When tubular cells are exposed to high glucose levels, tofofloxacin (3–30 nM) treatment for 24 hours inhibits the production of oxidative stress and the expression of the monocyte chemoattractant protein-1 (MCP-1) gene[2]. Treatment with tofofloxacin (3–30 nM; 8 days; tubular epithelial cells) prevents the high glucose-induced apoptotic cell death[2].

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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 Ki 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. [1]

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Ninety percent of glucose filtered by the glomerulus is reabsorbed by a sodium-glucose cotransporter 2 (SGLT2), which is mainly expressed on S1 and S2 segment of renal proximal tubules. Since SGLT-2-mediated glucose reabsorption is increased under diabetic conditions, selective inhibition of SGLT2 is a potential therapeutic target for the treatment of diabetes. We have recently shown that an inhibitor of SGLT2 has anti-inflammatory and antifibrotic effects on experimental diabetic nephropathy partly by suppressing advanced glycation end products formation and oxidative stress generation in the kidney. However, the direct effects of SGLT2 inhibitor on tubular cell damage remain unclear. In this study, we investigated the effects of Tofogliflozin, a highly selective inhibitor of SGLT2 on oxidative stress generation, inflammatory and proapoptotic reactions in cultured human proximal tubular cells exposed to high glucose. Tofogliflozin dose-dependently suppressed glucose entry into tubular cells. High glucose exposure (30 mM) for 4 and 24 h significantly increased oxidative stress generation in tubular cells, which were suppressed by the treatment of tofogliflozin or an antioxidant N-acetylcysteine (NAC). Monocyte chemoattractant protein-1 (MCP-1) gene expression and apoptotic cell death were induced by 4 h- and 8 day-exposure to high glucose, respectively, both of which were also blocked by tofogliflozin or NAC. The present study suggests that SGLT2-mediated glucose entry into tubular cells could stimulate oxidative stress and evoke inflammatory and proapoptotic reactions in this cell type. Blockade of glucose reabsorption in tubular cells by SGLT2 inhibitor might exert beneficial effects on tubulointerstitial damage in diabetic nephropathy. [2]

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The metabolism and drug-drug interaction (DDI) risk of Tofogliflozin, a potent and highly specific sodium-glucose co-transporter 2 inhibitor, were evaluated by in vitro studies using human liver microsomes, human hepatocytes, and recombinant human CYPs. 2. The main metabolite of tofogliflozin was the carboxylated derivative (M1) in human hepatocytes, which was the same as in vivo. The metabolic pathway of tofogliflozin to M1 was considered to be as follows: first, tofogliflozin was catalyzed to the primary hydroxylated derivative (M4) by CYP2C18, CYP4A11 and CYP4F3B, then M4 was oxidized to M1. 3. Tofogliflozin had no induction potential on CYP1A2 and CYP3A4. Neither tofogliflozin nor M1 had inhibition potential on CYPs, with the exception of a weak CYP2C19 inhibition by M1. 4. Not only are multiple metabolic enzymes involved in the tofogliflozin metabolism, but the drug is also excreted into urine after oral administration, indicating that tofogliflozin is eliminated through multiple pathways. Thus, the exposure of tofogliflozin would not be significantly altered by DDI caused by any co-administered drugs. Also, tofogliflozin seems not to cause significant DDI of co-administered drugs because tofogliflozin has no CYP induction or inhibition potency, and the main metabolite M1 has no clinically relevant CYP inhibition potency [4].

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In vitro activity: Tofogliflozin is the potent and most selective inhibitor of SGLT2; the selectivity of tofogliflozin toward SGLT2 is 2900 times that toward SGLT1. Tofogliflozin dose-dependently inhibited glucose entry into tubular cells, tofogliflozin suppressed high glucose-induced ROS generation, MCP-1 gene induction and apoptosis in tubular cells and an antioxidant NAC mimicked the effects of tofogliflozin on high glucoseexposed tubular cells Kinase Assay: Tofogliflozin (also known as CSG-452) hydrate is a novel, very potent and highly selective inhibitor of sodium/glucose cotransporter 2 (SGLT2) with Ki values of 2.9, 14.9, and 6.4 nM for human, rat, and mouse respectively. Tofogliflozin competitively inhibited SGLT2 in cells that overexpress SGLT2. The selectivity of tofogliflozin towards human SGLT2 versus human SGLT1, SGLT6, and sodium/myo-inositol transporter 1 is the highest among the tested SGLT2 inhibitors under clinical trials. Long-term inhibition of renal SGLT2 by tofogliflozin not only preserved pancreatic beta-cell function, but also prevented kidney dysfunction in a mouse model of type 2 diabetes. These findings suggest that long-term use of tofogliflozin in patients with type 2 diabetes may prevent progression of diabetic nephropathy. Cell Assay: Tubular cells are treated with or without 3 nM or 30 nM tofogliflozin under serum-free BM containing 10 μg/ml transferrin and GA-1 000 for 24 h at 37 °C. Then, the cells are washed with PBS and incubated with Hanks’ balanced salt solution (HBSS) containing 100 μM of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG), a fluorescent derivative of glucose, in the absence of tofogliflozin for 15 min. Culture medium is removed and replaced with HBSS, and fluorescence intensity in the cells is analyzed in an ARVO fluorescent plate reader.

A single oral administration of this compound lowers blood glucose levels in Zucker diabetic rats with increased renal glucose clearance and treatment for 4 weeks with this compound improves glucose tolerance in db/db mice. Tofogliflozin treatment lowers urine volume compared with the untreated control group at 8 weeks of treatment. Tofogliflozin treatment increases renal glucose clearance levels compared with untreated db/db mice, whereas losartan treatment has no effect on this parameter. Tofogliflozin treatment reduces the threshold of glucose reabsorption in db/db mice and increases the UGE, and then reduces the PG. Tofogliflozin treatment significantly and dose-dependently elevates the total beta-cell mass, suggesting that beta-cell loss is prevented. Tofogliflozin suppresses plasma glucose and glycated Hb and preserves pancreatic beta-cell mass and plasma insulin levels.

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The treatment of obese diabetic mice with tofogliflozin (0.1–10 mg/kg; oral administration; once daily; for 4 weeks; db/db mice) improves hyperglycemia and, consequently, ameliorates glucose intolerance[1].

