Title page Effects of the Combination of a Dipeptidyl ...jpet.aspetjournals.org/content/jpet/early/2006/11/08/jpet.106... · JPET #112011 3 Abstract Several combination therapies

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Title page

Effects of the Combination of a Dipeptidyl Peptidase IV Inhibitor and an Insulin

Secretagogue on Glucose and Insulin Levels in Mice and Rats

Kazuto Yamazaki, Nobuyuki Yasuda, Takashi Inoue, Eiichi Yamamoto, Yukiko Sugaya,

Tadashi Nagakura, Masanobu Shinoda, Richard Clark, Takao Saeki and Isao Tanaka

Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3, Tokodai, Tsukuba, Ibaraki

300-2635, Japan

JPET Fast Forward. Published on November 8, 2006 as DOI:10.1124/jpet.106.112011

Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on November 8, 2006 as DOI: 10.1124/jpet.106.112011

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Running title page

Running title: Combination of DPP-IV inhibitor and insulin secretagogue

Corresponding author: Kazuto Yamazaki; Tsukuba Research Laboratories, Eisai Co.,

Ltd., 5-1-3, Tokodai, Tsukuba, Ibaraki 300-2635, Japan; Tel: +81-29-847-5817, Fax:

+81-29-847-5367; E-mail: [email protected]

Number of text pages: 23

Number of tables: 0

Numbers of figures: 8

Numbers of references: 31

Number of words in the Abstract: 208

Number of words in the Introduction: 405

Number of words in the Discussion: 836

Abbreviations: ANOVA, analysis of variance; AUC, area under the curve; DPP-IV,

dipeptidyl peptidase IV; E3024,

3-but-2-ynyl-5-methyl-2-piperazin-1-yl-3,5-dihydro-4H- imidazo[4,5-d]pyridazin-4-one

tosylate; GIP, glucose-dependent insulinotropic polypeptide; GLP-1, glucagon-like

peptide-1; GPCR, guanine nucleotide-binding protein-coupled receptor; OGTT, oral

glucose tolerance test; P32/98,

di-[(2S,3S)-2-amino-3-methyl-pentanoic-1,3-thiazolidide] fumarate; SUR1,

sulfonylurea receptor type 1; KATP channel, ATP-sensitive potassium channel; PKA,

protein kinase A; cAMP-GEFII, cAMP-regulated guanine nucleotide exchange factor

II; Rim2, Rab3-interacting molecule 2 (GIP)

Recommended section: Endocrine and Diabetes


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Abstract

Several combination therapies have been tried for treating of type 2 diabetes in order to

control more effectively fasting hyperglycemia and postprandial hyperglycemia. In this

study, we have examined the effects of combinating a novel, selective and competitive

dipeptidyl peptidase IV (DPP-IV) inhibitor, E3024

(3-but-2-ynyl-5-methyl-2-piperazin-1-yl-3,5-dihydro-4H-imidazo[4,5-d]pyridazin-4-on

e tosylate) with a representative of one of two types of insulin secretagogues, i.e. either

glybenclamide (a sulfonylurea) or nateglinide (a rapid-onset/short-duration insulin

secretagogue), on glucose and insulin levels in an oral glucose tolerance test (OGTT)

using mice fed a high-fat diet. In addition, we have investigated the effects of these

combinations on blood glucose levels in fasting rats. Two-way analysis of variance

showed that the combination of E3024 and glybenclamide improved glucose tolerance

additively, and also caused a synergistic increase in insulin levels in the OGTT in mice

fed a high-fat diet. In a similar way, the combination of E3024 and nateglinide

ameliorated glucose tolerance additively and raised insulin levels additively. In fasting

rats, co-administration of E3024 with glybenclamide or nateglinide treatment did not

affect the glucose-lowering effects of the insulin secretagogues. Therefore, a DPP-IV

inhibitor in combination with glybenclamide or nateglinide may be a promising option

for the treatment of type 2 diabetes, and particularly for controlling postprandial

hyperglycemia in the clinic.


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Introduction

Dipeptidyl peptidase IV (DPP-IV) degrades active glucagon-like peptide-1

(GLP-1 (7-36)amide and GLP-1 (7-37)), which is an incretin released from L cells in

the intestine after meal intake that promotes insulin secretion in a glucose-dependent

manner. GLP-1 has an antidiabetic action in patients with type 2 diabetes (Gutniak et al.,

1994; Nauck et al., 1993). DPP-IV cleaves GLP-1 rapidly so the latter’s half-life is only

1-2 min. Accordingly, the prevention of GLP-1 inactivation by DPP-IV inhibition is

currently being actively explored as a novel approach to the treatment of type 2 diabetes

(Deacon, et al. 2004). DPP-IV inhibition leads to blood glucose-lowering effects in

animal models of diabetes (Perderson et al., 1998; Reimer et al., 2002; Burkey et al.,

2005), and in patients with type 2 diabetes (Ahren et al., 2002; 2005).

