Sodium/glucose cotransporter 2 (SGLT2)

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].

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.\n

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\n\nLong-term administration\nThe 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.\n

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\n\nTofogliflozin was dissolved at 0.6 mg/ml in saline and diluted serially. [5]
\nInfusion Protocols with Blood and Urine Collection [5]
\n\nUGE under hyperglycemic conditions induced by glucose titration (protocol 1). [5]
\nEach 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.\n

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\n\nUGE under hypo- and euglycemic conditions induced by glucose clamp (protocol 2). [5]
\nEach 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.\n

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\n\nEffects of acute UGE induced by Tofogliflozin or phlorizin on plasma glucose levels and EGP (protocol 3). [5]
\nEach 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.\n

Metabolic profile of tolagliflozin in humans [4] Figure 1 shows the metabolic profile of [14C] tolagliflozin after incubation with human hepatocytes for 5 hours. [14C] tolagliflozin is metabolized into carboxylated derivatives (M1), secondary hydroxylated derivatives, epimers-1 and-2 (M2 and M3), primary hydroxylated derivatives (M4) and ketone derivatives (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 structures of each metabolite are shown in Figure 2. Identification of metabolic enzymes [4] To clarify whether the conversion of [14C] tolagliflozin to M2/M3 or M4 is catalyzed by CYP enzymes, we used human liver microsomes to assess cofactor requirements and the role of 1 mM 1-aminobenzotriazole (a nonspecific CYP inhibitor). Metabolites are generated in the presence of the NADPH-generating system, and 1-aminobenzotriazole almost completely inhibits the generation of M2/M3 or M4 (Table 1).

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CYP Induction and Inhibition[4]
In vitro CYP induction and inhibition studies were conducted at concentrations close to or higher than the maximum plasma concentrations (Cmax) observed in clinical studies; the Cmax values for tylosin and M1 were 489 and 189 ng/mL (1.27 and 0.45 μM), respectively.
In the concentration range of 0.5–50 μM, tylosin did not induce CYP1A2 and CYP3A4 activities (Table 3). The free concentration of tylosin may be lower than the nominal value because it is bound to FBS in the incubation mixture. Results for the positive control inducer are shown in Supplementary Material 3 (Table S1).
Torpagliflozin showed irreversible inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, or CYP3A activities (IC50 > 50 μM for each CYP). M1 showed no inhibitory effect on any CYP isoenzyme except CYP2C19 (IC50 = 27.1 ± 6.5 μM) (IC50 > 50 μM for each CYP) (Table 4). Results for the positive control inhibitor are shown in Supplementary Material 3 (Table S2). Furthermore, the time-dependent inhibition of CYP1A2, CYP2C9, and CYP3A activities was assessed; no inhibition was observed in the range of 10–100 μM for torpagliflozin and 10–50 μM for M1 (Table 5). Results for the positive control inhibitor are shown in Supplementary Material 3 (Table S3).

