mSGLT2 ( IC50 = 2 nM ); rSGLT2 ( IC50 = 3.7 nM ); hSGLT2 ( IC50 = 4.4 nM )
In CHO-hSGLT2 cells, canagliflozin inhibits Na+-dependent 14C-AMG uptake with an IC50 of 4.4±1.2 nM. Rat and mouse SGLT2 have IC50 values of 2.0 nM and 3.7 nM, respectively, in CHO-mSGLT2 and CHO-rSGLT2 cells. With an IC50 of 684±159 nM and >1,000 nM, respectively, canagliflozin inhibits the absorption of 14C-AMG in CHO-hSGLT1 and mSGLT1 cells[1].

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In vitro activity: Canagliflozin is a newly discovered thiophene-ringed C-glucoside. In a concentration-dependent manner, canagliflozin inhibits 14C-AMG uptake that is dependent on Na+. In CHO-hSGLT1 and mSGLT1 cells, canagliflozin inhibits 14C-AMG uptake with IC50 values of 0.7 μM and >1 μM, respectively. Less than 50% of L6 myoblasts' facilitative (non-Na+-linked) GLUT-mediated 2H-2-DG uptake is inhibited by canagliflozin. Currents in oocytes injected with sham are unaffected by canagliflozin (10 μM) or phlorizin (3 mM) when combined with 50 μM DNJ. DMSO and Canagliflozin 10 μM inhibit DNJ-induced currents by 15.6% and 23.4%, respectively, in oocytes that have received SGLT3 injections. [1]

In CHO-hSGLT2 cells, canagliflozin inhibits Na+-dependent 14C-AMG uptake with an IC50 of 4.4±1.2 nM. Rat and mouse SGLT2 have IC50 values of 2.0 nM and 3.7 nM, respectively, in CHO-mSGLT2 and CHO-rSGLT2 cells. With an IC50 of 684±159 nM and >1,000 nM, respectively, canagliflozin inhibits the absorption of 14C-AMG in CHO-hSGLT1 and mSGLT1 cells[1].

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In vitro activity: Canagliflozin is a newly discovered thiophene-ringed C-glucoside. In a concentration-dependent manner, canagliflozin inhibits 14C-AMG uptake that is dependent on Na+. In CHO-hSGLT1 and mSGLT1 cells, canagliflozin inhibits 14C-AMG uptake with IC50 values of 0.7 μM and >1 μM, respectively. Less than 50% of L6 myoblasts' facilitative (non-Na+-linked) GLUT-mediated 2H-2-DG uptake is inhibited by canagliflozin. Currents in oocytes injected with sham are unaffected by canagliflozin (10 μM) or phlorizin (3 mM) when combined with 50 μM DNJ. DMSO and Canagliflozin 10 μM inhibit DNJ-induced currents by 15.6% and 23.4%, respectively, in oocytes that have received SGLT3 injections. [1]

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In a concentration-dependent manner, Canagliflozin inhibited Na+-dependent 14C-AMG uptake in Chinese Hamster Ovary (CHO) cells expressing human SGLT2 (hSGLT2) with an IC50 of 4.4±1.2 nM. Similar potent inhibition was observed against rat and mouse SGLT2. [1]
Canagliflozin inhibited Na+-dependent 14C-AMG uptake in CHO cells expressing human SGLT1 (hSGLT1) with an IC50 of 684±159 nM, showing selectivity for SGLT2 over SGLT1. Inhibition of mouse SGLT1 was very weak (IC50 >1,000 nM). [1]
At a concentration of 10 µM, Canagliflozin inhibited the facilitative (non-Na+-linked) GLUT-mediated 3H-2-deoxy-D-glucose (2-DG) uptake in L6 myoblast cells by less than 50%. [1]
When tested on human SGLT3 expressed in Xenopus oocytes using a two-electrode voltage clamp, Canagliflozin (10 µM) did not cause statistically significant inhibition of the currents induced by 50 µM 1-deoxynojirimycin (DNJ) compared to the DMSO control. [1]

In DIO mice, canagliflozin (30 mg/kg) therapy for 4 weeks lowers body weight increase, respiratory exchange ratio, and blood glucose (BG) levels[1]. as canagliflozin (3 mg/kg) is administered for three weeks, rats treated with ZF experience a loss in body weight due to an increase in urine glucose excretion (UGE) without a significant change in total food consumption as compared to vehicle-treated rats[1].

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Canagliflozin exhibits strong anti-hyperglycemic effects in high-fat diet fed KK (HF-KK) mice. Male SD rats given oral canagliflozin at 30 mg/kg for 24 hours experience an increase in glucose excretion of 3,696 mg per 200 g body weight. After oral administration, pharmacokinetic studies show a significantly higher exposure of canagliflozin. Male SD rats were given intravenous and oral doses of 3 and 10 mg/kg, respectively.The results show that the oral bioavailability was 85%, the po, t1/2, and AUCf0−in were 35,980 ng·h/mL, 5.2 hours, and po, respectively. Therefore, after oral dosing of canagliflozin, inhibition of SGLT2 in renal tubules is likely to continuously suppress glucose reabsorption. The broad UGE would indicate both the high potency of SGLT2 inhibition and the excellent pharmacokinetic characteristics of canagliflozin in vivo. The novel compound could be useful as an anti-diabetic agent because SGLT2 in the renal tubules reabsorbs most of the filtered glucose. In hyperglycemic high-fat diet-fed KK (HF-KK) mice, a single oral administration of canagliflozin at a dose of 3 mg/kg significantly lowered blood glucose levels without affecting food intake. After six hours, the blood glucose level is 48% lower than in the vehicle. Conversely, in normoglycemic mice, canagliflozin has a negligible effect on blood glucose levels. Canagliflozin would therefore reduce the risk of hypoglycemia while controlling hyperglycemia in the treatment of type 2 diabetes. [2]