E3024

(3-but-2-ynyl-5-methyl-2-piperazin-1-yl-3,5-dihydro-4H-imidazo[4,5-d]pyridazin-4-on

e tosylate) is a novel, selective and competitive DPP-IV inhibitor, discovered in our

laboratories (Yasuda et al., 2006). Demuth et al. (2005) have classified 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

non-peptidic heterocyclic inhibitors (e.g. sitagliptin (MK-0431) (Kim et al., 2005)).

E3024 belongs to the third group, and has a novel, imidazopyridazinone structure.

Insulin secretagogues used in the clinic are categorized into the following two

groups: sulfonylureas and rapid-onset/short-duration insulinotropic agents (Krentz and

Bailey, 2005). The former class includes glybenclamide and glyburide, while the latter

includes nateglinide and repaglinide. The sulfonylureas target fasting hyperglycemia,

while the rapid-acting insulin secretagogues improve postprandial hyperglycemia (Van


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Gaal and De Leeuw, 2003).

Recently, the importance of reducing not only fasting hyperglycemia, but also

postprandial hyperglycemia, has been demonstrated. Fasting hyperglycemia increases a

risk of microvascular complications (UK Prospective Diabetes Study (UKPDS) Group,

1998), while postprandial hyperglycemia has shown to be an independent risk factor for

the development of macrovascular complications (Monnier, 2000; Ceriello, 2000;

Temelkokova-Kurktschiev, 2000). Several combination therapies employing agents

with complementary mode of actions have been examined to address improvement of

fasting hyperglycemia and postprandial hyperglycemia. In this study, we examined the

effects of a combination of E3024 and glybenclamide, and that of E3024 and

nateglinide on glucose and insulin levels in an oral glucose tolerance test in mice fed a

high-fat diet, and further studied the effects of these combinations on blood glucose

levels in fasting rats to explore their possibilities as novel combination therapies.


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Materials and Methods

Chemicals. E3024 was synthesized in our laboratories. Glybenclamide

(4-[2-(5-chloro-2-methoxybenzoylamino)ethyl](N-cyclohexylcarbamoyl)benzene

sulfonamide) was purchased from Sigma (St. Louis, MO). Nateglinide

((-)-N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine) was purchased from

Yamanouchi Pharmaceutical Co., Ltd. (Tokyo, Japan). The chemical structure of E3024

is indicated in Figure 1. GLP-1 (7-36)amide and human glucose-dependent

insulinotropic polypeptide (GIP) were purchased from Bachem AG (Bubendorf,

Switzerland) and Peptide Institute, Inc. (Osaka, Japan), respectively.

Animals. Ten-week-old male C57BL/6NCrj mice and five-week-old male Wistar rats

were purchased from Charles River Japan (Tokyo, Japan). The mice and rats were

provided with a commercial diet (MF, Oriental Yeast, Tokyo, Japan) and water ad

libitum and were kept under conventional conditions of controlled temperature,

humidity and lighting (22 ± 2°C, 55 ± 5% and a 12-hr light/dark cycle with lights on at

07:00 a.m.). All procedures were conducted according to the Eisai Animal Care

Committee’s guideline.

Effects of the combination of E3024 and glybenclamide or nateglinide on blood

glucose levels in an oral glucose tolerance test (OGTT). Mice were fed a high-fat diet

(D12492 Rodent Diet with 60 kcal% fat; Research Diets, Inc., NJ) for four weeks from

11 weeks of age, and 28 mice were selected based on body weight and randomly

divided into four groups. The reason why we used the high-fat diet-fed mouse model is

that this 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


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inhibitors. E3024 (3 mg/kg), glybenclamide (1 mg/kg), a mixture of E3024 (3 mg/kg)

and glybenclamide (1 mg/kg), or vehicle (0.5% methylcellulose (MC)) alone was orally

administered to overnight-fasted mice 0.5 hr prior to an oral glucose load (2 g/kg).

Blood samples were collected from the tail vein 0.5 hr before the glucose load, and 0,

0.5, 1 and 2 hr after the glucose load, and blood glucose and plasma insulin levels were

determined. The same OGTT procedure was performed for the combination of E3024

and nateglinide, but the dose of nateglinide was 50 mg/kg.

Effects of the combination of glybenclamide and nateglinide on blood glucose

levels in the OGTT. Mice were fed a high-fat diet for five weeks from 11 weeks of age,

and 20 mice were selected based on body weight and randomly divided into four groups.