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Tofogliflozin suppressed plasma glucose and glycated Hb and preserved pancreatic beta-cell mass and plasma insulin levels. No improvement of glycaemic conditions or insulin level was observed with losartan treatment. Although the urinary albumin/creatinine ratio of untreated db/db mice gradually increased from baseline, tofogliflozin or losartan treatment prevented this increase (by 50-70%). Tofogliflozin, but not losartan, attenuated glomerular hypertrophy. Neither tofogliflozin nor losartan altered matrix expansion.
Conclusions and implications: Long-term inhibition of renal SGLT2 by Tofogliflozin not only preserved pancreatic beta-cell function, but also prevented kidney dysfunction in a mouse model of type 2 diabetes. These findings suggest that long-term use of tofogliflozin in patients with type 2 diabetes may prevent progression of diabetic nephropathy. [3]

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To evaluate the renoprotective effects of long-term SGLT2 inhibition more precisely, we compared the effects of Tofogliflozin (a highly specific SGLT2 inhibitor) with the effects of losartan, (angiotensin II receptor antagonist), on renal and beta-cell functions, together with a quantitative analysis of glomerular and islet beta-cell mass. We demonstrated that long-term SGLT2 inhibition with tofogliflozin prevented not only loss of islet β-cells, but also the progression of renal impairment in db/db mice.

In this study, sustained blood glucose-lowering effects and stably reduced glycated Hb levels were observed over 4–8 weeks of treatment with Tofogliflozin together with a significant increase in glucose clearance (Figure 1A, B, E), suggesting that stable long-term glycaemic control can be achieved by tofogliflozin treatment. Based on the measured concentrations of tofogliflozin in plasma in the mice (0.015% tofogliflozin group) and the protein-binding properties of tofogliflozin, we estimated the unbound tofogliflozin concentrations to be between 120 and 350 nM. These concentrations are about 24–70 times the IC50 value of tofogliflozin against mouse SGLT2 (5.0 nM) and one-fifteenth to one-fifth of its IC50 value against mouse SGLT1 (1800 nM; Suzuki et al., 2012). Therefore, the unbound concentrations of tofogliflozin mentioned earleir are sufficient to inhibit mouse SGLT2 almost completely, but not to inhibit mouse SGLT1. [3]

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To understand the risk of hypoglycemia associated with urinary glucose excretion (UGE) induced by sodium-glucose cotransporter (SGLT) inhibitors, it is necessary to know the relationship between the ratio of contribution of SGLT2 vs. SGLT1 to renal glucose reabsorption (RGR) and the glycemic levels in vivo. To examine the contributions of SGLT2 and SGLT1 in normal rats, we compared the RGR inhibition by Tofogliflozin, a highly specific SGLT2 inhibitor, and phlorizin, an SGLT1 and SGLT2 (SGLT1/2) inhibitor, at plasma concentrations sufficient to completely inhibit rat SGLT2 (rSGLT2) while inhibiting rSGLT1 to different degrees. Under hyperglycemic conditions by glucose titration, tofogliflozin and phlorizin achieved ≥50% inhibition of RGR. Under hypoglycemic conditions by hyperinsulinemic clamp, RGR was reduced by 20-50% with phlorizin and by 1-5% with tofogliflozin, suggesting the smaller contribution of rSGLT2 to RGR under hypoglycemic conditions than under hyperglycemic conditions. Next, to evaluate the hypoglycemic potentials of SGLT1/2 inhibition, we measured the plasma glucose (PG) and endogenous glucose production (EGP) simultaneously after UGE induction by SGLT inhibitors. Tofogliflozin (400 ng/ml) induced UGE of about 2 mg·kg⁻¹·min⁻¹ and increased EGP by 1-2 mg·kg⁻¹·min⁻¹, resulting in PG in the normal range. Phlorizin (1,333 ng/ml) induced UGE of about 6 mg·kg⁻¹·min⁻¹ and increased EGP by about 4 mg·kg⁻¹·min⁻¹; this was more than with tofogliflozin, but the minimum PG was lower. These results suggest that the contribution of SGLT1 to RGR is greater under lower glycemic conditions than under hyperglycemic conditions and that SGLT2-selective inhibitors pose a lower risk of hypoglycemia than SGLT1/2 inhibitors. [5]

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In this study, we examined the contributions of SGLT2 and SGLT1 under different glycemic conditions by comparing the inhibitory effects of Tofogliflozin, a highly specific SGLT2 inhibitor, and phlorizin, an SGLT1/2 inhibitor, on RGR with glucose titration and clamp protocols in normal rats. In particular, we conducted these experiments under fixed plasma concentrations of each SGLT inhibitor to evaluate the relationship between the inhibitory activities estimated from the plasma concentration and the inhibition of RGR.

Under hyperglycemic conditions (protocol 1), over 50% inhibition of RGR was achieved by Tofogliflozin (≥133 ng/ml) and phlorizin (≥400 ng/ml) (Fig. 3). Based on the actual plasma concentrations (Table 1) and the protein-binding properties of tofogliflozin (21), we estimated the unbound tofogliflozin concentrations at 133 ng/ml (actual mean concentration: 168 ng/ml) and at 400 ng/ml (actual mean concentration: 474 ng/ml) to be 70 and 196 nM, respectively. Considering the IC50 values of tofogliflozin against rSGLT1 and rSGLT2 (rSGLT1, 8,200 nM; rSGLT2, 15 nM) calculated from its inhibitory activities on the sodium-dependent uptake of α-methyl-d-glucopyranoside (AMG), a nonmetabolizable glucose analog, in COS-7 cells overexpressing rSGLT1 or rSGLT2, the unbound concentrations of tofogliflozin mentioned above are relevant concentrations to inhibit rSGLT2 almost completely but not rSGLT1 [5].

In Vitro SGLT2 Inhibition and SGLT2 Selectivity. [1]
The inhibitory activities of Tofogliflozin and phlorizin against human, rat, and mouse SGLT2 were examined in cells (CHO, COS-7) overexpressing each SGLT2 by evaluating sodium-dependent AMG uptake. Analysis using Lineweaver-Burk plots showed that both compounds inhibited AMG uptake in a substrate competitive inhibition manner (Fig. 2), and Ki values of phlorizin for human, rat, and mouse SGLT2 inhibition were 13.6 ± 1.4, 39.4 ± 0.8, and 13.8 ± 0.7 nM, respectively. Tofogliflozin inhibited each...

