Pharmaceutical composition comprising enagliflozin metabolite and use thereof

Abstract

The present invention relates to a pharmaceutical composition comprising an enagliflozin metabolite and a use thereof. The Enagliflozin M1 metabolite of the chemical formula 1 is an SGLT1 / SGLT2 dual inhibitor, and shows a pharmacological mechanism different from that of Enagliflozin serving as an SGLT2 selective inhibitor. A pharmaceutical composition comprising an Enagliflozin M1 metabolite as an active ingredient as a dual SGLT1 / SGLT2 inhibitor can be used for preventing or treating diabetes or heart failure.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition comprising enagliflozin metabolites and uses thereof. Background Art

[0002] Sodium-glucose cotransporter 2 (SGLT2) is a transporter that, together with sodium-glucose cotransporter 1 (SGLT1), is responsible for glucose reabsorption in the kidneys, and SGLT2 plays a large role. Therefore, when SGLT2 inhibitors inhibit the SGLT2 transporter, glucose excretion into the urine increases, ultimately lowering blood sugar levels, and in addition, calories contained in blood sugar are excreted, resulting in a weight-loss effect.

[0003] One of the drugs developed as SGLT2 inhibitors that can be effectively used as a therapeutic agent for type 2 diabetes due to this action is enagliflozin, which is represented by the following structural formula (chemical formula A) and disclosed in Korean Patent Publication No. 2014-0022086 (Patent Document 1).

[0004] [Chemical formula A]

[0005]

[0006] (2S,3R,4R,5S,6R)-2-(7-chloro-6-(4-cyclopropylbenzyl)-2,3-dihydrobenzofuran-4-yl)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

[0007] Enagagliflozin is a drug that selectively inhibits SGLT2 to treat type 2 diabetes. It shows the same or better efficacy with only 0.3 mg, which is less than 1 / 30 of the dosage of existing SGLT2 inhibitors. Phase 3 clinical trials on patients with type 2 diabetes have proven that Enagagliflozin has superior glycated hemoglobin (HbA1c) and fasting blood sugar lowering effects and safety compared to existing commercially available drugs, and thus Enagagliflozin was approved for marketing.

[0008] According to non-patent document 1, it was confirmed that enagliflozin produces a total of five metabolites in human hepatocytes: M1, M2, M3, U1 and U2. M1 and M2 are produced by catalyzing enagliflozin via CYP3A4 and CYP2C19, and M3 is produced by hydroxylation of M1, which is catalyzed by CYP3A4. U1 is produced by catalysis of UGT2B7, and U2 is produced by catalysis of UGT1A4, UGT1A9 and UGT2B7.

[0009] Non-patent document 1 is a paper on the drug metabolism of enagliflozin in the liver and the description of metabolites and metabolic enzymes of enagliflozin, but does not disclose the activities exhibited by the metabolites of enagliflozin.

[0010] [Background Technology Literature]

[0011] [Patent Document]

[0012] Korean Patent Publication No. 2014-0022086

[0013] [Non-patent literature]

[0014] Ju-Hyun Kim et al., Pharmaceutics. 2020 Sep 11;12(9):865. doi:10.3390 / pharmaceutics12090865. Summary of the invention

[0015] [Technical issues]

[0016] The object of the present invention is to provide a pharmaceutical use of an enagliflozin metabolite.

[0017] [Technical solution]

[0018] In the process of studying the metabolites of enagliflozin, the present inventors confirmed that the enagliflozin M1 metabolite represented by Chemical Formula 1 is a dual inhibitor of sodium-glucose co-transporter 1 (SGLT1) / sodium-glucose co-transporter 2 (SGLT2), and exhibits a pharmacological mechanism different from that of enagliflozin as a selective SGLT2 inhibitor, thereby completing the present invention.

[0019] [Chemical formula 1]

[0020]

[0021] (2S,3R,4R,5S,6R)-2-(7-chloro-6-(4-cyclopropylbenzyl)-2-hydroxy-2,3-dihydrobenzofuran-4-yl)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

[0022] SGLT2 inhibitors target SGLT2, which is responsible for reabsorb- ing more than 90% of glucose filtered by the kidneys, whereas SGLT1 inhibitors target SGLT1, which is responsible for reabsorb- ing the remaining 10% of glucose in the intestinal epithelium.

