Characterization and comparison of SGLT2 inhibitors: Part 3. Effects on diabetic complications in type 2 diabetic mice

Author’s Accepted Manuscript Characterization and comparison of SGLT2 inhibitors: Part 3. Effects on diabetic complications in type 2 diabetic mice Atsuo Tahara, Toshiyuki Takasu, Masanori Yokono, Masakazu Imamura, Eiji Kurosaki www.elsevier.com/locate/ejphar

PII: DOI: Reference:

S0014-2999(17)30338-2 http://dx.doi.org/10.1016/j.ejphar.2017.05.019 EJP71209

To appear in: European Journal of Pharmacology Received date: 26 January 2017 Revised date: 5 May 2017 Accepted date: 10 May 2017 Cite this article as: Atsuo Tahara, Toshiyuki Takasu, Masanori Yokono, Masakazu Imamura and Eiji Kurosaki, Characterization and comparison of SGLT2 inhibitors: Part 3. Effects on diabetic complications in type 2 diabetic m i c e , European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2017.05.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Characterization and comparison of SGLT2 inhibitors: Part 3. Effects on diabetic complications in type 2 diabetic mice Atsuo Tahara*, Toshiyuki Takasu, Masanori Yokono, Masakazu Imamura, Eiji Kurosaki

Drug Discovery Research, Astellas Pharma Inc., Ibaraki, Japan

*Address correspondence to: Atsuo Tahara, Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan. Tel: +81 29 829 6292; fax: +81 29 852 5391. E-mail. [email protected] Abstract In this study, we investigated and compared the effects of all six sodium-glucose cotransporter (SGLT) 2 inhibitors commercially available in Japan on diabetes-related diseases and complications in type 2 diabetic mice. Following 4-week repeated administration to diabetic mice, all SGLT2 inhibitors showed significant improvement in diabetes-related diseases and complications, including obesity; abnormal lipid metabolism; steatohepatitis; inflammation; endothelial dysfunction; and nephropathy. While all SGLT2 inhibitors exerted comparable effects in reducing hyperglycemia, improvement of these diabetes-related diseases and complications was more potent with the two long-acting drugs (ipragliflozin and dapagliflozin) than with the four intermediate-acting four drugs (tofogliflozin, canagliflozin, empagliflozin, and luseogliflozin), albeit without statistical significance. These findings demonstrate that SGLT2 inhibitors alleviate 1

various diabetic pathological conditions in type 2 diabetic mice, and suggest that SGLT2 inhibitors, particularly long-acting drugs, might be useful not only for hyperglycemia but also in diabetes-related diseases and complications, including nephropathy in type 2 diabetes.

Keywords: Ipragliflozin; SGLT2 inhibitor; Hyperglycemia; Diabetes; Diabetic complications

1. Introduction According to the World Health Organization, approximately 180 million people worldwide currently have type 2 diabetes, a progressive metabolic disease characterized by chronic hyperglycemia and relative insulin deficiency as a result of impaired insulin secretion from pancreatic β-cells or insulin resistance. This number is estimated to double by 2030 (Cade, 2008). Type 2 diabetes is strongly associated with various metabolic disorders, such as obesity and dyslipidemia, and both micro-/macrovascular complications, including retinopathy, neuropathy, and nephropathy (microvascular), and ischemic heart disease, peripheral vascular disease, and cerebrovascular disease (macrovascular), resulting in serious organ and tissue damage in approximately one-third to half of diabetic patients (UK Prospective Diabetes Study, 1991). These are strongly associated with increased risk for cardiovascular and all-cause mortality in diabetic patients. Given the need for glycemic control in preventing diabetes-related metabolic disorders and complications, a number of antidiabetic drugs are currently available for the clinical treatment. In