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Tofogliflozin metabolism in human hepatocytes [4]
The incubation mixture contained pooled human hepatocytes (1 × 106 cells/mL), l-glutamine (0.292 g/L), penicillin/streptomycin (50 units/50 μg/mL), insulin (10−7 M), and dexamethasone (10−7 M) in William’s medium E. The incubation mixtures were pre-incubated in duplicate at 37 °C under 5% CO2 with sufficient mixing at 300 rpm by Rotamax120 (As One Corp., Osaka, Japan) for 15 min until the mixture was warmed. In the inhibition experiment, an inhibitor of ω- and (ω−1)-hydroxylation of lauric acid, 10-undecynoic acid (final concentration: 150 μM), was added to the incubation mixture before initiating pre-incubation, and blank vehicle was added in place of the inhibitor as a control sample. The reaction was initiated by the addition of [14C]tofogliflozin at the final concentration of 1 μM. The inhibitor and substrate were dissolved in dimethyl sulfoxide (DMSO) and the final DMSO concentration in the incubation mixture was less than 0.4%. The reaction was terminated by adding iced acetonitrile after 5 h incubation. The mixture was mixed and centrifuged at 5 °C for 5 min at 3000 rpm, and then the supernatant was collected as an analytical sample for HPLC. The radioactivity percentage of each peak was calculated from the following formula: percent of radioactivity (%) = radioactivity of metabolite peak/total peak radioactivity × 100. When the peak height of radioactivity was less than two-fold the height of the background, the peak was defined as not detected (ND) and the radioactivity (%) was not calculated. The inhibitory effect of 10-undecynoic acid on tofogliflozin metabolism is expressed as the percentage of inhibition as follows: inhibition (%) = (1 − I/C) × 100, where I and C represent the percentage of metabolite radioactivity in the presence and absence of an inhibitor, respectively. The value for inhibition (%) is defined as 100 when the percentage of radioactivity is ND in the presence of the inhibitor (I). [14C]Tofogliflozin and each metabolite in human hepatocytes incubations were identified by comparing the retention times and mass fragmentation patterns with those of authentic standards (Supplementary Material 1).

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Tofogliflozin metabolism in human liver microsomes [4]
The incubation mixture contained 1 mg/mL human liver microsomes, 0.1 M phosphate buffer (pH 7.4), and NADPH-generating system (1.3 mM NADP+, 3.3 mM glucose-6-phosphate, 3.3 mM MgCl2, and 0.4 units/mL glucose-6-phosphate dehydrogenase). The incubation mixtures were pre-incubated in duplicate at 37 °C for 15 min until warmed. An inhibitor, 1-aminobenzotriazole (final concentration: 1 mM), was added to the incubation mixture before initiating pre-incubation, and blank vehicle was added in place of the inhibitor as a control sample. The reaction was initiated by the addition of [14C]tofogliflozin at the final concentration of 10 μM. The inhibitor and substrate were dissolved in DMSO and the final DMSO concentration in the incubation mixture was set at less than 0.4%. The reaction was terminated by addition of iced acetonitrile after 1 h incubation. The mixture was mixed and centrifuged at 5 °C for 5 min at 15 000 rpm, and then the supernatant was collected as an analytical sample for HPLC. The inhibitory effect of 1-aminobenzotriazole was calculated as mentioned above.

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Tofogliflozin metabolism in rhCYPs [4]
Fourteen types of rhCYP (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP4A11, CYP4F2, and CYP4F3B) were used. The incubation mixtures contained 200 pmol CYP/mL, additional control microsomal proteins (used for adjusting final protein concentration to 4 mg/mL), the NADPH-generating system, and a buffer: 0.1 M Tris-HCl buffer (pH 7.5) was used for CYP2A6, CYP2C9, and CYP4A11, and 0.1 M potassium phosphate buffer (pH 7.4) for the other isoforms. The incubation of [14C]tofogliflozin (final concentration: 1 μM) and analytical sample preparation were carried out in duplicate by the same method as the human liver microsomes. [14C]Tofogliflozin was dissolved in DMSO and the final DMSO concentration in the incubation mixture was set at 0.2%. The obtained samples were analyzed by HPLC.

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CYP induction studies [4]
A CYP induction study was carried out using hepatocytes from three donors. The plated hepatocytes from each donor were cultured in the exposure medium, which consist of Modified Lanford’s medium containing 10 v/v% fetal bovine serum (FBS), and Tofogliflozin (0.5, 5, and 50 μM) for 48 h in a CO2 incubator set at 37 °C, 5% CO2, and 95% humidity. Tofogliflozin was dissolved in DMSO and the final DMSO concentration in each well was set at 0.1%. The exposure medium was renewed 24 h after incubation. After removal of the exposure medium, pre-warmed (37 °C) Hank's Balanced Salt Solution (HBSS) was added to wash. After removal of the HBSS, a pre-warmed substrate solution (37 °C) (1 μM 7-ethoxyresorufin for CYP1A2 or 100 μM testosterone for CYP3A4) was added and incubated in duplicate for 1 h in a CO2 incubator. An aliquot of exposure medium was mixed with iced 25% acetonitrile (for 7-ethoxyresorufin) or iced 25% methanol (for testosterone) to terminate the reaction. The reaction mixture was mixed and centrifuged at 5 °C for 10 min at 3000 rpm (Himac CF7D). Then, the supernatant was collected as an analytical sample for HPLC, and the residue was lysed by 1 M NaOH and mixed with HCl for neutralization, and then the protein concentration was determined with a BCA protein assay kit. The target substance (resorufin or 6β-hydroxytestosterone as the metabolite of 7-ethoxyresorufin or testosterone, respectively) was analyzed by HPLC (Supplementary Material 2).

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Reversible inhibition experiments to determine IC50 [4]
The incubation mixture contained 0.2 mg/mL human liver microsomes, 0.1 M phosphate buffer (pH 7.4), a CYP isoform-selective substrate (1 μM 7-ethoxyresorufin for CYP1A2, 100 μM bupropion for CYP2B6, 2 μM amodiaquine for CYP2C8, 5 μM diclofenac for CYP2C9, 20 μM S-mephenytoin for CYP2C19, 5 μM dextromethorphan for CYP2D6, and 5 μM midazolam, 10 μM nifedipine or 30 μM testosterone for CYP3A), and the test article, tofogliflozin or M1 (final concentration: 0.20, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, and 50 μM). Tofogliflozin and M1 were dissolved in DMSO and the final DMSO concentration in the incubation mixture was set at 0.2%. DMSO was added instead of the test article as a control sample. The incubation mixtures were pre-incubated in triplicate at 37 °C for 10 min. The reaction was initiated by the addition of NADPH (final concentration: 1 mM), and terminated after 5- to 30-min incubation (depending on the substrate used) by the addition of iced acetonitrile containing deuterium-midazolam and deuterium-dextrorphan as internal standards (IS). After terminating the reaction, the mixture was mixed and centrifuged (5800 × g, 10 min), and then the supernatant was collected to analyze the concentration of metabolite of each substrate by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Supplementary Material 2). The percentage of enzyme activity was calculated as (metabolite concentration observed in the incubation with the test article)/(metabolite concentration observed in the control sample) × 100. When an enzyme inhibition was observed, the IC50 value was estimated using the calculated inhibition percentage by the non-linear curve fitting procedure of Origin software. Inhibition (%) was calculated as follows: inhibition (%) = 100 − (percentage of enzyme activity).