[0023] Most SGLT2 inhibitors developed in the past are drugs with highly selective binding affinity to SGLT2, while enagliflozin developed by the applicant of the present invention is a drug that is also highly selective for SGLT2 compared to SGLT1 and has recently been approved as a therapeutic agent for type 2 diabetes.

[0024] As a dual SGLT1 / SGLT2 inhibitor, Lexicon Pharmaceuticals' sogliflozin has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of type 1 diabetes and heart failure (HF) in the European Union.

[0025] The present invention provides use of enagliflozin M1 metabolite as a SGLT1 / SGLT2 dual inhibitor.

[0026] Hereinafter, the present invention will be described in more detail.

[0027] The present invention provides the use of enagliflozin M1 metabolite or a pharmaceutically acceptable salt thereof as a SGLT1 / SGLT2 dual inhibitor, a pharmaceutical composition for preventing or treating diabetes or heart failure, a pharmaceutical composition comprising enagliflozin M1 metabolite or a pharmaceutically acceptable salt thereof as an active ingredient, and a method for preventing or treating diabetes or heart failure, the method comprising administering an effective amount of enagliflozin or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0028] The present invention provides a pharmaceutical composition for preventing or treating diabetes or heart failure, comprising the enagliflozin M1 metabolite represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0029] In addition, the present invention provides a SGLT1 / SGLT2 dual inhibitor, which comprises the enagliflozin M1 metabolite represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0030] As can be confirmed in the examples described in the present invention, the enagliflozin M1 metabolite has binding affinity to SGLT2 and also exhibits strong binding affinity to SGLT1. Therefore, the enagliflozin M1 metabolite is a candidate drug that can act on indications that may be treated with SGLT2 inhibitors and indications that may be treated with SGLT1 / SGLT2 dual inhibitors.

[0031] In the present invention, the diabetes may be type 1 diabetes or type 2 diabetes.

[0032] A representative indication that can be treated with SGLT2 inhibitors may be type 2 diabetes. In Example 3 described below, it was confirmed that the enagliflozin M1 metabolite has excellent anti-diabetic efficacy by an oral glucose tolerance test (OGTT) and urinary glucose excretion (UGE) in a normal rat model.

[0033] Representative indications that can be treated with SGLT1 / SGLT2 dual inhibitors may be type 1 diabetes and heart failure.

[0034] Since the enagliflozin M1 metabolite can simultaneously block SGLT2 present in the kidney and SGLT1 present in the small intestine, it can effectively act on type 1 diabetes with disordered insulin secretion. In the following Example 4, the anti-diabetic efficacy of the enagliflozin M1 metabolite in a rat model of type 1 diabetes was confirmed. It was verified by Example 4 that compared with enagliflozin or sotagliflozin, the enagliflozin M1 metabolite may be the most effective drug for type 1 diabetes.

[0035] In one embodiment, the pharmaceutical composition can be administered to a diabetic patient or a patient in the pre-diabetic stage.

[0036] The pharmaceutical composition comprising enagliflozin M1 metabolite or a pharmaceutically acceptable salt thereof as an active ingredient can be used in the form of single administration or combined administration.

[0037] The use of enagliflozin M1 metabolite alone is sufficient to prevent or treat diabetes and / or heart failure. However, since antidiabetic drugs or anti-heart failure drugs are administered in combination for various purposes and can exhibit complementary or synergistic effects when administered in combination, a pharmaceutical composition including enagliflozin M1 metabolite as an active ingredient can be administered in combination with another antidiabetic drug or anti-heart failure drug.

[0038] For example, it can be co-administered with known anti-diabetic drugs, including biguanide-based drugs such as metformin, dipeptidyl peptidase-4 (DPP4) inhibitors, sulfonylurea-based insulin secretagogues, and / or insulin.

[0039] In addition, another indication that can be treated with SGLT1 / SGLT2 dual inhibitors may be heart failure. The efficacy of sotagliflozin as a SGLT1 / SGLT2 dual inhibitor for preventing or treating heart failure has been confirmed by the US FDA approval.

[0040] The term "prevention" used in the present invention refers to any effect of inhibiting or delaying the onset of diabetes or heart failure by administering the pharmaceutical composition of the present invention.

[0041] Furthermore, the term "treatment" as used in the present invention refers to any effect of diabetes or heart failure that is improved or beneficially altered by administering the pharmaceutical composition of the present invention.