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recent years, inhibitors of sodium-glucose cotransporter (SGLT) 2, which exert antihyperglycemic effects via increases in urinary glucose excretion, have been proposed as novel antihyperglycemic agents for treating type 2 diabetes. Although many studies have focused on nonclinical and clinical pharmacologic effects of SGLT2 inhibitors (Kurosaki and Ogasawara, 2013; Nauck, 2014), most of these studies have focused on the effects of these drugs on hyperglycemia, the most important biological feature of diabetes, with few addressing their effects on diabetes-related diseases or complications. Even studies on diabetic complications such as obesity and nephropathy have focused on individual SGLT2 inhibitors, with none comparing several SGLT2 inhibitors at once. We previously investigated and compared the pharmacokinetics, pharmacodynamics, and pharmacologic effects of all six SGLT2 inhibitors commercially available in Japan in type 2 diabetic mice (Tahara et al., 2016a;b). Our findings showed that these SGLT2 inhibitors could be classified into two categories, namely long-acting (ipragliflozin and dapagliflozin) and intermediate-acting (tofogliflozin, canagliflozin, empagliflozin, and luseogliflozin). In addition, while all SGLT2 inhibitors exerted antidiabetic effects, including a reduction in hyperglycemia, these pharmacologic effects were slightly superior with the long-acting compared with intermediate-acting SGLT2 inhibitors. However, that study focused only on the effects on the major symptoms of type 2 diabetes, such as improvement in hyperglycemia, glucose tolerance, and insulin resistance and prevention of pancreatic exhaustion, necessitating additional detailed investigation of diabetes-related diseases and complications to differentiate various SGLT2 inhibitors in terms of pharmacologic effects. Here, we used data and samples from the above study 3

as a basis for additional evaluation and comparison of the effects on improvement in various type 2 diabetic pathologies.

2. Materials and methods 2.1. Materials Ipragliflozin (Tahara et al., 2012), dapagliflozin (Komoroski et al., 2009), tofogliflozin (Suzuki et al., 2012), canagliflozin (Liang et al., 2012), empagliflozin (Grempler et al., 2012), and luseogliflozin (Kojima et al., 2013) were synthesized at Astellas Pharma Inc. (Ibaraki, Japan) and suspended in 0.5% methylcellulose solution for oral administration. Doses of drugs are expressed as the free base form.

2.2. Animals Male C57BL/6 (normal) and KK/Ay type 2 diabetic mice were purchased from CLEA Japan at age 6 weeks (Kanagawa, Japan) and uniformly grouped by blood glucose levels at age 7 weeks. All animals were housed under conventional conditions with controlled temperature, humidity, and light (12-h light-dark cycle) and were provided with standard commercial diet and water. Starting from the day after grouping, animals received feed only during the active period (i.e. fed for 14 h

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from 19:00 to 9:00, and fasted during the rest of the 24-h period), and three days after grouping, drug administration was started. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Astellas Pharma Inc. Further, Astellas Pharma Inc., Tsukuba Research Center has been awarded Accreditation Status by the AAALAC International.

2.3. Repeated administration study Vehicle or each SGLT2 inhibitor (ipragliflozin and dapagliflozin: 0.1-1 mg/kg, tofogliflozin, canagliflozin, empagliflozin, and luseogliflozin: 1-10 mg/kg) was orally administered to diabetic mice (and vehicle was orally administered to normal mice) once daily (just before feeding at night) for 4 weeks. Body weight was measured weekly. Daily food consumption was calculated based on the weekly difference in food remaining and food supplied. On the morning after final drug administration (Day 30), blood samples were collected under nonfasting conditions, and tissues (liver, kidney, and adipose tissues) were isolated under isoflurane anesthesia.

2.4. Biochemical determinations Plasma lipid concentrations and hepatic lipid contents were measured in accordance with the method previously reported (Tahara et al., 2011). Levels of aminotransferases—alanine amino transferase (ALT) and aspartate amino transferase (AST)—were measured using the Transaminase CII test reagent (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Hepatic contents of 5

thiobarbituric acid reactive substances (TBARS) and 4-hydroxynonenal (HNE) protein adducts were measured in accordance with the method previously reported (Atkinson and Eisenbarth, 2001) using a TBARS Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA) and HNE Adduct Competitive enzyme-linked immunosorbent assay (ELISA) Kit (CELL BIOLABS, INC., San Diego, CA, USA), respectively. Plasma concentrations of cytokines, interleukin (IL)-1b, IL-6, monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor α (TNF-α), and c-reactive protein (CRP), as well as those of cellular adhesion molecules (CAMs), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin were measured using commercial ELISA kits (R&D Systems Inc., Minneapolis, MN, USA). Plasma and urinary creatinine levels were measured using Determiner L CRE (Kyowa Medex Co., Ltd, Tokyo, Japan). Creatinine clearance (µl/min) was calculated based on urinary and plasma creatinine levels and urine volume. Urinary concentration of albumin was measured using mouse albumin ELISA. Urinary N-acetyl-β-D-glucosaminidase (NAG) activity was measured using NAG Test Shionogi (Shionogi Co. Ltd, Osaka, Japan). Urinary concentrations of kidney injury molecule-1 (KIM-1), nephrin, and podocalyxin were measured using commercial ELISA kits (R&D Systems Inc.).