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In Zucker Diabetic Fatty (ZDF) rats, a single dose of Canagliflozin (1 mg/kg, p.o.) significantly lowered the renal threshold for glucose excretion (RTG) from 415±12 mg/dL to 94±10 mg/dL during a graded glucose infusion study. The relationship between blood glucose (BG) and urinary glucose excretion (UGE) remained a threshold relationship, with virtually no UGE when BG was below the lowered RTG. [1]
In diabetic db/db mice, single oral doses of Canagliflozin (0.1, 1, and 10 mg/kg) dose-dependently reduced non-fasting blood glucose levels. The effect was rapid, with significant reductions observed at 1 hour post-dose for the 1 and 10 mg/kg groups. The maximal effect was seen at 6 hours. [1]
In ZDF rats treated orally with Canagliflozin (3, 10, or 30 mg/kg) for 4 weeks, fed plasma glucose and glycated hemoglobin (HbA1c) levels were significantly reduced compared to the vehicle group in a dose-dependent manner. [1]
After 4 weeks of treatment in ZDF rats, Canagliflozin improved beta-cell function as assessed by an oral glucose tolerance test (OGTT). The insulin-to-glucose ratio (AUC insulin / AUC glucose) during the OGTT was significantly increased (4- to 6-fold) compared to vehicle-treated rats. [1]
In Diet-Induced Obese (DIO) mice treated with Canagliflozin (30 mg/kg, p.o.) for 4 weeks, body weight gain was reduced compared to vehicle-treated mice. Fed blood glucose levels and the respiratory exchange ratio (RER) were also significantly decreased. [1]
In Zucker Fatty (ZF) rats treated with Canagliflozin (3 mg/kg, p.o.) for 3 weeks, body weight gain and feeding efficiency (body weight gain/food intake) were reduced. Urinary glucose excretion (UGE) was significantly increased, while fed blood glucose, epididymal fat pad weight, liver weight, and the respiratory exchange ratio (RER) were significantly decreased. No significant change in total food intake was observed. [1]

Sodium-Dependent Glucose Uptake in CHO cells Expressing human SGLT1 and SGLT2. Parental Chinese hamster ovary-K (CHOK) cells expressing human SGLT1 and SGLT21 were used in these experiments. For the uptake assay, cells were seeded into 24-well plates, and were post-confluent on the day of assay. Cells were rinsed one time with 400 µL Assay Buffer (137 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 50 mM HEPES, 20 mM Tris Base, pH 7.4), and were pre-incubated with the solutions of compounds (250 µL) for 10 min at 37 °C. The transport reaction was initiated by addition of 50 µL alpha methyl-D-glucopyranoside (AMG) / 14C-AMG solution (16.7 µCi; final concentration, 0.3 mM for CHOK-SGLT1 and 0.5 mM for CHOK-SGLT2, respectively) and incubated for 120 min at 37 °C. After the incubation, the AMG uptake was halted by aspiration of the incubation mixture followed by immediate washing three times with PBS. The cells were solubilized in 0.3 N NaOH of 300 µL and the radioactivity associated with the cells was monitored by a liquid scintillation counter. Inhibitory concentration of 50% (IC50) was calculated by nonlinear least squares analysis using a four-parameter logistic model.[2]

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Two-electrode Voltage Clamp Recording of Oocytes Expressing Human SGLT3 [1]
The functional effects of canagliflozin on human SGLT3 were studied by 2-electrode voltage clamp electrophysiology using OpusXpress 6000A. Stage V–VI oocytes were injected with 50 nl of either human SGLT3 mRNA (at 1 ng/nl) or distilled water (control) and incubated at 18°C in a calcium-free solution (92 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, 0.05 mg/ml gentamicin at pH 7.5) for 4–6 days before recording. The extracellular recording solution contained 92 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES at pH 7.5. Injected oocytes were impaled with 2 microelectrodes filled with 3 M KCl (resistance of ∼0.5–3 MΩ) and voltage clamped to −120 mV, at which continuous recordings were made (filtered at 5 kHz and sampled at 625 Hz). To establish the baseline in the absence of agonist, oocytes were first perfused for 85 seconds with a control buffer (92 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES at pH 7.5). Next, 50 µM 1-deoxynojirimycin (DNJ) was applied for 160 seconds, followed by co-application of imino sugars 1-deoxynojirimycin (DNJ, 50 µM) with either canagliflozin (10 µM) or dimethyl sulfoxide (DMSO) (0.1%) for 160 seconds. Finally, phlorizin (3 mM) was applied in the presence of 50 µM DNJ for 160 seconds. All experiments were performed at 22°C. Currents in the presence of 50 µM DNJ were subtracted from leak currents (currents in control buffer alone) to obtain DNJ-induced currents (IDNJ). Effects of compounds were calculated as: % Inhibition = 100×(IDNJ−Icmpd)/IDNJ, where Icmpd is the DNJ-induced and leak-subtracted current in the presence of a compound or DMSO. Due to lack of effect at the highest dose tested, a dose-response relationship was not examined.

