Inhibitory effect of novel oral hypoglycemic agent nateglinide (AY4166) on peptide transporters PEPT1 and PEPT2

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Abstract

The novel oral hypoglycemic agent nateglinide (AY4166) is a nonsulfonylurea insulin secretagogue, and its pharmacokinetic features include rapid absorption and elimination. As nateglinide is a dipeptide-like drug, we investigated the interaction of nateglinide with peptide transporters PEPT1 and PEPT2, which mediate the absorption of various peptide-like drugs. Nateglinide exhibited a potent inhibitory effect on [14C]glycylsarcosine uptake by the human colon adenocarcinoma cell line Caco-2 and rat PEPT-transfectants. Kinetic analysis revealed that these inhibitory effects were noncompetitive. Na+-coupled alanine or threonine uptake by Caco-2 cells was not inhibited by nateglinide, suggesting that the inhibitory effect of nateglinide on peptide transporters was not due to nonspecific interaction. There was little uptake of [14C]nateglinide by peptide transporters. Various sulfonylureas, such as glibenclamide, also inhibited [14C]glycylsarcosine uptake by rat PEPT-transfectants. In conclusion, nateglinide as well as sulfonylureas inhibit the transport activity of PEPT1 and PEPT2, although nateglinide itself is not transported by these transporters.

Introduction

Impaired glucose-induced insulin secretion and insulin resistance are hallmarks of Type 2 diabetes (noninsulin-dependent diabetes mellitus). To compensate for defective insulin secretion, sulfonylureas have been widely used for more than 40 years in the treatment of Type 2 diabetes. However, there are several disadvantages to sulfonylurea therapy, such as excess hypoglycemia between meals due to the long duration of action of these agents.

Nateglinide (AY4166, Fig. 1) has been exploited as a new class of oral hypoglycemic agent Shinkai et al., 1988, Shinkai et al., 1989. Although the mechanism of insulin secretion induced by nateglinide was the same as that of sulfonylureas Fujitani and Yada, 1994, Akiyoshi et al., 1995, nateglinide exhibited a quicker and shorter lasting hypoglycemic effect than sulfonylureas due to its rapid absorption and elimination Sato et al., 1991, Ikenoue et al., 1997. Because of these pharmacokinetic features, it was suggested that nateglinide could be beneficial in clinical use to prevent postprandial hyperglycemia without causing prolonged hypoglycemia in Type 2 diabetes patients.

Peptide transporters (PEPT1 and PEPT2) mediate the efficient absorption of a wide variety of oral peptide-like drugs in the small intestine and kidney (Inui and Terada, 1999). For example, oral β-lactam antibiotics Saito et al., 1995, Terada et al., 1997a, Terada et al., 1997b, anticancer agent bestatin (Saito et al., 1996) and angiotensine-converting enzyme inhibitors (Boll et al., 1994) are transported by these transporters. Furthermore, intestinal PEPT1 has been utilized to improve the intestinal absorption of poorly absorbed pharmacologically active agents by chemically converting to substrates for PEPT1 Hu et al., 1989, Tsuji et al., 1990, Balimane et al., 1998, Ganapathy et al., 1998, Han et al., 1998, Sawada et al., 1999b. Because these peptide-like drugs are pharmacologically independent each other, the pharmacokinetic profiles and therapeutic efficacy may be affected in coadministration of other peptide-like drugs. Therefore, in order to evaluate the appropriate usage of peptide-like drugs, especially newly developed peptide-like drugs, it should be important to investigate their interaction with peptide transporters.

Nateglinide is a novel oral hypoglycemic agent and a dipeptide-like drug, and therefore, the present study was carried out to examine the interaction of nateglinide with peptide transporters using the human colon adenocarcinoma cell line Caco-2, and rat PEPT1- or rat PEPT2-expressing stable transfectant. Furthermore, the effects of other hypoglycemic agents, sulfonylureas, on peptide transporters were also investigated.

Section snippets

Materials

Nateglinide and [14C]nateglinide (9.00 MBq/mmol) were supplied by Ajinomoto (Yokohama, Japan). [14C]glycylsarcosine (1.78 GBq/mmol) was obtained from Daiichi Pure Chemicals (Ibaraki, Japan), and [3H]alanine (2.07 TBq/mmol) and [3H]threonine (485 GBq/mmol) were from Amersham Int. (Buckinghamshire, UK). Glycylsarcosine, tolbutamide and chlorpropamide were obtained from Sigma (St. Louis, MO). Glibenclamide was purchased from Wako (Kyoto, Japan). All other chemicals used were of the highest purity

Inhibitory effects of nateglinide on glycylsarcosine uptake by peptide transporters

Caco-2 cells have been used as a model for studying the functions of peptide transporters Inui et al., 1992, Saito and Inui, 1993, and it was reported that human PEPT1 was expressed in Caco-2 cells (Liang et al., 1995). First, we examined the effects of nateglinide on [14C]glycylsarcosine uptake by Caco-2 cells and rat PEPT1- or PEPT2-expressing transfectant (LLC-rPEPT1 and LLC-rPEPT2 cells, respectively). As shown in Fig. 2, [14C]glycylsarcosine uptake was not affected in the presence of 0.05

Discussion

The present study have clearly demonstrated that nateglinide have the inhibitory effects on the glycylsarcosine uptake by PEPT1 and PEPT2. The inhibitory effects of this agent on both transporters were characterized as kinetically noncompetitive. Recently, we found that [14C]glycylsarcosine uptake by PEPTs was inhibited by glibenclamide in a noncompetitive fashion, and it was suggested that this agent was bound to a site distinct from the glycylsarcosine binding site (Sawada et al., 1999a). An

Acknowledgements

This work was supported in part by a Grant-in-Aid for Scientific Research (B) and a Grant-in-Aid for Scientific Research on Priority Areas (No. 296) from the Ministry of Education, Science, Sports and Culture of Japan, and by grants from the Uehara Memorial Foundation and from the Japan Research Foundation for Clinical Pharmacology.

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