SGLT2 ( IC50 = 2 nM )

The in vitro potency and selectivity of Bexagliflozin (EGT1442) and EGT1474 for human SGLT1 and SGLT2 were evaluated in cell-based AMG uptake assays in the presence of 25% human plasma. Both EGT1442 and EGT1474 inhibited SGLT1 and SGLT2-mediated sodium-dependent AMG uptake in a dose-dependent manner. The IC50 values for SGLT1 and SGLT2 inhibition and the SGLT2 selectivity are summarized in Table 1. EGT1442 exhibited an IC50 of 2 nM with a 2435-fold selectivity ratio against SGLT2 compared to SGLT1 whereas EGT1474 showed an IC50 of 3 nM and selectivity ratio of 2123 against SGLT2.

In normal rats and dogs a saturable urinary glucose excretion was produced with an ED(50) of 0.38 and 0.09mg/kg, respectively. Following chronic administration to db/db mice, EGT1442 dose-dependently reduced HbA(1c) and blood glucose concentration without affecting body mass or insulin level. Additionally, EGT1442 significantly prolonged the median survival of SHRSP rats. EGT1442 significantly prolongs the median survival of stroke prone rats fed a high salt diet. [1]

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EGT1442 lowers blood glucose levels in a dose-dependent manner after a 2 g/kg glucose challenge (Fig. 2A). The baseline-corrected blood glucose AUC values observed following administration of 0.1, 0.3, 1, 3 and 10 mg/kg EGT1474 were 74.2%, 70.7%, 75.9%, 64.8%, and 26.2% of control AUC, respectively (Fig. 2B). The results indicate that EGT1442 can significantly lower blood glucose in rats and can blunt the normal physiological response to a high dietary glucose load. EGT1442 can significantly increase (F(5,41) = 10.3; p < 0.0001) the urinary glucose excretion dose dependently with an IC50 of 0.38 mg/kg (Fig. 3A). A trend toward increased urine output with increased dose was seen at all time points (Fig. 3B). Compared to the control group, a statistically significant increase of water consumption was observed at all levels, except for the 0.1 mg/kg cohort (Fig. 3C). The increased water consumption may reflect a compensatory mechanism to maintain hydration in the presence of a higher urine output. No difference in food consumption was observed between treated and control groups.[1]

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EGT1442 shows some activity to reduce blood glucose levels after a 2 g/kg glucose challenge in healthy dogs (Fig. 4A). The latter effect reached statistical significance at the lower dose levels but did not, in general, show dose-dependency. EGT1442 induces a significant glucosuria in healthy dogs. The glucosuria is dose-dependent, with an ED50 of 0.09 mg/kg (Fig. 4B). Since SGLT2 mediated glucose transport is coupled to sodium transport, we evaluated the Na+, K+ as well as Cl− level in the urine. Trends toward higher excretions of Na+, K+, and Cl− were observed, likely explained by increased urine volume (Fig. 4C). A trend toward increased urine output with increasing dose was seen for the full 24 h interval (Fig. 4D). As in the rat study, a trend toward increased water consumption in treated dogs was noted but no difference in food consumption was observed between treated and control groups. [1]

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To further investigate the in vivo efficacy of EGT1442, we utilized a well established mouse genetic model of diabetes. Non-fasting blood glucose levels were reduced significantly in all treated groups from day 1 to day 28 and in a dose-dependent manner (Fig. 5A). Consistent with the observed reductions in blood glucose concentration, significant (p < 0.0001) dose-dependent reductions in HbA1c levels were seen in 0.3–3 mg/kg EGT1474 treated groups (equals to 0.2–2 mg/kg EGT1442) (Fig. 5B). After 4 weeks administration, the reductions in HbA1c were 0.51%, 1.08%, 1.13% and 1.37% in 0.1, 0.3, 1 and 3 mg/kg groups compared to vehicle control, respectively. No obvious changes in plasma insulin levels were seen in any treated groups when compared to vehicle control groups or pre-dose evaluations (Fig. 5C). Large amounts of urinary glucose were excreted by both vehicle control and EGT1442 treated mice (Fig. 5D). However, no obvious increase in UGE was found in EGT1442 groups after 2 or 4 week treatment compared to concurrent control group.[1]

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EGT1442 reduced blood glucose levels dose-dependently at all indicated times compared to vehicle control rats during the oral glucose tolerance test performed on day 30 (Fig. 6A). Therefore, EGT1442 significantly improved tolerance to oral glucose challenge in these mice.

