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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Compounds and Dosing[2]
Empagliflozin [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]
Animal Studies [2]
Male 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.
Urinary 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.
Glucose 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.

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Hyperinsulinaemic–Euglycaemic Clamp [2]
Animals 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.

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Pharmacokinetics and pharmacodynamics of Empagliflozin in beagle dogs and ZDF rats [1]
Animals 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™.

Absorption, Distribution and Excretion
Following oral administration, peak plasma concentrations are reached in approximately 1.5 hours (Tmax). At steady-state, plasma AUC and Cmax were 1870 nmol·h/L and 259 nmol/L, respectively, following therapy with empagliflozin 10mg daily and 4740 nmol·h/L and 687 nmol/L, respectively, following therapy with empagliflozin 25mg daily. Administration with food does not significantly affect the absorption of empagliflozin.
After oral administration of radiolabeled empagliflozin approximately 41.2% of the administered dose was found eliminated in feces and 54.4% eliminated in urine. The majority of radioactivity in the feces was due to unchanged parent drug while approximately half of the radioactivity in urine was due to unchanged parent drug.
The estimated apparent steady-state volume of distribution is 73.8 L.
Apparent oral clearance was found to be 10.6 L/h based on a population pharmacokinetic analysis.

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Metabolism / Metabolites
Empagliflozin undergoes minimal metabolism. It is primarily metabolized via glucuronidation by 5'-diphospho-glucuronosyltransferases 2B7, 1A3, 1A8, and 1A9 to yield three glucuronide metabolites: 2-O-, 3-O-, and 6-O-glucuronide. No metabolite represented more than 10% of total drug-related material.

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Biological Half-Life
The apparent terminal elimination half-life was found to be 12.4 h based on population pharmacokinetic analysis.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of empagliflozin during breastfeeding. Empagliflozin is an uncharged molecule that is 86% protein bound in plasma, so it is unlikely to pass into breastmilk in clinically important amounts. The manufacturer does not recommend empagliflozin 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
Empagliflozin is approximately 86.2% protein-bound in plasma.

[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-glycosyl compound consisting of a beta-glucosyl residue having a (4-chloro-3-{4-[(3S)-tetrahydrofuran-3-yloxy]benzyl}phenyl group at the anomeric centre. A sodium-glucose co-transporter 2 inhibitor used 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 and a hypoglycemic agent. It is a C-glycosyl compound, an aromatic ether, a tetrahydrofuryl ether and a member of monochlorobenzenes.
Empagliflozin is an inhibitor of sodium-glucose co-transporter-2 (SGLT2), the transporters primarily responsible for the reabsorption of glucose in the kidney. It is used clinically as an adjunct to diet and exercise, often in combination with other drug therapies, for the management of type 2 diabetes mellitus. The first known inhibitor of SGLTs, phlorizin, was isolated from the bark of apple trees in 1835 and researched extensively into the 20th century, but was ultimately deemed inappropriate for clinical use given its lack of specificity and significant gastrointestinal side effects. Attempts at overcoming these limitations first saw the development of O-glucoside analogs of phlorizin (e.g. [remogliflozin etabonate]), but these molecules proved relatively pharmacokinetically unstable. The development of C-glucoside phlorizin analogs remedied the issues observed in the previous generation, and led to the FDA approval of [canagliflozin] in 2013 and both [dapagliflozin] and empagliflozin in 2014. As the most recently approved of the "flozin" drugs, empagliflozin carries the highest selectivity for SGLT2 over SGLT1 (approximately 2700-fold). Empagliflozin was further approved by the EMA in March 2022 and Health Canada in April 2022, making it the first and only approved treatment in Europe and Canada for adults with symptomatic chronic heart failure regardless of ejection fraction.
Empagliflozin is a Sodium-Glucose Cotransporter 2 Inhibitor. The mechanism of action of empagliflozin is as a Sodium-Glucose Transporter 2 Inhibitor.
Empagliflozin is an orally available competitive inhibitor of sodium-glucose co-transporter 2 (SGLT2; SLC5A2) with antihyperglycemic activity. Upon oral administration, empagliflozin selectively and potently inhibits SGLT2 in the kidneys, thereby suppressing the reabsorption of glucose in the proximal tubule. Inhibition of SGLT2 increases urinary glucose excretion by the kidneys, resulting in a reduction of plasma glucose levels in an insulin-independent manner. Inhibition of SGLT2 in the kidneys also suppresses the renal reabsorption of 1,5-anhydroglucitol (1,5AG). This lowers serum 1,5AG and neutrophil 1,5-anhydroglucitol-6-phosphate (1,5AG6P) levels, which may improve neutropenia and neutrophil dysfunction in patients with glycogen storage disease type Ib (GSD Ib). SGLT2, a transport protein exclusively expressed in the proximal renal tubules, mediates approximately 90% of renal glucose reabsorption from tubular fluid.
See also: Empagliflozin; METformin Hydrochloride (component of); Empagliflozin; Linagliptin (component of) ... View More ...

