Acyclovir prodrug for the intestinal di/tri-peptide transporter PEPT1: comparison of in vivo bioavailability in rats and transport in Caco-2 cells

https://doi.org/10.1016/j.ejps.2004.08.005Get rights and content

Abstract

It has previously been shown that the prodrug Glu(acyclovir)-Sar has a high affinity for PEPT1 in Caco-2 cells. However, affinity does not necessarily lead to translocation by the transporter which is necessary for achieving an increased oral bioavailability. Therefore i.v. and p.o. doses of Glu(acyclovir)-Sar, acyclovir and valacyclovir were given to rats and the collected blood samples were analysed via LC–MS–MS. Furthermore, Caco-2 cell monolayers were exposed apically to Glu(acyclovir)-Sar, acyclovir, and valacyclovir and the concentration of drug and prodrugs in the cell extracts were determined and taken as a measure for intracellular accumulation. In addition, bi-directional transport studies of Glu(acyclovir)-Sar across Caco-2 cell monolayers and in vitro metabolism studies of Glu(acyclovir)-Sar in various media of rat origin were performed. For these purposes HPLC-UV analysis was applied. Oral administration of Glu(acyclovir)-Sar to rats resulted in low bioavailabilities of acyclovir (<2%) and intact prodrug (<5%). Studies performed on Caco-2 cell monolayers showed that in contrast to valacyclovir Glu(acyclovir)-Sar did not result in a detectable amount of acyclovir or Glu(acyclovir)-Sar in the cell extracts. Bi-directional flux across Caco-2 cell monolayers apical to basolateral (FluxA→B) and basolateral to apical (FluxB→A) was measured and the FluxB→A/FluxA→B ratios of approximately 0.8 indicate that apical efflux mechanisms may not explain this lack of intracellular accumulation. These data indicate that Glu(acyclovir)-Sar may not be translocated by PEPT1.

Introduction

The oral bioavailability of the antiviral drug acyclovir is limited (de Miranda et al., 1981, de Miranda and Blum, 1983). One approach for improving the oral bioavailability of acyclovir is to design prodrugs that have affinity for and are transported via the di/tri-peptide transporter, PEPT1, situated in the small intestine. This may be achieved by reversibly linking the drug to stabilised di-peptide pro-moieties. Previous studies have shown that the prodrug Glu(acyclovir)-Sar has a high affinity for PEPT1 (Thomsen et al., 2003). Furthermore, the release profile of acyclovir from the prodrug appears favourable. Acyclovir is released quantitatively at pH 7.4 (half-life t1/2 ∼ 5.5 h) and in 80% human plasma (t1/2 ∼ 1.2 h) yet the prodrug remains stable at pH 6 (t1/2 ∼ 6 days) corresponding to the pH of the upper small intestine and is relative stable towards enzymatic activity in 10% porcine intestinal homogenate (t1/2 ∼ 45 min). Glu(acyclovir)-Sar therefore appears to be a promising candidate for further investigation (Thomsen et al., 2003). However, affinity for PEPT1 was measured as the ability to inhibit Gly-Sar uptake. Therefore, high affinity does not necessarily imply that the compound is translocated by the transporter and translocation is necessary for achieving an increased oral bioavailability. The aim of the present study is therefore to further evaluate Glu(acyclovir)-Sar as an oral drug delivery system by investigating in vivo bioavailability in rats, intracellular accumulation in and bi-directional transport across Caco-2 cell monolayers and in vitro metabolism in various media of rat origin. For comparison, acyclovir and valacyclovir, an acyclovir prodrug that is transported by PEPT1 (Beauchamp et al., 1992, de Vrueh et al., 1998, Han et al., 1998), are included in the studies.

Section snippets

Chemicals

Prodrugs were synthesised as previously described (Thomsen et al., 2003) and valacyclovir hydrochloride was purified from Zelitrex® tablets 500 mg (Glaxo Wellcome) by simple water extraction. The purity of the final compound was >95% determined by 1H NMR and HPLC-UV. Acyclovir, glycylproline (Gly-Pro), 2-(N-morpholino)etanesulfonic acid (MES), N-2-[hydroxyethyl]-piperazine N′[2-ethanesulfonate] (HEPES) and bovine serum albumin (BSA) were purchased from Sigma. For the animal study Hypnorm

In vivo bioavailability in rats

Pharmacokinetic parameters are listed in Table 1. The oral bioavailability of Glu(acyclovir)-Sar in relation to i.v. administration of the prodrug was <5%. Oral administration of Glu(acyclovir)-Sar lead to blood levels of acyclovir and prodrug that were generally below the limit of quantification.

Intracellular accumulation in Caco-2 cells

Concentration of acyclovir, Glu(acyclovir)-Sar, and valacyclovir in Caco-2 cell extracts was investigated in the presence and absence of 20 mM Gly-Pro (Fig. 1). Neither prodrug nor acyclovir could be

Discussion

It has previously been demonstrated that Glu(acyclovir)-Sar has a high affinity for PEPT1 in Caco-2 cells (Thomsen et al., 2003). However, affinity for PEPT1 was measured as the ability to inhibit Gly-Sar uptake. High affinity does therefore not necessarily imply that the compound is translocated by the transporter. In vivo bioavailability and pharmacokinetics of the prodrug Glu(acyclovir)-Sar in rats was therefore investigated. Oral administration of Glu(acyclovir)-Sar resulted in a very low

Conclusion

Oral administration of Glu(acyclovir)-Sar to rats resulted in low bioavailabilities of acyclovir (<2%) and intact prodrug (<5%). Intracellular accumulation and bi-directional transport studies in Caco-2 cell monolayers indicate that the prodrug Glu(acyclovir)-Sar may not be translocated by PEPT1 even though it has high affinity for the transporter.

Acknowledgements

We thank Janni K. K. Sørensen, Helle Bach, AnnBritt Nielsen, Susanne N. Sørensen and Bettina Dinitzen for their excellent technical assistance. The Danish Medical Research Council supported this work via The Centre for Drug Design and Transport and project Grant #22–01–0310.

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