Assay for human SGLT1 and SGLT2[1]
A DNA fragment encoding human SGLT2 was inserted between the Hind III and Not I sites of the pEAK15 expression vector. Plasmids harboring the desired cDNA inserts were identified by restriction enzyme analysis. A plasmid bearing the human SGLT1 coding sequence in the pDream 2.1 expression vector was purchased from GenScript Corporation. Human SGLT1 expression plasmid DNA was transfected into COS-7 cells using Lipofectamine 2000 according to the manufacturer's suggested protocol. Transfected cells were cryopreserved in DMEM containing 10% DMSO at −195 °C (liquid nitrogen). Plasmid containing human SGLT2 was linearized with Nsi I and purified by agarose gel electrophoresis. Using Lipofectamine 2000, DNA was transfected into 293.ETN cells and cultured in DMEM containing 10% FBS at 37 °C under 5% CO2 for 24 h. Transfectants were then selected in the same growth medium supplemented with puromycin for two weeks. Puromycin-resistant cells were recovered and seeded on a fresh 96-well plate (single cell per well) and cultured in the presence of puromycin until cells become confluent. Puromycin-resistant clones were evaluated for SGLT2 activity in methyl-α-d-[U-14C]glucopyranoside (AMG) uptake assay. Clones that exhibited the highest signal to background ratio were propagated and cryopreserved in DMEM containing 10% DMSO at −195 °C. Cells expressing SGLT1 or SGLT2 were seeded on poly-d-lysine coated 96-well Scintiplates in DMEM containing 10% FBS (1 × 105 cells per well in100 μl medium) and incubated at 37 °C under 5% CO2 for 48 h prior to the assay. Cells were washed with 150 μL of either sodium buffer (137 mM NaCl, 5.4 mM KCl, 2.8 mM CaCl2, 1.2 mM MgCl2, 10 mM Tris/Hepes, pH 7.2) or sodium free buffer (137 mM N-methylglucamine, 5.4 mM KCl, 2.8 mM CaCl2, 1.2 mM MgCl2, 10 mM Tris/Hepes, pH 7.2) twice. Either 50 μL of the sodium free buffer containing 40 μCi/mL α-methyl-d-glucopyranoside or 50 μL of the sodium buffer containing 8 μCi/mL AMG, 25% human plasma and the test compounds at indicated concentrations were added to each well of a 96-well plate and incubated at 37 °C with shaking for 2 or 1.5 h for SGLT1 and SGLT2, respectively. Cells were washed with 150 μL of wash buffer (137 mM N-methylglucamine, 10 mM Tris/Hepes, pH 7.2) twice and AMG uptake was quantitated using a TopCount scintillation counter

Urinary glucose excretion and oral glucose tolerance test in rats[1]
Bexagliflozin (EGT1442) (delivered as its co-crystal form EGT1474) was administered orally to overnight fasted rats by gavage at different doses (0.1, 0.3, 1, 3 and 10 mg/kg of EGT1474). Control rats were given the vehicle 10% PEG400 only. One hour post dosing, glucose solution (2 g/kg, 10 mL/kg) was administered by oral gavage. Blood glucose concentration was measured before dosing, before the glucose challenge, 15, 30, 60, and 120 min post-glucose challenge. Urine was collected from 0 to 4 h, 4 to 8 h and 8 to 24 h post-dosing for glucose measurement. Urine volumes, food and water consumptions were also recorded. Blood glucose levels were determined using a glucometer . The glucose concentration in urine was determined using a HITACHI 7080 automatic biochemistry analyzer.[1]

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Urinary glucose excretion and urinary electrolytes in dogs[1]
Bexagliflozin (EGT1442) (delivered as its co-crystal form EGT1474) was administered orally to overnight-fasted dogs by gavage at different doses (0.03, 0.1, 0.3, 1 and 3 mg/kg of EGT1474). Control dogs were given the vehicle 10% PEG400 only. One hour post dosing, glucose solution (2 g/kg, 5 ml/kg) was administered by gavage. The blood glucose concentration was quantitated by means of an automatic biochemistry analyzer for samples collected before dosing, before glucose challenge, and 15, 30, 60, and 120 min post glucose challenge. Urine was collected from 0 to 4 h, 4 to 8 h and 8 to 24 h post dosing for electrolyte analysis (Na+, K+ and Cl−) and glucose measurement. Food and water consumptions were also recorded. Urine glucose concentrations were measured using an Integra 400 Plus automatic biochemistry analyzer. Urine Na+, K+, Cl− concentrations were determined with an HC9883 HANGCHUANG Electrolyte Analyzer.[1]