Glybenclamide (1 mg/kg), nateglinide (50 mg/kg), a mixture of glybenclamide (1

mg/kg) and nateglinide (50 mg/kg), or vehicle (0.5% MC) alone was orally

administered to overnight-fasted mice 0.5 hr prior to the oral glucose load (2 g/kg).

Blood samples were collected from the tail vein 0.5 hr before the glucose load, and 0,

0.5, 1 and 2 hr after the glucose load, and blood glucose levels were determined.

Effects of the combination of glybenclamide and GLP-1 on blood glucose levels in

the OGTT. Mice were fed a high-fat diet for six weeks from 11 weeks of age, and 24

mice were selected based on body weight and randomly divided into four groups.

Before the glucose load (2 g/kg), glybenclamide (1 mg/kg) or vehicle (0.5% MC) alone

was orally administered to overnight-fasted mice. GLP-1 (7-36)amide (1 µg) or saline

was intraperitoneally injected immediately after the glucose administration. Blood

samples were collected from the tail vein 0.5 hr before the glucose load, and 0, 0.5, 1

and 2 hr after the glucose load, and blood glucose levels were determined.


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Effects of the combination of glybenclamide and GIP on blood glucose levels in the

OGTT. Mice were fed a high-fat diet for six weeks from 11 weeks of age, and 20 mice

were selected based on body weight and randomly divided into four groups. Before the

glucose load (2 g/kg), glybenclamide (1 mg/kg) or vehicle (0.5% MC) alone was orally

administered to overnight-fasted mice. GIP (1 µg) or saline was intraperitoneally

injected immediately after the glucose administration. Blood samples were collected

from the tail vein 0.5 hr before the glucose load, and 0, 0.5, 1 and 2 hr after the glucose

load, and blood glucose levels were determined.

Effects of the combination of E3024 and glybenclamide or nateglinide on fasting

blood glucose levels in normal rats. Six-week-old rats were used. E3024 (3 mg/kg),

glybenclamide (1 mg/kg), a mixture of E3024 (3 mg/kg) and glybenclamide (1 mg/kg),

or vehicle (0.5% MC) alone was orally administered to overnight-fasted rats. Blood

samples were collected from the tail vein before (0 hr), and 1, 2 and 3 hr after the drug

administration in order to determine the glucose levels. The same procedures were

performed for the combination of E3024 and nateglinide, but the dose of nateglinide

was 50 mg/kg.

Blood glucose and plasma insulin determination. Blood samples (10 µL) were

collected from the tail vein and mixed with 140 µL of 0.6 mol/L perchloric acid. After

centrifugation (3,000 rpm; 10 min; room temperature), the supernatants were assayed

for glucose using an enzymatic assay kit (Glucose CII-test WAKO, Wako Pure

Chemicals) with a microplate spectrophotometer (SpectraMax; Molecular Devices, CA).

Blood samples (approximately 50 µL) were collected from the tail vein with


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heparinized capillary tubes and centrifugated (7,000 rpm; 5 min; 4°C). The supernatants

were assayed for plasma insulin levels using an enzyme-linked immunosorbent assay kit

(Ultra sensitive rat insulin ELISA kit, Morinaga Institute of Biological Science,

Kanagawa, Japan) and mouse insulin (Morinaga Institute of Biological Science) as a

standard with the microplate spectrophotometer.

Statistical analysis. Data are expressed as the mean ± standard error of the mean

(S.E.M.). To determine the integrated glucose response to the oral glucose challenge,

the area under the curve (AUC) of delta blood glucose after the oral glucose load was

calculated using a trapezoidal rule. To examine main effects and interaction in the

combination studies, two-way analysis of variance (ANOVA) was performed on the

AUC values and plasma insulin levels at 0.5 hr. The Tukey multiple comparison test

was used to examine the difference between the two groups.

In the studies on effects of the combination of E3024 and glybenclamide or

nateglinide on fasting blood glucose levels, we performed two-way ANOVA with

respect to the values of delta blood glucose at 2 hr (that is, the differences in blood

glucose levels between 0 and 2 hr) in the combination with glybenclamide, and those at

1 hr in the combination with nateglinide.

A probability (p) value <0.05 (two-sided) was considered statistically significant.

Statistical analyses were performed using an SAS software package version 8.1 (SAS

Institute Japan Ltd., Tokyo, Japan).


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Results

Figure 2 illustrates the changes of blood glucose levels (A) and the AUC of delta

blood glucose (B) in the OGTT using mice fed the high-fat diet that were treated with

E3024 (3 mg/kg) and/or glybenclamide (1 mg/kg). Two-way ANOVA indicated

significant main effects of both E3024 and glybenclamide on the AUC values. No

significant interaction was seen between the effects of E3024 and glybenclamide (Fig.

