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ENDOCRINE AND DIABETES

7-But-2-ynyl-9-(6-methoxy-pyridin-3-yl)-6-piperazin-1-yl-7,9-dihydro-purin-8-one Is a Novel Competitive and Selective Inhibitor of Dipeptidyl Peptidase IV with an Antihyperglycemic Activity

Tsukuba Research Laboratories, Eisai Co., Ltd., Ibaraki, Japan

Received August 21, 2006; accepted September 13, 2006.

	Abstract

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7-But-2-ynyl-9-(6-methoxy-pyridin-3-yl)-6-piperazin-1-yl-7,9-dihydro-purin-8-one (ER-319711) is a novel dipeptidyl peptidase (DPP)-IV inhibitor discovered in our laboratories. In this study, we have characterized this DPP-IV inhibitor in vitro and in vivo as an antidiabetic agent. The trifluoroacetate salt form of ER-319711, ER-319711-15, inhibited human DPP-IV with an IC₅₀ value of 0.089 µM, whereas its IC₅₀ values toward human DPP8 and DPP9 were >100 µM. Inhibition kinetic pattern analysis indicated that ER-319711-15 inhibited DPP-IV in a competitive manner. ER-319711-15 (1 mg/kg) reduced glucose excursion in an oral glucose tolerance test (OGTT) using Zucker fa/fa rats, with significant increases in plasma insulin and active glucagon-like peptide-1 levels. In an OGTT using mice fed a high-fat diet in which ER-319711-15 (0.1–10 mg/kg) was orally administered at 0 h, and glucose was loaded at 0 and 5 h, this compound improved glucose tolerance dose dependently at both 0- and 5-h glucose loading. Next, we compared efficacy of ER-319711-15, E3024, a competitive DPP-IV inhibitor having an imidazopyridazinone structure, or vildagliptin, a slow-binding and long-acting DPP-IV inhibitor, at the same dose, 10 mg/kg, in the same procedures. At the first glucose challenge, all compounds lowered area under the curve (AUC) values of delta blood glucose between 0 and 2 h significantly to the same degree. At the second glucose load, the AUC values between 5 and 7 h were significantly decreased by ER-319711-15 and vildagliptin, but not by E3024. Therefore, ER-319711 might be a potent, competitive, and selective DPP-IV inhibitor with an antihyperglycemic activity.

ER-319711 is a novel DPP-IV inhibitor discovered in our laboratories. Demuth et al. (2005) categorized DPP-IV inhibitors based on their mode of inhibition and structures as follows: reversible product analog inhibitors (e.g., P32/98; Sorbera et al., 2001), covalently modifying product analog inhibitors [e.g., vildagliptin (LAF237); Villhauer et al., 2003)], and reversible nonpeptidic heterocyclic inhibitors [e.g., sitagliptin (MK-0431); Kim et al., 2005)]. ER-319711 belongs to the third group, and is a novel, 8-oxo-purine derivative.

In this study, we have characterized the inhibition mode for DPP-IV and enzyme selectivity toward DPP-IV, DPP8, and DPP9 of the newly discovered DPP-IV inhibitor, and we have investigated its antihyperglycemic activity using Zucker fa/fa rats and mice fed a high-fat diet. The high-fat diet-fed mouse model is considered to be a robust model for impaired glucose tolerance and early type 2 diabetes (Winzell and Ahrén, 2004), both of which are targets of DPP-IV inhibitors.

	Materials and Methods

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Chemicals. The trifluoroacetate salt form of ER-319711 (ER-319711-15), E3024, and 1-[[(3-hydroxy-1-adamantyl)amino]acetyl]-2-cyano-(S)-pyrrolidine (vildagliptin) were synthesized in our laboratories. Lys[Z(NO₂)] pyrrolidide was purchased from Bachem (Bubendorf, Switzerland), which is a nonselective DPP-IV/DPP8/DPP9 inhibitor (Lankas et al., 2005), used as a reference. The chemical structures of these compounds are shown in Fig. 1.

