SGLT; hSGLT2 (IC50 = 7.4 nM); hSGLT1 (IC50 = 1876 nM); rSGLT2 (IC50 = 6.73 nM); rSGLT1 (IC50 = 1166 nM); mSGLT2 (IC50 = 5.64 nM); mSGLT1 (IC50 = 1380 nM)

In a dose-dependent manner, Ipragliflozin (1-50 μM) considerably suppresses the proliferation of the human breast cancer cell line MCF-7. Ipragliflozin initially reduced breast cancer cell proliferation, but this effect was entirely eliminated when SGLT2 expression was knocked down using siRNA. This suggests that Ipragliflozin inhibits SGLT2 to minimize breast cancer cell proliferation. DNA production in MCF-7 cells was considerably decreased by high dosages (50 and 100 μM) of Ipragliflozin, as demonstrated by the BrdU assay [1].

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SGLT2 and SGLT1 inhibition assay [1]
Ipragliflozin concentration-dependently inhibited mouse, rat, and human SGLT2 activity at nanomolar concentrations (Table 1). For human SGLT2, the inhibitory effect of ipragliflozin was approximately five times greater than that of phlorizin, but for human SGLT1, it was only one ninth of that of phlorizin. Phloretin had very little potency to inhibit either SGLT. The ratios of selectivity (IC50 of human SGLT1/SGLT2) of ipragliflozin, phlorizin, and phloretin were 254, 6, and 3, respectively. In addition, ipragliflozin exhibited high selectivity for mouse and rat SGLT2. Dapagliflozin also potently and selectively inhibited mouse and human SGLT2 activity.

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Specificity assay [1]
To confirm the specificity, effects of Ipragliflozin on several representative receptors, channels, and transporters were examined using radioligand binding and enzyme assays. Ipragliflozin did not interact with various receptors, ion channels, and transporters such as adrenergic (α1, α2, and β), muscarinic (M1, M2, and non-selective), angiotensin (AT1 and AT2), calcium channel (L-type and N-type), potassium channel (KATP and SKCa), sodium channel (site 2), cholecystokinin (CCKA and CCKB), dopamine (D1, D2, and transporter), endothelin (ETA and ETB), gamma-aminobutyric acid (GABAA and GABAB), glutamate (AMPA, kainate, and NMDA), serotonin (5-HT1, 5HT2B, and transporter), histamine (H1, H2, and H3), and neurokinin (NK1, NK2, and NK3), exhibiting IC50 values >3,000 nM.

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Stability of Ipragliflozin against mouse intestinal glucosidases [1]
The in vitro biological stability of ipragliflozin and phlorizin against glucosidases was examined using mucosal homogenates of mouse small intestine. While ipragliflozin was not degraded at all (Fig. 2a), phlorizin was quickly degraded in mouse mucosal homogenates into its aglycon, phloretin (Fig. 2b).

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Cancer is currently one of the major causes of death in patients with type 2 diabetes mellitus. We previously reported the beneficial effects of the glucagon-like peptide-1 receptor agonist exendin-4 against prostate and breast cancer. In the present study, we examined the anti-cancer effect of the sodium-glucose cotransporter 2 (SGLT2) inhibitor Ipragliflozin using a breast cancer model. In human breast cancer MCF-7 cells, SGLT2 expression was detected using both RT-PCR and immunohistochemistry. Ipragliflozin at 1-50 μM significantly and dose-dependently suppressed the growth of MCF-7 cells. BrdU assay also revealed that ipragliflozin attenuated the proliferation of MCF-7 cells in a dose-dependent manner. Because the effect of ipragliflozin against breast cancer cells was completely canceled by knocking down SGLT2, ipragliflozin could act via inhibiting SGLT2. We next measured membrane potential and whole-cell current using the patch clamp technique. When we treated MCF-7 cells with ipragliflozin or glucose-free medium, membrane hyperpolarization was observed. In addition, glucose-free medium and knockdown of SGLT2 by siRNA suppressed the glucose-induced whole-cell current of MCF-7 cells, suggesting that ipragliflozin inhibits sodium and glucose cotransport through SGLT2. Furthermore, JC-1 green fluorescence was significantly increased by ipragliflozin, suggesting the change of mitochondrial membrane potential. These findings suggest that the SGLT2 inhibitor ipragliflozin attenuates breast cancer cell proliferation via membrane hyperpolarization and mitochondrial membrane instability. [2]

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Ipragliflozin, also known as L-Proline, has been shown to be stable against intestinal glucosidases and to potently and selectively inhibit SGLT2 in humans, rats, and mice at nanomolar concentrations [5].

Ipragliflozin exhibits hypoglycemic properties. The rise in blood glucose levels is dose-dependently inhibited by ipragliflozin (0.1–1 mg/kg). This effect was significant at doses of 0.3 and 1 mg/kg in STZ-induced type 1 diabetic rats, and significant at all tested doses in KK-Ay type 2 diabetic mice [1]. In type 1 diabetic rats induced by streptozotocin, ipragliflozin (0.3 and 1 mg/kg) demonstrated antidiabetic effects at repeated doses [1].