2.5. Histopathology Preparation of specimens and histopathological examination were performed at CMIC Bioresearch Center Co., Ltd. (Yamanashi, Japan). Sagittal slices of renal tissue fixed in 10% neutral buffered formalin were embedded in paraffin, and 2-μm sections were cut for 6

morphological study. These sections were stained with hematoxylin and eosin, and periodic acid Schiff (PAS). All tissue samples were evaluated by an independent investigator blinded to the group to which the mouse belonged. All glomeruli and the entire microscopic area in each specimen were examined. A semiquantitative scoring system was utilized to evaluate histopathological changes (glomerulosclerosis, tubular dilatation, cell infiltration, and basophilic change) according to the percentage of affected renal tissue; 0: no lesion, 1: very slight (<10%), 2: slight (10%≤X<25%), 3: moderate (25%≤X<50%), 4: marked (≥50%) (Uehara et al., 1993).

2.6. Statistical analysis The experimental results are expressed as the mean ± standard error of means (S.E.M.) or standard deviation (S.D.). Significance of differences between normal and diabetic vehicle groups was assessed using Student’s t-test, while that between the vehicle and drug-treated groups was assessed using one-way ANOVA followed by post-hoc Tukey’s and Dunnett’s multiple comparison test. For the comparison of histopathological scores, Wilcoxon rank sum test was used to analyze the differences between normal and diabetic vehicle groups, while Steel’s multiple comparison test was used for comparisons between the vehicle and drug-treated groups. A value of P<0.05 was considered to be significant. Statistical and data analyses were conducted using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA).

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3. Results Following 4-week repeated administration to type 2 diabetic mice, despite differing effective doses, all SGLT2 inhibitors exerted dose-dependent, significant effects on reduction in hyperglycemia through increased urinary glucose excretion with comparable potency (Fig. 1A,B; published data) (Tahara et al., 2016b). In contrast, the amplitude of blood glucose excursion, as determined from standard deviation (S.D.) of blood glucose levels for 24 h, was smaller with the long-acting inhibitors (ipragliflozin, dapagliflozin) than with the intermediate-acting inhibitors (tofogliflozin, canagliflozin, empagliflozin, and luseogliflozin) (Fig. 1C; published data) (Tahara et al., 2016b). Diabetic mice displayed conditions characteristic of type 2 diabetic pathology, such as obesity, abnormal lipid metabolism, steatohepatitis, inflammation, endothelial dysfunction, and nephropathy. None of the SGLT2 inhibitors influenced food consumption throughout the study period, but all exhibited significant improvements or trends toward improvement in obesity parameters (body and visceral fat [sum of epididymal, perirenal, retroperitoneal, and mesenteric fat] weights) and abnormal lipid metabolism parameters (plasma levels of triglycerides, cholesterol, and non-esterified fatty acids [NEFAs]) (Fig. 2). The steatohepatitis parameters, liver weight, hepatic lipid (triglycerides and cholesterol) contents, plasma levels of ALT/AST, and hepatic contents of TBARS and 4-HNE protein adducts, were also significantly decreased or showed a decreasing trend (Fig. 3). Significant improvement or a trend toward improvement were also noted