2B). E3024 and glybenclamide significantly reduced the AUC values, compared with

that of the vehicle treatment. The AUC value of the combination group was

significantly lower than those of the other groups. Figure 2C shows the changes of

plasma insulin levels in the mice. Two-way ANOVA indicated significant main effects

of E3024 and glybenclamide on plasma insulin levels 0.5 hr after the oral glucose load.

In addition, a significant interaction was detected between the effects of E3024 and

glybenclamide. The insulin level of the E3024 plus glybenclamide treatment was

significantly higher than those of the other three groups.

Figure 3 shows the changes of blood glucose levels (A) and the AUC of delta

blood glucose (B) in the OGTT using mice fed the high-fat diet that were treated with

E3024 (3 mg/kg) and/or nateglinide (50 mg/kg). Two-way ANOVA indicated

significant main effects of both E3024 and nateglinide on the AUC values. No

significant interaction was seen between the effects of E3024 and nateglinide (Fig. 3B).

E3024 and nateglinide significantly decreased the AUC values in comparison with that

of the vehicle treatment. The AUC value of the combination group was significantly

lower than those of the vehicle- and nateglinide-treated groups. Figure 3C. shows the

changes of plasma insulin levels in the mice. Two-way ANOVA indicated significant

main effects of both E3024 and nateglinide on plasma insulin levels 0.5 hr after the oral

glucose load. No significant interaction between the effects of E3024 and nateglinide


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was seen. The combination resulted in levels significantly higher than any other group.

Effects of the combination of glybenclamide and GLP-1 on glucose excursions in

the OGTT are shown in Figure 4A. Two-way ANOVA indicates significant main

effects of both glybenclamide and GLP-1 on the AUC values, but there was no

significant interaction between the effects of glybenclamide and GLP-1 (Fig. 4B). Both

GLP-1 alone and glybenclamide alone caused significant reduction of the AUC,

compared with the control. The AUC values of the GLP-1 plus glybenclamide treatment

were significantly lower than those of treatment with either GLP-1 or glybenclamide

alone. Likewise, we also observed an additive effect of improvement of glucose

tolerance by the combination of glybenclamide and GIP from results of two-way

ANOVA (Figs. 5A and 5B).

We examined the effects of the combination of glybenclamide and nateglinide on

glucose tolerance in the OGTT (Figs. 6A and 6B). Two-way ANOVA shows significant

main effects of both glybenclamide and nateglinide, and significant interaction between

their effects (Fig. 6B). The AUC values were almost the same for the groups treated

with glybenclamide alone, nateglinide alone or with both.

Figure 7 shows the changes of fasting blood glucose levels in normal rats that

were treated with E3024 (3 mg/kg) and/or glybenclamide (1 mg/kg) (Fig. 7A).

Two-way ANOVA indicated that there is a significant main effect of glybenclamide on

delta blood glucose values at 2 hr, whereas the main effect of E3024 was not significant

(Fig. 7B). There was no significant interaction. In the case of the combination of E3024

and nateglinide, a clear reduction in blood glucose levels was detected in both the

nateglinide alone, and the E3024 plus nateglinide groups (Fig. 8A). As for the

combination of E3024 and glybenclamide, two-way ANOVA demonstrated a

significant main effect of nateglinide on delta blood glucose values at 1 hr, whereas the


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main effect of E3024 was not significant (Fig. 8B). Neither was there a significant

interaction.


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Discussion

Takasaki et al. (2004) reported that a long-acting DPP-IV inhibitor, K579

((S)-1-[4-methyl-1-(2-pyrimidinyl)-4-piperidylamino]acetyl-2-pyrrolidinecarbonitrile),

significantly suppressed blood glucose elevation in glybenclamide-pretreated rats, but

they did not measure insulin concentrations. The insulinotropic effect of glybenclamide

was further amplified by intravenous infusion of GLP-1 in perfused rat pancreas, and in

NIDDM patients (Gutniak et al., 1996). Our study demonstrated that the combination of

glybenclamide and GLP-1 additively improved glucose tolerance in the OGTT in mice

fed a high-fat diet, and previously reported that E3024 elevated plasma active GLP-1

levels in the OGTT using Zucker fa/fa rats (Yasuda et al. 2006). Taken together these

findings suggest that DPP-IV inhibition may augment the glucose-lowering effects of

glybenclamide through the presence of active GLP-1 which has escaped from

inactivation by DPP-IV.