View larger version (11K): [in this window] [in a new window]   Fig. 1. Chemical structures of ER-319711 (A), E3024 (B), and Lys[Z(NO2)] pyrrolidide (C).

Inhibitory Kinetic Analysis. Recombinant human DPP-IV activity was measured at varied concentrations of ER-319711-15 (0.1, 1, 10, and 100 µM). For each concentration, measurements were conducted in the presence of varied concentrations of glycyl-L-proline p-nitroanilide tosylate (0.006, 0.024, 0.10, 0.39, 1.56, 6.25, 25, and 100 mM). Three separate experiments were performed in triplicate. The inhibitory pattern was evaluated by the Eadie-Hofstee plot using a curve-fitting program (GraphPad Software Inc., San Diego, CA).

Animals. Male Crlj:ZUC-Lepr^fa (Zucker fa/fa) rats and male C57BL/6NCrlCrlj mice were purchased from Charles River Japan (Tokyo, Japan). The rats and mice were provided with a commercial diet (MF; Oriental Yeast, Tokyo, Japan) and water ad libitum, and they were kept under conventional conditions of controlled temperature, humidity, and lighting (22 ± 2°C, 55 ± 5%, and a 12-h light/dark cycle with lights on at 7:00 AM). All procedures were conducted according to the Eisai Animal Care Committee's guideline.

Plasma Insulin and Active GLP-1 Levels in an Oral Glucose Tolerance Test OGTT Using Zucker fa/fa Rats. ER-319711-15 (1 mg/kg) or vehicle (0.5% methyl cellulose; MC) was orally administered to overnight-fasted Zucker fa/fa rats 0.5 h before oral glucose load (2 g/kg). Blood samples were drawn from the tail vein –0.5, 0, 0.5, 1, 2, and 3 h after the glucose load. In addition, approximately 250 µl of blood samples was collected with heparinized capillary tubes at 0, 0.5, 1, and 2 h. After centrifugation, supernatants were assayed for plasma insulin and active GLP-1 levels. Plasma insulin levels were determined using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Morinaga Institute of Biological Science, Kanagawa, Japan) and rat insulin as a standard with the microplate spectrophotometer. Plasma active GLP-1 levels were determined using an ELISA kit [GLP-1(7-36) Active ELISA kit; Linco Research, Inc., St. Charles, MO].

OGTT Using Mice Fed a High-Fat Diet: Dose Dependence of Efficacy of ER-319711-15. Mice were fed a high-fat diet (D12492 [GenBank] Rodent Diet with 60 kcal% fat) for 4 weeks from 11 weeks of age, and 42 mice were selected based on absolute body weight and randomly divided into seven groups. ER-319711-15 (0.1, 0.3, 1, 3, and 10 mg/kg), vildagliptin (10 mg/kg) as a positive control, or vehicle (0.5% MC) alone was orally administered to overnight-fasted mice at the same time of oral glucose administration (2 g/kg) (0 h). After 5 h, glucose was orally administered again. Blood samples were collected from the tail vein 0, 0.5, 1, 2, 5, 5.5, 6, and 7 h after the compound treatment.

OGTT Using Mice Fed a High-Fat Diet: Comparison of Efficacy among E3024, ER-319711-15, and Vildagliptin. Mice were fed a high-fat diet (D12492 [GenBank] Rodent Diet with 60 kcal% fat; Research Diets, Inc., New Brunswick, NJ) for 4 weeks from 11 weeks of age, and 24 mice were selected based on body weight and randomly divided into four groups. E3024 (10 mg/kg), ER-319711-15 (10 mg/kg), vildagliptin (10 mg/kg), or vehicle (0.5% MC) alone was orally administered to overnight-fasted mice at the same time of oral glucose administration (2 g/kg) (0 h). After 5 h, glucose was orally administered again. Blood samples were collected from the tail vein 0, 0.5, 1, 2, 5, 5.5, 6, and 7 h after the compound treatment.