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In vitro pharmacological activity of metabolites against hSGLT2[4]
The IC50 values of tolpagliflozin, M1, M2, M3, M4 and M5 against hSGLT2 were 0.0039, 2.7, 0.015, 0.014, 0.0049 and 0.016 μM, respectively (Table 6). The in vitro inhibitory efficacy of all metabolites was lower than that of tolpagliflozin.
It has been reported that after oral administration of 20 mg [14C] tolpagliflozin to healthy subjects, the area under the plasma concentration-time curve (AUC0-24h) were: tolpagliflozin 1814 ng·h/mL, M1 2215 ng·h/mL, M2/M3 225 ng·h/mL, M5 136 ng·h/mL, and M1 was the major metabolite in urine and feces. These results indicate that the major metabolite of tolpagliflozin in humans is M1. This study investigated the metabolism of [14C]toggliflozin using human hepatocytes, finding that the major metabolite of toggliflozin in vitro is M1, consistent with in vivo results (Figure 1). Furthermore, according to human hepatocyte experiments (Supplementary Material 4), M1 is generated from M4. On the other hand, considering the structure of the metabolites, it is expected that toggliflozin will be converted to M5 via M2/M3 (Figure 2). M4 is an intermediate of the major metabolite M1 of toggliflozin; therefore, enzymes involved in the generation of M4 are crucial for the metabolism of toggliflozin. In this study, we focused on the generation of M4 and identified the metabolic enzymes responsible for the conversion of toggliflozin to M4 using recombinant human cytochrome P450 (rhCYP), human liver microsomes, and human hepatocytes. Table 1 shows that certain CYP enzymes catalyze the oxidation of [14C]toggliflozin. Reactions with 14 rhCYPs showed that the conversion of toragliflozin to M2/M3 is catalyzed by CYP2C18, CYP3A4, and CYP3A5, while the conversion to M4 is catalyzed by CYP2C18, CYP4A11, and CYP4F3B (Table 2). Furthermore, in human hepatocytes, 10-undecynyl acid almost completely inhibited the conversion of toragliflozin to M4. 10-Undecynyl acid is a known inhibitor of ω- and (ω-1)-hydroxylation of laurate, and it inhibits CYP4A11 and CYP4F3B because it inhibits the metabolism of [14C]laurate (a substrate of CYP4A11) and [3H]leukotriene B4 (a substrate of CYP4F3B), but does not inhibit the metabolism of diclofenac (a substrate of CYP2C18) (Supplementary Material 5). In vitro experimental results appear to be somewhat inconsistent: data using rhCYP indicate that toragliflozin is primarily converted to M4 via CYP2C18 (Table 2), but inhibition assays using human hepatocytes suggest it is primarily converted via CYP4A11 and/or CYP4F3B. Due to the lack of background data on these uncommon drug-metabolizing enzymes, we can only offer limited interpretations, but we believe that CYP4A11 and/or CYP4F3B may contribute more to the conversion of toragliflozin to M4 than CYP2C18, as hepatocyte enzyme activity can reflect in vivo conditions. Furthermore, although it is currently unclear which enzymes are involved in the conversion of M4 to M1, alcohol dehydrogenases and aldehyde dehydrogenases are likely key enzymes given the structure of the metabolites. This study found that multiple enzymes are involved in the metabolism of toragliflozin. Considering that the overall clearance of toragliflozin depends not only on multiple metabolic enzymes but also on 15.5% urinary excretion, it is not expected that any concomitant medications will alter toragliflozin exposure. This study using recombinant human cytochrome P450 (rhCYP) showed that CYP3A4/5 are involved in the metabolism of tonagliflozin to M2/M3; however, in humans, the major metabolite is clearly not M2/M3 or M5, but M1. This means that CYP3A4/5 is not the major metabolic enzyme. In fact, it has been reported that the effect of ketoconazole on tonagliflozin exposure is not clinically significant. The lack of induction of CYP1A2 and CYP3A4 by tonagliflozin is a desirable characteristic (Table 3). Furthermore, tonagliflozin and M1 showed irreversible inhibition of most CYP isoenzymes (IC50 > 50 μM for each CYP), with only M1 showing a weak inhibitory effect on CYP2C19 (IC50 = 27.1 μM) (Table 4). Bjornsson et al. noted that for reversible inhibition, no interaction occurs if the ratio of Cmax to the inhibition constant (Ki) is less than 0.1. According to reports, after oral administration of 20 mg [14C] toggliflozin to healthy subjects, the Cmax values of toggliflozin and M1 were 489 ng/mL and 189 ng/mL (1.27 μM and 0.45 μM), respectively. Therefore, the Cmax/IC50 values of toggliflozin and M1 for each CYP enzyme were calculated to be less than 0.025 and 0.017, respectively, indicating that the inhibitory efficacy of toggliflozin and M1 was not clinically significant. Neither toggliflozin nor M1 showed time-dependent inhibitory efficacy against CYP1A2, CYP2C9, and CYP3A (Table 5). These results suggest that the inhibitory efficacy of toggliflozin against CYP enzymes is not clinically significant. Despite the lack of in vitro data, we believe that toggliflozin has no inhibitory/inducing effect on its metabolism-related enzymes (CYP2C18, CYP4A11, and CYP4F3B) because the exposure levels in healthy subjects did not change significantly after repeated administration of 20 mg toggliflozin for 7 consecutive days. Furthermore, it has been reported that no transporter-related drug interactions induced by tonagliflozin and M1 are expected when evaluating the inhibitory effects of tonagliflozin and M1 on transporters: human multidrug resistance protein (MDR) 1, organic anion transporter (OAT) 1, OAT3, organic cation transporter (OCT) 2, and organic anion transport polypeptide (OATP) 1B1 (data not shown). Given that the fp values of tonagliflozin and M1 in human plasma are 0.17 and 0.45 (concentration of 0.1 μg/mL, as their Cmax is shown in Table 6), and their AUC0-24h values are 1814 and 2215 ng·h/mL, respectively, M1 is not expected to show additional therapeutic efficacy, as the in vivo inhibitory potency of M1 against tonagliflozin is estimated to be 0.005. M2/M3 and M5 were also considered not to affect in vivo efficacy: the AUC0–24h values of M2/M3 and M5 were 225 and 136 ng·h/mL, respectively, and even with their fp values of 1, the in vivo potency of M2/M3 and M5 relative to tonaggliflozin was estimated to be 0.2 and 0.1, respectively. Since the exposure of M4 was undetectable, it was considered not to affect in vivo efficacy.
Conclusion[4]
The overall elimination of tonaggliflozin is mediated not only by a variety of metabolic enzymes but also by urinary excretion, suggesting that the exposure of tonaggliflozin is not easily affected by any concomitant drugs. In addition, tonaggliflozin does not appear to cause significant drug interactions with concomitant drugs because it has no inducing effect on CYP and tonaggliflozin and its major metabolite M1 have no clinically relevant inhibitory effect 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.