Glybenclamide binds to the sulfonylurea receptor type 1 (SUR1) of adenosine

triphosphate (ATP)-sensitive potassium (KATP) channels in pancreatic β-cell membranes,

and closes this channel. The resultant reduction in K+ permeability generates membrane

depolarization, opening of Ca2+ channels, elevation of [Ca2+]i, and eventually initiation

of Ca2+-dependent exocytosis of insulin-containing granules (Aguilar-Bryan et al.,

1995). Nateglinide also binds to SUR1, but another binding site, specific to nateglinide,

has been proposed (Fujita et al., 1996). On the other hand, GLP-1 binds to the GLP-1

receptor, a guanine nucleotide-binding protein-coupled receptor (GPCR), in pancreatic

β-cell membranes, and activates adenyl cyclase, leading to an increase in cAMP

production and activation of the cAMP-dependent protein kinase (protein kinase A;

PKA) pathway. This pathway is thought to potentiate glucose-induced insulin release.

In addition, the cAMP-GEFII·Rim pathway has been reported to be a PKA-independent


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mechanism for the potentiation of insulin secretion by GLP-1 (Kashima et al., 2001;

Gromada et al., 2004). Therefore, as the mechanisms of insulin secretion are different

for theses insulin secretagogues and GLP-1, synergistic or additive insulin release is

triggered with the combination of E3024 and glybenclamide or nateglinide.

GIP is the other incretin, released from K cells in the upper portion of the small

intestine after ingestion of glucose and fat (Yip and Wolfe, 2000). Like GLP-1,

GIP-(1-42), the active form of GIP, potentiates glucose-dependent insulin secretion, and

GIP-(1-42) is degraded by DPP-IV to inactive GIP-(3-42). Hansotia et al. (2004)

showed that DPP-IV inhibitors lowered glucose and increased plasma insulin in OGTT

in both GLP-1 receptor knockout mice and GIP receptor knockout mice. On the other

hand, the DPP-IV inhibitor actions were eliminated in double incretin receptor knockout

mice. Thus, both GLP-1 and GIP contribute to the improvement of glucose tolerance

elicited by DPP-IV inhibitors. Interestingly, both GLP-1 and GIP simulates insulin

secretion via GPCR activation of adenyl cyclase, coupled with the production of cAMP

(Ehses et al. 2002). The present study suggested that GIP is also be involved in the

combination effect of a DPP-IV inhibitor and an insulin secretagogue. Employing the

incretin receptor deficient mice and double knockout mice, we could document more

clearly and precisely the involvement of GLP-1 and/or GIP in the combination effect.

The combination of sulfonylures and nateglinide is not recommended, because

the “fast on-fast-off” mode of action of nateglinide is negated by the sustained action of

sulfonylureas (Van Gaal and De Leeuw, 2003). In this study, the combination of

glybenclamide and nateglinide did not show efficacy on glucose excursions in mice.

Also in in vitro study, simultaneous exposure of isolated rat islets to nateglinide and

glybenclamide did not cause a further increase of insulin release in comparison with that

produced by either agent alone (Ikenoue et al., 1997). Thus, the combination of


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glybenclamide and nateglinide may not be of use. However, E3024 could be combined

with either agent: the combination of E3024 and a sulfonylurea could reduce both

fasting and postprandial glucose levels, while that of E3024 and a rapid-acting insulin

secretagogue could enhance postprandial glucose-lowering effects.

We examined the combination of E3024 and glybenclamide or nateglinide on

fasting blood glucose levels in normal rats. E3024 alone did not reduce fasting blood

glucose levels, probably because E3024 induces insulin secretion in a

glucose-dependent fashion. Moreover, E3024 did not affect the blood-lowering effect of

either glybeclamide or nateglinide under fasting conditions. Therefore, the risk of

hypoglycemia may not be increased by combining E3024 and glybenclamide or

nateglinide, as compared with treatment using either glybenclamide or nateglinide alone,

despite the greater hypoglycemic effects seen for the combination compared with the

single treatment.

This is the first report on the statistically evident effectiveness of the combination

of E3024, a DPP-IV inhibitor, and glybenclamide, and the combination of E3024 and

nateglinide, presented with data for the accompanying changes in insulin levels. E3024

plus glybenclamide improved glucose tolerance additively, with insulin levels increased

synergistically in the OGTT in mice. Likewise, the combination of E3024 and

nateglinide ameliorated glucose tolerance additively with additively elevated insulin

levels. E3024 did not affect the glucose-lowering effect of glybenclamide or nateglinide

in fasting normal rats. These findings of our study suggest the combination of a DPP-IV

inhibitor and glybenclamide or nateglinide may be a promising option for the treatment

of type 2 diabetes, and especially for controlling postprandial hyperglycemia.


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Acknowledgements

We express thanks to S. Yoshikawa, K. Kira, E. Emori, F. Matsuura, H. Ikuta, Y.

Takeuchi, Y. Kitahara, M. Bando, and Y. Sakuma for assistance and suggestions.

Thanks are also due to I. Hishinuma and J. Nagakawa for valuable advice and

encouragement.