Blood Glucose Determination. Blood samples (10 µl) were collected from the tail vein and mixed with 140 µl of 0.6 M perchloric acid. After centrifugation, the supernatants were assayed for glucose using an enzymatic assay kit (Glucose CII-test WAKO; Wako Pure Chemicals, Osaka, Japan) with the microplate spectrophotometer.

Statistical Analysis. Data are expressed as the mean ± S.E.M. To determine the integrated glucose response to the glucose challenge, the area under the curve (AUC) of delta blood glucose after the glucose load was calculated using a trapezoidal rule (between 0 and 2 h and between 5 and 7 h). Differences in the AUC values of delta blood glucose in an OGTT using mice between the vehicle- and E3024-, ER-319711- and vildagliptin-treated groups were determined by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test.

Differences in the AUC values of delta blood glucose in an OGTT using mice between the groups treated with vehicle or ER-319711 at several doses were determined by one-way ANOVA, followed by Dunnett's multiple comparison test. Differences in the AUCs between the vehicle- and vildagliptin-treated groups were determined by unpaired Student's t test. The dose responsiveness was evaluated using regression analysis.

In the study using Zucker fa/fa rats, differences in the AUC values of delta blood glucose between 0 and 3 h and delta plasma insulin levels at 0.5 h (that is, the differences between 0 and 0.5 h) of the vehicle- and ER-319711-treated groups were analyzed using unpaired Student's t test. Differences in delta plasma active GLP-1 levels at 0.5 h of the vehicle- and ER-319711-treated groups were determined using Mann-Whitney U-test.

A p value <0.05 (two-sided) was considered statistically significant. Statistical analyses were performed using SAS software package version 8.1 (SAS Institute Japan Ltd., Tokyo, Japan).

	Results

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Table 1 summarizes IC₅₀ and 95% CI values of ER-319711-15 and Lys[Z(NO₂)] pyrrolidide toward DPP-IV, DPP8, and DPP9, and ratios of IC₅₀ values toward DPP8 or DPP9 to those toward DPP-IV. The mean IC₅₀ value of ER-319711-15 toward DPP-IV was 0.087 µM, but no inhibitory activity was observed for DPP8 or DPP9 at up to 100 µM. Lys[Z(NO₂)] pyrrolidide had an inhibitory effect toward DPP-IV with an IC₅₀ value of 0.214 µM. But this inhibitor also had strong inhibitory activities toward both DPP8 and DPP9 with IC₅₀ values of 0.013 and 0.064 µM, respectively. Its IC₅₀ values toward DPP8 and DPP9 were more potent than toward DPP-IV by 16- and 3-fold, respectively (Table 1). To elucidate DPP-IV inhibition kinetic pattern of ER-319711, we conducted inhibition kinetics analysis with human recombinant DPP-IV. As illustrated in Fig. 2, ER-319711-15 showed a competitive inhibition pattern well fitted to the Eadie-Hofstee plot.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Inhibition kinetics of dipeptidyl peptidase IV by ER-319711-15 (the trifluoroacetate salt form of ER-319711). Different concentrations of ER-319711-15 (0, 0.03, 0.1, 0.3, 1, and 3 µM) were incubated in the presence of various concentrations of glycyl-L-proline p-nitroanilide tosylate (0.006, 0.024, 0.10, 0.39, 1.56, 6.25, 25, and 100 mM). Initial rates of the reaction were measured, and the results were expressed as the Eadie-Hofstee plot. Data represent the mean ± S.E.M. of three separate experiments performed in triplicate.