The effect of canagliflozin on the activity of the glucose transporter 1 (GLUT1) is examined in rat skeletal muscle cell line L6 cells. The culture medium used for the cells is Dulbecco's modified Eagle's medium, which contains 5.6 mM glucose and 10% fetal bovine serum. The cells are seeded in 24-well plates at a density of 3 × 10⁵ cells/well and are cultured for 24 hours at 37 °C in an atmosphere of 5% CO2. The cells are pre-incubated with the solutions of Canagliflozin (250 μL, 10 μM) for 5 minutes at room temperature after being rinsed twice with Kreb's ringer phosphate HEPES buffer (pH 7.4, 150 mM NaCl, 5 mM KCl, 1.25 mM MgSO₄, 1.25 mM CaCl2, 2.9 mM Na²HPO₄, 10 mM HEPES). 50 μL of 4.5 mM 2-DG (a GLUTs substrate)/3H-2-DG (0.625 μCi) is added to start the transport reaction, which is then incubated for 15 minutes at room temperature. Aspiration of the incubation mixture stops the uptake of 2-DG. The cells are instantly cleaned three times in ice-cold PBS. Radioactivity is measured using liquid scintillation after samples are extracted using 0.3 N NaOH.

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Cell-based Assays [1]
Sodium-dependent Glucose Uptake in Chinese Hamster Ovary (CHO) Cells Expressing SGLT1 and SGLT2 Co-transporters Parental CHO-K (CHOK) cells (commonly used mammalian cells for gene overexpression studies) expressing human or mouse SGLT1 and SGLT2 were utilized in this study. Cells were seeded into 96-well plates. Cells were then washed one time with 0.15 ml assay buffer (137 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 50 mM HEPES, pH 7.4) at 37°C. After the assay buffer was removed, 50 µl of fresh assay buffer with 5 µl of canagliflozin (0.3–300 nM) was added, followed by 10 minutes of incubation. Then, 5 µl of 6 mM alpha-methyl-d-glucopyranoside (AMG, a selective SGLT1/2 substrate)/14C-AMG (0.07 µCi) was added to the cells and incubated at 37°C for 2 hours. Next, the cells were washed 3 times with 0.15 ml ice-cold phosphate-buffered saline (PBS). After the final wash was aspirated, 50 µl of microscint 20 was added. The plate was counted by TopCount.

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The 2-deoxy-glucose (2-DG) Uptake in L6 Myoblast Cells [1]
Cells from the rat skeletal muscle cell line, L6, was used to test the effect of canagliflozin on glucose transporter 1 (GLUT1) activity. Cells were maintained in Dulbecco's modified Eagle's medium containing 5.6 mM glucose supplemented with 10% fetal bovine serum, were seeded in 24-well plates at a density of 3.0×105 cells/well and cultured for 24 hours in an atmosphere of 5% CO2 at 37°C. Cells were rinsed twice with Kreb's ringer phosphate HEPES buffer (pH 7.4, 150 mM NaCl, 5 mM KCl, 1.25 mM MgSO4, 1.25 mM CaCl2, 2.9 mM Na2HPO4, 10 mM HEPES) and were pre-incubated with the solutions of canagliflozin (250 µl, 10 uM) for 5 minutes at room temperature. The transport reaction was initiated by adding 50 µl of 4.5 mM 2-DG (a substrate for GLUTs)/3H-2-DG (0.625 µCi) followed by incubation for 15 minutes at room temperature. The 2-DG uptake was halted by aspiration of the incubation mixture. Cells were immediately washed 3 times with ice-cold PBS. Samples were extracted with 0.3 N NaOH, and radioactivity was determined by liquid scintillation.

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SGLT1/SGLT2 Inhibition Assay in CHO Cells: Chinese Hamster Ovary-K (CHOK) cells expressing human, rat, or mouse SGLT1 or SGLT2 were seeded in 96-well plates. Cells were washed with assay buffer and then incubated with assay buffer containing various concentrations of Canagliflozin for 10 minutes. Subsequently, a mixture of 14C-labeled alpha-methyl-D-glucopyranoside (AMG) and unlabeled AMG was added, and cells were incubated for 2 hours to allow uptake. After incubation, cells were washed extensively with ice-cold phosphate-buffered saline (PBS) to stop the reaction and remove extracellular radioactivity. Cell-associated radioactivity was measured using a scintillation counter. Inhibition curves were generated, and IC50 values were calculated. [1]
GLUT Inhibition Assay in L6 Myoblasts: Rat L6 myoblast cells were seeded in 24-well plates and cultured. Cells were rinsed and pre-incubated with a solution containing Canagliflozin (10 µM) for 5 minutes at room temperature. The transport reaction was initiated by adding a mixture of 3H-labeled 2-deoxy-D-glucose (2-DG) and unlabeled 2-DG. After a 15-minute incubation at room temperature, the reaction was stopped by aspiration. Cells were washed with ice-cold PBS, lysed with NaOH, and the radioactivity was measured by liquid scintillation counting to assess GLUT-mediated glucose uptake. [1]
SGLT3 Functional Assay in Oocytes: Stage V-VI Xenopus oocytes were injected with human SGLT3 mRNA or water (sham control) and incubated. For electrophysiological recording, oocytes were impaled with two microelectrodes and voltage-clamped. After establishing a baseline, oocytes were perfused with 50 µM 1-deoxynojirimycin (DNJ, an SGLT3 agonist) to induce currents, followed by co-application of DNJ with either Canagliflozin (10 µM) or DMSO control. Finally, phlorizin (3 mM) was applied in the presence of DNJ as a reference inhibitor. The DNJ-induced currents were recorded, and the effect of compounds was calculated as percentage inhibition relative to the DNJ-induced current. [1]

