SGLT-2 ( IC50 = 3.1 nM ); SGLT-5 ( IC50 = 1.1 μM ); SGLT-6 ( IC50 = 2 μM ); SGLT-1 ( IC50 = 8.3 μM ); SGLT-4 ( IC50 = 11 μM )

Dogs receive a high exposure to empagliflozin; 24 hours after receiving 5 mg/kg of the drug, plasma concentrations were measured, and they were >100-fold above the IC50. In ZDF rats, the total plasma clearance of empagliflozin is 43 mL/min/kg, whereas in dogs, it is only 1.8 mL/min/kg. The C_max values for empagliflozin in ZDF rats and dogs are 167 nM and 17254 nM, in that order. [1] The half-lives of terminal elimination are 1.5 hours for ZDF rats and 6.3 hours for dogs. The bioavailability of empagliflozin in ZDF rats is 33.2%, whereas it is 89.0% in dogs. Long-term empagliflozin treatment improves metabolic syndrome characteristics and glycaemic control in diabetic rats. In [2]

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Empagliflozin is a potent, selective sodium glucose co-transporter-2 inhibitor that is in development for the treatment of type 2 diabetes. This series of studies was conducted to assess the in vivo pharmacological effects of single or multiple doses of Empagliflozin in Zucker diabetic fatty rats. Single doses of Empagliflozin resulted in dose-dependent increases in urinary glucose excretion and reductions in blood glucose levels. After multiple doses (5 weeks), fasting blood glucose levels were reduced by 26 and 39% with 1 and 3 mg/kg empagliflozin, respectively, relative to vehicle. After 5 weeks, HbA1c levels were reduced (from a baseline of 7.9%) by 0.3 and 1.1% with 1 and 3 mg/kg empagliflozin, respectively, versus an increase of 1.1% with vehicle. Hyperinsulinaemic-euglycaemic clamp indicated improved insulin sensitivity with empagliflozin after multiple doses versus vehicle. These findings support the development of empagliflozin for the treatment of type 2 diabetes. [2]

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Pharmacokinetics of Empagliflozin in ZDF rats and beagle dogs [1]
Pharmacokinetic parameters in rats and dogs are summarised in Table 3. High exposure of Empagliflozin was achieved in dogs, with plasma concentrations >100-fold above IC50 measured 24 h after administration of 5 mg/kg empagliflozin (data not shown). Despite only moderate CL and BA of empagliflozin in rats, acceptable exposure was also achieved (Table 3). Thus, in beagle dogs and ZDF rats, plasma concentrations well above the SGLT-2 IC50 can be achieved with low oral doses of empagliflozin.

This assay for sodium-dependent monosaccharide transport inhibition uses stable cell lines that overexpress hSGLT-1, -2, -4, -5, or -6 or rSGLT-1 or -2. Pre-incubation of cells is conducted for 25 minutes at 37°C in 200 μL uptake buffer (10 mM HEPES, 137 mM NaCl, 5.4 mM KCl, 2.8 mM CaCl₂, 1.2 mM MgCl2, 50 μg/ml Gentamycin, 0.1% BSA). Half an hour before the uptake experiment starts, 10 μM Cytochalasin B and the test compound are added at various concentrations. The addition of 0.6 μCi [¹⁴C]-labeled monosaccharide, such as [¹⁴C]-labeled AMG, glucose, fructose, mannose, or myo-inositol, to 0.1 mM AMG (or the corresponding non-radioactive monosaccharide) initiates the uptake reaction. The cells are incubated at 37°C for 60 minutes (hSGLT-5), 90 minutes (hSGLT-4), or 4 hours (hSGLT-2) before being washed three times with 300 μL PBS and lysed in 0.1 N NaOH for five minutes while being shaken intermittently. After mixing the lysate with 200 μL MicroScint 40 and shaking it for 15 minutes, the radioactivity is measured using the TopCount NXT. Prior to the addition of uptake buffer, cells are pre-incubated in pre-treatment buffer (uptake buffer containing choline chloride instead of NaCl) for 25 minutes in the case of the SGLT-4 and SGLT-5 tests.