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The pharmacological profile of Ipragliflozin (ASP1941; (1S)-1,5-anhydro-1-C-{3-[(1-benzothiophen-2-yl)methyl]-4-fluorophenyl}-D: -glucitol compound with L: -proline (1:1)), a novel SGLT2 selective inhibitor, was investigated. In vitro, the potency of ipragliflozin to inhibit SGLT2 and SGLT1 and stability were assessed. In vivo, the pharmacokinetic and pharmacologic profiles of ipragliflozin were investigated in normal mice, streptozotocin-induced type 1 diabetic rats, and KK-A(y) type 2 diabetic mice. Ipragliflozin potently and selectively inhibited human, rat, and mouse SGLT2 at nanomolar ranges and exhibited stability against intestinal glucosidases. Ipragliflozin showed good pharmacokinetic properties following oral dosing, and dose-dependently increased urinary glucose excretion, which lasted for over 12 h in normal mice. Single administration of ipragliflozin resulted in dose-dependent and sustained antihyperglycemic effects in both diabetic models. In addition, once-daily ipragliflozin treatment over 4 weeks improved hyperglycemia with a concomitant increase in urinary glucose excretion in both diabetic models. In contrast, ipragliflozin at pharmacological doses did not affect normoglycemia, as was the case with glibenclamide, and did not influence intestinal glucose absorption and electrolyte balance. These results suggest that ipragliflozin is an orally active SGLT2 selective inhibitor that induces sustained increases in urinary glucose excretion by inhibiting renal glucose reabsorption, with subsequent antihyperglycemic effect and a low risk of hypoglycemia. Ipragliflozin has, therefore, the therapeutic potential to treat hyperglycemia in diabetes by increasing glucose excretion into urine.[1]

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Effect of Ipragliflozin on urinary glucose excretion in normal mice [1]
In normal mice, ipragliflozin (0.01–10 mg/kg) dose-dependently and significantly increased urinary glucose excretion, and this effect was still apparent 12–18 h after administration at doses of ≥0.3 mg/kg (Fig. 4a). Urine volume was also significantly increased at doses of 3 and 10 mg/kg (Fig. 4b).

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Effect of single administration of Ipragliflozin in diabetic animals In STZ-induced type 1 diabetic rats and KK-Ay type 2 diabetic mice, ipragliflozin (0.1−1 mg/kg) dose-dependently lowered blood glucose levels, and this effect was significant at all tested doses (Figs. 5a and 6a). During the oral glucose tolerance test (OGTT) 12 h after dosing, ipragliflozin (0.1–1 mg/kg) dose-dependently inhibited increases in blood glucose levels. In STZ-induced type 1 diabetic rats, this effect was significant at doses of 0.3 and 1 mg/kg (Fig. 5b), and in KK-Ay type 2 diabetic mice, the effect was significant at all tested doses (Fig. 6b).

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Effects of repeated administration of Ipragliflozin in STZ-induced type 1 diabetic rats [1]
Compared to the normal control rats, STZ-induced type 1 diabetic rats had significantly higher mean levels of HbA1c, blood glucose, and urinary glucose excretion and significantly lower plasma insulin levels and pancreatic insulin content under non-fasting conditions (Table 2). Repeated administration of ipragliflozin (0.3 and 1 mg/kg) for 4 weeks significantly reduced the levels of HbA1c and blood glucose. Plasma insulin level was not significantly changed, but pancreatic insulin content was significantly increased at a dose of 1 mg/kg. Urinary glucose excretion was increased dose-dependently, and this was significant at the 1 mg/kg dose. Ipragliflozin did not affect body weight or food intake (data not shown) throughout the study.

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Effects of repeated administration of Ipragliflozin in KK-Ay type 2 diabetic mice [1]
Repeated administration of ipragliflozin (0.3 and 1 mg/kg) for 4 weeks reduced HbA1c and blood glucose levels, with concomitant increases in urinary glucose excretion (Table 3). In addition, urinary albumin excretion was significantly decreased. Ipragliflozin treatment did not affect body weight or food intake (data not shown) throughout the study.

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Effect of Ipragliflozin on fasting blood glucose levels in normal mice [1]
In normal mice, ipragliflozin (0.03–100 mg/kg) dose-dependently inhibited the increase in blood glucose level after glucose loading, and this effect was significant at doses ≥0.1 mg/kg (Fig. 7a). Glibenclamide (0.3–300 mg/kg) also dose-dependently inhibited the increase in blood glucose levels; this effect was significant at doses ≥3 mg/kg (Fig. 7c). In overnight-fasted mice, ipragliflozin (0.03–100 mg/kg) dose-dependently reduced blood glucose levels, but this effect was only significant at doses ≥10 mg/kg, which are 100-fold higher than that in the OGTT (Fig. 7b). Glibenclamide (0.3–300 mg/kg) also dose-dependently reduced fasting blood glucose levels at the same doses as in the OGTT (Fig. 7d). Ipragliflozin did not alter plasma insulin levels under fasting conditions but significantly reduced the increase in plasma insulin levels under glucose loading conditions. In contrast, glibenclamide significantly increased plasma insulin levels under both conditions (data not shown).