Tolpagliflozin has been used in clinical trials to investigate the treatment and prevention of type 2 diabetes. Sodium/glucose cotransporter 2 (SGLT2) is a major mediator of renal glucose reabsorption and an emerging molecular target in diabetes treatment. We have discovered a novel, highly effective, and selective SGLT2 inhibitor—tolpagliflozin (CSG452)—and investigated its efficacy and pharmacological properties as an antidiabetic drug. Tolpagliflozin competitively inhibits SGLT2 in SGLT2-overexpressing cells, with Ki values of 2.9 nM, 14.9 nM, and 6.4 nM for human, rat, and mouse SGLT2, respectively. Among all SGLT2 inhibitors currently in clinical development, tolpagliflozin exhibits higher selectivity for human SGLT2 than for human SGLT1, SGLT6, and sodium/inositol transporter 1. Furthermore, no interaction between tolpagliflozin and any receptor was observed in a series of tests detecting glucose-related physiological processes such as glucose uptake, glucose oxidation, glycogen synthesis, hepatic glycogen production, glucose-stimulated insulin secretion, and glucosidase responses. A single gavage administration of toggliflozin increased renal glucose clearance and reduced blood glucose levels in Zucker diabetic obese rats. Toggliflozin also improved postprandial blood glucose variability in GK rats. In db/db mice, 4 weeks of continuous toggliflozin treatment reduced glycated hemoglobin levels and improved glucose tolerance in an oral glucose tolerance test 4 days after the last administration. No reduction in blood glucose was observed in blood glucose-normal SD rats treated with toggliflozin. These results suggest that toggliflozin inhibits SGLT2 in a specific manner, reduces blood glucose levels by increasing renal glucose clearance, and improves the pathological status of type 2 diabetes at the risk of hypoglycemia. [1]

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Recently, we discovered a potent and highly selective SGLT2 inhibitor, toggliflozin (Sato et al., 2010). Because the number of patients with familial renal diabetes is relatively small, concerns about the safety of long-term use of SGLT2 inhibitors are generally low (this is often attributed to the milder nature of the disease in these patients). Therefore, in-depth, multi-dimensional analysis of these emerging drugs is of great significance for drug development, especially for type 2 diabetes. In this study, we investigated the pharmacological characteristics of tolagliflozin (CSG452) in vitro and in vivo, evaluating not only its selectivity for other SGLTs but also its effects on glucose-related physiological processes, such as glucose uptake, glucose oxidation, glycogen synthesis, hepatic glucose production, glucose-stimulated insulin secretion, and glucosidase responses. We found that tolagliflozin has high specificity for SGLT2 (the highest selectivity for SGLT2 relative to other SGLT members among the SGLT2 inhibitors we tested), and it improves the pathological condition of type 2 diabetes by inhibiting renal glucose reabsorption, with a low risk of hypoglycemia. [1]