[0042] The dosage of enagliflozin M1 metabolite, which can be used to prevent or treat patients with diabetes and / or heart failure or patients at risk of diabetes and / or heart failure, is not particularly limited and can be appropriately adjusted according to the severity of the disease, weight, age, gender, whether the patient has other complications, etc.

[0043] Enagagliflozin M1 metabolite or a pharmaceutically acceptable salt thereof can be administered orally or parenterally. The administration cycle can be appropriately adjusted by a clinician based on the effect of its administration on preventing or treating diabetes and / or heart failure in patients with diabetes and / or heart failure or patients at risk of diabetes and / or heart failure.

[0044] The pharmaceutical composition according to the present invention may further contain a pharmaceutically acceptable carrier in addition to the enagliflozin M1 metabolite of Chemical Formula 1 or a pharmaceutically acceptable salt thereof, which is an active ingredient and may be formulated together with the carrier.

[0045] The term "pharmaceutically acceptable carrier" used in the present invention refers to a carrier or diluent that does not stimulate living organisms and does not inhibit the biological activity and properties of the administered compound. Pharmaceutically acceptable carriers in compositions formulated into liquid solutions, which are sterile and biocompatible carriers, include saline solutions, sterile water, Ringer's solution, buffered saline, albumin injection solutions, glucose solutions, maltodextrin solutions, glycerol, ethanol, and one or more mixtures of these components. In addition, other conventional additives such as antioxidants, buffers, and antibacterial agents may be added as needed. In addition, diluents, dispersants, surfactants, adhesives, and lubricants may be further added to prepare the composition into injectable preparations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

[0046] In the present invention, the pharmaceutical composition may have a dosage form for oral administration, such as a tablet or capsule. In a specific embodiment of the present invention, the pharmaceutical composition may have a dosage form of a tablet.

[0047] When the dosage form is for oral administration, the pharmaceutical composition of the present invention may include excipients, disintegrants and binders as additives.

[0048] Examples of excipients include lactose (including hydrates), dextrin, mannitol, sorbitol, starch, microcrystalline cellulose (e.g. Celphere TM ), silicified microcrystalline cellulose (e.g. Prosolv TM), calcium phosphate hydrate, anhydrous calcium phosphate, calcium carbonate, sugar or a mixture thereof. In a specific embodiment of the present invention, the preferred excipient is microcrystalline cellulose.

[0049] Examples of disintegrants include croscarmellose, croscarmellose sodium, sodium starch glycolate and low-substituted hydroxypropylcellulose. In a particular embodiment of the present invention, the preferred excipient is croscarmellose sodium.

[0050] Examples of binders include polyvinylpyrrolidone, povidone, gelatin, starch, sucrose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylalkylcellulose (e.g. hydroxypropylmethylcellulose), and mixtures thereof. In a specific embodiment of the present invention, the preferred binder is hydroxypropylcellulose.

[0051] Examples of other additives include lubricants, colorants, and the like.

[0052] Lubricants include stearic acid, stearates (e.g., magnesium stearate), light anhydrous silicic acid, talc, corn starch, carnauba wax, magnesium silicate, synthetic aluminum silicate, hydrogenated oil, white lead, titanium dioxide, microcrystalline cellulose, Macrogol 4000 and Macrogol 6000, isopropyl myristate, calcium hydrogen phosphate, and mixtures thereof.

[0053] In the present invention, the pharmaceutical composition may have a dosage form for parenteral administration. For example, a preparation for parenteral administration containing the composition of the present invention as an active ingredient may be prepared in an injectable form such as subcutaneous injection, intravenous injection or intramuscular injection, but is not limited thereto.

[0054] For the preparation of injectable preparations, the composition of the present invention may be mixed with a stabilizer or a buffer in water to prepare a solution or suspension, and these solutions or suspensions may be formulated in the form of unit administration in an ampoule or a vial.

[0055] Alternatively, the composition of the present invention can be formulated into various parenteral preparations such as eye drops, microneedles, patches, and sustained-release preparations (depots).