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Effect of Ipragliflozin on gastrointestinal carbohydrate contents in normal mice [1]
Following liquid-meal loading in normal mice, gastrointestinal disaccharide (sucrose and maltose) and monosaccharide (glucose and fructose) contents increased significantly (Fig. 8). Voglibose (1 mg/kg) significantly increased gastrointestinal disaccharide content (Fig. 8a, b), decreased monosaccharide content (Fig. 8c, d), and significantly inhibited the increase in blood glucose levels (data not shown). In contrast, ipragliflozin (0.3–30 mg/kg) did not significantly affect gastrointestinal disaccharide content, even at the highest dose. In addition, ipragliflozin did not significantly affect gastrointestinal fructose content, and although it did dose-dependently increased glucose content, this effect was only significant at the maximum dose of 30 mg/kg. Ipragliflozin dose-dependently inhibited the increase in blood glucose levels, and this effect was significant at all tested doses (data not shown).

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Effect of Ipragliflozin on plasma and urinary parameters in KK-Ay type 2 diabetic mice [1]
In type 2 diabetic mice, the loop diuretic, furosemide (10 mg/kg), significantly increased urinary electrolyte (Na+, K+, and Cl−) excretion and urine volume, with a concomitant decrease in urinary osmolality (Table 4). This marked diuresis induced by electrolyte excretion also induced a significant decrease in plasma electrolyte concentrations and a significant increase in plasma osmolality. The vasopressin V1A/V2 receptor antagonist, YM471 (3 mg/kg), significantly increased urine volume without increasing electrolyte excretion and significantly decreased urinary osmolality. This marked water diuresis induced significant increases in plasma electrolyte concentrations and osmolality. In contrast, ipragliflozin (1 mg/kg) markedly increased urinary glucose excretion, with a concomitant slight increase in urine volume, and significantly decreased blood glucose levels, but did not significantly affect plasma or urinary electrolyte balance.

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Sodium-glucose cotransporter (SGLT) 2 plays an important role in renal glucose reabsorption, and inhibition of renal SGLT2 activity represents an innovative strategy for the treatment of hyperglycemia in diabetic patients. The present study investigated the antidiabetic effects of Ipragliflozin, a SGLT2-selective inhibitor, in streptozotocin-nicotinamide-induced mildly diabetic mice, which exhibited a mild decline in glucose tolerance associated with the loss of early-phase insulin secretion. Oral administration of ipragliflozin increased urinary glucose excretion in a dose-dependent manner, an effect which was significant at doses of 0.3 mg/kg or higher and lasted over 12 h. In addition, ipragliflozin dose-dependently improved hyperglycemia and glucose intolerance with concomitant decreases in plasma insulin levels without causing hypoglycemia. Once-daily dosing of ipragliflozin (0.1 - 3 mg/kg) for 4 weeks attenuated hyperglycemia, glucose intolerance, and impaired insulin secretion. These results suggest that the SGLT2-selective inhibitor ipragliflozin increases urinary glucose excretion by inhibiting renal glucose reabsorption, improves hyperglycemia in streptozotocin-nicotinamide-induced mildly diabetic mice, and may be useful for treating type 2 diabetes.[6]

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The sodium-glucose cotransporter 2 (SGLT2) is responsible for most glucose reabsorption in the kidney and has been proposed as a novel therapeutic target for the treatment of type 2 diabetes. In the present study, the therapeutic effects of SGLT2 selective inhibitor Ipragliflozin were examined in high-fat diet and streptozotocin-nicotinamide-induced type 2 diabetic mice which exhibit impaired insulin secretion, insulin resistance, hyperlipidemia, hepatic steatosis, and obesity. Single administration of ipragliflozin dose-dependently increased urinary glucose excretion, reduced blood glucose and plasma insulin levels, and improved glucose intolerance. Four-week repeated administration of ipragliflozin improved not only glucose tolerance, hyperglycemia, and hyperinsulinemia but also impaired insulin secretion, hyperlipidemia, hepatic steatosis, and obesity with a concomitant increase in urinary glucose excretion. In addition, ipragliflozin reduced plasma and liver levels of oxidative stress biomarkers (thiobarbituric acid reactive substances and protein carbonyl) and inflammatory markers (interleukin 6, tumor necrosis factor α, monocyte chemotactic protein-1, and c-reactive protein), and improved liver injury as assessed by plasma levels of aminotransferases. These results demonstrate that SGLT2 selective inhibitor ipragliflozin improves not only hyperglycemia but also diabetes/obesity-associated metabolic abnormalities in type 2 diabetic mice and suggest that ipragliflozin may be useful in treating type 2 diabetes with metabolic syndrome.[7]

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In mice that are healthy, Ipragliflozin (L-Proline) exhibits good pharmacokinetic qualities following oral treatment and enhances urine glucose excretion in a dose-dependent manner that lasts for more than 12 hours [5]. Ipagliflozin taken orally causes a dose-dependent increase in urine glucose excretion; this effect is most pronounced at dosages of 0.3 mg/kg and lasts longer than 12 hours [6]. Ipagliflozin dose-dependently improves glucose intolerance, lowers blood glucose and plasma insulin levels, and enhances urine glucose excretion [7].