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Time-dependent inhibition experiments [4]
The incubation mixture contained 1 mg/mL human liver microsomes, 0.1 M phosphate buffer (pH 7.4), and the test article, Tofogliflozin (final concentration: 10 to 100 μM) or M1 (final concentration: 10 to 50 μM). Tofogliflozin and M1 were dissolved in DMSO and the final DMSO concentration in the incubation mixture was set at 0.25%. DMSO was added in place of the test article as a control sample. The incubation mixtures were warmed to 37 °C over 10 min, then the pre-incubation of the test article was initiated by the addition of NADPH (final concentration: 1 mM). At timepoints of 0.5- to 15-min pre-incubation (0.5, 2.5, 4.5, 8, and 15 min for CYP1A2 and CYP2C9, 0.5, 3, 6, 10, and 14.5 min for CYP3A), aliquots were transferred with fresh NADPH to pre-warmed substrate solution; the final concentration of microsomes and NADPH were 0.1 mg/mL and 1.1 mM, respectively. The substrates used were 1 μM 7-ethoxyresorufin or 10 μM tacrine for CYP1A2; 20 or 25 μM diclofenac for CYP2C9; and 10 μM midazolam for CYP3A. Incubations were performed in singlicate (tofogliflozin) or triplicate (M1). The reaction was allowed to proceed for 10 or 15 min (depending on the substrate used), and then terminated by addition of iced acetonitrile containing deuterium-midazolam and deuterium-dextrorphan as IS. After terminating the reaction, the mixture was mixed and centrifuged (5800 × g, 10 min), and then the supernatant was collected to analyze the concentration of metabolite of each substrate by LC-MS/MS (Supplementary Material 2). The relative metabolite formation rates for each substrate were calculated as follows: relative metabolite formation rate = (metabolite concentration after incubation)/(metabolite concentration in the 0.5-min pre-incubated control sample) × 100. The natural logarithm of the relative metabolite formation rate was plotted against the pre-incubation time, and then the slope as a rate constant for the loss of enzyme activity (kobs, min−1) was estimated.

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Protein binding to human plasma [4]
Blood was collected from healthy volunteers working in-house (5 men who had fasted for at least 8 h) using a syringe, and the blood samples were placed in sample tubes treated with sodium heparin. Informed consent was obtained from all individuals before testing, and the study protocol was approved by the human research ethics committee. The heparinized blood samples were centrifuged (3000 rpm by himac CF7D for 20 min at 4 °C) and plasma samples were obtained. The plasma samples from five donors were mixed and pooled on ice, then used within a day. A protein binding assay was performed using the equilibrium dialysis method. A dialysis membrane that had been immersed in phosphate buffered saline (PBS, pH 7.4) and had swollen sufficiently was placed at the boundary between the two cells of the equilibrium dialysis apparatus and fixed with bolts. The plasma sample containing M1 and PBS was added in triplicate to the equilibrium dialysis device and incubated in a shaking-incubator (37 °C, shaking frequency: 40 rpm). M1 was dissolved in purified water and the final concentration of M1 was set at 0.1 and 1 μg/mL. The plasma sample and PBS were collected at 20 h after incubation, then the concentrations of M1 were measured using a LC-MS/MS system. The free fraction in plasma (fP) and the percentage of protein binding were calculated as follows: where CB and CP are the concentrations of M1 in PBS and plasma samples, respectively.

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SGLT inhibition studies [4]
An in vitro SGLT2 inhibition experiment was conducted as described in the literature. In brief, human SGLT2 (hSGLT2) cDNA was cloned into pcDNA3.1, and the constructed expression vector was introduced into CHO-K1 cells to establish a cell line expressing hSGLT2. The cells expressing SGLT2 were cultured in 96-well plates for 4 d and washed twice with sodium-free buffer containing 140 mM choline chloride, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)/Tris buffer (pH 7.4). The cells were incubated in duplicate with 1 mM [14C]AMG and a test article (Tofogliflozin or its metabolites) in sodium-free or sodium-containing buffer (sodium-free buffer containing 140 mM NaCl) at 37 °C for 40 min. The test articles were dissolved in DMSO and the final DMSO concentration in each well was set at 0.4%. The incubation was terminated by washing twice with sodium-free buffer containing 10 mM non-radiolabeled AMG. The cells were lysed with 0.1 w/v% sodium dodecyl sulfate, 0.1 w/v% Triton X-100, and 1 M NaOH, and the radioactivity of each sample was measured by TopCount-NXT (PerkinElmer Inc., Boston, MA). Sodium-dependent AMG uptake was calculated by subtracting the level of AMG incorporated into the sodium-free buffer from that of AMG in the sodium-containing buffer. IC50 values were calculated by multiple logistic regressions using SAS preclinical package.

RT-PCR[2]
Cell Types: Tubular epithelial cells
Tested Concentrations: 3 nM and 30 nM
Incubation Duration: 24 hrs (hours)
Experimental Results: Inhibited MCP -1 gene expression in tubular cells induced by high glucose exposure.

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Apoptosis Analysis[2]
Cell Types: Tubular epithelial cells
Tested Concentrations: 3 nM and 30 nM
Incubation Duration: 8 days
Experimental Results: Inhibited the apoptotic cell death induced by high glucose.

Animal/Disease Models: db/db mice[1] ]
Doses: 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 10 mg/kg
Route of Administration: Oral administration; one time/day; for 4 weeks
Experimental Results: Observed acute blood glucose reduction, dose-dependently decreased glycated hemoglobin, Dramatically prevented the decrease of IRI levels at doses of 3 and 10 mg/kg, and no difference in food intake or body weight.