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Drug Indication
Empagliflozin is indicated as an adjunct to diet and exercise to improve glycemic control in patients aged 10 years and older with type 2 diabetes. It is used either alone or in combination with [metformin] or [linagliptin]. It is also indicated to reduce the risk of cardiovascular death in adult patients with both type 2 diabetes mellitus and established cardiovascular disease, either alone or as a combination product with metformin. An extended-release combination product containing empagliflozin, metformin, and linagliptin was approved by the FDA in January 2020 for the improvement of glycemic control in adults with type 2 diabetes mellitus when used adjunctively with diet and exercise. Empagliflozin is also approved to reduce the risk of cardiovascular mortality and hospitalization due to heart failure in adult patients with heart failure, either alone or in combination with metformin. It is also indicated in adults to reduce the risk of sustained decline in eGFR, end-stage kidney 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 1 diabetes.
Type 2 diabetes mellitusJardiance is indicated for the treatment of adults with insufficiently controlled type 2 diabetes mellitus as an adjunct to diet and exerciseas monotherapy when metformin is considered in addition to other medicinal products for the treatment of diabetesFor study results with respect to combinations of therapies, effects on glycaemic control, and cardiovascular and renal events, and the populations studied, see sections 4. 4, 4. 5 and 5. 1. of the annex. Heart failureJardiance is indicated in adults for the treatment of symptomatic chronic heart failure.  Chronic kidney diseaseJardiance is indicated in adults for the treatment of chronic kidney disease.  
Treatment of type II diabetes mellitus
Treatment of ischaemic heart disease
Treatment of type I diabetes mellitus
Treatment of chronic kidney disease
Prevention of cardiovascular events in patients with chronic heart failure

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Mechanism of Action
The vast majority of glucose filtered through the glomerulus is reabsorbed within the proximal tubule, primarily via SGLT2 (sodium-glucose linked co-transporter-2) which is responsible for ~90% of the total glucose reabsorption within the kidneys. Na⁺/K⁺-ATPase on the basolateral membrane of proximal tubular cells utilize ATP to actively pump Na+ ions into the interstitium surrounding the tubule, establishing a Na⁺ gradient within the tubular cell. SGLT2 on the apical membrane of these cells then utilize this gradient to facilitate secondary active co-transport of both Na+ and glucose out of the filtrate, thereby reabsorbing glucose back into the blood – inhibiting this co-transport, then, allows for a marked increase in glucosuria and decrease in blood glucose levels. Empagliflozin is a potent inhibitor of renal SGLT2 transporters located in the proximal tubules of the kidneys and works to lower blood glucose levels via an increase in glucosuria. Empagliflozin also appears to exert cardiovascular benefits - specifically in the prevention of heart failure - independent of its blood glucose-lowering effects, though the exact mechanism of this benefit is not precisely understood. Several theories have been posited, including the potential inhibition of Na⁺/H⁺ exchanger (NHE) 1 in the myocardium and NHE3 in the proximal tubule, reduction of pre-load via diuretic/natriuretic effects and reduction of blood pressure, prevention of cardiac fibrosis via suppression of pro-fibrotic markers, and reduction of pro-inflammatory adipokines.

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Aims: Empagliflozin is a selective sodium glucose cotransporter-2 (SGLT-2) inhibitor in clinical development for the treatment of type 2 diabetes mellitus. This study assessed pharmacological properties of empagliflozin in vitro and pharmacokinetic properties in vivo and compared its potency and selectivity with other SGLT-2 inhibitors. Methods: [(14)C]-alpha-methyl glucopyranoside (AMG) uptake experiments were performed with stable cell lines over-expressing human (h) SGLT-1, 2 and 4. Two new cell lines over-expressing hSGLT-5 and hSGLT-6 were established and [(14)C]-mannose and [(14)C]-myo-inositol uptake assays developed. Binding kinetics were analysed using a radioligand binding assay with [(3)H]-labelled empagliflozin and HEK293-hSGLT-2 cell membranes. Acute in vivo assessment of pharmacokinetics was performed with normoglycaemic beagle dogs and Zucker diabetic fatty (ZDF) rats. Results: Empagliflozin has an IC(50) of 3.1 nM for hSGLT-2. Its binding to SGLT-2 is competitive with glucose (half-life approximately 1 h). Compared with other SGLT-2 inhibitors, empagliflozin has a high degree of selectivity over SGLT-1, 4, 5 and 6. Species differences in SGLT-1 selectivity were identified. Empagliflozin pharmacokinetics in ZDF rats were characterised by moderate total plasma clearance (CL) and bioavailability (BA), while in beagle dogs CL was low and BA was high. Conclusions: Empagliflozin is a potent and competitive SGLT-2 inhibitor with an excellent selectivity profile and the highest selectivity window of the tested SGLT-2 inhibitors over hSGLT-1. Empagliflozin represents an innovative therapeutic approach to treat diabetes. [1]

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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.)