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Radioligand binding assays using [3H]-labelled Empagliflozin [1]
Membranes (60 µg/well) were assayed in a 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.4) containing 137 mM NaCl in the presence or absence of 20 mM glucose and indicated concentrations of [3H]-Empagliflozin in 96-well plates at room temperature for 2 h. Incubations were stopped by rapid filtration through GF/B Filterplates impregnated with polyethyleneimine 0.5% and pre-wetted with 0.9% NaCl solution, and washed four times with 0.9% NaCl solution (4 °C) using a Harvester Filtermate 96. Filterplates were dried for 2 h and 50 µl of Microscint 20 was added to each well. Radioactivity retained on the filters was measured using the TopCount NXT. In parallel, the actual amount of activity used in the assays was determined by adding the same amount of [3H]-Empagliflozin that was added per well in the radioligand binding studies and 4 ml Ultima Gold Scintilator into 5 ml vials and measuring using a Tricarb 2900TR. Non-specific [3H]-Empagliflozin -binding was determined in the presence of 30 µM dapagliflozin.
Kinetic binding parameters were determined in the presence or absence of 20 mM glucose (see the Supporting Information for more details). Graphpad Prism 5.0 was used for calculating the equilibrium dissociation constant (Kd) using a nonlinear regressions for a single binding site model, and for calculating the association rate constant (Kon) and the dissociation rate constant values by means of a global fitting procedure using the ‘association and then dissociation’ nonlinear regression.

Empagliflozin treatment competitively binds to SGLT-2 over glucose at low doses, as demonstrated by tests conducted on a panel of human cell lines that overexpress SGLT-1, 2, 4, 5, and 6. In human proximal tublular cell (PTC) cell line HK2 cells, Empagliflozin treatment for 72 h inhibits the expression of SGLT-2 which in turn reversed high glucose induced TLR4 expression, NF-κB binding, IL-6 secretion, AP-1 binding and CIV expression.

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Sodium/glucose cotransporter 2 (SGLT2) inhibitors are oral hypoglycemic agents used to treat patients with diabetes mellitus. SGLT2 inhibitors block reabsorption of filtered glucose by inhibiting SGLT2, the primary glucose transporter in the proximal tubular cell (PTC), leading to glycosuria and lowering of serum glucose. We examined the renoprotective effects of the SGLT2 inhibitor Empagliflozin to determine whether blocking glucose entry into the kidney PTCs reduced the inflammatory and fibrotic responses of the cell to high glucose. We used an in vitro model of human PTCs. HK2 cells (human kidney PTC line) were exposed to control 5 mM, high glucose (HG) 30 mM or the profibrotic cytokine transforming growth factor beta (TGFβ1; 0.5 ng/ml) in the presence and absence of Empagliflozin for up to 72 h. SGLT1 and 2 expression and various inflammatory/fibrotic markers were assessed. A chromatin immunoprecipitation assay was used to determine the binding of phosphorylated smad3 to the promoter region of the SGLT2 gene. Our data showed that TGFβ1 but not HG increased SGLT2 expression and this occurred via phosphorylated smad3. HG induced expression of Toll-like receptor-4, increased nuclear deoxyribonucleic acid binding for nuclear factor kappa B (NF-κB) and activator protein 1, induced collagen IV expression as well as interleukin-6 secretion all of which were attenuated with empagliflozin. Empagliflozin did not reduce high mobility group box protein 1 induced NF-κB suggesting that its effect is specifically related to a reduction in glycotoxicity. SGLT1 and GLUT2 expression was not significantly altered with HG or empagliflozin. In conclusion, empagliflozin reduces HG induced inflammatory and fibrotic markers by blocking glucose transport and did not induce a compensatory increase in SGLT1/GLUT2 expression. Although HG itself does not regulate SGLT2 expression in our model, TGFβ increases SGLT2 expression through phosphorylated smad3 [3].

Mice: Male C57BL/6J mice (10 weeks of age) are used. The mice in the experimental group receive 3 or 10 mg/kg of Empagliflozin orally once a day for 8 days via oral gavage; the vehicle group receives the same volume of HEC alone.