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References

Aguilar-Bryan L, Nichols CG, Wechsler SW, Clement JP, IV, Boyd AE, III, Gonzalez

G, Herrera-Sosa H, Nguy K, Bryan J and Nelson DA (1995) Cloning of the β cell

high-affinity sulfonylurea receptor: a regulator of insulin secretion. Science

268:423-426.

Ahrén B, Simonsson E, Larsson H, Landin-Olsson M, Torgeirsson H, Jansson P-A,

Sandqvist M, Båvenholm P, Efendic S, Eriksson JW, Dickinson S and Holmes D

(2002) Inhibition of dipeptidyl peptidase IV improves metabolic control over a

4-week study period in type 2 diabetes. Diabetes Care 25:869-875.

Ahrén B, Pacini G, Foley JE and Schweizer A (2005) Improved meal-related β-cell

function and insulin sensitivity by the dipeptidyl peptidase-IV inhibitor vildagliptin

in metformin-treated patients with type 2 diabetes over 1 year. Diabetes Care

28:1936-1940.

Burkey BF, Li X, Bolognese L, Balkan B, Mone M, Russell M, Hughes TE and Wang

PR (2005) Acute and chronic effects of the incretin enhancer vildagliptin in insulin

resistant rats. J Pharmacol Exp Ther 315:688-695.

Ceriello A (2000) The post-prandial state and cardiovascular disease: relevance to

diabetes mellitus. Diabetes Metab Res Rev 16:125-132.

Deacon CF, Ahrén B and Holst JJ (2004) Inhibitors of dipeptidyl peptidase IV: a novel

approach for the prevention and treatment of Type 2 diabetes? Expert Opin

Investig Drugs 13:1091-1102.

Demuth H-U, McIntosh CH and Pederson RA (2005) Type 2 diabetes - therapy with

dipeptidyl peptidase IV inhibitors. Biochim Biophys Acta 1751:33-44.

Ehses JA, Pelech SL, Pederson RA and McIntosh CHS (2002) Glucose-dependent

insulintropic polypeptide activates the Raf-Mek1/2-ERK1/2 module via a cyclic


at ASPE



Dow

nloaded from


JPET #112011

18

AMP/cAMP-dependent protein kinase/Rap1-mediated pathway. J Biol Chem

277:37088-37097.

Fujita T, Seto Y, Kondo N and Kato R (1996) Studies on the

N-[(trans-4-isopropylcyclohexyl)-carbonyl]-D-phenylalanine (A-4166) receptor in

HIT T-15 cells. Displacement of [3H]glibenclamide. Biochem Pharmacol

52:407-411.

Gromada J, Brock B, Schmitz O and Rorsman P (2004) Glucagon-like peptide-1:

regulation of insulin secretion and therapeutic potential. Basic Clin Phamacol

Toxicol 95:252-262.

Gutniak MK, Linde B, Holst JJ and Efendic S (1994) Subcutaneous injection of the

incretin hormone glucagon-like peptide 1 abolishes postprandial glycemia in

NIDDM. Diabetes Care 17:1039-1044.

Gutniak MK, Juntti-Berggren L, Hellström PM, Guenifi A, Holst JJ and Efendic S

(1996) Glucagon-like peptide I enhances the insulinotropic effect of glibenclamide

in NIDDM patients and in the perfused rat pancreas. Diabetes Care 19:857-863.

Hansotia T, Baggio LL, Delmeire D, Hinke SA, Yamada Y, Tsukiyama K, Seino Y,

Holst JJ, Schuit F and Drucker DJ (2004) Double incretin receptor knockout

(DIRKO) mice reveal an essential role for the enteroinsular axis in transducing the

glucoregulatory actions of DPP-IV inhibitors. Diabetes 53:1326-1335.

Holst JJ and Gromada J (2004) Role of incretin hormones in the regulation of insulin

secretion in diabetic and nondiabetic humans. Am J Physiol Endocrinol Metab

287:E199-E206.

Ikenoue T, Akiyoshi M, Fujitani S, Okazaki K, Kondo N and Maki T (1997)

Hypoglycaemic and insulinotropic effects of a novel oral antidiabetic agent,

(-)-N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine (A-4166). Br J


at ASPE



Dow

nloaded from


JPET #112011

19

Pharmacol 120:137-145.

Kashima Y, Miki T, Shibasaki T, Ozaki N, Miyazaki M, Yano H and Seino S (2001)

Critical role of cAMP-GEFII·Rim2 complex in incretin-potentiated insulin

secretion. J Biol Chem 276:46046-46053.

Kim D, Wang L, Beconi M, Eiermann GJ, Fisher MH, He H, Hickey GJ, Kowalchick

JE, Leiting B, Lyons K, Marsilio F, McCann ME, Patel RA, Petrov A, Scapin G,

Patel SB, Roy RS, Wu JK, Wyvratt MJ, Zhang BB, Zhu L, Thornberry NA and

Weber AE (2005) (2R)-4-Oxo-4-[3-(trifluoromethyl)-5,6-

dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-a

mine: a potent, orally active dipeptidyl peptidase IV inhibitor for the treatment of

type 2 diabetes. J Med Chem 48:141-151.