[0056] The composition of the present invention is applied in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" refers to an amount sufficient to treat a disease, and the effective dose level can be determined according to factors including the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of application, the route of administration, the excretion rate, the treatment cycle, and the drugs used simultaneously, as well as other factors well-known in the medical field. The composition of the present invention can be applied as a single therapeutic agent or in combination with other therapeutic agents, and can be applied sequentially or simultaneously with conventional therapeutic agents, and can be applied in a single dose or multiple doses. In other words, the total effective amount of the composition of the present invention can be applied to the patient in a single dose, or can be applied by applying a graded treatment regimen of multiple doses over a long period of time. Importantly, by considering all of the above factors, the amount that can achieve the maximum effect with a minimum amount without side effects can be applied, which can be easily determined by a person of ordinary skill in the art.

[0057] The appropriate daily dose of enagliflozin determined during clinical trials is 0.1 mg to 0.5 mg, and based on the results of the Examples described below, it is expected that enagliflozin M1 metabolite can also be used at a dose of 0.1 mg to 0.5 mg similar to enagliflozin.

[0058] When the pharmaceutical composition is formulated into a unit dosage form, the content of the active ingredient in the pharmaceutical composition may be 0.1 to 0.5 mg.

[0059] The pharmaceutical composition of the present invention may be administered once to three times a day, for example, once a day, but is not limited thereto.

[0060] [Beneficial Effects]

[0061] The pharmaceutical composition comprising the enagliflozin M1 metabolite of the present invention as an active ingredient can be effectively used for preventing or treating diabetes or heart failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Shown is a graph illustrating the SGLT1 activity inhibitory effects of enagliflozin, M1, M2, dapagliflozin and empagliflozin on methyl α-D-glucopyranoside (AMG) uptake by Chinese hamster ovary (CHO)-SGLT1 cells.

[0063] Figure 2 Shown is a graph illustrating the inhibitory effects of enagliflozin, M1, M2, dapagliflozin and empagliflozin on the SGLT2 activity of AMG uptake by CHO-SGLT2 cells.

[0064] Figure 3 A graph illustrating changes in the inhibitory effects of enagliflozin, M1, M2, dapagliflozin, and empagliflozin on SGLT1 activity according to the pretreatment time of the drugs is shown.

[0065] Figure 4 A graph illustrating changes in the inhibitory effects of enagliflozin, M1, M2, dapagliflozin, and empagliflozin on SGLT2 activity according to the pretreatment time of the drugs is shown.

[0066] Figure 5 The results of comparison of the ability of enagliflozin, M1, M2, dapagliflozin and empagliflozin to restore the activity of SGLT1 are shown.

[0067] Figure 6 The results of comparison of the ability of enagliflozin, M1, M2, dapagliflozin and empagliflozin to restore the activity of SGLT2 are shown.

[0068] Figure 7 The results of comparison of the binding affinity and dissociation degree of enagliflozin, M1, M2 and dapagliflozin to SGLT1 are shown.

[0069] Figure 8 The results of comparison of the binding affinity and dissociation degree of enagliflozin, M1, M2 and dapagliflozin to SGLT2 are shown.

[0070] Fig. 9 Shown are graphs illustrating changes in blood sugar levels over time after administration of enagliflozin, M1, or M2 in normal rats.

[0071] Fig.10 Shown is a graph illustrating the area under the blood glucose concentration-time curve (AUC) after administration of enagliflozin, M1, or M2 in normal rats.

[0072] Fig.11 A graph showing non-fasting blood glucose levels observed up to 72 hours after administration of test substances in streptozotocin (STZ)-induced type 1 diabetic rats.

[0073] Fig.12 Shown is a graph illustrating blood glucose levels in STZ-induced type 1 diabetic rats according to an oral glucose tolerance test.

[0074] Fig.13 Shown is a graph illustrating the area under the blood glucose concentration-time curve (AUC) in STZ-induced type 1 diabetic rats according to an oral glucose tolerance test.

[0075] Fig.14 Shown are graphs illustrating changes over time in the levels of inflammatory response-related cytokines after administration of a test substance in STZ-induced type 1 diabetic rats. DETAILED DESCRIPTION

[0076] The advantages and features of the present invention and methods for achieving them will become clear with reference to the experimental examples and preparation examples described in detail below. However, the present invention is not limited to the experimental examples and preparation examples disclosed below, but can be implemented in various different forms, and the embodiments are provided only to ensure that the disclosure of the present invention is complete and to fully inform the scope of the present invention to those skilled in the art to which the present invention belongs.