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\n\nCompounds and Dosing[2]
\nEmpagliflozin [BI 10773] was administered by oral gavage using 0.5% hydroxyethylcellulose as a vehicle. The compound was moistened with Polysorbat 80 (polyoxyethylene sorbitan monooleate) before solution in vehicle (final concentration: 0.015%).[2]
\n\nAnimal Studies [2]
\nMale Zucker diabetic fatty (ZDF) rats (ZDF-Leprfa/Crl) were housed in groups at controlled temperature and humidity conditions, with a 12-h light/dark cycle, and ad libitum access to food [diet 2437, containing 4.5% sucrose] and water. \n
\nUrinary glucose excretion (UGE) and blood glucose levels were assessed in ZDF rats (10 and 13 weeks old, respectively) after single doses of Empagliflozin . Food was withdrawn during the experiments. Urine was collected for 24 h after dosing with 1 or 3 mg/kg Empagliflozin or vehicle using metabolic cages. Blood samples were obtained by tail bleed at 0.5, 1, 2, 3, 4, 5 and 7 h after dosing with 0.1, 0.3, 1 or 3 mg/kg Empagliflozin or vehicle.\n
\nGlucose homeostasis was investigated in a 5-week study in 12-week-old ZDF rats given multiple, once-daily doses of Empagliflozin . Blood samples (tail bleed) were taken from overnight fasted animals 16 h after dosing with 0.3, 1 or 3 mg/kg Empagliflozin or vehicle. Oral glucose tolerance tests (OGTTs) were performed on days 2 and 37. Overnight fasted animals were challenged with 2 g/kg glucose 16 h after administration of 3 mg/kg Empagliflozin . Blood samples were taken before and 0.25, 0.5, 1, 1.5, 2 and 3 h after glucose load.\n

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\nHyperinsulinaemic–Euglycaemic Clamp [2]
\nAnimals from the 5-week study were subjected to a hyperinsulinaemic–euglycaemic clamp 4 days after last dose [basal fasting blood glucose levels: 11.8 ± 0.8 mM (vehicle) and 9.0 ± 0.5 mM (3 mg/kg Empagliflozin )]. Indwelling catheters were inserted in the right jugular vein (for insulin/glucose infusion) and in the right carotid artery (for blood sampling). Overnight fasted rats received a primed-continuous infusion of insulin at 48 mU/kg/min (10 ml/h) for 5 min, followed by 4.8 mU/kg/min (1 ml/h) for up to 120 min. Isotopically labelled glucose was infused at 10 mg/kg/min during the prime phase and at 1 mg/kg/min during the continuous phase [d-Glucose-(13C6), 99 atom% 13C]. After the prime phase, a variable infusion of a 7.5% (w/v) glucose solution was administered to clamp the blood glucose concentration at 5 mM. The rate of endogenous glucose appearance, Ra, was calculated using the formula: Ra = GIR*/SA–GIR, where GIR* = glucose infusion rate of (13C)-glucose; SA = specific activity of (13C)-glucose in blood during steady state [(13C)-glucose / ([12C]-glucose + [13C]-glucose)]; and GIR = total glucose infusion rate [(12C)-glucose + (13C)-glucose]. The rate of endogenous glucose disappearance, Rd, was calculated as Rd = GIR +Ra.\n

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\n\nPharmacokinetics and pharmacodynamics of Empagliflozin in beagle dogs and ZDF rats [1]
\nAnimals were fasted overnight before and for 2 (rats) or 4 (dogs) h after dosing, and serial blood samples taken up to 24 or 48 h after dosing from rats and dogs, respectively. See the Supporting Information for details of dosing. Pharmacokinetic parameters were calculated by non-compartmental methods as follows. Area under the plasma concentration-time curve (AUC0−t) to the last quantifiable time point was calculated using the linear trapezoidal method. AUC0−t was extrapolated to infinity (AUC0−∞) using log-linear regression of the terminal portion of the individual curves to estimate the terminal elimination half-life (t½). Area under the moment curve (AUMC0−∞) was calculated in a manner similar to AUC0−∞. Mean residence time was calculated as AUMC0−∞/AUC0−∞, total plasma clearance (CL) as dose/AUC0−∞, and steady state volume of distribution (Vss) as (Dose × AUMC0−∞)/(AUC0−∞× AUC0−∞). Apparent bioavailability (BA) was calculated as (oral AUC0−∞/oral dose)/(intravenous AUC0−∞/intravenous dose) × 100. Maximum concentration (Cmax) and time of maximum concentration (tmax) are also reported. Individual and mean pharmacokinetic parameters were calculated using Kinetica version 4.41 or ToxKin™ version 3. The means and standard deviations (SDs) of the plasma concentrations were calculated using Microsoft Excel 2002 or ToxKin™.\n