SGLT2 and SGLT1 inhibition assay [1]
Human, rat, and mouse SGLT2 and SGLT1 full-length complementary deoxyribonucleic acid sequences were cloned and stably transfected into Chinese hamster ovary (CHO) cells using standard techniques as described previously (Katsuno et al. 2007). Cells were seeded into 96-well plates at a density of 3 × 104 cells/well in Ham’s F12 medium containing 10% fetal bovine serum. The cells were used 1 day after plating. Test compounds were initially dissolved in dimethyl sulfoxide and diluted to the desired concentration with sodium assay buffer (140 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 5 mM Tris–HCl, pH 7.4). After the medium was removed, the cells were preincubated in 100 μl choline assay buffer (NaCl in sodium assay buffer was replaced with the same concentration of choline chloride) at 37°C for 20 min. They were then incubated in the test compound solution (25 μl) containing 14C-AMG (2.2 μCi/ml) and nonlabeled AMG (final concentration 55 μM) at 37°C for 2 h. Cells were washed twice with 200 μl ice-cold wash buffer (choline assay buffer containing 10 mM AMG) and then solubilized in 0.5% sodium dodecyl sulfate (SDS) solution (25 μl). The cell lysate was mixed with 75 μl MicroScint MS-40, and radioactivity was measured using a Top Count Microplate Scintillation Counter.

Cell Viability Assay[2]
Cell Types: MCF-7 human breast cancer cell lines
Tested Concentrations: 1, 10, 50 μM
Incubation Duration: 24, 48, 72, 96 hrs (hours)
Experimental Results: diminished the number of MCF-7 cells in a dose- dependent manner.

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Cell culture and cell proliferation assays [2]
The MCF-7 and MDA-MB-231 human breast cancer cell lines were purchased from the American Type Culture Collectio. All cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cell prolif‐ eration assays were performed as described previously with minor modifications. Briefly, cells were see‐ ded in 12-well tissue culture plates and maintained in complete medium with 0–50 μM Ipragliflozin. Cell proliferation was analyzed 0–4 days later by cell count‐ ing using a hemocytometer.

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Bromodeoxyuridine (BrdU) assays [2]
The BrdU incorporation assay was performed using Cell Proliferation ELISA kits. Briefly, MCF-7 cells were plated at 5,000 cells/well in 96-well culture plates in complete media. After attaining 60%–70% conflu‐ ence, cells were treated with media containing 10% FBS with 0–100 μM Ipragliflozin for 24 h. BrdU solution (10 μM) was added during the last 2 h of stimulation. The cells were dried and fixed, and cellular DNA was dena‐ tured with FixDenat solution for 30 min at room temperature. A peroxidase-conjugated mouse anti-BrdU monoclonal antibody was added to the culture plates and cells were incubated for 90 min at room temperature. Tetramethyl‐ benzidine substrate was added and the plates were incu‐ bated for 15 min at room temperature. The absorbance of samples was measured using a microplate reader at 450– 620 nm. Mean data are expressed as the ratio relative to the proliferation of control (untreated) cells.

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Small interfering (si)RNA knockdown of SGLT2 and cell proliferation assay [2]
To knock down SGLT2, we used SGLT-2 siRNA, which was designed to target human SGLT2; control siRNA was used as a negative control. MCF-7 cells were plated at 2 × 105 cells/well in six-well plates and transfected with 10 nmol/L SGLT-2 siRNA or negative control siRNA using MISSION siRNA Transfection Reagent. Seventy-two hours after transfection, cells were subject‐ ed to cell proliferation assays. Briefly, cells were detached and re-plated in 24-well tissue culture plates in complete media with or without 10 μM Ipragliflozin. At 0–4 days after treatment, cells were collected and coun‐ ted using a hemocytometer.

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Mitochondrial permeability potential [2]
Mitochondrial membrane potential (ΔΨm) was exam‐ ined using a JC-1 mitochondrial membrane potential detection kit, according to the company’s instruc‐ tions. MCF-7 cells treated with or without 10 μM Ipragliflozin were stained with the cationic dye JC-1, which exhibits potential-dependent accumulation in mitochondria. At low membrane potential, JC-1 exists as a monomer and produces green fluorescence (emission at 527 nm). At high membrane potential and polarization, JC-1 forms J aggregates and produces red fluorescence (emission at 590 nm).

Animal/Disease Models: Single Administration[1] Streptozotocin (STZ; 50 mg/kg)-induced type 1 diabetic rats and KK-Ay type 2 diabetic mice
Doses: 0.1-1 mg/kg
Route of Administration: Single oral administration in the fed condition. Blood glucose levels were then measured for 8 h under fasting conditions.
Experimental Results: Dose-dependently lowered blood glucose levels, and this effect was significant at all tested doses.