Animal/Disease Models: Diet-induced obese, insulin resistantmice (DIO) Mice[1]
Doses: 30 mg/kg
Route of Administration: po (oral gavage); daily; 4 weeks
Experimental Results: decreased BG levels, respiratory exchange ratio, and body weight gain.

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Animal/Disease Models: Male Zucker fatty (ZF) obese, insulin resistant rats[1]
Doses: 3 mg /kg
Route of Administration: po (oral gavage); daily; 3 weeks
Experimental Results: UGE was increased with no significant change in total food intake compared with that in vehicle-treated rats, leading to a decrease in body weight.\\n\\n

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\\nAnimals and canagliflozin Administration [1]
\\nFour rodent models were used in these experiments: (1) male C57BL/6J mice fed with a high-fat diet (D-12492 with 60 kcal% fat) (diet-induced obese, insulin resistantmice [DIO]); (2) male C57BL/ksj-db/db hyperglycemic mice; (3) male Zucker fatty (ZF) obese, insulin resistant rats; and (4) male ZDF obese, hyperglycemic rats. Canagliflozin was formulated in 0.5% hydroxypropyl methylcellulose and administrated via oral gavage at 10 ml/kg.\\n

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\\n\\nReduction of Hyperglycemia in Diabetic Rodent Models [1]
\\nTo examine the effect of canagliflozin on hyperglycemia, single doses of canagliflozin (0.1, 1, and 10 mg/kg) were administered to overnight-fasted db/db mice. BG levels were monitored at 0, 0.5, 1, 3, 6, and 24 hours after dosing. Canagliflozin was also administered to ZDF rats at varying doses (3–30 mg/kg) for 4 weeks to evaluate its effect on BG control and pancreatic beta-cell function. BG levels were monitored weekly, and HbA1c, plasma glucose, and insulin levels were determined at the end of the 4-week treatment. An oral glucose tolerance test (OGTT) (2 mg/kg of body weight, given by gavage) was conducted in ZDF rats after 4 weeks of treatment. Blood was sampled at 0, 30, 60, and 120 minutes after glucose challenge from the tail vein for measurement of BG levels using a glucometer and plasma insulin using ELISA method.\\n

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\\n\\nBody Weight Control Studies in Obese Mice and Rats [1]
\\nThe effects of canagliflozin on body weight gain were evaluated in DIO mice and ZF rats. DIO mice received a 4-week treatment of canagliflozin at 30 mg/kg. Body weight, food intake, and BG levels were monitored weekly. UGE and indirect calorimetry were conducted in the fourth week of treatment during the compound treatment. In another study, ZF rats were treated with canagliflozin at 3 mg/kg for 3 weeks. Body weight, food intake, and BG were measured weekly during the 19-day treatment period. UGE, body fat, and indirect calorimetric studies were conducted at the end of this study.\\n

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\\n\\nUrinary Glucose Excretion (UGE) Study. [1]
\\n Male Sprague-Dawley (SD) rats aged 4-5 weeks were used for experiments at 6 weeks of age after acclimation period. The animals were divided into experimental groups matched for body weight (n = 2-3). The compounds were prepared in vehicles as suspension or solution. UGE studies were performed after two-day acclimation period in metabolic cages. The compounds (canagliflozin) or vehicle were orally administered at a dose of 30 mg/kg in 0.2% CMC/0.2% Tween 80. Urine samples were collected for 24 hours using metabolic cages to measure urinary glucose excretion. Urine glucose contents were determined by an enzymatic assay kit (UGLU-L). All animals were allowed free access to a standard pellet diet (CRF1) and tap water. \\n

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\\n\\nSingle Oral Dosing Study. [1]
\\n Male KK/Ta Jcl mice aged 9 weeks were kept on a standard diet (CRF-1; 5.7% (w/w) fat, 3.59 kcal/g), 20-week-old mice were fed with a high-fat diet (60 kcal%) for 4 weeks. The experiment was carried out at the age of 24 weeks. Male C57BL/6N mice aged 11 weeks were also used in this study. The animals were divided into experimental groups matched for body weight and blood glucose levels, which were measured in the fed state on the day of the experiment. The compounds (canagliflozin; 3 mg/kg) or vehicle (0.2% CMC/0.2% Tween 80) were orally administered at a volume of 10 mL/kg. The blood samples were collected from the tail vein before and at 1, 2, 4, 6 and 24 hr after the administration. The blood glucose level was determined using commercially available kits based on the glucose oxidase method. Data are expressed as means ± SEM. Area under the curve for blood glucose levels (AUCglucose 0-6 hr) was calculated by the trapezoidal rule. \\n