Absorption, Distribution and Excretion
Following oral administration, peak plasma concentrations are reached at approximately 1.5 hours (Tmax). At steady state, after daily treatment with 10 mg empagliflozin, the plasma AUC and Cmax are 1870 nmol·h/L and 259 nmol/L, respectively; after daily treatment with 25 mg empagliflozin, the plasma AUC and Cmax are 4740 nmol·h/L and 687 nmol/L, respectively. Co-administration with food has no significant effect on the absorption of empagliflozin. Approximately 41.2% of the administered dose of radiolabeled empagliflozin is excreted in feces, and 54.4% in urine. The majority of the radioactive material in feces originates from unmetabolized parent drug, while approximately half of the radioactive material in urine originates from unmetabolized parent drug. The estimated apparent steady-state volume of distribution is 73.8 L. Based on population pharmacokinetic analysis, the apparent oral clearance is 10.6 L/h.

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Metabolism/Metabolites
Empagliflozin has minimal metabolism. It is primarily metabolized via glucuronidation by 5'-bisphosphoglucuronyltransferases 2B7, 1A3, 1A8, and 1A9, producing three glucuronide metabolites: 2-O-, 3-O-, and 6-O-glucuronide. No metabolites constitute more than 10% of the total drug-related substances.

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Biological Half-Life
Based on population pharmacokinetic analysis, the apparent terminal elimination half-life is 12.4 hours.
The apparent terminal elimination half-life is 12.4 hours.

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
There is currently no information regarding the clinical use of empagliflozin during lactation. Empagliflozin is an uncharged molecule with a plasma protein binding rate of 86%, making it unlikely to enter breast milk in clinically significant amounts. Due to the theoretical risk to the developing kidneys of the infant, the manufacturer does not recommend the use of empagliflozin during lactation. Especially when breastfeeding newborns or premature infants, other medications should be preferred.
◉ Effects on Breastfed Infants
No published information found as of the revision date.
◉ Effects on Lactation and Breast Milk
No published information found as of the revision date.

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Protein Binding
The plasma protein binding rate of empagliflozin is approximately 86.2%.

[1]. Diabetes Obes Metab . 2012 Jan;14(1):83-90.

[2]. Diabetes Obes Metab . 2012 Jan;14(1):94-6.

[3]. PLoS One . 2013;8(2):e54442.