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in plasma levels of proinflammatory cytokines (IL-1b, IL-6, MCP-1, and TNF-a) and CRP as inflammatory parameters (Fig. 4) and CAMs (ICAM-1, VCAM-1, and E-selectin) as endothelial dysfunction parameters (Fig. 5). Diabetic mice displayed markedly increased urinary albumin excretion, along with renal hypertrophy and increased creatinine clearance, representing diabetic nephropathy characterized by glomerular hyperfiltration (Fig. 6). All SGLT2 inhibitors were associated with dose-dependent, significant decreases in urinary albumin excretion as well as improvements in renal hypertrophy and decreases in creatinine clearance. Renal tubular injury markers (urinary NAG activity and excretion of KIM-1) and glomerular podocyte injury markers (urinary excretions of nephrin and podocalyxin) were also significantly decreased or showed a decreasing trend. Further, renal tissue lesions (glomerulosclerosis, tubular dilatation, cell infiltration, and basophilic change) showed significant improvement or a trend toward improvement (Fig. 7). Although we noted no marked or statistically significant differences in these effects among the six SGLT2 inhibitors, comparison at doses producing comparable reduction in hyperglycemia (ipragliflozin and dapagliflozin, 0.3 mg/kg; tofogliflozin, canagliflozin, empagliflozin, and luseogliflozin, 3 mg/kg) demonstrated the slight superiority of the long-acting drugs to the intermediate-acting drugs in amplitude of blood glucose excursion (an index of daily blood glucose control) and improvement in type 2 diabetic pathology, obesity (visceral fat weight), abnormal lipid metabolism (plasma triglycerides level), steatohepatitis (hepatic triglyceride content and plasma ALT level), inflammation (plasma IL-6 level), endothelial dysfunction (plasma ICAM-1 level), and 9

nephropathy (urinary albumin excretion and glomerulosclerosis score) (Fig. 8). In addition, improvements in all other diabetic parameters were also slightly starker with the long-acting drugs, than with the intermediate-acting drugs.

4. Discussion The incidence of type 2 diabetes has dramatically increased as lifestyles involving excessive calorie intake and little exercise have become more prevalent. Obesity and abnormal lipid metabolism are commonly associated with insulin resistance and hyperinsulinemia and are the major environmental risk factors for the development of type 2 diabetes (Kahn et al., 2006). Therefore, basic management of type 2 diabetes involves caloric restriction and exercise to promote the reduction of body fat mass. Various antidiabetic drugs are available for the clinical control of hyperglycemia in type 2 diabetic patients; however, insulin and peroxisome proliferator-activated receptor-g agonists aggravate obesity, and other drugs are also not expected to improve obesity or abnormal lipid metabolism, except glucagon-like peptide-1 (GLP-1) analogue, which has been reported to improve these symptoms via delayed gastric emptying and central anorectic action (Hermansen and Mortensen, 2007; Inoue et al., 2014). In the present study, diabetic mice presented with hyperphagia, obesity, and hyperlipidemia, and repeated administration of SGLT2 inhibitors

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improved obesity and abnormal lipid metabolism without affecting food consumption. In addition, we determined visceral fat weight as the sum of epididymal, perirenal, retroperitoneal, and mesenteric fat weights, and the obesity-improving effects of SGLT2 inhibitors did not considerably differ by site of adipose tissue. A previous study confirmed using dual-energy X-ray absorptiometry and computed tomography that SGLT2 inhibitor-induced reduction in body weight in obese rats was accompanied by reduced visceral and subcutaneous fat masses but not lean mass or bone mineral content (Yokono et al., 2014). In addition, these improvements in obesity and lipid abnormalities were thought to be dependent on promoting the use of fatty acids instead of glucose as an energy source via SGLT2 inhibitor-induced urinary glucose excretion with steady calorie loss. These findings suggest that SGLT2 inhibitors have therapeutic potential for improving obesity and lipid abnormalities in type 2 diabetes patients. Type 2 diabetes is commonly associated with insulin resistance, obesity, hypertension, and lipid abnormalities, and a recent study noted that these diseases are also closely associated with nonalcoholic fatty liver disease (NAFLD) (Marchesini et al., 2001). NAFLD is a chronic progressive liver disease characterized by hepatic steatosis accompanied by necroinflammation and hepatocellular injury (Angulo, 2002). Incidence of NAFLD is strongly suggested to be associated with type 2 diabetes and obesity and has risen dramatically over the past several decades (Shams et al., 2011). In addition, NAFLD is also associated with poor glycemic control and increased risk of cardiovascular and chronic kidney diseases in type 2 diabetic patients (Fruci et al., 2013). However, the mechanisms involved in the development and progression of NAFLD are poorly understood. In 11