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\nAcute Blood Glucose Lowering in db/db Mice: Overnight-fasted db/db mice were administered a single oral dose of Canagliflozin (0.1, 1, or 10 mg/kg) or vehicle (0.5% hydroxypropyl methylcellulose, 10 mL/kg) via gavage. Blood glucose levels were monitored at 0, 0.5, 1, 3, 6, and 24 hours after dosing from the tail vein. [1]
\nChronic Study in ZDF Rats: ZDF rats were treated orally with Canagliflozin (3, 10, or 30 mg/kg) or vehicle (0.5% hydroxypropyl methylcellulose) once daily for 4 weeks. Body weight and non-fasting blood glucose were monitored weekly. At the end of the treatment, fed plasma glucose, HbA1c, and plasma insulin were measured. An oral glucose tolerance test (OGTT) was performed by administering glucose (2 g/kg body weight) via gavage. Blood samples were collected from the tail vein at 0, 30, 60, and 120 minutes post-glucose challenge for blood glucose and plasma insulin measurement. [1]
\nRenal Threshold for Glucose (RTG) Study in ZDF Rats: ZDF rats were anesthetized. A catheter was inserted into the jugular vein for glucose or insulin infusion, and another catheter was introduced into the bladder for urine collection. In one experiment, a graded glucose infusion (GGI) was performed to raise blood glucose. In a separate experiment, insulin was first infused to lower blood glucose, followed by a GGI. Rats were pretreated with either vehicle or Canagliflozin (1 mg/kg). Blood and urine samples were collected every 5 minutes during the infusion to measure glucose and creatinine for RTG calculation. [1]
\nBody Weight Study in Diet-Induced Obese (DIO) Mice: DIO mice were treated orally with Canagliflozin (30 mg/kg) or vehicle once daily for 4 weeks. Body weight and food intake were monitored weekly. In the fourth week, urinary glucose excretion (UGE) over 4 hours and indirect calorimetry (to measure respiratory exchange ratio and oxygen consumption) were conducted. [1]
\nBody Weight Study in Zucker Fatty (ZF) Rats: ZF rats were treated orally with Canagliflozin (3 mg/kg) or vehicle once daily for 3 weeks. Body weight and food intake were monitored. At the end of the study, UGE over 4 hours and indirect calorimetry were measured. Epididymal fat pad and liver weights were determined during necropsy. [1]
\nGeneral Drug Formulation: For all in vivo studies, Canagliflozin was formulated in 0.5% (w/v) hydroxypropyl methylcellulose suspension and administered orally via gavage at a volume of 10 mL/kg body weight. [1]

Absorption, Distribution and Excretion
Bioavailability and Steady-State Concentration The absolute oral bioavailability of canagliflozin is approximately 65% on average. Steady-state concentrations are reached after 4 to 5 days with daily doses of 100 mg to 300 mg. Effect of Food on Absorption Taking canagliflozin with a high-fat meal has no significant effect on its pharmacokinetic parameters. This drug can be taken without food intake. Nevertheless, because canagliflozin may reduce postprandial plasma glucose excretion by prolonging intestinal glucose absorption, it is recommended to take this drug before the first meal of the day. Following a single oral dose of radiolabeled canagliflozin in healthy subjects, the proportions of canagliflozin or its metabolites detected in feces and urine were as follows: Feces 41.5% unmetabolized radiolabeled drug; 7.0% hydroxylated metabolites; 3.2% O-glucuronide metabolites. Urine Approximately 33% of the ingested radiolabeled dose is detected in urine, usually as an O-glucuronide metabolite. Less than 1% of the dose is excreted in urine as unmetabolized drug. The drug is widely distributed throughout the body. The mean steady-state volume of distribution after a single intravenous injection of canagliflozin in healthy subjects is 83.5 L. The clearance of canagliflozin after intravenous injection in healthy subjects is approximately 192 mL/min. Renal clearance of canagliflozin at 100 mg and 300 mg doses ranges from 1.30 to 1.55 mL/min. /Breast Milk/ Canagliflozin is distributed in rat milk; it is unknown whether the drug is distributed in human milk. Canagliflozin is an oral hypoglycemic agent used to treat type 2 diabetes. It works by inhibiting sodium-glucose cotransporter 2, blocking the reabsorption of glucose in the proximal renal tubules. This article describes the biotransformation and distribution of canagliflozin in vivo following a single oral administration of [(14)C]canagliflozin in intact mice and bile duct cannulated (BDC) mice, rats, intact dogs, and humans. Fecal excretion is the primary route of elimination of the radioactive material from the drug in both animals and humans. In BDC mice and rats, most of the radioactive material is excreted via bile. The amount of radioactive material excreted in urine in animals ranges from 1.2% to 7.6% of the administered [(14)C]canagliflozin dose, while in humans it is approximately 33%. The primary metabolic pathway for canagliflozin elimination is oxidation in animals and direct glucuronidation in humans. Unmetabolized canagliflozin is the major component in systemic circulation in all species. In human plasma, the two pharmacologically inactive O-glucuronide conjugates, M5 and M7, account for 19% and 14% of total drug exposure, respectively, and are considered the major human metabolites. In repeated-dose safety studies in mice and rats, plasma concentrations of M5 and M7 were lower than in humans taking the maximum recommended dose of 300 mg canagliflozin. However, bile metabolite analysis in rodents showed significant exposure to M5 and M7 in the livers of mice and rats. The pharmacological inactivity and high water solubility of M5 and M7 support glucuronidation of canagliflozin as a safe detoxification route. The mean absolute oral bioavailability of canagliflozin is approximately 65%. Concomitant administration of canagliflozin with a high-fat meal does not affect its pharmacokinetics; therefore, INVOKANA can be taken with or without food. However, given that INVOKANA can delay intestinal glucose absorption, thereby reducing postprandial glycemic variability, it is recommended to take it before the first meal of the day. Following a single intravenous infusion of canagliflozin in healthy subjects, the mean steady-state volume of distribution was 119 L, indicating extensive tissue distribution. Canagliflozin is extensively bound to plasma proteins (99%), primarily albumin. Protein binding was not correlated with canagliflozin plasma concentration. Plasma protein binding was not significantly altered in patients with impaired renal or hepatic function. Following a single oral dose of [14C] canagliflozin in healthy subjects, 41.5%, 7.0%, and 3.2% of the radioactive dose were recovered in feces, respectively, and excreted as canagliflozin, hydroxylated metabolites, and O-glucuronide metabolites. Enterohepatic circulation of canagliflozin was negligible. Approximately 33% of the radioactive dose was excreted in the urine, primarily as O-glucuronide metabolites (30.5%). Less than 1% of canagliflozin was excreted unchanged in the urine. Renal clearance of canagliflozin at 100 mg and 300 mg doses ranged from 1.30 to 1.55 mL/min. The mean systemic clearance after intravenous administration of canagliflozin in healthy subjects was approximately 192 mL/min.