Empagliflozin is a C-glycoside compound consisting of a β-glucose residue with an anomeric carbon center linked to a (4-chloro-3-{4-[(3S)-tetrahydrofuran-3-yloxy]benzyl}phenyl]. It is a sodium-glucose cotransporter 2 (SGLT2) inhibitor used as adjunctive therapy to improve glycemic control in adults with type 2 diabetes. It is both an SGLT2 subtype inhibitor and a hypoglycemic agent. It is a C-glycoside compound belonging to the aromatic ether, tetrahydrofuran ether, and monochlorobenzene classes. Empagliflozin is a sodium-glucose cotransporter 2 (SGLT2) inhibitor. SGLT2 is a transport protein in the kidneys primarily responsible for glucose reabsorption. Clinically, it is often used in combination with other drugs as an adjunct to diet and exercise for the treatment of type 2 diabetes. Phlorin, the first known SGLT inhibitor, was isolated from apple bark in 1835 and extensively studied in the 20th century, but was ultimately deemed unsuitable for clinical use due to its lack of specificity and significant gastrointestinal side effects. To overcome these limitations, O-glucoside analogs of phlorin were first developed (e.g., ramogliptin epoxetine), but these molecules proved to be relatively pharmacokineticly unstable. The development of C-glucoside phlorin analogs addressed the problems observed in previous generations of drugs and led to the FDA approval of canagliflozin in 2013, and dapagliflozin and empagliflozin in 2014. As the most recently approved phlorin... Empagliflozin is a class of drugs with higher selectivity for SGLT2 than for SGLT1 (approximately 10%). Empagliflozin received approval from the European Medicines Agency (EMA) in March 2022 and from Health Canada in April 2022, becoming the first and currently only approved drug in Europe and Canada for the treatment of symptomatic chronic heart failure in adults (regardless of ejection fraction). Empagliflozin is a sodium-glucose cotransporter 2 inhibitor. Its mechanism of action is as a sodium-glucose cotransporter 2 inhibitor. Empagliflozin is an orally effective competitive inhibitor of sodium-glucose cotransporter 2 (SGLT2; SLC5A2) with hypoglycemic activity. After oral administration, empagliflozin selectively and potently inhibits SGLT2 in the kidneys, thereby inhibiting the reabsorption of glucose in the proximal tubules. Inhibition of SGLT2 increases renal excretion of glucose in the urine, leading to a decrease in SGLT2... It can lower plasma glucose levels in an insulin-independent manner. Inhibition of SGLT2 in the kidneys also inhibits renal reabsorption of 1,5-dehydroglucistol (1,5AG). This reduces serum 1,5AG and neutrophil 1,5-dehydroglucistol-6-phosphate (1,5AG6P) levels, thereby improving neutropenia and neutrophil dysfunction in patients with type Ib glycogen storage disease (GSD Ib). SGLT2 is a transporter protein expressed only in the proximal tubules, mediating approximately 90% of renal glucose reabsorption. The reabsorption of glucose in the renal tubular fluid.
See also: Empagliflozin; Metformin Hydrochloride (ingredient); Empagliflozin; Linagliptin (ingredient)...See more...

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Drug Indications
Empagliflozin is indicated as an adjunct to diet and exercise. It is used to improve glycemic control in patients aged 10 years and older with type 2 diabetes. It can be used alone or in combination with metformin or linagliptin. In addition, it is indicated to reduce the risk of cardiovascular death in adult patients with both type 2 diabetes and a history of cardiovascular disease, and can be used alone or in combination with metformin. Combined use. In January 2020, the U.S. Food and Drug Administration (FDA) approved a sustained-release combination formulation containing empagliflozin, metformin, and linagliflozin for improving glycemic control in adults with type 2 diabetes, in conjunction with diet and exercise. Empagliflozin is also approved to reduce the risk of cardiovascular death and hospitalization for heart failure in adults, and can be used alone or in combination with metformin. Furthermore, it is indicated for reducing the risk of persistently declining eGFR, end-stage renal disease, cardiovascular death, and hospitalization in adults with chronic kidney disease at risk of progression. Empagliflozin is not approved for use in patients with type 2 diabetes. Type 1 diabetes.
Type 2 diabetes Jardiance is indicated for the treatment of type 2 diabetes in adults with poorly controlled blood glucose levels, as an adjunct to diet and exercise; it can also be used as monotherapy with metformin or in combination with other diabetes medications. For information on combination therapy, blood glucose control, cardiovascular and renal events, and study population results, please see Annex Sections 4.4, 4.5, and 5.1. Heart failure Jardiance is indicated for the treatment of symptomatic chronic heart failure in adults. Chronic kidney disease Jardiance is indicated for the treatment of chronic kidney disease in adults.
Treatment of type 2 diabetes
Treatment of ischemic heart disease
Treatment of type 1 diabetes Type 2 Diabetes Mellitus Treatment of Chronic Kidney Disease Prevention of Cardiovascular Events in Patients with Chronic Heart Failure Mechanism of Action The vast majority of glucose filtered by the glomerulus is reabsorbed in the proximal tubule, primarily via SGLT2 (sodium-glucose cotransporter 2), which is responsible for approximately 90% of glucose reabsorption in the kidney. Na+/K+-ATPases on the basolateral membrane of proximal tubular cells actively pump Na+ ions into the peritubular interstitium using ATP, thus establishing a Na+ gradient within the tubular cells. SGLT2 on these cell apical membranes then utilize this gradient to promote a secondary active cotransport of Na+ and glucose, thereby reabsorbing glucose back into the bloodstream. Inhibition of this cotransport leads to a significant increase in urinary glucose and a decrease in blood glucose levels. Empagliflozin is a potent renal SGLT2 inhibitor. SGLT2 The transporter protein is located in the proximal tubule of the kidney and lowers blood glucose levels by increasing urinary glucose. Empagliflozin also appears to have cardiovascular benefits—particularly in the prevention of heart failure—although the exact mechanism of this benefit is not fully understood. Several theories have been proposed, including possible inhibition of the Na+/H+ exchanger (NHE)1 in the myocardium and NHE3 in the proximal tubule; reduction of preload and blood pressure through diuresis/natriuresis; prevention of myocardial fibrosis by inhibiting profibrotic markers; and reduction of pro-inflammatory adipokines.