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Anti-hyperglycemic and HbA1c-lowering effects in db/db mice[1]
Db/db mice were dosed daily with the vehicle 10% PEG400 water solution or Bexagliflozin (EGT1442) at different doses (0.1, 0.3, 1 and 3 mg/kg) by gavage administration for 30 days. Animals were dosed between 10:00 AM and 12:00 PM daily. The body weights were measured every 4 days and the dosage was adjusted according to the most recent body weight. The volume of administration was 10 mL/kg body weight. All animals were observed daily and any abnormal findings were recorded. Blood glucose concentrations were determined on days −3, 0, 1, 7, 14, 21 and 28 at 6 h post-dose. Plasma insulin was determined on day −1 and day 29. HbA1c levels were determined on day −1 and day 29. On day 30, an oral glucose tolerance test (OGTT) was performed. Mice were fasted overnight prior to treatment with vehicle or compound. One hour after administration of the test article or vehicle, glucose solution (1 g/kg, 10 mL/kg) was delivered by gavage. Blood glucose concentration was determined by glucometer measurement immediately before drug administration, before glucose challenge, and at 15, 30, 60, and 120 min after glucose challenge. Urine was collected from mice placed in metabolic cages for 24 h following dosage on days −5, 13 and 27 to permit glucose measurements. Urine volumes were also recorded.[1]

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The effects of chronic Bexagliflozin (EGT1442) administration on the survival of SHRSP rats[1]
Prior to experiment initiation, 7-week old SHRSP rats were assigned to 3 groups of 15 males each, based on body weights. During this week, daily water consumption was determined in order to calculate appropriate drug concentrations for the drinking water. The following week (Day 1), rats were switched to a stroke-promoting diet (Japanese diet 4%NaCl; Zeigler Brothers, PA) containing reduced potassium and protein and elevated NaCl. Animals received water bottles containing either no compound, EGT1442 (3.0 mg/kg) or the sodium channel blocker amiloride (1.0 mg/kg). Body weights were measured 2 times per week, and dosages were adjusted weekly. Water consumption was likewise measured 2 times per week, while food consumption was measured weekly. Daily values were estimated. Urine samples were analyzed by test strips once every two weeks. Additionally, all animals were observed twice daily and any abnormal findings were recorded. Rats were observed for 2 month using the cycle as described above.

Absorption, Distribution and Excretion
Healthy subjects and adult patients with type 2 diabetes mellitus given bexagliflozin have similar pharmacokinetic profiles. In a fasted state, the mean Cmax and AUC0-∞ of bexagliflozin were 134 ng/mL and 1,162 ng·h/mL, respectively. Bexagliflozin does not follow a time-dependent pharmacokinetic profile, and after multiple doses, approximately up to 20% is accumulated in plasma. The peak plasma concentration of bexagliflozin is reached between 2 and 4 hours after oral administration. This timing can be delayed if bexagliflozin is taken after a meal or with medications that slow gastric emptying. Between single doses of 3 mg and 90 mg (0.15 to 4.5 times the recommended dose), the plasma Cmax and AUC of bexagliflozin increase in a dose-proportional manner. Compared to dosing in the fasted state, consuming a standard high-fat, high-caloric meal leads to a 31% and 10% higher Cmax and AUC, respectively. Under these conditions, the median Tmax was increased to 5 hours. The effects of food on bexagliflozin pharmacokinetics are not considered clinically relevant.
Bexagliflozin is mainly eliminated through feces and urine. In healthy subjects given an oral [14C]-bexagliflozin solution, 91.6% of input radioactivity was recovered. Of this amount, 51.1% was recovered in feces, mainly as the parent compound, while 40.5% was recovered in urine, mostly as the 3'-O-glucuronide. The proportions of input radioactivity recovered as bexagliflozin in urine and feces were 1.5% and 28.7%, respectively.
Bexagliflozin has an apparent volume of distribution of 262 L.
Population pharmacokinetic modeling has shown that the apparent oral clearance of bexagliflozin is 19.1 L/h.

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Metabolism / Metabolites
Bexagliflozin is metabolized in the liver mainly by UGT1A9 and, to a lesser extent, CYP3A. In healthy volunteers given an oral [14C]-bexagliflozin solution, the 3'-O-glucuronide, a pharmacologically inactive metabolite, constituted 32.2% of the parent compound AUC. The rest of the bexagliflozin metabolites contributed less than 10% of the parent AUC. None of the metabolites are expected to have clinically relevant pharmacological effects.

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Biological Half-Life
Bexagliflozin has an apparent terminal elimination half-life of approximately 12 hours.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of bexagliflozin during breastfeeding. Bexagliflozin is 93% protein bound in plasma, so it is unlikely to pass into breastmilk in clinically important amounts. The manufacturer does not recommend bexagliflozin during breastfeeding because of a theoretical risk to the infant's developing kidney. An alternate drug may be preferred, especially while nursing a newborn or preterm infant.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Approximately 93% of bexagliflozin is bound to plasma protein.

[1]. Pharmacol Res . 2011 Apr;63(4):284-93.

