these compounds reversibly to stabilized dipeptide pro-moieties with affinity for the human intestinal di/tri-peptide transporter, hPepT1. In the present study, novel l-Glu-Sar and d-Glu-Ala ester prodrugs of acyclovir and 1-(2-hydroxyethyl)-linked thymine were synthesized and their affinities for hPepT1 in Caco-2 cells were determined. Furthermore, the degradation of the prodrugs was investigated in various aqueous and biological media and compared to the corresponding hydrolysis of the prodrug valaciclovir. Affinity studies showed that the l-Glu-Sar prodrugs had high affinity for hPepT1 (K_i∼0.2–0.3 mM), whereas the d-Glu-Ala prodrugs had poor affinity (K_i∼50 mM). The pH-rate profiles of the prodrugs d-Glu[1-(2-hydroxyethyl)thymine]-Ala and l-Glu[acyclovir]-Sar showed specific base catalyzed degradation at pH above 4.5 and 5.5, respectively. This implicates that the degradation rates at pH∼7.4 (t_1/2∼3.5 and 5.5 h) are approximately 25 times faster than at upper small intestinal pH∼6.0. In 10% porcine intestinal homogenate and 80% human plasma the half-lives of the l-Glu-Sar prodrugs were approximately between 45 and 90 min indicating a limited enzyme catalyzed degradation. In contrast, valaciclovir underwent extensive enzyme catalyzed hydrolysis in 10% porcine intestinal homogenate (t_1/2∼1 min). In conclusion, l-Glu-Sar may potentially function as pro-moiety for purine and pyrimidine analogues, where release of parent compound primarily is controlled by a specific base catalyzed hydrolysis. Acyclovir is quantitatively released at the relevant pH 7.4, whereas the 1-(2-hydroxyethyl)-linked thymine is released instead of the parent compound thymine.

Introduction

Many purine and pyrimidine analogues such as acyclovir, ganciclovir and 5-fluorouracil may, for different reasons, have a limited bioavailability after oral administration. The oral bioavailability of acyclovir as such is approximately 20% [1], whereas the bioavailability of acyclovir administered as the l-valine ester prodrug, valaciclovir, increases 3–5-fold [2], [3], [4]. This increased bioavailability of acyclovir has retrospectively been shown to be caused by valaciclovir’s affinity for the human di/tri-peptide transporter, hPepT1, which is located in the apical enterocytic membrane in the upper small intestine [5], [6], [7]. However, esterfication with a single amino acid may not be a suitable approach for increasing the bioavailability of all purine and pyrimidine analogues. A more general approach may be to reversibly link the analogues to stabilized dipeptide pro-moieties with affinity for hPepT1. Previous studies made on various benzyl alcohols (BnO), used as model drugs, and reversibly linked to the carboxylic acid of the N-terminal side chain of either l-Asp-Sar, d-Asp-Ala or d-Glu-Ala show that the resulting model prodrugs, such as d-Glu(BnO)-Ala or l-Asp(BnO)-Sar, have affinity for hPepT1 in Caco-2 cells [8], [9]. Furthermore, the studies demonstrate that d-Glu(BnO)-Ala and d-Asp(BnO)-Ala are transported across Caco-2 cells by an hPepT1 mediated process [9]. Release studies made on these model ester prodrugs indicate that the model drug release is primarily controlled by a specific base catalyzed hydrolysis at pH 5–10. The model prodrugs may be relatively stable in the lumen of the upper small intestine at pH∼6 and yet release the model drug at a reasonable rate at intracellular and blood pH∼7.4 [10], [11], [12].

In the present study, novel l-Glu-Sar and d-Glu-Ala prodrugs of acyclovir and 1-(2-hydroxyethyl)-linked thymine have been synthesized (Fig. 1) with the aim of studying their affinities for hPepT1 in Caco-2 cells. A second aim was to investigate the degradation of the prodrugs in various aqueous and biological media to further evaluate the potential of the stabilized dipeptides as pro-moieties for purine and pyrimidine analogues. For comparison, the affinity of valaciclovir for hPepT1 and release of acyclovir from valaciclovir is investigated.