[0077] [Example]

[0078] [Example 1] Evaluation of the inhibitory effects of enagliflozin metabolites on SGLT1 and SGLT2

[0079] According to the preliminary study of the applicant of the present invention, it is confirmed that compared with the competing drugs dapagliflozin and ipragliflozin, enagliflozin (also referred to as DWP16001 in the following examples) has a higher inhibitory effect on SGLT2 than SGLT1, and does not show changes in inhibitory effect after pretreatment for up to two hours. The Dixon diagram and Replot results of the inhibition evaluation based on the substrate concentration show that the inhibition of SGLT2 shows a reversible competitive inhibition mechanism. However, when the intracellular drug concentration is quantified after 24 hours of culture, it is confirmed that the amount of enagliflozin remaining in the cell is significantly higher than that of dapagliflozin and ipragliflozin. In addition, in the case of enagliflozin, the recovery ability of intracellular SGLT2 activity is delayed over time, but this does not occur in the competing drugs dapagliflozin and ipragliflozin. These results show that enagliflozin may have better drug efficacy persistence than dapagliflozin and ipragliflozin.

[0080] The present inventors intended to evaluate whether the same results were observed for the M1 and M2 metabolites of enagliflozin (referred to herein as M1 and M2, respectively).In addition, the present inventors intended to evaluate and compare dapagliflozin and empagliflozin, which have been actively clinically studied.

[0081] Example 1-1. Evaluation of the inhibitory activity of enagliflozin metabolites on SGLT subtypes

[0082] (1) Evaluation of SGLT1 inhibition

[0083] The cell line overexpressing SGLT1 (CHO-SGLT1 stable cells) and mock cells (CHO-mock cells) were cultured until they reached 80% confluence, and then the cells were plated at 1×10 5 Cells / well were seeded in 96-well plates.

[0084] After 24 hours, the medium was removed and preincubation buffer [10 mM hydroxyethylpiperazineethanesulfonic acid (HEPES), 5 mM Tris, 140 mM choline chloride, 2 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , pH 7.4], and the cells were incubated for one hour.

[0085] One hour later, the preincubation buffer was removed and a mixture containing 10 μM [ 14 C] methyl α-D-pyranoglucoside (AMG), SGLT1 substrate and various concentrations of inhibitory evaluation substances in incubation buffer [10 mM HEPES, 5 mM Tris, 140 mM NaCl, 2 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , pH 7.4] and allowed to react in a thermo-micromixer at 37°C for two hours.

[0086] The concentrations of inhibitory evaluation substances used in the experiments are described below.

[0087] Enagagliflozin: 1, 10, 100, 500, 1000, 5000, 20000, 50000 nM

[0088] M1: 1,10,100,500,1000,5000,20000,50000nM

[0089] M2: 1,10,100,500,1000,5000,20000,50000nM

[0090] Dapagliflozin: 1, 10, 100, 500, 1000, 5000, 20000, 50000 nM

[0091] Empagliflozin: 1, 10, 100, 500, 1000, 5000, 20000, 50000 nM

[0092] For reference, the M2 metabolite has the following compound name and structure represented by Chemical Formula 2.

[0093] Compound name: (2S,3R,4R,5S,6R)-2-(7-chloro-6-(4-(1-hydroxycyclopropyl)benzyl)-2,3-dihydrobenzofuran-4-yl)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol)

[0094] [Chemical formula 2]

[0095]

[0096] After the preset time, wash the cells with 200 μL of ice-cold wash buffer [10 mM HEPES, 5 mM Tris, 140 mM choline chloride, 2 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 100 mM AMG, pH 7.4] and washed the cells twice.

[0097] After removing the culture medium, 40 μL of 10% sodium dodecyl sulfate (SDS) solution was added to lyse the cells, and then the sample was transferred to a measurement plate of a liquid scintillation counter and 150 μL of OptiPhase The mixture solution was isotopically measured and the cells were incubated overnight to ensure that the cell solution was evenly mixed with the mixture solution.

[0098] [ 14 C] AMG in the samples was measured using a liquid scintillation counter.

[0099] In cell lines overexpressing SGLT1, 14 The intracellular uptake rate of C]AMG was compared with that in CHO-mock cells according to the substrate [ 14 The IC value with 50% inhibition was calculated by plotting the intracellular uptake rate of C]AMG (% of control) and the concentration of the inhibitory evaluation substance applied. 50 Value (maximum inhibitory concentration). At this time, the inhibitory effect SigmoidE was used max Model, and WinNonlin program (version 2.0) was used. All data values ​​are expressed as the mean ± standard deviation (SD) of 3 independent experiments.