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Metabolism/Metabolites
Canagliflozin is primarily metabolized via O-glucuronidation.
It is primarily eliminated via glucuronidation by the enzymes UGT1A9 and UGT2B4, producing two inactive O-glucuronide metabolites. In the human body, the oxidative metabolism of canagliflozin by the hepatic cytochrome P44 enzyme CYP3A4 is negligible (approximately 7%). O-glucuronidation is the main metabolic elimination pathway of canagliflozin, primarily carried out by UGT1A9 and UGT2B4, producing two inactive O-glucuronide metabolites. CYP3A4-mediated (oxidative) metabolism of canagliflozin in the human body is minimal (approximately 7%).

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Biological Half-Life
In a clinical study, the terminal half-life of canagliflozin at a 100 mg dose was 10.6 hours, and at a 300 mg dose, it was 13.1 hours.

Toxicity Summary
Identification and Use: Canagliflozin is an oral renal sodium/glucose cotransporter 2 (SGLT2) inhibitor that causes glycosuria and provides a unique mechanism for lowering blood glucose levels in patients with diabetes. Human Exposure and Toxicity: Canagliflozin is used to treat type 2 diabetes. It lowers blood glucose primarily by increasing urinary glucose excretion through inhibition of sodium-glucose cotransporter 2 (SGLT2) in the kidneys. Data from randomized clinical trials lasting up to 52 weeks indicate that canagliflozin is generally well tolerated. The most common adverse reaction is genital fungal infection, occurring in 11–15% of women receiving canagliflozin, compared to 2–4% in women randomized to glimepiride or sitagliptin. In men, the corresponding rates were 8–9% and 0.5–1%, respectively. The incidence of urinary tract infection (UTI) was slightly higher (5–7%) after treatment with canagliflozin compared to placebo (4%). The risk of hypoglycemia associated with canagliflozin is slightly higher than with placebo, but this risk increases significantly when used in combination with insulin or sulfonylureas (SU), in patients with chronic kidney disease (CKD), and in elderly patients. Patients using canagliflozin, especially those with CKD, may experience worsening renal function and hyperkalemia. Canagliflozin offers two major advantages: mild weight loss (average 2-4 kg) and lower blood pressure due to its osmotic diuretic effect. However, the latter may cause orthostatic hypotension and dizziness in susceptible individuals. Another noteworthy adverse effect of canagliflozin is an average 8% increase in plasma low-density lipoprotein cholesterol (LDL-C) levels compared to placebo. Elderly patients may discontinue use of the drug due to adverse effects such as urinary frequency, genital fungal infections, and urinary tract infections. Animal studies: A two-year rat study (at doses of 10, 30, and 100 mg/kg) evaluated the carcinogenicity of canagliflozin. The results showed an increased incidence of pheochromocytoma, renal tubular tumors, and testicular interstitial cell tumors in rats. Interstitial cell tumors were associated with elevated luteinizing hormone levels, while pheochromocytomas were likely associated with glucose malabsorption and calcium homeostasis disturbances. Renal tubular tumors may also be associated with glucose malabsorption. In mice, administration of canagliflozin at doses of 10, 30, or 100 mg/kg did not increase the incidence of tumors. In a toxicity study in pupal rats, researchers administered canagliflozin directly to pupal rats from day 21 (PND 21) to day 90 (PND 90) at doses of 4, 20, 65, or 100 mg/kg. The results showed increased kidney weight in all dose groups, and the incidence and severity of renal pelvis and tubular dilatation increased in a dose-dependent manner. The exposure in the lowest tested dose group was equal to or greater than 0.5 times the maximum clinical dose of 300 mg. The renal pelvis dilatation observed in pupal rats did not completely reverse within a one-month recovery period. In studies of administering canagliflozin to pregnant rats or rabbits during organogenesis, or to mothers from day 6 of gestation (GD 6) to PND 21, and to pups in utero and throughout lactation, no similar effects on developing kidneys were observed. Canagliflozin at doses up to 100 mg/kg (approximately 14 and 18 times the clinical dose of 300 mg for males and females, respectively) had no effect on the ability of rats to mate, reproduce, or maintain offspring, although slight changes were observed in some reproductive parameters at the highest dose (decreased sperm velocity, increased number of abnormal sperm, slightly fewer corpus luteum, reduced implantation sites, and reduced pup number). In the Ames assay, canagliflozin was not mutagenic regardless of metabolic activation. In in vitro mouse lymphoma assays, canagliflozin was mutagenic, but only after metabolic activation. In both the oral micronucleus assay and the oral comet assay in rats, canagliflozin did not show mutagenicity or chromosome breakage. Effects during pregnancy and lactation ◉ Overview of use during lactation: There is currently no information on the clinical use of canagliflozin during lactation. Canagliflozin is an uncharged molecule that binds to proteins up to 99% in plasma, making it unlikely to enter breast milk in clinically significant amounts. Due to the theoretical risk to the developing kidneys of infants, the manufacturer does not recommend the use of canagliflozin during lactation. Especially in breastfeeding newborns or premature infants, alternative medications may be necessary. ◉ Effects on breastfed infants: As of the revision date, no relevant published information was found. ◉ Effects on lactation and breast milk: As of the revision date, no relevant published information was found. Protein binding: Canagliflozin is primarily bound to albumin. The plasma protein binding rate of this drug is 99%.