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Animal/Disease Models: Repeated Administration[1] Streptozotocin (STZ; 50 mg/kg)- induced type 1 diabetic rats
Doses: 0.3 and 1 mg/kg
Route of Administration: Administration orally one time/day (at night) for 4 weeks.
Experimental Results: Dramatically decreased the levels of HbA1c and blood glucose. Pancreatic insulin content was Dramatically increased at a dose of 1 mg/kg. Urinary glucose excretion was increased dose-dependently, and this was significant at the 1 mg/kg dose.\

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Stability against mouse intestinal glucosidases [1]
Under ether anesthesia, the small intestine was removed from overnight fasted normal mice, washed with cold saline, excised, and rinsed with phosphate buffer (48 mM NaCl, 5.4 mM KCl, 28 mM Na2HPO4, 43 mM NaH2PO4, 35 mM mannitol, 10 mM glucose, pH 6.5). The mucosal tissue was scraped off gently using a slide glass, homogenized with phosphate buffer, and used for the stability study. Test compounds were initially dissolved in acetonitrile at a concentration of 5 mM and then diluted to 100 μM with the phosphate buffer. Mucosal homogenates (5 mg/ml, 100 μl) were preincubated at 37°C in microtubes. Thereafter, each compound solution (100 μl, final concentration 50 μM) was added and incubated at 37°C for varying time periods. The reaction was stopped by the addition of ice-cold acetonitrile (200 μl), then 200 μl of methyl tert-butyl ether was added, and the mixture was centrifuged (15,000 rpm, 10 min). The supernatant was transferred into a tube and evaporated in a vacuum centrifugal concentrator. The residue was dissolved in mobile phase for use as the assay sample. Concentrations of compound in the assay sample were analyzed using a high-performance liquid chromatography (HPLC) with an ultraviolet detector (265 nm for Ipragliflozin and 280 nm for phlorizin and phloretin) and a 4.6 × 250-mm reversed-phase ODS-80Ts column. The column temperature was maintained at 60°C, 20 mM ammonium acetate/acetonitrile [20:80 (v/v)] was used as the mobile phase, and the flow rate was 1.5 ml/min.

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Pharmacokinetics [1]
After oral administration of ipragliflozin (3 mg/kg) or phlorizin (100 mg/kg) to non-fasted normal mice, blood was withdrawn from the abdominal vena cava under ether anesthesia. The plasma concentrations of Ipragliflozin or phlorizin were measured using HPLC. Acetonitrile (100 μl) and methyl tert-butyl ether (100 μl) were added to the plasma samples (100 μl), mixed, and then centrifuged (15,000 rpm, 10 min). The supernatant was transferred into a tube and evaporated in a vacuum centrifugal concentrator. The residue was dissolved in HPLC mobile phase, 0.1% formic acid solution/acetonitrile [55:45 (v/v)] for use as the assay sample. Concentrations of ipragliflozin or phlorizin in the assay samples were measured as described above.

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Effect of Ipragliflozinon urinary glucose excretion in normal mice [1]
Ipragliflozin (0.01–10 mg/kg) was administered to non-fasted normal mice, and spontaneously voided urine was collected for 24 h after administration while the animals were kept in metabolic cages. After the urine volume had been measured, the glucose concentration in the urine was measured using the Glucose CII test reagent.

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Effect of single administration of Ipragliflozin in diabetic animals [1]
To investigate its antihyperglycemic effect, ipragliflozin (0.1–1 mg/kg) was administered to STZ-induced type 1 diabetic rats and KK-Ay type 2 diabetic mice in the fed condition. Blood glucose levels were then measured for 8 h under fasting conditions, in order to eliminate the influence of feeding during the experiment. To evaluate sustainability, ipragliflozin (0.1–1 mg/kg) was administered to both types of diabetic animals, which were then fasted for 12 h (overnight). A glucose solution (2 g/kg) was subsequently administered orally, and blood glucose levels were measured as described above.

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Effect of repeated administration of Ipragliflozin in STZ-induced type 1 diabetic rats [1]
Ipragliflozin (0.3 and 1 mg/kg) was administered to STZ-induced type 1 diabetic rats once daily (at night) for 4 weeks. Body weight and food intake were measured every week. After drug administration on day 26, rats were transferred to metabolic cages and spontaneously voided urine was collected for 24 h. On the morning after the final drug administration on day 28, blood samples were collected under non-fasting conditions, and the pancreas was isolated under ether anesthesia. Blood and urinary glucose concentrations were measured as described above. The pancreas was homogenized by adding acid–ethanol solution (75% ethanol, 23.5% purified water, and 1.5% concentrated hydrochloric acid) and incubating at 4°C for 1 h to extract the insulin. Subsequently, the culture was centrifuged and the supernatant was used as a measurement sample. Plasma and pancreatic insulin concentrations were measured using an enzyme-linked immunosorbent assay (ELISA) kit. Hemoglobin A1c (HbA1c) levels were measured using a DCA2000 System.

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Effect of repeated administration of Ipragliflozin in KK-Ay type 2 diabetic mice [1]
Ipragliflozin (0.3 and 1 mg/kg) was administered to KK-Ay type 2 diabetic mice once daily (at night) for 4 weeks. Body weight and food intake were measured every week. On the morning after the drug administration on day 28, blood samples were collected under non-fasting conditions. After the drug administration on day 30, mice were transferred to metabolic cages and spontaneously voided urine was collected for 24 h. Blood and urinary glucose concentrations, HbA1c, and plasma insulin levels were measured as described above. Urinary albumin concentration was measured using a mouse albumin ELISA.

