416 Original Article

Efficacy of Mitiglinide Combined with Dapagliflozin in Streptozotocin-nicotinamide-induced Type 2 Diabetic Rats and in Zucker Fatty Rats

Authors

K. Akahane1, T. Inoue1, A. Yokoyama1, A. Yaguchi1, K. Ojima1, S. Kiguchi1, K. Maruyama1, M. Kobayashi1, Y. Mori2

Affiliations

1

Key words ▶ mitiglinide ● ▶ dapagliflozin ● ▶ glinide ● ▶ SGLT2 inhibitor ●

Abstract

Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1387719 Published online: September 4, 2014 Drug Res 2015; 65: 416–421 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence K. Akahane Kissei Pharmaceutical Co., Ltd., 4365-1 Kashiwabara Hotaka Azumino-City Nagano-Pref. 399-8304 Japan Tel.:  + 81/(0)263/82 8820 Fax:  + 81/(0)263/82 8826 [email protected]. co.jp

 Pharmacology Research Laboratory R&D, Kissei Pharmaceutical Co., Ltd., Nagano, Japan  Division of Diabetes and Endocrinology, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, Japan



The efficacy of the combination of the rapid-acting insulin secretagogue mitiglinide and the sodium-glucose cotransporter 2 (SGLT2) inhibitor dapagliflozin was explored in streptozotocinnicotinamide-induced type 2 diabetic (STZ-NA) rats and in Zucker fatty (ZF) rats. The STZ-NA rats were prepared at 8 weeks of age. At 9 weeks of age, the combination study was conducted by oral glucose tolerance test (OGTT). At 13 weeks of age, ZF rats were dosed orally with dapagliflozin once daily up to the 22nd day. At days 15 and 22, the combination study was conducted by OGTT. In 2 different animal models, plasma glucose levels were strongly suppressed by the combination of mitiglinide and dapagliflozin as compared with either drug alone. The urinary glucose

Introduction



It is reported that there were 366 million people with diabetes in 2011 and that this number is expected to rise to 552 million by 2030 [1]. In addition to exercise therapy and insulinization, oral antihyperglycemic drugs such as sulfonylureas, biguanides, α-glucosidase inhibitors, rapidacting insulin secretagogues, thiazolidinediones, and dipeptidyl peptidase 4 (DPP-4) inhibitors are employed in the treatment of diabetes. These drugs are used not only as monotherapy but also in various combinations [2]. Prevention of the complications caused by hyperglycemia is a major goal of the diabetic treatment with these drugs. However, it is important for the treatment with combination therapy using drugs with different mechanisms of action to consider whether the combination of these drugs can supplement the weak point of each drug, such as side effects, with antihyperglycemic effect.

Akahane K et al. Combination of Antidiabetic Agents …  Drug Res 2015; 65: 416–421

excretion was drastically elevated in the dapagliflozin group, but the combination with mitiglinide suppressed it about 50 %. In STZ-NA rats, the plasma insulin secretion by the combination of both drugs was about at the same level as in the mitiglinide group. In ZF rats, the plasma insulin secretion by the combination of both drugs was less than mitiglinide group. Thus, in 2 different animal models, the combination of mitiglinide and dapagliflozin showed stronger antihyperglycemic action accompanied by less insulin secretion than mitiglinide alone, and reduced the urinary glucose excretion as compared with dapagliflozin used alone. These results indicate that the combination of mitiglinide and dapagliflozin can be a promising combination for the treatment of diabetic patients.

Mitiglinide is classified as a drug of the glinide type, and it causes a rapid onset of insulin secretion and has a reducing effect on postprandial hyperglycemia [3]. Since its fast action on the pancreatic β-cells in the islet of Langerhans is of short duration, mitiglinide is less likely to cause hypoglycemia when compared with sulfonylureas, and the load to the pancreatic β-cells is reported to be low [4]. The normal pancreatic insulin response to glucose shows a biphasic profile. In the first phase, the insulin level rises within the first 10 min of the presence of the glycemic stimulus, which phase is followed by a second phase of release lasting for as long as this stimulus is present. In patients with typical type 2 diabetes (T2DM), the loss of the first phase and a reduced and delayed second phase in insulin secretion are observed. Mitiglinide improves the first phase by causing a rapid onset of insulin secretion, and brings the pattern of insulin secretion closer to the physiological one [5]. Mitiglinide is used in combination with antidiabetes