[1]. Effect of canagliflozin on renal threshold for glucose, glycemia, and body weight in normal and diabetic animal models. PLoS One. 2012;7(2):e30555.

[2]. Discovery of canagliflozin, a novel C-glucoside with thiophene ring, as sodium-dependent glucose cotransporter 2 inhibitor for the treatment of type 2 diabetes mellitus. J Med Chem. 2010 Sep 9;53(17):6355-60.

Canagliflozin hydrate is the hemihydrate form of canagliflozin. It treats type 2 diabetes by inhibiting sodium-glucose cotransporter 2 (SGLT2). It is both a hypoglycemic agent and an SGLT2 inhibitor. Its main component is canagliflozin. Canagliflozin is a C-glycoside containing a thiophene ring, an orally effective SGLT2 inhibitor with hypoglycemic activity. Canagliflozin can also reduce weight and has a low risk of hypoglycemia. It is a glycoside SGLT2 inhibitor that promotes urinary glucose excretion by inhibiting renal glucose reabsorption. It is used to control blood glucose levels in patients with type 2 diabetes. Drug Indications Invokana is indicated for the treatment of poorly controlled type 2 diabetes in adults as an adjunct to diet and exercise; it can also be used as monotherapy in patients who are not suitable for metformin due to intolerance or contraindications; or in combination with other diabetes medications. For results regarding combination therapy, glycemic control, cardiovascular and renal events, and the study population, please see Sections 4.4, 4.5, and 5.1. Canagliflozin is a C-glycoside compound (in its hemihydrate form) that treats type 2 diabetes by inhibiting sodium-glucose cotransporter type 2 (SGLT2). It has a hypoglycemic effect and is also an SGLT2 inhibitor. It is a C-glycoside compound belonging to the thiophene class and organofluorine compounds. Canagliflozin, also known as Invokana, is a sodium-glucose cotransporter 2 (SGLT2) inhibitor used to treat type 2 diabetes in conjunction with lifestyle modifications, including diet and exercise. It was initially approved by the FDA in 2013 for the treatment of diabetes and later in 2018 for reducing the risk of cardiovascular events in patients with type 2 diabetes. Canagliflozin was the first oral hypoglycemic agent approved for the prevention of cardiovascular events in patients with type 2 diabetes. Cardiovascular disease is the most common cause of death in these patients. Anhydrous canagliflozin is a sodium-glucose cotransporter 2 (SGLT2) inhibitor. The mechanism of action of anhydrous canagliflozin is as a SGLT2 inhibitor and a P-glycoprotein inhibitor. Canagliflozin is a C-glycoside containing a thiophene ring, an orally effective SGLT2 inhibitor with hypoglycemic activity. Canagliflozin also helps with weight loss and has a low risk of hypoglycemia. Anhydrous canagliflozin is the anhydrous form of canagliflozin, a C-glycoside containing a thiophene ring, an orally effective SGLT2 inhibitor with hypoglycemic activity. Canagliflozin also helps with weight loss and has a low risk of hypoglycemia. It is a glucosinolate SGLT2 inhibitor that stimulates glucose excretion in urine by inhibiting renal glucose reabsorption. It is used to control blood glucose levels in patients with type 2 diabetes.