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Effect of Ipragliflozin on fasting blood glucose levels in normal mice [1]
In order to investigate their effects on postprandial hyperglycemia, ipragliflozin (0.03–100 mg/kg) or glibenclamide (0.3–300 mg/kg) was administered to normal mice that had been fasted overnight. After 30 min, glucose solution (2 g/kg) was administered orally, and blood glucose levels were measured. To investigate their effects on hypoglycemia, ipragliflozin (0.03–100 mg/kg) or glibenclamide (0.3–300 mg/kg) was administered to normal mice fasted overnight, and blood glucose levels were measured.

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Effect of Ipragliflozin on gastrointestinal carbohydrate contents in normal mice [1]
After fasting for 24 h, mice received ipragliflozin (0.3–30 mg/kg) or voglibose (1 mg/kg). After 15 min, a liquid meal (ENSURE·H: carbohydrates 206 mg/ml, fats 53 mg/ml, proteins 53 mg/ml) was given orally at 20 ml/kg. Control mice were given purified water instead of a liquid meal. At 1 h after the liquid meal or water administration, blood glucose levels were measured, and gastrointestinal tracts (stomach, upper and lower small intestine, cecum, and lower large intestine) were isolated under ether anesthesia. Isolated gastrointestinal tracts were homogenized with purified water (5 ml) and centrifuged (3,000 rpm, 10 min) to retrieve the supernatant. Glucose concentration was measured as described above. Sucrose and maltose concentrations were measured by a previously described method with minor modifications (Dörner 1977). Fructose concentration was measured using a fructose assay kit.

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Effect of Ipragliflozin on plasma and urinary parameters in KK-Ay type 2 diabetic mice [1]
Ipragliflozin (1 mg/kg), furosemide (10 mg/kg), or YM471 (3 mg/kg) was administered to non-fasted diabetic mice, which were then transferred to metabolic cages. The mice were allowed free access to food and water, and spontaneously voided urine samples were collected for 8 h. Thereafter, blood samples were collected from the tail vein for the determination of blood glucose level. Under ether anesthesia, urine was collected from the bladder, and blood samples were collected from the abdominal vena cava. The volume of spontaneously voided urine combined with the urine in the bladder was measured. Blood and urine samples were centrifuged (15,000 rpm, 10 min), after which the supernatants were used for the determination of several parameters. Plasma and urine osmolalities were measured using a freezing point depression osmometer. Plasma and urine electrolyte (Na+, K+, and Cl−) concentrations were determined using a flame photometer, and the urinary electrolyte excretion was calculated as the product of the urine electrolyte concentration and the urine volume.

Pharmacokinetics [1]
After oral administration of Ipragliflozin (3 mg/kg) to normal mice, plasma concentrations of ipragliflozin reached a maximum at 1 h and then gradually decreased (Fig. 3). Obvious plasma concentrations were detected even 8 h after administration. In contrast, when phlorizin (100 mg/kg) was administered orally, plasma drug concentrations were low and rapidly eliminated.

[1]. Pharmacological profile of ipragliflozin (ASP1941), a novel selective SGLT2 inhibitor, in vitro and in vivo. Naunyn Schmiedebergs Arch Pharmacol. 2012 Apr;385(4):423-36.

[2]. SGLT2 inhibitor ipragliflozin attenuates breast cancer cell proliferation. Endocr J. 2020 Jan 28;67(1):99-106.

[3]. Discovery of Ipragliflozin (ASP1941): a novel C-glucoside with benzothiophene structure as a potent and selective sodium glucose co-transporter 2 (SGLT2) inhibitor for the treatment of type 2 diabetes mellitus. Bioorg Med Chem. 2012 May 15;20(10):3263-79

[4]. Tofogliflozin, a potent and highly specific sodium/glucose cotransporter 2 inhibitor, improves glycemic control in diabetic rats and mice. J Pharmacol Exp Ther. 2012 Jun;341(3):692-701.

[5]. Pharmacological profile of ipragliflozin (ASP1941), a novel selective SGLT2 inhibitor, in vitro and in vivo. Naunyn Schmiedebergs Arch Pharmacol. 2012 Apr;385(4):423-36.

[6]. Antidiabetic effects of SGLT2-selective inhibitor ipragliflozin in streptozotocin-nicotinamide-induced mildly diabetic mice. J Pharmacol Sci. 2012;120(1):36-44.

[7]. Effects of SGLT2 selective inhibitor ipragliflozin on hyperglycemia, hyperlipidemia, hepatic steatosis, oxidative stress, inflammation, and obesity in type 2 diabetic mice. Eur J Pharmacol. 2013 Sep 5;715(1-3):246-55.

Ipragliflozin is a glycoside.
Ipragliflozin is under investigation in Type 2 Diabetes and Diabetes Mellitus, Type 2.