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Background and Objectives: Although inhibition of renal sodium-glucose cotransporter 2 (SGLT2) has a stable hypoglycemic effect in patients with type 2 diabetes, the impact of SGLT2 inhibition on renal dysfunction in type 2 diabetes remains to be determined. To more accurately assess the renal protective effects of SGLT2 inhibitors, we compared the effects of toragliflozin (a specific SGLT2 inhibitor) and losartan (an angiotensin II receptor antagonist) on renal and β-cell function in db/db mice.
Experimental Methods: The effects of toragliflozin or losartan treatment for 8 weeks on renal and β-cell function were investigated by quantitative image analysis of glomerular size, mesangial matrix expansion, and pancreatic β-cell number in db/db mice. Blood glucose, glycated hemoglobin, and insulin levels, as well as urinary albumin and creatinine levels, were measured simultaneously.
In previous studies, we reported that toragliflozin had no direct effect on insulin secretion stimulated by isolated islet glucose (Suzuki et al., 2012). Imaging analysis showed a significant increase in β-cell count in db/db mice treated with toragliflozin (Figures 7D and E), suggesting that the preserved number of islet cells may help maintain plasma insulin secretion. Preservation of β-cell function in db/db-SGLT2−/− mice is associated with an increase in β-cell count and a decrease in β-cell apoptosis (Jurczak et al., 2011). Plasma toragliflozin concentrations measured in the 0.015% toragliflozin group were sufficient to specifically inhibit mSGLT2. Therefore, the increase in β-cell count in db/db mice treated with toragliflozin is likely due to a mechanism similar to that in SGLT2−/− db/db mice. Since there are currently no human clinical studies directly investigating the effects of SGLT2 inhibitors on β-cell loss, their beneficial role in type 2 diabetes remains to be determined.
This study has some limitations. First, due to the lack of hemodynamic data in db/db mice, the previously discussed mechanisms by which losartan and toragliflozin exert their renal protective effects through hemodynamic processes remain speculative. Secondly, although the mechanism by which tolagliflozin reduces ACR is thought to be closely related to its blood glucose reduction, it remains to be clarified how much the effect of tolagliflozin depends on the reduction of glucose toxicity and how much is independent of glucose. In summary, we have demonstrated that long-term use of tolagliflozin to inhibit SGLT2 can prevent renal and pancreatic dysfunction in a type 2 diabetic mouse model. Further studies are needed to evaluate the therapeutic value of tolagliflozin in protecting renal function and β cells in patients with type 2 diabetes. [3] Under elevated blood glucose conditions, the inhibitory effect of tolagliflozin on RGR reaches saturation at 133–400 ng/ml, approximately 60% (Figure 3A), at which point rSGLT2 is expected to be almost completely inhibited, while rSGLT1 is not inhibited. Conversely, no saturation was observed in the inhibitory effect of phlorizin on RGR at concentrations of 400–1333 ng/ml (Figure 3B), resulting in a stronger inhibitory effect of 1333 ng/ml phlorizin on RGR than that of 400 ng/ml tonagliflozin (73 ± 5%, 1333 ng/ml phlorizin; 61 ± 5%, 400 ng/ml tonagliflozin; P < 0.05). At a concentration of 1333 ng/ml phlorizin, rSGLT2 was expected to be almost completely inhibited, while rSGLT1 was also significantly inhibited by approximately 50%. Therefore, the difference in RGR inhibition rate (%) between phlorizin (1333 ng/ml) and tonagliflozin (400 ng/ml) is attributed to the partial inhibition of rSGLT1 by phlorizin. In conclusion, under elevated blood glucose conditions, rSGLT2 contributes approximately 60% to the RGR in rats.
Under hypoglycemic and normglycemic conditions, using the glucose clamp technique (Scheme 2), phlorizin at concentrations of 400 ng/ml and 1333 ng/ml reduced the renal growth rate (RGR) by approximately 25-35% and 50-60%, respectively. At these concentrations, rSGLT2 was almost completely inhibited, while rSGLT1 was partially inhibited. In contrast, even at concentrations considered to almost completely inhibit rSGLT2, terazosin only slightly reduced the RGR (1-5%) under hypoglycemic conditions (Figure 6). Since the actual concentrations of phlorizin and terazosin remained at the same level (Table 2), and plasma glucose levels and creatinine clearance remained stable during the RGR inhibition assay (Tables 3 and 4), the minimal inhibitory effect of