[0100] (2) Evaluation of SGLT2 inhibition

[0101] This evaluation was performed in the same manner as the evaluation of SGLT1 inhibition, except that a cell line overexpressing SGLT2 (CHO-SGLT2 stable cells) was used, and the inhibitory evaluation substances described below were used.

[0102] The concentrations of inhibitory evaluation substances are described below.

[0103] Enagagliflozin: 0.01, 0.05, 0.2, 2, 10, 50, 100, 500 nM

[0104] M1: 0.01,0.05,0.2,2,10,50,100,500nM

[0105] M2: 0.01,0.05,0.2,2,10,50,100,500nM

[0106] Dapagliflozin: 0.01, 0.05, 0.2, 2, 10, 50, 100, 500 nM

[0107] Empagliflozin: 0.01, 0.05, 0.2, 2, 10, 50, 100, 500 nM

[0108] (3) Evaluation of the inhibitory activity of enagliflozin and its M1 and M2 metabolites in cell lines overexpressing SGLT1 and SGLT2

[0109] The inhibitory activities of enagliflozin, M1, M2, dapagliflozin and empagliflozin on SGLT1 or SGLT2 were evaluated and are shown in Tables 1 and Figure 1 and Figure 2 middle.

[0110] [Table 1] Inhibitory effects of enagliflozin, M1, M2, dapagliflozin and empagliflozin on SGLT1 or SGLT2 activity

[0111]

[0112] The inhibition constants derived from the evaluation results were similar to those previously studied, and enagliflozin was evaluated to have a stronger inhibitory effect and better selectivity against SGLT2 compared to dapagliflozin and empagliflozin, as determined by the ratio of the inhibition constants for SGLT1 and SGLT2. In addition, both enagliflozin metabolites M1 and M2 showed inhibitory effects on SGLT2.

[0113] Specifically, unlike enagliflozin, M2, dapagliflozin, and empagliflozin, which selectively inhibit SGLT2, metabolite M1 showed excellent SGLT1 activity inhibition (IC 50 value of about 28.4 nM), and also showed the IC 50 The very low value of the ratio confirms its potential as a dual inhibitor of both SGLT1 and SGLT2.

[0114] Example 1-2. Evaluation of the inhibitory mechanism of enagliflozin and its M1 and M2 metabolites on SGLT1 and SGLT2

[0115] (1) Comparative evaluation of the effects of drug exposure time on inhibitory changes in cell lines overexpressing SGLT1

[0116] CHO-SGLT1 cells were pretreated with different concentrations of enagliflozin, M1, M2, dapagliflozin and empagliflozin in sodium-free pretreatment buffer (Na-negative pretreatment buffer) for 1 hour or 2 hours, and then the concentration-dependent inhibitory effect of enagliflozin on AMG uptake into CHO-SGLT1 cells was evaluated and compared with the unpretreated group. All data values ​​are expressed as the mean ± SD of four independent experiments.

[0117] As a result of the evaluation, as shown in Table 2 and Figure 3 As shown, enagliflozin, M2, dapagliflozin, and empagliflozin did not show any significant changes or increases in inhibition with increasing exposure time (p>0.05). On the other hand, the inhibition constant of M1 increased significantly with increasing exposure time (p<0.05).

[0118] [Table 2] IC of SGLT1 activity of enagliflozin, M1, M2, dapagliflozin and empagliflozin at different pretreatment times 50 (nM) value (nM) change

[0119]

[0120] P values ​​were evaluated by one-way analysis of variance (ANOVA).

[0121] (2) Evaluation of the effect of substrate AMG concentration on the inhibitory effect in cell lines overexpressing SGLT1

[0122] To determine the inhibitory mechanism and inhibition constant (Ki) of SGLT1, the concentration-dependent inhibitory effects of enagliflozin, M1, M2, dapagliflozin and empagliflozin were evaluated by varying the concentration of the substrate AMG in the CHO-SGLT1 cell line.