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Long-term administration The db/db mice were randomly allocated into four dietary treatment groups matched for both 24 h urinary albumin excretion and body weight at 8 weeks of age. The db/db mice were kept on the standard diet or on a diet containing 0.005 or 0.015% tofogliflozin or 0.045% losartan for 8 weeks. The tofogliflozin content was determined according to previous pharmacokinetic data (Suzuki et al., 2012) and the estimated food consumption of db/db mice in order to inhibit SGLT2 completely, but not affect SGLT1. The db/ + m mice were kept on the standard diet. Blood glucose, glycated Hb, plasma insulin, plasma creatinine, urinary glucose, urinary creatinine and urinary albumin levels were measured periodically. Blood samples were collected from the tail vein or inferior vena cava to measure blood glucose, glycated Hb, plasma insulin and plasma creatinine levels. Metabolic cages were used to collect urine to measure urinary glucose, urinary creatinine, and urinary albumin excretion. At the end of 8 weeks’ treatment, animals were killed by whole blood collection from the abdominal aorta under anaesthesia with isoflurane. The kidneys and pancreas were isolated for the histological analysis described later. As part of these studies a separate group of db/db mice (16 weeks of age, n = 9) was kept on the diet containing 0.015% Tofogliflozin for 4 days, then three mice each were killed at 10:00, 15:00 and 20:00 h on day 4 by whole blood collection from the abdominal aorta under anaesthesia and the plasma samples were obtained by centrifugation to determine plasma tofogliflozin concentrations. Urine and plasma samples were stored at −80°C until use.

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Tofogliflozin was dissolved at 0.6 mg/ml in saline and diluted serially. [5]
Infusion Protocols with Blood and Urine Collection [5]
UGE under hyperglycemic conditions induced by glucose titration (protocol 1). [5]
Each animal was infused with saline at a rate of 15 ml·kg−1·h−1 through vein catheter V1 and 10 ml·kg−1·h−1 through vein catheter V2 for 60 min. Next, the infusion of Tofogliflozin or phlorizin solution was started at a rate of 2 ml/kg (bolus) plus 15 ml·kg−1·h−1 through vein catheter V1 without changing the constant infusion of saline at 10 ml·kg−1·h−1 through vein catheter V2. The concentrations of the tofogliflozin and phlorizin solutions used were determined on the basis of pharmacokinetic parameters obtained from separate pharmacokinetic studies (data not shown) to maintain plasma concentrations of 4, 13.3, 40, 133, or 400 ng/ml for tofogliflozin and 40, 133, 400, or 1,333 ng/ml for phlorizin. Namely, the infusion rate needed to achieve a target plasma concentration of Tofogliflozin of 400 ng/ml was 1.2 mg/kg (bolus) and 0.5 mg·kg−1·h−1 (constant), and that to achieve a target plasma concentration of phlorizin of 1,333 ng/ml was 0.15 mg/kg (bolus) and 2.8 mg·kg−1·h−1 (constant). After 60 min of tofogliflozin or phlorizin infusion, infusion of glucose solutions (10, 20, 30, 40, and 50%) was started at 10 ml·kg−1·min−1 in a stepwise manner from 10% at 30-min intervals through vein catheter V2 to raise the plasma glucose concentration to above 400 mg/dl. A blood sample (0.25 ml) was collected every 15 min with a heparinized syringe; the plasma glucose level in the sample was checked with a plasma glucose monitoring system, and then a plasma sample was obtained by centrifugation to determine plasma glucose and creatinine levels and Tofogliflozin or phlorizin concentrations. Urine was collected at 30-min intervals after glucose infusion to preweighed polyethylene sample tubes through the bladder catheter. The catheter was flushed with 0.5 ml saline to minimize the residual urine. Urine volume was determined by subtracting the weight of the preweighed sample tube from the sampled urine plus tube weight, with the specific gravity of sampled urine as 1. Urine and plasma samples were stored at −80°C until use.

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UGE under hypo- and euglycemic conditions induced by glucose clamp (protocol 2). [5]
Each animal was infused with saline at the rate of 15 ml·kg−1·h−1 through vein catheter V1 and 10 ml·kg−1·h−1 through vein catheter V2 for 90 min. Next, insulin (40 mU·kg−1·min−1 for 3 min; 20 mU·kg−1·min−1, constant) infusion was started through vein catheter V3. After 30 min of insulin infusion, infusion of Tofogliflozin or phlorizin solution was started at a rate of 2 ml/kg (bolus) and 15 ml·kg−1·h−1 (constant) through vein catheter V1 without changing the constant infusion of saline at 10 ml·kg−1·h−1 through vein catheter V2. The concentrations of Tofogliflozin and phlorizin solution used were determined as in protocol 1. After infusion of tofogliflozin or phlorizin solution for 60 min, glucose (20%) infusion was started through vein catheter V2 at a variable infusion rate based on a formula calculated to raise the plasma concentration to around 100 mg/dl. After this glucose infusion, blood (0.01 ml) was sampled from the jugular vein every 5–10 min, the plasma glucose levels were measured using Accu-check Aviva, and the glucose infusion rate was adjusted based on the same formula. Additional blood samples (0.25 ml) and urine samples were collected and prepared in the same manner as protocol 1. In this protocol, we defined UGE under hypoglycemic conditions as that during the last 30 min of the insulin plus tofogliflozin or insulin plus phlorizin infusion period and the UGE under euglycemic conditions as that during the last 30 min of insulin plus tofogliflozin or phlorizin with glucose infusion as indicated in Fig. 4.

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Effects of acute UGE induced by Tofogliflozin or phlorizin on plasma glucose levels and EGP (protocol 3). [5]
Each animal was infused with saline at the rate of 25 ml·kg−1·h−1 through vein catheter V1 and [U-13C]glucose (99%) saline solution at 0.14 mg·kg−1·min−1 through vein catheter V2. After a basal infusion period of 150 min, infusion of Tofogliflozin (bolus, 1.2 mg/kg; constant, 0.5 mg·kg−1·h−1) or phlorizin (bolus, 0.15 mg/kg; constant, 2.8 mg·kg−1·h−1) was started at the rate of 2 ml/kg (bolus) and 25 ml·kg−1·h−1 (constant) through vein catheter V1. Blood and urine samples were collected and prepared in the same manner as protocol 1 for 120 min from the start of Tofogliflozin or phlorizin infusion.

Human metabolite profile of Tofogliflozin [4]
The metabolite profile of [14C]tofogliflozin after 5 h incubation with human hepatocytes is shown in Figure 1. [14C]Tofogliflozin was metabolized to the carboxylated derivative (M1), the secondary hydroxylated derivatives, epimer-1 and epimer-2 (M2 and M3), the primary hydroxylated derivative (M4), and the ketone derivative (M5). The radioactivity of each metabolite after incubation was 30.9% for M1, 0.6% for M2/M3, 1.1% for M4, and 3.7% for M5. The chemical structure of each metabolite is shown in Figure 2.