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SGLT2, which is specifically expressed in the kidneys, plays an important role in renal glucose reabsorption (Jabbour and Goldstein 2008). In contrast, SGLT1 is highly expressed in the small intestine and mediates dietary glucose absorption (Pajor and Wright 1992). In order to elucidate the in vivo effect of ipragliflozin against intestinal SGLT1, we examined its effect on gastrointestinal carbohydrate absorption. α-Glucosidase inhibitors, such as voglibose, inhibit the small intestinal disaccharidases, sucrase and maltase (Matsuo et al. 1992). This slows the absorption of carbohydrates from the small intestine, thereby lowering postprandial hyperglycemia (Baron 1998). In this study, voglibose induced a significant increase in intestinal disaccharide content (by delaying disaccharide digestion) that inhibited the increase in blood glucose levels. Thus, α-glucosidase inhibitors are effective at preventing postprandial hyperglycemia by this mechanism. However, osmotic water retention induced by accumulation of intestinal disaccharide content can cause gastrointestinal symptoms such as soft feces or diarrhea (Vichayanrat et al. 2002). Ipragliflozin, however, did not affect intestinal disaccharide or fructose content, but at the maximum dose of 30 mg/kg, it did significantly increase intestinal glucose content. It is known that gastrointestinal expression of SGLTs and absorption of glucose depend mainly on SGLT1 (Turk et al. 1991) and that gastrointestinal drug concentrations immediately after oral administration are at a very high level (Masaoka et al. 2006). Thus, although ipragliflozin shows a 245-fold higher selectivity for mouse SGLT2 versus SGLT1, the significant increase in gastrointestinal glucose content with the highest dose of ipragliflozin is thought to be due to the inhibition of glucose absorption via SGLT1 in the small intestine. Nevertheless, gastrointestinal glucose elevation was only significant at a dose which is 100 times higher than that which significantly decreased postprandial hyperglycemia. It is therefore considered that therapeutic doses of ipragliflozin would not inhibit intestinal SGLT1, nor affect intestinal carbohydrate digestion and absorption. As such, the risk of gastrointestinal symptoms should be low. During the course of both acute and chronic experiments, no gastrointestinal side effects indicative of significant intestinal SGLT1 inhibition were noted at any dose.

Higher doses of Ipragliflozin slightly increased urine volume along with a significant increase in urinary glucose excretion. Since sodium-ion transportation accompanies the glucose transport promoted by SGLT2, we evaluated the effect of Ipragliflozin on plasma and urinary electrolyte balance. In this experiment, the loop diuretic, furosemide, induced a marked diuresis with concomitant increase in urinary electrolyte excretion, and decreased plasma concentrations of electrolytes including sodium. This furosemide-induced hyponatremia may be a severe adverse reaction (Sonnenblick et al. 1993). The vasopressin V1A/V2 receptor antagonist, YM471, also exerted a potent aquaretic effect and increased plasma electrolyte concentrations. In contrast, ipragliflozin (1 mg/kg) markedly increased urinary glucose excretion with a concomitant slight increase in urine volume, but did not affect plasma or urinary electrolyte balance. These results suggest that ipragliflozin would increase urinary glucose excretion without inducing either a marked osmotic diuresis or an electrolyte imbalance at pharmacological doses.

Mutations in the SGLT2 gene lead to the rare disorder, familial renal glucosuria, where glucose reabsorption in the kidneys is severely impaired and large amounts of glucose are excreted in the urine (Francis et al. 2004; Magen et al. 2005). Despite the severe glucosuria, patients appear to have a benign condition, with no serious adverse events or problems in kidney function. Familial renal glucosuria is also not associated with hypoglycemia and generally has no significant clinical manifestations (Santer et al. 2003). In this study, chronic administration of pharmacological doses of ipragliflozin did not induce significant adverse effects in diabetic models. Based on these findings, Ipragliflozin seems to be an effective and safe drug for the treatment of hyperglycemia.

In conclusion, the present study shows that Ipragliflozin is a potent selective SGLT2 inhibitor, possesses good pharmacokinetic characteristics, and exhibits a sustained antihyperglycemic effect by increasing urinary glucose excretion, without inducing hypoglycemia or insulin secretion. These results indicate the potential usefulness of ipragliflozin for further development as a therapeutic agent for hyperglycemia in patients with diabetes. Clinical trials with ipragliflozin are currently in progress, and proof-of-concept studies should clarify the suitability of ipragliflozin for the treatment of diabetes (Kashiwagi et al. 2010). [1]