Krentz AJ and Bailey CJ (2005) Oral antidiabetic agents. Current role in type 2 diabetes

mellitus. Drugs 65:385-411.

Monnier L (2000) Is postprandial glucose a neglected cardiovascular risk factor in type

2 diabetes? Eur J Clin Invest 30(Suppl. 2):3-11.

Nauck MA, Kleine N, Ørskov C, Holst, JJ, Willms B and Creutzfeldt W (1993)

Normalization of fasting hyperglycaemia by exogenous glucagon-like peptide 1

(7-36 amide) in type 2 (non-insulin-dependent) diabetic patients. Diabetologia

36:741-744.

Pederson RA, White HA, Schlenzig D, Pauly RP, McIntosh CH and Demuth H-U

(1998) Improved glucose tolerance in Zucker fatty rats by oral administration of

the dipeptidyl peptidase IV inhibitor isoleucine thiazolidide. Diabetes

47:1253-1258.

Reimer MK, Holst JJ and Ahrén B (2002) Long-term inhibition of dipeptidyl peptidase

IV improves glucose tolerance and preserves islet function in mice. Eur J


at ASPE



Dow

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JPET #112011

20

Endocrinol 146:717-727.

Sorbera LA, Revel L and Castañer L (2001) P32/98. Antidiabetic dipeptidyl peptidase

IV inhibitor. Drugs Future 26:859-864.

Takasaki K, Nakajima T, Ueno K, Nomoto Y and Higo K (2004) Effects of

combination treatment with dipeptidyl peptidase IV inhibitor and sulfonylurea on

glucose levels in rats. J Pharmacol Sci 95:291-293.

Temelkova-Kurktschiev TS, Koehler C, Henkel E, Leonhardt W, Fuecker K and

Hanefeld M (2000) Postchallenge plasma glucose and glycemic spikes are more

strongly associated with atherosclerosis than fasting glucose or HbA1c level.

Diabetes Care 23:1830-1834.

UK Prospective Diabetes Study (UKPDS) Group (1998) Intensive blood-glucose

control with sulphonylureas or insulin compared with conventional treatment and

risk of complications in patients with type 2 diabetes (UKPDS 33). Lancet

352:837-853.

Van Gaal LF and De Leeuw IH (2003) Rational and options for combination therapy in

the treatment of Type 2 diabetes. Diabetologia 46(Suppl. 1): M44-M50.

Villhauer EB, Brinkman JA, Naderi GB, Burkey BF, Dunning BE, Prasad K, Mangold

BL, Russell ME and Hughes TE (2003) 1-[[(3-Hydroxy-1-

adamantyl)amino]acetyl]-2-cyano-(S)-pyrrolidine: a potent, selective, and orally

bioavailable dipeptidyl peptidase IV inhibitor with antihyperglycemic properties. J

Med Chem 46:2774-2789.

Winzell, MS and Ahrén B (2004) The high-fat diet-fed mouse. A model for studying

mechanisms and treatment of impaired glucose tolerance and type 2 diabetes.

Diabetes 53 Suppl. 3:S215-S219.

Yasuda N, Nagakura T, Inoue T, Yamazaki K, Katsutani N, Takenaka O, Clark R,


at ASPE



Dow

nloaded from


JPET #112011

21

Matsuura F, Emori E, Yoshikawa S, Kira K, Ikuta H, Okada T, Saeki T, Asano O

and Tanaka I (2006) E3024, 3-but-2-ynyl-5-methyl-2-piperazin-1-yl-3,5-

dihydro-4H-imidazo[4,5-d]pyridazin-4-one tosylate, is a novel, selective and

competitive dipeptidyl peptidase IV inhibitor. Eur J Pharmacol 548:181-187.

Yip RG and Wolfe MM (2000) GIP biology and fat metabolism. Life Sci 66:91-103.


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Legends for figures

Figure 1. Chemical structure of E3024, 3-but-2-ynyl-5-methyl-2-piperazin-1-yl-3,5-

dihydro-4H- imidazo[4,5-d]pyridazin- 4-one tosylate.

Figure 2. Effects of a combination of E3024 (3 mg/kg) and glybenclamide (1 mg/kg) on

blood glucose (A and B) and plasma insulin levels (C) in an oral glucose tolerance

test using mice fed a high-fat diet. The compound(s) and glucose (2 g/kg) were

orally administered at -0.5 and 0 hr, respectively. Changes of blood glucose levels

and AUC values of delta blood glucose values between 0 and 2 hr are indicated in

A and B, respectively. Values are expressed as the mean ± S.E.M. of seven mice.