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Identification of metabolic enzymes [4]
To clarify whether the conversion of [14C]Tofogliflozin to M2/M3 or M4 was catalyzed by CYP enzymes, the cofactor requirement and the effect of 1 mM 1-aminobenzotriazole (a non-specific CYP inhibitor) were evaluated using human liver microsomes. The metabolites were generated in the presence of NADPH-generating system, and the formation of M2/M3 or M4 was almost completely inhibited by 1-aminobenzotriazole (Table 1).

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CYP induction and inhibition [4]
The in vitro CYP induction and inhibition studies were performed at concentrations near to or higher than the maximum plasma concentration (Cmax) observed in clinical study; the Cmax values of tofogliflozin and M1 were 489 and 189 ng/mL (1.27 and 0.45 μM), respectively.
CYP1A2 and CYP3A4 activity was not induced by Tofogliflozin in the concentration range of 0.5–50 μM (Table 3). The free concentration of tofogliflozin might be lower than the nominal value due to the protein binding to FBS in the incubation mixture. The results of positive control inducers are shown in Supplementary Material 3 (Table S1).
Tofogliflozin exhibited no reversible inhibition potential on CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, or CYP3A activity (IC50 of each CYP > 50 μM). M1 also did not inhibit CYP isoforms (IC50 of each CYP > 50 μM) except for CYP2C19 (IC50 = 27.1 ± 6.5 μM) (Table 4). The results of positive control inhibitors are shown in Supplementary Material 3 (Table S2). Moreover, time-dependent inhibition of CYP1A2, CYP2C9, and CYP3A activity were estimated; no inhibition potency was observed at the concentration range of 10–100 μM of tofogliflozin and at the range of 10–50 μM of M1 (Table 5). The results of positive control inhibitors are shown in Supplementary Material 3 (Table S3).

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In vitro pharmacological activity of the metabolites towards hSGLT2 [4]
The IC50 values towards hSGLT2 were 0.0039, 2.7, 0.015, 0.014, 0.0049, and 0.016 μM for Tofogliflozin, M1, M2, M3, M4, and M5, respectively (Table 6). In vitro inhibition potency of all metabolites was lower than that of tofogliflozin.

It was reported that the area under plasma concentration–time curves from time 0 to 24 h (AUC0–24h) after oral administration of 20 mg [14C]Tofogliflozin to healthy subjects was 1814, 2215, 225, and 136 ng h/mL for tofogliflozin, M1, M2/M3, and M5, respectively, and that M1 was found to be the major metabolite in urine and feces. These results suggested that the main metabolite of tofogliflozin was M1 in human. In the present study, the metabolism of [14C]tofogliflozin was investigated using human hepatocytes, and the in vitro main metabolite of tofogliflozin was found to be M1, the same as in vivo (Figure 1). Additionally, M1 was generated from M4 according to a human hepatocyte experiment (Supplementary Material 4). On the other hand, considering the metabolite structures, it was expected that tofogliflozin would be converted to M5, via M2/M3 (Figure 2). M4 is the intermediate of the main metabolite of tofogliflozin, M1; thus, the enzymes involved in M4 formation are important for tofogliflozin metabolism. In this study, we focused on M4 formation, and metabolic enzymes which were responsible for converting tofogliflozin to M4 were identified using rhCYP, human liver microsomes, and human hepatocytes.

Table 1 shows that the [14C]Tofogliflozin oxidation was catalyzed by certain CYP enzymes. The reactions with 14 types of rhCYP suggested that the conversion of tofogliflozin to M2/M3 was catalyzed by CYP2C18, CYP3A4, and CYP3A5 and that the conversion of tofogliflozin to M4 was catalyzed by CYP2C18, CYP4A11, and CYP4F3B (Table 2). Moreover, the conversion of tofogliflozin to M4 was almost completely inhibited by 10-undecynoic acid in human hepatocytes. 10-Undecynoic acid, which is known as an inhibitor of ω- and (ω-1)-hydroxylation of lauric acid, inhibits CYP4A11 and CYP4F3B, because it inhibited the metabolism of [14C]lauric acid (substrate of CYP4A11) and [3H]leukotriene B4 (substrate of CYP4F3B) but did not inhibit the metabolism of diclofenac (substrate of CYP2C18) (Supplementary Material 5). There seemed to be some inconsistency in the in vitro experimental results: the data using rhCYP suggested that tofogliflozin was converted to M4 mainly by CYP2C18 (Table 2), but the inhibition experiment using human hepatocytes suggested it was converted mainly by CYP4A11 and/or CYP4F3B. The lack of background data for these uncommon drug metabolizing enzymes means that we can only make a limited interpretation, but we consider the contribution of CYP4A11 and/or CYP4F3B to the conversion of tofogliflozin to M4 would be higher than that of CYP2C18, because hepatocytes have enzymatic activities that reflect in vivo condition. Moreover, although it is unclear what enzymes are involved in the conversion of M4 to M1, alcohol dehydrogenase and aldehyde dehydrogenase might be the ones, considering the structures of the metabolites. In the present study, it was found that multiple enzymes were involved in tofogliflozin metabolism. Considering that the overall elimination of tofogliflozin depends not only on multiple metabolic enzymes but also on 15.5% of urinary excretion, the exposure of tofogliflozin is not expected to be altered by any co-administered drugs.

This study using rhCYPs suggested that CYP3A4/5 contributed to the metabolism of Tofogliflozin to M2/M3; nevertheless, the main metabolite in human was unequivocally not M2/M3 or M5, but was M1. This means that CYP3A4/5 is not the main metabolic enzyme. Indeed, it was reported that the influence of ketoconazole on tofogliflozin exposure was not clinically relevant.