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ion in human breast cancer cells, which is not expressed in human normal mammary gland. Our findings revealed that the SGLT2 inhibitor Ipragliflozin attenuated breast cancer cell proliferation and DNA synthesis (Fig. 1). The dose of ipragliflozin that attenuated breast cancer cell proliferation, 1–10 μM, was similar to its pharmacologi‐ cal concentration in serum, suggesting that our data generally mirror clinical conditions. Furthermore, orally administrated ipragliflozin distributes into glandular tis‐ sues in similar or higher concentrations compared with serum (unpublished data by Astellas Pharma). Although growth was also suppressed at a lower dose of ipragliflo‐ zin (Fig. 2A), ipragliflozin at a high dose, 50–100 μM, reduced DNA synthesis in the BrdU assay (Fig. 2C). These findings suggest that ipragliflozin attenuated breast cancer cell proliferation through not only inhibit‐ ing DNA synthesis but also other mechanisms, such as cell death including apoptosis. We examined apoptosis by TUNEL assay, however, reproducible apoptosis was not observed. Further examination using other methods is required. We focused on the sodium transport by SGLT2 because sodium uptake is emerging as a mech‐ anism of cancer biology including breast cancer. Ipragliflozin shut down sodium uptake through SGLT2 and induced membrane hyperpolarization of MCF-7 cells. We also found that ipragliflozin induced instability of ΔΨm, which may lead to apoptosis and necrosis of host cells. The mechanism by which SGLT2 induces mitochondrial membrane instability may involve either inhibition of glucose or inhibition of sodium transport. Intracellular sodium could alter ΔΨm via sodiumcalcium and sodium-hydrogen exchangers. However, low glucose might not regulate ΔΨm; glucose transporter 1 is also expressed in breast cancer cells and is an important energy regulator and therapeutic target. Further experiments may reveal other effects of SGLT2 inhibitors on cancer cells and explore combina‐ tion treatment with SGLT2 inhibitor, metformin and GLP-1. A meta-analysis and case report suggested anticancer effects of SGLT2 inhibitors. In conclusion, in this study, we showed that the SGLT2 inhibitor Ipragliflozin attenuates breast cancer cell proliferation via membrane hyperpolarization and mitochondrial membrane instability [2]

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A series of C-glucosides with various heteroaromatics has been synthesized and its inhibitory activity toward SGLTs was evaluated. Upon screening several compounds, the benzothiophene derivative (14a) was found to have potent inhibitory activity against SGLT2 and good selectivity versus SGLT1. Through further optimization of 14a, a novel benzothiophene derivative (14h; ipragliflozin, ASP1941) was discovered as a highly potent and selective SGLT2 inhibitor that reduced blood glucose levels in a dose-dependent manner in diabetic models KK-A(y) mice and STZ rats. [3]

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Sodium/glucose cotransporter 2 (SGLT2) is the predominant mediator of renal glucose reabsorption and is an emerging molecular target for the treatment of diabetes. We identified a novel potent and selective SGLT2 inhibitor, tofogliflozin (CSG452), and examined its efficacy and pharmacological properties as an antidiabetic drug. Tofogliflozin competitively inhibited SGLT2 in cells overexpressing SGLT2, and K(i) values for human, rat, and mouse SGLT2 inhibition were 2.9, 14.9, and 6.4 nM, respectively. The selectivity of tofogliflozin toward human SGLT2 versus human SGLT1, SGLT6, and sodium/myo-inositol transporter 1 was the highest among the tested SGLT2 inhibitors under clinical development. Furthermore, no interaction with tofogliflozin was observed in any of a battery of tests examining glucose-related physiological processes, such as glucose uptake, glucose oxidation, glycogen synthesis, hepatic glucose production, glucose-stimulated insulin secretion, and glucosidase reactions. A single oral gavage of tofogliflozin increased renal glucose clearance and lowered the blood glucose level in Zucker diabetic fatty rats. Tofogliflozin also improved postprandial glucose excursion in a meal tolerance test with GK rats. In db/db mice, 4-week tofogliflozin treatment reduced glycated hemoglobin and improved glucose tolerance in the oral glucose tolerance test 4 days after the final administration. No blood glucose reduction was observed in normoglycemic SD rats treated with tofogliflozin. These findings demonstrate that tofogliflozin inhibits SGLT2 in a specific manner, lowers blood glucose levels by increasing renal glucose clearance, and improves pathological conditions of type 2 diabetes with a low hypoglycemic potential. [4]

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C26H30FNO7S

519.5823

519.173

951382-34-6

Ipragliflozin;761423-87-4

57339444

White to off-white solid powder

2.297

6

10

5

36

628

6

C1C[C@H](NC1)C(=O)O.C1=CC=C2C(=C1)C=C(S2)CC3=C(C=CC(=C3)[C@H]4[C@@H]([C@H]([C@@H]([C@H](O4)CO)O)O)O)F

TUVGWWULBZIUBS-FVYIYGEMSA-N

InChI=1S/C21H21FO5S.C5H9NO2/c22-15-6-5-12(21-20(26)19(25)18(24)16(10-23)27-21)7-13(15)9-14-8-11-3-1-2-4-17(11)28-14;7-5(8)4-2-1-3-6-4/h1-8,16,18-21,23-26H,9-10H2;4,6H,1-3H2,(H,7,8)/t16-,18-,19+,20-,21+;4-/m10/s1

(2S,3R,4R,5S,6R)-2-[3-(1-benzothiophen-2-ylmethyl)-4-fluorophenyl]-6-(hydroxymethyl)oxane-3,4,5-triol;(2S)-pyrrolidine-2-carboxylic acid

Ipragliflozin L-proline; Ipragliflozin L-proline [JAN]; UNII-M6N3GK48A4; M6N3GK48A4; Ipragliflozin L-proline (JAN); DTXSID20241771; IPRAGLIFLOZIN L-PROLINE [MI]; ...; 951382-34-6;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~20 mg/mL (~38.49 mM)

Solubility in Formulation 1: ≥ 2 mg/mL (3.85 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2 mg/mL (3.85 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)