Poor oral absorption is one of the limiting factors in utilizing the full potential of polar
antiviral agents. The
neuraminidase target site requires a polar chemical structure for high affinity binding, thus limiting oral efficacy of many high affinity
ligands. The aim of this study was to overcome this poor oral absorption barrier, utilizing
prodrug to target the apical brush border
peptide transporter 1 (PEPT1).
Guanidine oseltamivir carboxylate (GOCarb) is a highly active polar
antiviral agent with insufficient oral bioavailability (4%) to be an effective therapeutic agent. In this report we utilize a carrier-mediated targeted
prodrug approach to improve the oral absorption of GOCarb. Acyloxy(alkyl)
ester based
amino acid linked
prodrugs were synthesized and evaluated as potential substrates of mucosal transporters, e.g., PEPT1.
Prodrugs were also evaluated for their chemical and enzymatic stability. PEPT1 transport studies included [(3)H]
Gly-Sar uptake inhibition in Caco-2 cells and cellular uptake experiments using HeLa cells overexpressing PEPT1. The intestinal membrane permeabilities of the selected
prodrugs and the parent
drug were then evaluated for epithelial cell transport across Caco-2 monolayers, and in the in situ rat intestinal jejunal perfusion model.
Prodrugs exhibited a pH dependent stability with higher stability at acidic pHs. Significant inhibition of uptake (IC(50) <1 mM) was observed for l-valyl and l-isoleucyl
amino acid prodrugs in competition experiments with [(3)H]
Gly-Sar, indicating a 3-6 times higher affinity for PEPT1 compared to
valacyclovir, a well-known PEPT1 substrate and >30-fold increase in affinity compared to GOCarb. The l-valyl
prodrug exhibited significant enhancement of uptake in PEPT1/HeLa cells and compared favorably with the well-absorbed
valacyclovir. Transepithelial permeability across Caco-2 monolayers showed that these
amino acid prodrugs have a 2-5-fold increase in permeability as compared to the parent
drug and showed that the l-valyl
prodrug (P(app) = 1.7 × 10(-6) cm/s) has the potential to be rapidly transported across the epithelial cell apical membrane. Significantly, only the parent
drug (GOCarb) appeared in the basolateral compartment, indicating complete activation (hydrolysis) during transport. Intestinal rat jejunal permeability studies showed that l-valyl and l-isoleucyl
prodrugs are highly permeable compared to the orally well absorbed
metoprolol, while the parent
drug had essentially zero permeability in the jejunum, consistent with its known poor low absorption.
Prodrugs were rapidly converted to parent in cell homogenates, suggesting their ability to be activated endogenously in the epithelial cell, consistent with the transport studies. Additionally, l-valyl
prodrug was found to be a substrate for valacyclovirase (K(m) = 2.37 mM), suggesting a potential cell activation mechanism. Finally we determined the oral bioavailability of our most promising candidate, GOC-l-Val, in mice to be 23% under fed conditions and 48% under fasted conditions. In conclusion, GOC-l-Val
prodrug was found to be a very promising
antiviral agent for oral delivery. These findings indicate that the carrier-mediated
prodrug approach is an excellent strategy for improving oral absorption of polar
neuraminidase inhibitors. These promising results demonstrate that the oral
peptide transporter-mediated
prodrug strategy has enormous promise for improving the oral mucosal cell membrane permeability of polar, poorly absorbed
antiviral agents and treating
influenza via the oral route of administration.