Bexagliflozin is a C-glycosyl comprising of beta-D-glucose in which the anomeric hydroxy group is replaced by a 4-chloro-3-({4-[2-(cyclopropyloxy)ethoxy]phenyl}methyl)phenyl group. It is a sodium-glucose co-transporter 2 (SGLT2) inhibitor indicated as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus. It has a role as a sodium-glucose transport protein subtype 2 inhibitor, a hypoglycemic agent and an antihypertensive agent. It is a C-glycosyl compound, an aromatic ether, a member of monochlorobenzenes, a diether and a member of cyclopropanes.
Bexagliflozin is a highly specific and potent sodium-glucose co-transporter 2 (SGLT2) inhibitor. Similar to other SGLT2 inhibitors, bexagliflozin contains three basic moieties: glucose, two benzene rings and a methylene bridge. SGLT2 is responsible for 60% to 90% of renal glucose re-uptake, and unlike other isoforms such as SGLT1, SGLT2 is mainly expressed in the kidney. By inhibiting SGLT2, bexagliflozin reduces renal reabsorption of filtered glucose and increases urinary glucose excretion, which reduces blood glucose levels independently of insulin sensitivity. In January 2023, bexagliflozin was approved by the FDA for the treatment of adults with type 2 diabetes. Its use is not recommended in patients with type 1 diabetes since it may increase their risk of diabetic ketoacidosis.
Bexagliflozin is a Sodium-Glucose Cotransporter 2 Inhibitor. The mechanism of action of bexagliflozin is as a Sodium-Glucose Transporter 2 Inhibitor.

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Drug Indication
Bexagliflozin is indicated as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus.
Treatment of type II diabetes mellitus

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Mechanism of Action
Bexagliflozin is a highly selective sodium–glucose co-transporter 2 (SGLT2) inhibitor. SGLT2 is located in the proximal renal tubule, a part of the kidney where most reabsorption takes place, and they transport glucose and sodium from the tubular lumen to the epithelium. By inhibiting SGLT2, bexagliflozin reduces glucose reabsorption in the kidney and promotes its excretion in urine. Therefore, in patients with type 2 diabetes mellitus (T2DM), bexagliflozin reduces blood glucose levels independently of insulin sensitivity. Aside from improving glycemic control, bexagliflozin may also reduce body weight, systolic blood pressure, and albuminuria. The mechanism of action for these other effects have not been fully elucidated, but it is possible that they depend on the initial natriuresis caused by bexagliflozin, followed by a change in tissue sodium handling.

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Pharmacodynamics
Healthy subjects and adults with type 2 diabetes mellitus given single or multiple doses of bexagliflozin had dose-dependent increases in urinary glucose excretion (UGE) accompanied by increases in urine volume. A 20 mg bexagliflozin dose can provide near-maximal UGE, and elevated UGE values are maintained with multiple-dose administration. Bexagliflozin does not cause clinically significant QTc interval prolongation at 5 times the recommended dose. The use of bexagliflozin may cause ketoacidosis, volume depletion, urosepsis, pyelonephritis, necrotizing fasciitis of the perineum and genital mycotic infections. There is also an increased incidence of lower limb amputation in patients treated with bexagliflozin compared to those receiving a placebo. In addition, the use of bexagliflozin in patients treated with insulin and insulin secretagogues may increase the risk of hypoglycemia.

C24H29CLO7

464.94

464.16

C, 62.00; H, 6.29; Cl, 7.62; O, 24.09

1118567-05-7

1118567-05-7; 1118567-48-8 (diproline)

25195624

Solid powder

1.4±0.1 g/cm3

671.0±55.0 °C at 760 mmHg

359.6±31.5 °C

0.0±2.2 mmHg at 25°C

1.642

3.92

4

7

9

32

569

5

ClC1C([H])=C([H])C(=C([H])C=1C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])OC([H])([H])C([H])([H])OC1([H])C([H])([H])C1([H])[H])[C@@]1([H])C([H])(C([H])(C([H])(C([H])(C([H])([H])O[H])O1)O[H])O[H])O[H]

BTCRKOKVYTVOLU-SJSRKZJXSA-N

InChI=1S/C24H29ClO7/c25-19-8-3-15(24-23(29)22(28)21(27)20(13-26)32-24)12-16(19)11-14-1-4-17(5-2-14)30-9-10-31-18-6-7-18/h1-5,8,12,18,20-24,26-29H,6-7,9-11,13H2/t20-,21-,22+,23-,24+/m1/s1

(2S,3R,4R,5S,6R)-2-[4-chloro-3-[[4-(2-cyclopropyloxyethoxy)phenyl]methyl]phenyl]-6-(hydroxymethyl)oxane-3,4,5-triol

THR1442; EGT-1442; THR-1442; EGT1442; THR 1442; EGT 1442; Bexagliflozin; BRENZAVVY; EGT0001442;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO: ~93 mg/mL (~200.0 mM)
Water: ~93 mg/mL
Ethanol: ~23 mg/mL

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

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

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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

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

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

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