terazosin on RGR is attributed to the lack of inhibition of rSGLT1. In SGLT2 knockout mice, studies have suggested that SGLT1 contributes more to RGR under normglycemic conditions. Our results not only strongly support these views but also indicate that SGLT1 plays a dominant role in RGR under hypoglycemic conditions. To assess whether this dominant role of SGLT1 is only observable with complete SGLT2 inhibition, the actual glucose concentration gradient across different segments of the proximal tubules needed to be measured. Finally, we compared urinary glucose excretion (UGE) and endogenous glucose production (EGP) to evaluate the hypoglycemic potential of the SGLT inhibitor. Under normoglycemic conditions, toragliflozin induced both increased urinary glucose excretion (UGE) and endogenous glucose production (EGP), while plasma glucose concentration decreased slightly. Even 120 minutes after infusion of toragliflozin at a concentration of 400 ng/ml, plasma glucose levels remained above 100 mg/dL. The increased EGP (1–2 mg·kg−1·min−1) was almost identical to the toragliflozin-induced UGE level. These results suggest that under normoglycemic conditions, toragliflozin-induced UGE can be fully compensated by the increase in EGP. Conversely, compared to thioglitazone, phlorizin induced a greater UGE under normal blood glucose conditions, likely due to its simultaneous inhibition of SGLT1 and SGLT2. In the phlorizin group, although the endogenous glucose production rate (EGP) was also increased (approximately 4 mg·kg⁻¹·min⁻¹, higher than the thioglitazone group), the decrease in plasma glucose levels was greater than that in the thioglitazone group. Since the phlorizin-induced urinary glucose excretion rate (UGE) (approximately 6 mg·kg⁻¹·min⁻¹) was significantly higher than the increase in EGP, it suggests that the increase in UGE caused by the dual inhibition of SGLT1 and SGLT2 was not fully compensated by the increase in EGP. Studies of phlorizin treatment in rats or humans under normal blood glucose conditions have not reported any actual hypoglycemic effect. Even in our experiment, no actual hypoglycemia was observed after continuous infusion of phlorizin for 120 minutes. However, phlorizin-induced urinary glucose excretion (UGE) levels were comparable to approximately 75% of basal endogenous glucose production (EGP) (Figure 7), suggesting that simultaneous inhibition of SGLT1 and SGLT2 may lead to excessive UGE under both hypoglycemic and normoglycemic conditions, resulting in persistent hypoglycemia. Further investigation is needed to understand the mechanisms of compensatory EGP increase and the long-term effects of persistent UGE induced by SGLT inhibitors. In this study, we investigated the risk of hypoglycemia resulting from SGLT1 inhibition accompanied by SGLT2 inhibition in normal rats. Although our results indicate that highly specific SGLT2 inhibitors have better efficacy, experiments under diabetic conditions are still needed to accurately assess the potential risks of these compounds. Furthermore, the mechanisms by which SGLT1 and SGLT2 contribute differently to RGR under different glycemic conditions need to be elucidated. In summary, the study found that SGLT1 contributes more to RGR under hypoglycemic conditions than under hyperglycemic conditions, while the selective SGLT2 inhibitor torpagliflozin exhibits stronger inhibitory effects on RGR under hyperglycemic conditions. This suggests that SGLT2 selective inhibitors, such as tolagliflozin, pose a lower risk of hypoglycemia compared to SGLT1/2 inhibitors. [5]

C22H26O6.H2O

404.45

404.184

C, 65.33; H, 6.98; O, 27.69

1201913-82-7

Tofogliflozin;903565-83-3

46908928

White to off-white solid powder

0.932

5

7

4

29

521

5

CCC1=CC=C(C=C1)CC2=CC3=C(CO[C@@]34[C@@H]([C@H]([C@@H]([C@H](O4)CO)O)O)O)C=C2.O

ZXOCGDDVNPDRIW-NHFZGCSJSA-N

InChI=1S/C22H26O6.H2O/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;1H2/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;hydrate

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.18 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 25.0 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: ≥ 2.5 mg/mL (6.18 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 25.0 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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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.18 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2.5 mg/mL (6.18 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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