In the OGTT using Zucker fa/fa rats, administration of ER-319711-15 (1 mg/kg) produced a significant decrease in glucose excursion (Fig. 3A), resulting in a significant decrease in the AUC values of delta blood glucose (Fig. 3B). We also determined plasma insulin and active GLP-1 levels. A clear elevation of plasma insulin concentrations was observed 0.5 h after glucose administration (Fig. 3C). Delta plasma insulin levels at 0.5 h were significantly higher in the ER-319711-treated rats than in the vehicle-treated rats (Fig. 3D). In the same way, a peak of plasma active GLP-1 levels was seen at 0.5 h in the ER-319711 group, whereas no peak was detected in the vehicle group (Fig. 3E). Figure 3F indicates delta plasma active GLP-1 levels at 0.5 h, showing a significant increase in active GLP-1 levels by the ER-319711-15 treatment.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Effects of ER-319711 on blood glucose, plasma insulin and active glucagon-like peptide-1 (GLP-1) levels in an oral glucose tolerance test in Zucker fa/fa rats. ER-319711-15 (the trifluoroacetate salt form of ER-319711; 1 mg/kg) or vehicle was orally administered 0.5 h before glucose load (2 g/kg). Changes of blood glucose (A) and AUC values of delta blood glucose between 0 and 3 h (B). Changes of plasma insulin levels (C) and delta plasma insulin levels between 0 and 0.5 h (D). Changes of plasma active GLP-1 levels (E) and delta plasma active GLP-1 levels between 0 and 0.5 h (F). Values are expressed as the mean ± S.E.M. *, p < 0.05 by Student's t test (B and D). *, p < 0.05 by Mann-Whitney U-test (F).

View larger version (37K): [in this window] [in a new window]   Fig. 4. Changes of blood glucose levels (A) and AUC values of delta blood glucose between 0 and 2 h and between 5 and 7 h (B) in an oral glucose tolerance test in mice fed a high-fat diet. ER-319711-15 (the trifluoroacetate salt form of ER-319711; 0.1, 0.3, 1, 3, and 10 mg/kg), vildagliptin (10 mg/kg), or vehicle was orally administered at 0 h with glucose (2 g/kg). At 5 h, glucose (2 g/kg) was orally loaded again. Values are expressed as the mean ± S.E.M. *, p < 0.05 versus vehicle group by Dunnett's multiple comparison test. #, p < 0.05 versus vehicle group by Student's t test.

In an OGTT using mice fed a high-fat diet, we examined the efficacy of antihyperglycemic effects between ER-319711-15 (10 mg/kg), E3024 (10 mg/kg), and vildagliptin (10 mg/kg) (Fig. 5, A and B). At simultaneous administration of a compound and glucose, ER-319711-15, E3024, and vildagliptin lowered glucose excursions to almost the same degree. At 5-h postglucose load, ER-319711-15 and vildagliptin reduced the AUC significantly, but a decrease in the AUC by E3024 was not significant.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Changes of blood glucose levels (A) and AUC values of delta blood glucose between 0 and 2 h and between 5 and 7 h (B) in an oral glucose tolerance test in mice fed a high-fat diet. E3024 (10 mg/kg), ER-319711-15 (the trifluoroacetate salt form of ER-319711; 10 mg/kg), vildagliptin (10 mg/kg), or vehicle was orally administered at 0 h with glucose (2 g/kg). At 5 h, glucose (2 g/kg) was orally loaded again. Values are expressed as the mean ± S.E.M. *, p < 0.05 versus vehicle group by the Turkey multiple comparison test.

	Discussion

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Kinetics study indicated that ER-319711-15 inhibited DPP-IV competitively. In an OGTT using Zucker fa/fa rats, we confirmed that ER-319711-15 improved glucose tolerance, accompanied with increases in both plasma insulin and intact GLP-1 levels. These are characteristic actions of DPP-IV inhibitors. In addition, ER-319711-15 ameliorated glucose tolerance in mice fed a high-fat diet, and this effect was dose-dependent.

A recent focus of research of DPP-IV inhibitors is on long-acting inhibitors, aiming at less dosing frequency. Vildagliptin, which is a slow-binding DPP-IV inhibitor, was dosed to patients with type 2 diabetes once daily, thereby improving glycemic control (Ristic et al., 2005). Following vildagliptin, K579 (Takasaki et al., 2004) and saxagliptin (BMS-477118) (Augeri et al., 2005) have been reported to be long-acting DPP-IV inhibitors, both of which also show slow-binding inhibition. In this study, we investigated efficacy of ER-319711-15, in comparison with E3024 and vildagliptin, in 0- and 5-h postdose OGTT using mice fed a high-fat diet, at the same dose of 10 mg/kg. E3024 is a selective and competitive DPP-IV inhibitor with an imidazopyridazinone structure discovered in our laboratories (Yasuda et al., 2006). In an OGTT with a simultaneous administration of the compound and glucose (0 h), all compounds showed drastic decreases in blood glucose to the same degree. In a 5-h postdose OGTT, we observed an equal, significant glucose-lowering effect between 10 mg/kg ER-319711-15 and 10 mg/kg vildagliptin. E3024, however, did not manifest significant decrease in AUC values. Thus, ER-319711-15 might be a longer acting competitive DPP-IV inhibitor than E3024.