A lack of induction potency of Tofogliflozin on CYP1A2 and CYP3A4 is a good characteristic (Table 3). Moreover, tofogliflozin and M1 showed no reversible inhibition potency on most CYP isoforms (IC50 of each CYP > 50 μM), except for a weak inhibition potency of CYP2C19 by M1 (IC50 = 27.1 μM) (Table 4). Bjornsson et al. mentioned that for reversible inhibition, an interaction does not occur if the ratio of Cmax to inhibition constant (Ki) is below 0.1. It was reported that Cmax values of tofogliflozin and M1 after oral administration of 20 mg [14C]tofogliflozin to healthy subjects were 489 and 189 ng/mL (1.27 and 0.45 μM), respectively. Thus, the Cmax/IC50 values for each CYP of tofogliflozin and M1 are calculated to be below 0.025 and 0.017, respectively, suggesting that the inhibition potency of tofogliflozin and M1 is not clinically relevant. Neither tofogliflozin nor M1 showed time-dependent inhibition potency on CYP1A2, CYP2C9, and CYP3A (Table 5). These indicate that the inhibition potency of tofogliflozin on CYPs is not clinically relevant. Although there are no in vitro data, we consider tofogliflozin has no inhibition/induction potency on the enzymes involved in its metabolism (CYP2C18, CYP4A11, and CYP4F3B), because there was no significant alteration of exposure after a 7-d repeated dose of tofogliflozin at 20 mg to healthy subjects. Additionally, it was reported that transporter-related DDIs caused by tofogliflozin and M1 would not be expected when the inhibitory effect of tofogliflozin and M1 on transporters was evaluated: human multidrug resistance (MDR) 1, organic anion transporter (OAT) 1, OAT3, organic cation transporter (OCT) 2 and organic anion transporting polypeptide (OATP) 1B1 (data not shown).

Considering that the fp of Tofogliflozin and M1 in human plasma were 0.17 and 0.45 (at 0.1 μg/mL because of its Cmax, Table 6), respectively, and their AUC0–24h values were 1814 and 2215 ng h/mL, respectively, it was expected that M1 would not show additional efficacy, because in vivo inhibition potency of M1 versus tofogliflozin was estimated to be 0.005. M2/M3 and M5 are also considered not to affect the in vivo efficacy: the AUC0–24h values of M2/M3 and M5 were 225 and 136 ng h/mL, respectively, and in vivo potency of M2/M3 and M5 versus tofogliflozin was estimated to be 0.2 and 0.1, respectively, even when their fp was 1. Because the exposure of M4 was undetectable, it was considered that it would not influence in vivo efficacy.

Conclusion [4]
The overall elimination of Tofogliflozin is mediated by not only multiple metabolic enzymes but also urinary excretion, suggesting that the exposure of tofogliflozin would not be easily altered by any co-administered drugs. Also, tofogliflozin seems not to cause significant DDI of co-administered drugs because it had no induction potency on CYP and neither tofogliflozin nor the main metabolite M1 had clinically relevant inhibition potency on CYP.

[1]. 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.

[2]. Tofogliflozin, A Highly Selective Inhibitor of SGLT2 Blocks Proinflammatory and Proapoptotic Effects of Glucose Overload on Proximal Tubular Cells Partly by Suppressing Oxidative Stress Generation. Horm Metab Res. 2016 Mar;48(3):191-5.

[3]. Tofogliflozin, a novel sodium-glucose co-transporter 2 inhibitor, improves renal and pancreatic function in db/db mice. Br J Pharmacol. 2013 Oct;170(3):519-31.

[4]. In vitro profiling of the metabolism and drug-drug interaction of tofogliflozin, a potent and highly specific sodium-glucose co-transporter 2 inhibitor, using human liver microsomes, human hepatocytes, and recombinant human CYP. Xenobiotica 2015 Mar;45(3):230-8.

[5]. Selective SGLT2 inhibition by tofogliflozin reduces renal glucose reabsorption under hyperglycemic but not under hypo- or euglycemic conditions in rats. Am J Physiol Endocrinol Metab. 2013 Feb 15;304(4):E414-23.

Tofogliflozin is a member of 2-benzofurans.
Tofogliflozin has been used in trials studying the treatment and prevention of Diabetes Mellitus Type 2.

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Tofogliflozin has been used in trials studying the treatment and prevention of Diabetes Mellitus Type 2.

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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. [1]

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Recently, we identified a potent and highly selective SGLT2 inhibitor, Tofogliflozin (Sato et al., 2010). The small number of patients with familial renal glucosuria limits confidence in the lower safety concern that is normally applied to long-term SGLT2 inhibition by virtue of the benign condition of the patient population, making intensive and multidimensional profiling of this emerging class of drugs of value in drug development, especially for T2D.
In the current study, we examined the pharmacological profiles of Tofogliflozin (CSG452), both in vitro and in vivo, including evaluation not only of its selectivity toward other SGLTs but also of its effect on glucose-related physiological processes, such as glucose uptake, glucose oxidation, glycogen synthesis, hepatic glucose production, glucose-stimulated insulin secretion, and glucosidase reactions. We found that tofogliflozin was highly specific to SGLT2 (its selectivity toward SGLT2 versus other SGLT members was the highest among the SGLT2 inhibitors we tested) and that it improved T2D pathological conditions by lowering blood glucose levels through the inhibition of renal glucose reabsorption, with a low risk of hypoglycemia. [1]

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Background and purpose: Although inhibition of renal sodium-glucose co-transporter 2 (SGLT2) has a stable glucose-lowering effect in patients with type 2 diabetes, the effect of SGLT2 inhibition on renal dysfunction in type 2 diabetes remains to be determined. To evaluate the renoprotective effect of SGLT2 inhibition more precisely, we compared the effects of Tofogliflozin (a specific SGLT2 inhibitor) with those of losartan (an angiotensin II receptor antagonist) on renal function and beta-cell function in db/db mice.

Experimental approach: The effects of 8-week Tofogliflozin or losartan treatment on renal and beta-cell function were investigated in db/db mice by quantitative image analysis of glomerular size, mesangial matrix expansion and islet beta-cell mass. Blood glucose, glycated Hb and insulin levels, along with urinary albumin and creatinine were measured

In an earlier study, we reported that Tofogliflozin had no direct effect on glucose-stimulated insulin secretion by isolated pancreatic islets (Suzuki et al., 2012). Imaging analysis revealed that beta-cell mass was significantly increased in tofogliflozin-treated db/db mice (Figure 7D, E), implying that preserved islet mass may contribute to maintaining the plasma insulin secretion. The preserved beta-cell function of db/db-SGLT2−/−mice was associated with increased beta-cell mass and reduced incidence of beta-cell apoptosis (Jurczak et al., 2011). The plasma tofogliflozin concentrations measured in the 0.015% tofogliflozin group were sufficient to specifically inhibit mSGLT2. Therefore, it is likely that increased beta-cell mass in tofogliflozin-treated db/db mice was caused by mechanisms similar to those in SGLT2-/- db/db mice. Because no human clinical studies have directly addressed the effect of SGLT2 inhibitors on beta-cell loss, this beneficial effect remains to be determined in human type 2 diabetes.