the present study, diabetic mice manifested the conditions representing the pathology of steatohepatitis, including increased hepatic lipid contents and elevated plasma ALT/AST levels, which were significantly improved or showed a trend toward improvement following administration of SGLT2 inhibitors. In addition, significant improvements were also noted in hepatic contents of TBARS and 4-HNE protein adducts, which are oxidative stress markers in the liver. Oxidative stress is known to lead to cell and tissue damage via production of reactive oxygen species such as active oxygen and free radicals, and oxidative stress markers including TBARS have been reported to be significantly elevated in type 2 diabetes with NAFLD (Narasimhan et al., 2010). Taken together, these finding suggest that SGLT2 inhibitors improve hepatic steatosis due mainly to improvement in hyperglycemia and hyperinsulinemia, which in turn can reduce diabetes-induced oxidative stress, thus attenuating hepatic injury. SGLT2 inhibitors therefore have the potential to prevent NAFLD in type 2 diabetes. Many studies have reported elevated circulating levels of various proinflammatory cytokines and inflammatory markers such as CRP in patients with type 2 diabetes (Akash et al., 2013). Diabetic conditions, such as hyperglycemia and dyslipidemia, provoke oxidative and endoplasmic reticulum stresses in various peripheral tissues, thereby inducing the generation of various proinflammatory mediators and causing further inflammation, which in turn contributes to the progressive worsening of diabetic symptoms such as insulin resistance and pancreatic β-cell dysfunction. Inflammation is therefore a major risk factor for type 2 diabetes. In the present study, diabetic mice displayed elevated plasma levels of proinflammatory cytokines and CRP, suggesting 12

chronic inflammatory symptoms. SGLT2 inhibitors significantly improved these inflammation markers due mainly to improvement of hyperglycemia and insulin resistance. These findings suggested that SGLT2 inhibitors improve inflammatory symptoms secondary to the antidiabetic effects, including reduction in hyperglycemia, thereby suppressing aggravation of diabetic pathology. Diabetes-associated macrovascular complications are major clinical problems, and diabetic patients have a 2- to 4-fold greater incidence of coronary artery disease and 10-fold greater incidence of peripheral vascular diseases (Laakso, 1999). Up to 75% of diabetic patients die from vascular disease, and endothelial dysfunction plays an early and prominent role in this disease process (Ding and Triggle, 2005). Despite the introduction of new therapies, reducing the high cardiovascular morbidity and mortality remains difficult. Endothelial dysfunction can be detected by measurement of elevated plasma levels of CAMs. Indeed, high plasma levels of CAMs have been observed in patients with coronary heart disease and carotid artery atherosclerosis (Meigs et al., 2004). The present study also identified elevated plasma levels of CAMs, suggestive of vascular endothelial dysfunction, in diabetic mice. SGLT2 inhibitors significantly decreased levels of these endothelial dysfunction markers. Our study is the first to examine the therapeutic effects of SGLT2 inhibitors against endothelial dysfunction in a model of type 2 diabetes. Recently, it has been reported that empagliflozin reduced heart failure hospitalization and cardiovascular death in patients with type 2 diabetes and high cardiovascular risk (Zinman et al., 2015). To confirm and expand these potentially important therapeutic and novel biological findings, additional and 13

detailed examinations should be undertaken. Chronic hyperglycemia is associated with a high incidence of not only the abovementioned macrovascular complications but also microvascular ones. Diabetic nephropathy is a major chronic microvascular complication of type 2 diabetes which ultimately progresses to end-stage renal disease requiring dialysis therapy. In the present study, type 2 diabetic mice exhibited microalbuminuria, the established parameter of early stage of nephropathy; glomerular hyperfiltration, as assessed by creatinine clearance; increases in urinary NAG activity and excretion of KIM-1, as markers of renal tubular injury; and increases in urinary excretions of nephrin and podocalyxin, as markers of glomerular podocyte injury. In addition, diabetic mice developed renal histopathology consistent with early phases of diabetic nephropathy, including glomerulosclerosis, tubular dilatation, cell infiltration, and basophilic changes. SGLT2 inhibitors exerted significant diabetic nephropathy-improving effects, which manifested as improvement in not only glomerular hyperfiltration and glomerulosclerosis but also renal tubular and glomerular podocyte injury and cell infiltration. The pathogenesis and progression of diabetic nephropathy involve many factors, although the detailed mechanism remains unclear (Sharma and Sharma, 2013). Accordingly, the nephropathy-improving effects of SGLT2 inhibitors observed in the present study are considered to be multifactorial and not limited to just a direct effect of reducing hyperglycemia. Further, many reports have suggested correlation between nephropathy and the parameters examined in the present study, as follows: · Inflammation: Chronic low-grade inflammation and activation of the innate immune system, 14