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received 20.03.2014 accepted 23.07.2014

2

Original Article 417

Materials and Methods



Materials

Mitiglinide was synthesized by Kissei Pharmaceutical Co., Ltd. (Nagano, Japan). Dapagliflozin was purchased from Shanghai Haoyuan Chemexpress Co., Ltd. (Shanghai, China). Mitiglinide was suspended in 0.5 % carboxymethyl cellulose (CMC) solution, whereas dapagliflozin was dissolved in distilled water (DW). Nicotinamide was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan); and streptozotocin, from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals were obtained from commercial sources.

Animals

Male Sprague-Dawley (SD) rats (SLC Inc., Shizuoka, Japan), Zucker fatty (ZF) rats, and Zucker lean rats (Charles River Laboratories Japan Inc., Kanagawa, Japan) were used for the present study. The rats were housed in an animal facility with controlled humidity, temperature, and light-dark cycles, and allowed food and tap water ad libitum. The present study was conducted according to the guidelines approved by the Laboratory Animal Committee of Kissei Pharmaceutical Co., Ltd.

STZ-NA rats

Diabetes was induced in 8-week-old SD rats by and intraperitoneal injection of nicotinamide (150 mg/kg) dissolved in saline followed 15 min later by an intravenous injection of streptozotocin (50 mg/kg) dissolved in citrate buffer (0.05 M, pH = 4.5). Age-matched non-diabetic animals (normal) were injected with vehicle alone. At 9 weeks of age, the oral glucose tolerance test (OGTT) was performed; and plasma glucose levels were measured. Then, the diabetic animals were divided into 4 groups such that the fasting glucose level and area-under the curve (AUC) from 0 to 1 h were similar among the groups.

Oral glucose tolerance test (OGTT, 1 g/kg) on STZ-NA rats

Animals were divided into the following 5 groups: Normal rats, 0.5 % CMC + DW; Control (STZ-NA rats), 0.5 % CMC + DW; Mitiglinide (STZ-NA rats), mitiglinide (1 mg/kg) + DW; Dapagliflozin (STZ-NA rats), 0.5 % CMC + dapagliflozin (0.1 mg/kg); Mitiglinide + Dapagliflozine (STZ-NA rats), mitiglinide (1 mg/kg) + dapagliflozin (0.1 mg/kg). The dose of mitiglinide and dapagliflozin referred to previous reports [16–18]. The OGTT was performed at 9 weeks of age. After an overnight fast, each drug was administered orally followed by administration of glucose solution (1 g/kg). Blood samples were collected immediately before dosing and at 0.25, 0.5, 1, 2, 3 and 5 h post dose from a tail vein (100 µL in each time point). Urine was collected over a 6-h period by using a metabolic cage. In brief, after an overnight fast, rats were individually placed into the metabolic cage, and the OGTT was performed. Plasma and urinary glucose levels were measured by using LabAssayTM Glucose (Wako Pure Chemical Industries, Ltd. Osaka, Japan). Plasma insulin was measured with a Rat Insulin ELISA Kit (Morinaga Institute of Biological Science, Inc., Kanagawa, Japan). Plasma samples at each time point were analyzed for the presence of glucose and insulin. The urine glucose and urine volume data were normalized by body weight.

ZF rats

At 13 weeks of age, the OGTT was performed; and the plasma glucose level was measured. Then, the ZF rats were divided into 4 groups such that the fasting glucose level and AUC from 0 to 1 h were similar among the groups. Zucker lean rats were used as the Normal group.

Oral glucose tolerance test (OGTT, 1 g/kg) on ZF rats

The animals were divided into the following 5 groups: Normal (Zucker lean), 0.5 % CMC + DW; Control (ZF), 0.5 % CMC + DW; Mitiglinide (ZF), mitiglinide (1 mg/kg) + DW; Dapagliflozin (ZF), 0.5 % MC + dapagliflozin (0.1 mg/kg); Mitiglinide + Dapagliflozine (ZF), mitiglinide (1 mg/kg) + dapagliflozin (0.1 mg/kg). Dapagliflozin was daily administered orally to the Dapagliflozin group and Mitiglinide + Dapagliflozine group up to 22nd day. The OGTT was performed at days 15 and 22 under the fasting condition. On day 15, mitiglinide was administered orally followed by the administration of glucose solution (1 g/kg). On day 22, each drug was administered orally followed by the administration of glucose solution (1 g/kg). Blood samples were collected immediately before dosing and at 0.25, 0.5, 1, 2, 3 and 5 h post dose from a tail vein at day 15. Urine was collected into a metabolic cage over a 6-h period on day 22. The urine glucose and urine volume data were normalized by body weight.