DPP-IV is a member of a family of serine peptidases including quiescent cell proline dipeptidase, DPP8, and DPP9. Acute and chronic administration of a DPP-IV inhibitor with DPP8/9 inhibition activity caused thrombocytopenia and splenomegaly in rats, and bloody diarrhea in dogs (Lankas et al., 2005). A selective DPP8/9 inhibitor caused the same signs, whereas no toxicity was observed in rats or dogs treated with a selective DPP-IV inhibitor, suggesting that inhibition of DPP8/9 leads to profound toxicity in preclinical studies. Lankas et al. (2005) suggested that preclinical assessment of clinical DPP-IV inhibitor candidates for DPP8/9 inhibition might be important. Our study showed that ER-319711-15 had an inhibitory activity for DPP-IV, but no activity for DPP8 or DPP9 at up to 100 µM. In contrast, Lys[Z(NO₂)] pyrrolidide inhibited not only DPP-IV but also DPP8 and DPP9 strongly, as reported previously (Lankas et al., 2005). Accordingly, ER-319711-15 is a specific inhibitor to DPP-IV and has a low risk of DPP8/9-related toxicity.

In summary, ER-319711-15 is a competitive and selective DPP-IV inhibitor with an 8-oxo-purine structure. Its glucose-lowering effect lasts longer than the effect of E3024. Thus, ER-319711 might be a novel potent DPP-IV inhibitor with an antihyperglycemic activity.

	Acknowledgements

 
We thank S. Yoshikawa, E. Emori, F. Matsuura, H. Ikuta, R. Clark, Y. Takeuchi, and Y. Kitahara for assistance and suggestions. We also thank J. Nagakawa and I. Hishinuma and for valuable advice and encouragement.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.112805.

ABBREVIATIONS: DPP, dipeptidyl peptidase; GLP-1, glucagon-like peptide-1; ER-319711, 7-but-2-ynyl-9-(6-methoxy-pyridin-3-yl)-6-piperazin1-yl-7,9-dihydro-purin-8-one; ER-319711-15, trifluoroacetate salt form of ER-319711; P32/98, di-[(2S,3S)-2-amino-3-methyl-pentanoic-1,3-thiazolidide] fumarate; OGTT, oral glucose tolerance test; E3024, 3-but-2-ynyl-5-methyl-2-piperazin-1-yl-3,5-dihydro-4H-imidazo[4,5-d]pyridazin-4-one tosylate; Lys[Z(NO₂)] pyrrolidide, 4-nitrobenzyl[(5S)-5-amino-6-oxo-6-(1-pyrrolidinyl)hexyl]carbamate; CI, confidence interval; MC, methyl cellulose; ELISA, enzyme-linked immunosorbent assay; AUC, area under the curve; ANOVA, analysis of variance; K579, (S)-1-[4-methyl-1-(2-pyrimidinyl)-4-piperidylamino]acetyl-2-pyrrolidinecarbonitrile; LAF237, vildagliptin; MK-0431, sitagliptin; BMS-477118, saxagliptin.

Address correspondence to: Dr. Kazuto Yamazaki; Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3, Tokodai, Tsukuba, Ibaraki 300-2635, Japan. E-mail: k5-yamazaki{at}hhc.eisai.co.jp

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This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.112805v1 319/3/1253    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Yamazaki, K. Articles by Tanaka, I. Search for Related Content PubMed PubMed Citation Articles by Yamazaki, K. Articles by Tanaka, I.