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Drug Indications
This drug is used in combination with diet and exercise to improve glycemic control in adults with type 2 diabetes.FDA Label
Invokana is indicated for the treatment of adult patients with poorly controlled type 2 diabetes as an adjunct to diet and exercise; it can be used as monotherapy when metformin is deemed unsuitable due to intolerance or contraindications; and it can also be used in combination with other diabetes medications. For results regarding combination therapy, glycemic control, cardiovascular and renal events, and studies in the study population, please see Sections 4.4, 4.5, and 5.1.
Treatment of Type 2 Diabetes Mellitus
Mechanism of Action
Sodium-glucose cotransporter 2 (SGLT2) is present in the proximal tubules of the kidney and is responsible for reabsorbing filtered glucose from the tubular lumen. Canagliflozin inhibits SGLT2 cotransporter. This inhibition leads to reduced reabsorption of filtered glucose and a lower renal glucose threshold (RTG), thereby increasing urinary glucose excretion.
Sodium-glucose cotransporter 2 (SGLT2) is expressed in the proximal tubules of the kidney and is responsible for reabsorbing most of the filtered glucose from the tubular lumen. Canagliflozin is an SGLT2 inhibitor. By inhibiting SGLT2, canagliflozin reduces the reabsorption of filtered glucose, lowers the renal glucose threshold (RTG), and thus increases urinary glucose excretion (UGE).
Background: Canagliflozin is a sodium-glucose cotransporter (SGLT) 2 inhibitor and is currently in clinical development for the treatment of type 2 diabetes mellitus (T2DM). Methods: The uptake of 14C-α-methylglucoside by Chinese hamster ovarian K cells expressing human, rat, or mouse SGLT2 or SGLT1 was analyzed; the uptake of 3H-2-deoxy-D-glucose by L6 myoblasts was analyzed; and two-electrode voltage-clamp recordings were performed on oocytes expressing human SGLT3. In this study, a gradient glucose infusion method was used to determine the urinary glucose excretion rate (UGE) and renal glucose excretion threshold (RT(G)) in Zucker diabetic obese (ZDF) rats at different blood glucose concentrations. Experimental subjects included a vector control group and a canagliflozin treatment group. This study aimed to elucidate the pharmacodynamic effects of canagliflozin in vitro and in preclinical models of type 2 diabetes and obesity. Results showed that canagliflozin 1 mg/kg treatment reduced RT(G) in ZDF rats from 415±12 mg/dl to 94±10 mg/dl, while maintaining the threshold relationship between blood glucose and UGE; UGE was almost unobserved when blood glucose was below RT(G). Canagliflozin dose-dependently reduced blood glucose concentration in acutely administered db/db mice. In ZDF rats treated with canagliflozin for 4 weeks, glycated hemoglobin (HbA1c) decreased and insulin secretion parameters improved. In the obese animal model, canagliflozin increased urinary glucose excretion (UGE) and decreased blood glucose (BG), body weight gain, epididymal fat, liver weight, and respiratory exchange rate. Conclusion: Canagliflozin reduced RT(G), increased urinary glucose excretion (UGE), improved glycemic control and β-cell function in a type 2 diabetic rodent model, and reduced body weight gain in the obese rodent model. [1]

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We found that C-glycoside 4 with a heterocyclic aromatic ring forms a more metabolically stable sodium-dependent glucose cotransporter 2 (SGLT2) inhibitor compared to O-glycoside 2 (T-1095). A novel thiophene derivative, 4b-3 (canagliflozin), is a highly potent and selective SGLT2 inhibitor that showed significant hypoglycemic effects in KK (HF-KK) mice fed a high-fat diet. [2]

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Canagliflozin is a sodium-glucose cotransporter 2 (SGLT2) inhibitor that is currently in clinical development for the treatment of type 2 diabetes mellitus (T2DM). [1]
Its main mechanism of action is to inhibit SGLT2 in the proximal renal tubules, thereby reducing renal reabsorption of glucose, lowering the renal glucose threshold, and increasing urinary glucose excretion, thereby lowering blood glucose in a non-insulin-dependent manner. [1] Preclinical studies have shown that, in addition to glycemic control, canagliflozin may improve β-cell function, reduce weight gain, decrease fat mass, and shift substrate utilization toward fatty acid oxidation. [1]

C48H52F2O11S2

907.05

906.291

C, 63.56; H, 5.78; F, 4.19; O, 19.40; S, 7.07

928672-86-0

Canagliflozin;842133-18-0

24997615

Off-white to yellow solid powder

5.872

9

15

10

63

574

10

CC1=C(C=C(C=C1)[C@H]2[C@@H]([C@H]([C@@H]([C@H](O2)CO)O)O)O)CC3=CC=C(S3)C4=CC=C(C=C4)F.CC1=C(C=C(C=C1)[C@H]2[C@@H]([C@H]([C@@H]([C@H](O2)CO)O)O)O)CC3=CC=C(S3)C4=CC=C(C=C4)F.O

VHOFTEAWFCUTOS-TUGBYPPCSA-N

InChI=1S/2C24H25FO5S.H2O/c2*1-13-2-3-15(24-23(29)22(28)21(27)19(12-26)30-24)10-16(13)11-18-8-9-20(31-18)14-4-6-17(25)7-5-14;/h2*2-10,19,21-24,26-29H,11-12H2,1H3;1H2/t2*19-,21-,22+,23-,24+;/m11./s1

(2S,3R,4R,5S,6R)-2-[3-[[5-(4-fluorophenyl)thiophen-2-yl]methyl]-4-methylphenyl]-6-(hydroxymethyl)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 (5.51 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 (5.51 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 (5.51 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: 0.5% CMC+0.25% Tween 80 :18 mg/mL

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