Increasing oral absorption of polar neuraminidase inhibitors: a prodrug transporter approach applied to oseltamivir analogue

Abstract

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.

Figure 1

Oseltamivir and its analogues

Figure 2

Apical to basolateral (A to B) apparent permeability (P_app in cm/sec) of valacyclovir, GOCarb and its amino acid prodrugs (0.25 mM) across Caco-2 monolayers as measured by LC/MS. Data presented as mean ± SEM, n = 3.

Figure 3

[³H]Gly-Sar Caco-2 competitive inhibition by GOCarb and its amino acid prodrugs. Uptake inhibition in Caco-2 Cells denoted by IC₅₀ values in mM. Data presented as Mean ± SEM, n = 3.

Figure 4

Direct uptake (mmol/mg of protein) for GOCarb and its amino acid prodrugs in wild type HeLa cells (control) and in PEPT1 over-expressing HeLa Cells. Data presented as Mean ± SEM, n = 3.

Figure 5

Ratio of GlySar uptake inhibition and PEPT1 uptake for amino acid GOCarb prodrugs to their parent showing increased affinity and increased uptake for the prodrugs.

Figure 6

The permeability coefficient (P_eff, cm/sec) obtained in in-situ rat perfusion study for metoprolol (reference standard), GOCarb and its amino acid prodrugs at 0.1mM. Acyclovir and valacyclovir P_eff values (0.01mM) were reported previously. Permeability was observed as disappearance of prodrug in perfusate. Data presented as Mean ± SEM, n = 4.

Figure 7

Oral bioavailability (%) comparison of GOCarb (GOC) and GOC-L-Val after oral administration of 10 mg /kg of GOCarb or GOC-L-Val.

Figure 8

A comparison of the GOCarb (GOC) plasma levels after oral administration of 10 mg eq GOC/kg of GOC-L-Val (□), GOC (Δ) or IV administration of 1 mg (eq. GOC)/kg GOC (◆) to fasted animals (n=5). The dashed line at 5 ng/ml is the approximate EC₅₀ for GOC versus the A/NWS/33 virus.

Scheme 1

Synthesis of L-valyl, L-isoleucyl and L-leucyl amino acid acyloxy ester prodrugs of GOCarb