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Objective: Empagliflozin is a selective sodium-glucose cotransporter 2 (SGLT-2) inhibitor currently in clinical development for the treatment of 2 Type 2 diabetes. This study aimed to evaluate its pharmacological properties. This study aimed to evaluate the in vitro activity and in vivo pharmacokinetic properties of empagliflozin and compare its potency and selectivity with other SGLT-2 inhibitors. Methods: [(14)C]-α-methylglucopyranoside (AMG) uptake assays were performed using cell lines stably overexpressing human SGLT-1, 2, and 4. Two new cell lines overexpressing hSGLT-5 and hSGLT-6 were established, and [(14)C]-mannose and [(14)C]-inositol uptake assays were developed. The binding kinetics of empagliflozin were analyzed using [(3)H]-labeled empagliflozin and HEK293-hSGLT-2 cell membranes as models by radioligand binding assay. Acute in vivo pharmacokinetic evaluation was performed using beagle dogs with normal blood glucose and Zucker diabetic fat (ZDF) rats. Results: Empagliflozin has… The IC50 of empagliflozin against human SGLT-2 was 3.1. nM. It binds to SGLT-2 in competition with glucose (half-life approximately 1 hour). Empagliflozin exhibits high selectivity for SGLT-1, 4, 5, and 6 compared to other SGLT-2 inhibitors. Species differences were found in SGLT-1 selectivity. In ZDF rats, empagliflozin was characterized by moderate total plasma clearance (CL) and bioavailability (BA), while in beagle dogs, CL was lower and BA was higher. Conclusion: Empagliflozin is a potent and competitive SGLT-2 inhibitor with excellent selectivity and the largest selective window for human SGLT-1 among the SGLT-2 inhibitors tested. Empagliflozin represents an innovative therapy for diabetes. [1]

C23H27CLO7

450.91

450.144

C, 61.26; H, 6.04; Cl, 7.86; O, 24.84

864070-44-0

Empagliflozin-d4; 2749293-95-4

11949646

White to off-white solid powder

1.4±0.1 g/cm3

664.5±55.0 °C at 760 mmHg

355.7±31.5 °C

0.0±2.1 mmHg at 25°C

1.628

3.38

4

7

6

31

558

6

O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1C=CC(Cl)=C(CC2C=CC(O[C@@H]3COCC3)=CC=2)C=1

OBWASQILIWPZMG-QZMOQZSNSA-N

InChI=1S/C23H27ClO7/c24-18-6-3-14(23-22(28)21(27)20(26)19(11-25)31-23)10-15(18)9-13-1-4-16(5-2-13)30-17-7-8-29-12-17/h1-6,10,17,19-23,25-28H,7-9,11-12H2/t17-,19+,20+,21-,22+,23-/m0/s1

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

BI1-0773; CE0108; CS0940; PB23119; 864070-44-0; JARDIANCE; Empagliflozin (BI 10773); UNII-HDC1R2M35U; VA10802; AJ93046; BI10773; BI-10773; BI 10773; Empagliflozin; trade name: Jardiance

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: 5 mg/mL (11.09 mM) in 0.5%HPMC (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.

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Solubility in Formulation 2: ≥ 2.87 mg/mL (6.36 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 3: ≥ 2.87 mg/mL (6.36 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 4: ≥ 2.08 mg/mL (4.61 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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 5: ≥ 2.08 mg/mL (4.61 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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 6: ≥ 2.08 mg/mL (4.61 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 7: 15% Captisol: 15 mg/mL

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