especially proinflammatory cytokines such as IL-1b, are closely involved in the development and progression of diabetic nephropathy (Navarro-Gonzalez and Mora-Fernandez, 2008). · Endothelial dysfunction: Plasma levels of CAMs including VCAM-1 were elevated in type 2 diabetes with microvascular complications including nephropathy, suggesting that systemic endothelial dysfunction in renal glomerular and tubular endothelial cells might be strongly involved in the development and progression of diabetic nephropathy (Koga et al., 1998). · Oxidative stress: Plasma levels of oxidative stress markers were increased in diabetic nephropathy patients (Pan et al., 2010). In the present study, renal content of TBARS was significantly increased in diabetic mice, and SGLT2 inhibitors significantly reduced (data not shown). Oxidative stress may therefore play an important role in the pathogenesis of diabetic nephropathy. Here, we showed that SGLT2 inhibitors induced improvement in the above nephropathy-aggravating factors, suggesting that these effects are mediated not only by reduction in hyperglycemia but also by other mechanisms, including improvement in inflammation, enhanced oxidative stress, and endothelial dysfunction. These diabetic pathology-improving effects observed in the present study with SGLT2 inhibitors were all slightly superior (not statistically significant advantage) with the long-acting drugs (ipragliflozin and dapagliflozin) compared with the intermediate-acting drugs (tofogliflozin, canagliflozin, empagliflozin, and luseogliflozin). In the previous study as well, while all of the examined SGLT2 inhibitors comparably increased urinary glucose excretion and reduced HbA1c 15

levels, the long-acting drugs were superior to the intermediate-acting drugs in protection of pancreatic exhaustion and improvement in insulin resistance and glucose tolerance, possibly due to differences in the quality of blood glucose control, as measured by the amplitude of daily blood glucose excursion. Favorable blood glucose control involving minimal daily blood glucose variability has been reported in many studies to be closely correlated with suppression in pathogenesis and progression of diabetic pathology, complications in particular, through improvement in inflammation and elevated oxidative stress (Nalysnyk et al., 2010; Yamazaki et al., 2014). These findings suggested that not only long-term blood glucose control but also daily favorable blood glucose control is crucial for improving various pathological conditions of type 2 diabetes, including inflammation and nephropathy. In conclusion, these results provide evidence that all of the SGLT2 inhibitors studied in the present study reduce hyperglycemia through increase in urinary glucose excretion as well as improve various pathological conditions of diabetes, such as obesity and nephropathy, in type 2 diabetic mice. SGLT2 inhibitors, especially long-acting drugs, might be useful for not only hyperglycemia but also diabetic complications including nephropathy in type 2 diabetes.

Acknowledgements The authors thank Drs. Yuichi Tomura, Hideaki Minoura, Yuka Hayashizaki, Shoji Takakura, Seiji Kaku, and Wataru Uchida (Astellas Pharma Inc.) and Drs. Hiroshi Tomiyama, Akira Tomiyama, Yoshihiko Haino, and Yoshinori Kondo (Kotobuki Pharmaceutical Co., Ltd.) for their 16

valuable comments and continuing encouragement.

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Yokono, M., Takasu, T., Hayashizaki, Y., Mitsuoka, K., Kihara, R., Muramatsu, Y., Miyoshi, S., Tahara, A., Kurosaki, E., Li, Q., Tomiyama, H., Sasamata, M., Shibasaki, M., Uchiyama, Y., 2014. SGLT2 selective inhibitor ipragliflozin reduces body fat mass by increasing fatty acid oxidation in high-fat diet-induced obese rats. Eur. J. Pharmacol. 727, 66-74. Zinman B, Wanner C, Lachin JM, Fitchett D, Bluhmki E, Hantel S, Mattheus M, Devins T, Johansen OE, Woerle HJ, Broedl UC, Inzucchi SE; EMPA-REG OUTCOME Investigators., 2015. Empagliflozin, Cardiovascular Outcomes, and Mortality in Type 2 Diabetes. N. Engl. J. Med. 50, 1844-1850.