Akahane K et al. Combination of Antidiabetic Agents …  Drug Res 2015; 65: 416–421

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drugs having different action mechanisms, such as α-glucosidase inhibitors, DPP-4 inhibitors, and so on. It is reported that these combinations appear to be more effective in controlling postprandial glucose excursion without inducing hypoglycemia in patients with T2DM than is either drug used alone [6–9]. Recently, SGLT2 inhibitors have been developed all over the world as a new type of diabetic drug. SGLT2 inhibitors are classified as an insulin-independent type of antihyperglycemic drug [10]. SGLT2 inhibitors inhibit the re-absorption of glucose from the urine by blocking the action of the SGLT2 transporter, which is expressed in the S1 segments of the renal proximal tubule, and excrete of excess glucose into the urine [11]. In clinical trials, SGLT2 inhibitors have been shown to improve hyperglycemia even when used as a monotherapy. In addition to the antihyperglycemic effect of these inhibitors, weight reduction is also reported [12]. However, SGLT2 inhibitors have been reported to increase the incidence of genital and the urinary tract infections owing to the excessive excretion of glucose into the urine. Furthermore, ketoacidosis and a reduction in lean muscle mass are causes for concern in the insulin secretory disorder patient treated with such inhibitors [11–15]. It is thought that considerable amount of glucose has been excreted at postprandial hyperglycemia because excreting action of glucose by SGLT2 inhibitor is dependent on the blood glucose. Therefore, the combination of mitiglinide, which can reduce postprandial hyperglycemia, and an SGLT2 inhibitor is thought to protect against excessive excretion of glucose into the urine. In addition, SGLT2 inhibitors reportedly have an improving action on insulin resistance [10]. There is a possibility that this potency might lead to enhance the efficacy of insulin secreted by mitiglinide. For these reasons, the present study was undertaken to evaluate the efficacy of the combination of the rapid-acting insulin secretagogue mitiglinide and an SGLT2 inhibitor, dapagliflozin, in streptozotocin-nicotinamide-induced type 2 diabetic rats (STZ-NA rats) and in Zucker fatty rats (ZF rats) which develop obesity and hyperinsulinemia.

Statistical analysis

perglycemic effect of the combination of mitiglinide and dapagliflozin was judged as an additive effect. In contrast to the plasma glucose levels, the plasma insulin levels of the STZ-NA rats were lower in the control group than in the normal group. Oral administration of mitiglinide caused insulin secretion close to that of the normal group. In contrast, dapagli▶  Fig. 2a, b). Furtherflozin hardly affected insulin secretion ( ● more, insulin secretion by the combination of both drugs was the same as that obtained by the mitiglinide alone. Because the onset of urination was different in individual animals, comparisons of urinary glucose levels were performed by ▶  Fig. 3). During the using urinary glucose excretion for 6 h (  ● OGTT, no significant difference in the urine volume over the 6-h period was observed among the groups (normal, 5.1 ± 0.7; control, 6.1 ± 0.5; mitiglinide, 5.9 ± 0.6; dapagliflozin, 7.2 ± 0.6 and mitiglinide + dapagliflozin, 4.6 ± 0.3 mL, respectively). The combined treatment with mitiglinide and dapagliflozin inhibited urine glucose excretion about 50 % as compared with that for the dapagliflozin group, which showed a great increase in urinary glucose level. The main effects on urinary glucose excretion in mitiglinide and dapagliflozin groups were statistically significant (F(1, 20) = 7.81, P = 0.0112 and F(1, 20) = 54.44, P 

Efficacy of Mitiglinide Combined with Dapagliflozin in Streptozotocin-nicotinamide-induced Type 2 Diabetic Rats and in Zucker Fatty Rats.

The efficacy of the combination of the rapid-acting insulin secretagogue mitiglinide and the sodium-glucose cotransporter 2 (SGLT2) inhibitor dapaglif...
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