Oseltamivir (Tamiflu) Is a Substrate of Peptide Transporter 1

Abstract

ABSTRACT:Oseltamivir, an ester-type prodrug of the neuraminidase inhibitor [3R,4R,5S]-4-acetamido-5-amino-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylate phosphate (Ro 64-0802), has been developed for the treatment of A and B strains of the influenza virus but has neuropsychiatric and other side effects. In this study, we characterized the transport across intestinal epithelial cells and the absorption of oseltamivir in rats. Uptake by Caco-2 cells (human carcinoma cell line) and HeLa cells transfected with peptide transporter 1 (HeLa/ PEPT1) was time-and temperature-dependent and was inhibited by typical PEPT1 inhibitors such as glycyl-sarcosine (Gly-Sar). The uptake by Caco-2 cells and HeLa/PEPT1 was saturable, with similar K m values. Oseltamivir absorption in adult rats was greatly reduced by simultaneous administration of milk, casein, or Gly-Sar. Furthermore, the plasma and brain concentrations of oseltamivir were higher in fasting than in nonfasting rats after oral administration. These results suggest that oseltamivir is a substrate of PEPT1 and that PEPT1 is involved in its intestinal absorption.Various transporters are expressed on apical and basolateral membranes of intestinal epithelial cells, serving to take up nutrients and to excrete xenobiotics into the lumen. Influx transporters are able to accept nutrients and also various drugs as substrates. In particular, peptide transporter 1 (PEPT1, SLC15A1), localized at brush-border membranes of human small intestine (Saito et al., 1995), plays important roles in the absorption of not only di-/tripeptides (Tamai et al., 1994) but also peptide-mimetic compounds, such as orally administered ␤-lactam antibiotics (Ganapathy et al., 1995;Sai et al., 1996) and the anticancer agent bestatin (Tomita et al., 1990;Inui et al., 1992). Several researchers recently found that intestinal PEPT1 can transport L-valine ester prodrugs, such as valacyclovir and valganciclovir (Balimane et al., 1998;Han et al., 1998;Sugawara et al., 2000). Therefore, such structural modification of drugs may result in increased intestinal absorption, mediated by PEPT1.Oseltamivir phosphate (oseltamivir), manufactured under the trade name Tamiflu as an ester-type prodrug of the neuraminidase inhibitor Ro 64-0802, has been developed for the treatment of A and B strains of the influenza virus. This drug has been reported to be associated with neuropsychiatric side effects (http://www.fda.gov/cder/drug/ infopage/tamiflu/QA20051117.htm and http://www.mhlw.go.jp/ english/index.html), which are likely to be caused by distribution of oseltamivir and/or its metabolite(s) to the central nervous system. We recently examined the possible role of P-glycoprotein (P-gp) as the determinant of brain distribution of oseltamivir and Ro 64-0802 both in vitro using LLC-GA5-COL150 cells, which overexpress human multidrug resistance (or resistant) 1 (MDR1) P-gp on the apical membrane, and in vivo using mdr1a/1b knockout mice (Morimoto et al., 2008). The permeability of oseltamivir in the basolateral-to-apical dire...

Species-Dependent Uptake of Glycylsarcosine but Not Oseltamivir in Pichia pastoris Expressing the Rat, Mouse, and Human Intestinal Peptide Transporter PEPT1

“…Therefore, the question is how can one reconcile the differences observed between our study and those of other investigators (Ogihara et al, 2009;Morimoto et al, 2011) regarding the PEPT1-mediated intestinal absorption of oseltamivir? In the first study (Ogihara et al, 2009), in vitro results in Caco-2 cells and hPEPT1-transfected HeLa cells indicated a time-and temperature-dependent uptake of oseltamivir, along with inhibition by typical PEPT1 inhibitors such as GlySar.…”

“…In the first study (Ogihara et al, 2009), in vitro results in Caco-2 cells and hPEPT1-transfected HeLa cells indicated a time-and temperature-dependent uptake of oseltamivir, along with inhibition by typical PEPT1 inhibitors such as GlySar. These investigators also reported that oseltamivir uptake was saturable with K m values of 6.5 and 8.6 mM, respectively, in Caco-2 and HeLa/hPEPT1 cells.…”

“…Once Pichia cells were validated for these three mammalian species, subsequent experiments focused on whether oseltamivir exhibited a difference in transport between human, mouse, and rat. This particular comparison was brought to the forefront because of recent conflicting studies suggesting a species difference between rodents and humans in the PEPT1-mediated intestinal absorption of oseltamivir (Ogihara et al, 2009;Morimoto et al, 2011). In the present study, we found that 1) GlySar was transported into yeast transformants expressing human, mouse, and rat PEPT1 and that dipeptide uptake was inhibited by known substrates of PEPT1 for all three mammalian species; 2) GlySar uptake was saturable for the mammalian species tested although some differences were observed in substrate affinity for PEPT1 (i.e., K m values); 3) oseltamivir inhibited the uptake of GlySar, in a dose-dependent manner, for human, rat, and mouse PEPT1, with some differences in inhibitory potential (i.e., IC 50 values); and 4) oseltamivir was not a substrate of human, mouse, and rat PEPT1, but instead exhibited nonspecific binding to the yeast vector and PEPT1 transformants.…”

“…In particular, it is believed that PEPT1 significantly improves the oral availability of the antiviral agent acyclovir by transforming the drug into a valine ester prodrug, which can then take advantage of the intestinally localized proton-coupled oligopeptide transporter protein (Han et al, 1998). Because of this structurefunction relationship and the concern regarding transporter-drug interactions, Ogihara et al, (2009) explored the possibility that oseltamivir may also be a substrate for PEPT1. These authors, using cell culture methods and in vivo rat studies, reported that oseltamivir was not only a substrate of PEPT1 but also that its intestinal absorption after oral dosing was markedly reduced when administered with milk.…”

Role of the Intestinal Peptide Transporter PEPT1 in Oseltamivir Absorption: In Vitro and In Vivo Studies

“…The in vitro transport characteristics of oseltamivir were examined in Caco-2 and hPEPT1 transfected HeLa cells (Ogihara et al, 2009). In these studies, a significant reduction of oseltamivir uptake at 4°C and in the presence of Gly-Sar and Trp-Gly was observed.…”