Figure legends Fig. 1. Effects of repeated administration of SGLT2 inhibitors on (A) urinary glucose excretion, (B) HbA1c levels, and (C) standard deviation (S.D.) of blood glucose levels for 24 h in type 2 diabetic mice. The values are the mean ± S.E.M. for six animals per group. *P<0.05 vs. normal group, #

P<0.05 vs. vehicle group. These figures are taken from previously reported data (Tahara et al.,

2016b).

Fig. 2 Effects of repeated administration of SGLT2 inhibitors on (A) food intake, (B) body weight, (C) total visceral fat weight, plasma levels of (D) triglycerides, (E) cholesterol, and (F) NEFAs in type 2 diabetic mice. The values are the mean ± S.E.M. for six animals per group. *P<0.05 vs. normal group, #P<0.05 vs. vehicle group. 22

Fig. 3 Effects of repeated administration of SGLT2 inhibitors on (A) liver weight, hepatic contents of (B) triglyceride and (C) cholesterol, plasma levels of (D) ALT and (E) AST, and hepatic contents of (F) TBARS and (G) 4-HNE protein adducts in type 2 diabetic mice. The values are the mean ± S.E.M. for six animals per group. *P<0.05 vs. normal group, #P<0.05 vs. vehicle group.

Fig. 4 Improvement in inflammatory parameters by repeated administration of SGLT2 inhibitors in type 2 diabetic mice. Effects of SGLT2 inhibitors on plasma levels of (A) IL-1b, (B) IL-6, (C) MCP-1, (D) TNF-a, and (E) CRP. The values are the mean ± S.E.M. for six animals per group. *P<0.05 vs. normal group, #P<0.05 vs. vehicle group.

Fig. 5 Improvement in endothelial dysfunction parameters by repeated administration of SGLT2 inhibitors in type 2 diabetic mice. Effects of SGLT2 inhibitors on plasma levels of (A) ICAM-1, (B) VCAM-1, and (C) E-selectin. The values are the mean ± S.E.M. for six animals per group. *P<0.05 vs. normal group, #P<0.05 vs. vehicle group.

Fig. 6 Improvement in diabetic nephropathy by repeated administration of SGLT2 inhibitors in type 2 diabetic mice. Effects of SGLT2 inhibitors on (A) urinary albumin excretion, (B) creatinine clearance, (C) kidney weight, urinary (D) activity of NAG and excretions of (E) KIM-1, (F) nephrin, and (G) podocalyxin. The values are the mean ± S.E.M. for six animals per group. 23

*P<0.05 vs. normal group, #P<0.05 vs. vehicle group. Fig. 7 Improvement in renal injury by repeated administration of SGLT2 inhibitors in type 2 diabetic mice. Effects of SGLT2 inhibitors on histopathological scores of (A) glomerulosclerosis, (B) tubular dilatation, (C) cell infiltration, and (D) basophilic change. The values are the mean ± S.D. for six animals per group. *P<0.05 vs. normal group, #P<0.05 vs. vehicle group. Representative light micrographs of renal tissue obtained from (E) normal, (F) diabetic vehicle-treated, and (G) ipragliflozin (1 mg/kg)-treated mice. PAS stain. Magnification: x25.

Fig. 8 Improvement in various diabetic parameters by repeated administration of SGLT2 inhibitors in type 2 diabetic mice. Effects of SGLT2 inhibitors at mid dose on (A) HbA1c level, (B) standard deviation (S.D.) of blood glucose level for 24 h, (C) total visceral fat weight, (D) plasma triglycerides level, (E) hepatic triglyceride content, plasma levels of (F) ALT, (G) IL-6, and (H) ICAM-1, (I) urinary albumin excretion, and (J) glomerulosclerosis score. The values are the mean ± S.E.M. or individual and median for six animals per group. *P<0.05 vs. normal group, #P<0.05 vs. vehicle group. Figures (A and B) are taken from previously reported data (Tahara et al., 2016b).

24

A Urinary glucose excretion (mg/day)

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Fig. 1

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Body weight (g)

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 42

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Fig. 8