Results of two-way ANOVA on the AUC values and plasma insulin levels are

indicated in insets (B and C, respectively). Results of the Tukey multiple

comparison test are as follows: *, p<0.05 vs. vehicle group; #, p<0.05 vs. E3024

group; $, p<0.05 vs. glybenclamide group.

Figure 3. Effects of a combination of E3024 (3 mg/kg) and nateglinide (50 mg/kg) on

blood glucose (A and B) and plasma insulin levels (C) in an oral glucose tolerance

test using mice fed a high-fat diet. The compound(s) and glucose (2 g/kg) were

orally administered at -0.5 and 0 hr, respectively. Changes of blood glucose levels

and AUC values of delta blood glucose values between 0 and 2 hr are indicated in

A and B, respectively. Values are expressed as the mean ± S.E.M. of seven mice.

Results of two-way ANOVA on the AUC values and plasma insulin levels are

indicated in insets (B and C, respectively). Results of the Tukey multiple

comparison test are as follows: *, p<0.05 vs. vehicle group; #, p<0.05 vs. E3024

group; $, p<0.05 vs. nateglinide group.

Figure 4. Effects of a combination of glybenclamide (1 mg/kg) and GLP-1 (1 µg) on

blood glucose levels in and oral glucose tolerance test using mice fed a high-fat


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diet. Glybenclamide was orally administered at -0.5 hr, and glucose (2 g/kg; p.o.)

and GLP-1 (1 µg; i.p.) treatment were conducted at 0 hr. Changes of blood glucose

levels and AUC values of delta blood glucose values between 0 and 2 hr are

indicated. Values are expressed as the mean ± S.E.M. of six mice. Results of

two-way ANOVA on the AUC values are indicated in an inset. Results of the

Tukey multiple comparison test are as follows: *, p<0.05 vs. vehicle + saline

group; #, p<0.05 vs. vehicle + GLP-1 group; $, p<0.05 vs. glybenclamide + saline

group.

Figure 5. Effects of a combination of glybenclamide (1 mg/kg) and GIP (1 µg) on blood

glucose levels in and oral glucose tolerance test using mice fed a high-fat diet.

Glybenclamide was orally administered at -0.5 hr, and glucose (2 g/kg; p.o.) and

GIP (1 µg; i.p.) treatment were conducted at 0 hr. Changes of blood glucose levels

and AUC values of delta blood glucose values between 0 and 2 hr are indicated.

Values are expressed as the mean ± S.E.M. of six mice. Results of two-way

ANOVA on the AUC values are indicated in an inset. Results of the Tukey

multiple comparison test are as follows: *, p<0.05 vs. vehicle + saline group; #,

p<0.05 vs. vehicle + GIP group.

Figure 6. Effects of a combination of glybenclamide (1 mg/kg) and nateglinide (50

mg/kg) on blood glucose levels in and oral glucose tolerance test using mice fed a

high-fat diet. The compound(s) and glucose (2 g/kg) were orally administered at

-0.5 and 0 hr, respectively. Changes of blood glucose levels and AUC values of

delta blood glucose values between 0 and 2 hr are indicated. Values are expressed

as the mean ± S.E.M. of six mice. Results of two-way ANOVA on the AUC values

are indicated in an inset (B). Results of the Tukey multiple comparison test are as

follows: *, p<0.05 vs. vehicle group.


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Figure 7. Effects of a combination of E3024 (3 mg/kg) and glybenclamide (1 mg/kg) on

fasting blood glucose levels in normal rats. Changes of blood glucose levels (A)

and delta blood glucose values between 0 and 2 hr (B) are indicated. Results are

expressed as the mean ± S.E.M. of six rats. Results of two-way ANOVA on the

delta blood glucose levels at 2 hr are indicated in an inset (B). Results of the Tukey

multiple comparison test are as follows: *, p<0.05 vs. vehicle group; #, p<0.05 vs.

E3024 group.

Figure 8. Effects of a combination of E3024 (3 mg/kg) and nateglinide (50 mg/kg) on

fasting blood glucose levels in normal rats. Changes of blood glucose levels (A)

and delta blood glucose values between 0 and 1 hr (B) are indicated. Results are

expressed as the mean ± S.E.M. of six rats. Results of two-way ANOVA on the

delta blood glucose levels at 1 hr are indicated in an inset (B). Results of the Tukey

multiple comparison test are as follows: *, p<0.05 vs. vehicle group; #, p<0.05 vs.

E3024 group.


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Title page Effects of the Combination of a Dipeptidyl ...jpet.aspetjournals.org/content/jpet/early/2006/11/08/jpet.106... · JPET #112011 3 Abstract Several combination therapies