Several study limitations should be considered in this study. First, the lack of haemodynamic data from the db/db mice means that the mechanisms of the renoprotective effects of losartan and tofogliflozin through their haemodynamic effects discussed earlier are speculative. Second, although the mechanisms underlying the reduction of ACR with Tofogliflozin are considered to be closely related to its lowering of blood glucose, exactly how much of the action of tofogliflozin is dependent on the decrease of glucose toxicity and how much independent of glucose, remains to be elucidated.

In conclusion, we have provided evidence for prevention of kidney and pancreatic dysfunctions in a mouse model of type 2 diabetes by long-term SGLT2 inhibition with Tofogliflozin. Further studies are required to evaluate the therapeutic usefulness of tofogliflozin for preservation of renal function and beta-cells in patients with type 2 diabetes. [3]

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The inhibitory effect of Tofogliflozin on RGR was saturated at about 60% at 133–400 ng/ml under hyperglycemic conditions (Fig. 3A), where rSGLT2 was expected to be inhibited almost completely but not rSGLT1. In contrast, no saturation was observed in the inhibitory effect on RGR by phlorizin at 400–1,333 ng/ml (Fig. 3B), resulting in greater RGR inhibition at 1,333 ng/ml phlorizin than at 400 ng/ml tofogliflozin (73 ± 5%, phlorizin 1,333 ng/ml, 61 ± 5%, tofogliflozin 400 ng/ml; P < 0.05). At 1,333 ng/ml phlorizin, rSGLT2 was expected to be inhibited almost completely with a substantial rSGLT1 inhibition by about 50%. Therefore, the difference in RGR inhibition (%) between phlorizin (1,333 ng/ml) and tofogliflozin (400 ng/ml) was attributed to the partial inhibition of rSGLT1 by phlorizin. Taken together, the contribution of rSGLT2 to the RGR under hyperglycemic conditions was assumed to be about 60% in rats.

Under hypo- and euglycemic conditions with glucose clamp (protocol 2), phlorizin reduced RGR by about 25–35% and 50–60% at 400 and 1,333 ng/ml, respectively, where rSGLT2 is almost totally inhibited and rSGLT1 is partially inhibited. In contrast, Tofogliflozin minimally (1–5%) reduced RGR under hypoglycemic conditions even at the concentrations supposed to inhibit rSGLT2 almost completely (Fig. 6). Because the actual concentrations of phlorizin and tofogliflozin were maintained at the same levels (Table 2) and the plasma glucose levels and creatinine clearance were stable during the measurement of RGR inhibition (Tables 3 and 4), the minimal inhibition of RGR with tofogliflozin is the result of the absence of rSGLT1 inhibition. In SGLT2 knockout mice, a greater contribution of SGLT1 to RGR under euglycemic conditions has been proposed. Our results not only strongly support these suggestions but also suggest the dominant role of SGLT1 in RGR under hypoglycemic conditions. To evaluate whether this dominant role of SGLT1 is observed only under complete SGLT2 inhibition or not, it will be necessary to measure the actual glucose concentration gradient along the different segments of the proximal tubules.

Finally, we compared the UGE and EGP simultaneously to evaluate the hypoglycemic potentials of the SGLT inhibitors. Under euglycemic conditions, Tofogliflozin-induced UGE and EGP were increased together with a slight decrease in plasma glucose concentration. Even after 120 min of tofogliflozin infusion at 400 ng/ml, the plasma glucose levels were maintained above 100 mg/dl. The increased EGP (1–2 mg·kg−1·min−1) was nearly the same as the UGE level induced with tofogliflozin. These results suggest that UGE induction with tofogliflozin under euglycemic conditions can be fully compensated for by the increase of EGP.

In contrast, compared with Tofogliflozin, phlorizin induced greater UGE under euglycemic conditions, which may be the result of the dual inhibition of both SGLT1 and SGLT2. In the phlorizin group, although the EGP was also increased (by about 4 mg·kg−1·min−1, which was greater than in the tofogliflozin group), the plasma glucose was decreased more than in the Tofogliflozin group. Because the level of UGE induced with phlorizin (about 6 mg·kg−1·min−1) was apparently greater than the increased level of EGP, it is suggested that the induction of UGE with the dual inhibition of both SGLT1 and SGLT2 was not fully compensated for by the increase in EGP. The actual blood glucose-lowering effects have not been mentioned in studies in rats or humans treated with phlorizin under euglycemic conditions. Even in our experiment, actual hypoglycemia was not observed with continuous infusion of phlorizin for 120 min. However, the level of UGE observed with phlorizin, which was comparable to about 75% of the basal EGP (Fig. 7), suggests that dual inhibition of both SGLT1 and SGLT2 may pose a risk of excessive UGE under hypo- and euglycemic conditions, which may lead to sustained hypoglycemia. Further studies are required to understand the mechanisms of compensatory EGP increase and the long-term effects of sustained UGE induction by SGLT inhibitors.

In this study we examined the potential risk of hypoglycemia resulting from SGLT1 inhibition accompanying SGLT2 inhibition in normal rats. Although our results suggest the better profile of highly specific SGLT2 inhibition, experiments under diabetic conditions will be needed to precisely examine the potential risk of these compounds. Moreover, the mechanism that regulates the differential contributions of SGLT1 and SGLT2 to RGR under different glycemic conditions will need to be clarified.

In conclusion, the contribution of SGLT1 to RGR was found to be greater under lower glycemic conditions than under hyperglycemic conditions, and selective SGLT2 inhibition by Tofogliflozin exhibited greater reduction of RGR preferentially under hyperglycemic conditions. This suggests that SGLT2-selective inhibitors, such as tofogliflozin, carry a lower risk of causing hypoglycemia than SGLT1/2 inhibitors. [5]

C22H26O6

386.43824

386.173

C, 68.38; H, 6.78; O, 24.84

903565-83-3

1201913-82-7;Tofogliflozin hydrate

46908929

Typically exists as solid at room temperature

0.996

4

6

4

28

521

5

O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@]21OCC3=CC=C(CC4=CC=C(CC)C=C4)C=C23

VWVKUNOPTJGDOB-BDHVOXNPSA-N

InChI=1S/C22H26O6/c1-2-13-3-5-14(6-4-13)9-15-7-8-16-12-27-22(17(16)10-15)21(26)20(25)19(24)18(11-23)28-22/h3-8,10,18-21,23-26H,2,9,11-12H2,1H3/t18-,19-,20+,21-,22+/m1/s1

(3S,3'R,4'S,5'S,6'R)-5-[(4-ethylphenyl)methyl]-6'-(hydroxymethyl)spiro[1H-2-benzofuran-3,2'-oxane]-3',4',5'-triol

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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