[0123] The uptake rate of AMG and the concentrations of enagliflozin, M1, M2, dapagliflozin and empagliflozin were plotted in a Dixon diagram, and the AMG concentration and the Dixon slope were represented as a graph. In addition, the results were represented as a Lineweaver-Burk (LB) graph, and the inhibitor concentration and the LB slope were represented as a graph (not shown). The inhibition mechanism can be confirmed and the inhibition constant can be calculated. The inhibition constant derived from the slope of the LB graph and the inhibition mechanism is shown in Table 3.

[0124] [Table 3] Ki values ​​of enagliflozin, M1, M2, dapagliflozin and empagliflozin for SGLT1 activity

[0125] Compound Ki(nM) Inhibition Mechanism Enagagliflozin 495 Competition Mechanism M1 84.8 Competition Mechanism M2 1872 Competition Mechanism Dapagliflozin 502 Competition Mechanism Empagliflozin 1708 Competition Mechanism

[0126] As shown in Table 3, the Ki value of the M1 metabolite for SGLT1 activity was 84.8 nM, which was the smallest value among the tested substances. The smaller the Ki value, the higher the affinity. These results indicate that the affinity of M1 for SGLT1 is higher than that of the tested substances, and therefore it has a strong inhibitory effect on SGLT1.

[0127] (3) Comparative evaluation of inhibitory changes according to drug exposure time in cell lines overexpressing SGLT2

[0128] CHO-SGLT2 cells were pretreated with different concentrations of enagliflozin, M1, M2, dapagliflozin, and empagliflozin in sodium-free pretreatment buffer for 1 or 2 hours, and then the concentration-dependent inhibitory effect of enagliflozin on AMG uptake into CHO-SGLT2 cells was evaluated and compared with the unpretreated group. All data values ​​are expressed as the mean ± SD of four independent experiments.

[0129] The evaluation results are shown in Table 4 and Figure 4 As shown, even with the increase in exposure time, the inhibitory effects of enagliflozin, M1, M2, dapagliflozin and empagliflozin did not increase or change significantly (p>0.05).

[0130] [Table 4] IC of enogliflozin, M1, M2, dapagliflozin and empagliflozin for SGLT2 activity according to pretreatment time 50 (nM) value changes

[0131]

[0132] P values ​​were evaluated by one-way ANOVA.

[0133] (4) Inhibition evaluation based on substrate AMG concentration in cell lines overexpressing SGLT2

[0134] To determine the inhibitory mechanism and inhibition constant (Ki) of SGLT2, the concentration-dependent inhibitory effects of enagliflozin, M1, M2, dapagliflozin and empagliflozin were evaluated by varying the concentration of the substrate AMG in the CHO-SGLT2 cell line.

[0135] The uptake rate of AMG and the concentrations of enagliflozin, M1, M2, dapagliflozin and empagliflozin are plotted in a Dixon diagram, and the AMG concentration and the Dixon slope are represented as a graph. In addition, the results are represented in an LB diagram, and the inhibitor concentration and the LB slope are represented as a graph (not shown). The inhibition mechanism can be confirmed and the inhibition constant can be calculated. The inhibition constant derived from the slope of the LB diagram and the inhibition mechanism is shown in Table 5.

[0136] [Table 5] Ki values ​​of enogliflozin, M1, M2, dapagliflozin and empagliflozin for SGLT2 activity

[0137]

[0138]

[0139] Example 1-3. Comparative evaluation of the ability to restore SGLT1 and SGLT2 activity after 24-hour pretreatment with enagliflozin and metabolites

[0140] (1) Comparative evaluation of the activity recovery ability of enagliflozin and its metabolites after washout in cell lines overexpressing SGLT1

[0141] When the cell line overexpressing SGLT1 (CHO-SGLT1 stable cells) was cultured to 80% confluence, the cells were plated at 1×10 5 The cells were seeded in 96-well plates at a density of 10 cells / mL. After 24 hours, the medium was removed and replaced with medium containing different concentrations of the evaluation substances, and the cells were cultured for 24 hours.

[0142] The concentrations of the evaluation substances used in the experiments are as follows.

[0143] Enagagliflozin: 50, 500, 5000, 20000 nM

[0144] M1: 50,500,5000,20000nM

[0145] M2: 50,500,5000,20000nM

[0146] Dapagliflozin: 50,500,5000,20000nM

[0147] Empagliflozin: 50, 500, 5000, 20000 nM

[0148] Preincubation buffer (10 mM HEPES, 5 mM Tris, 140 mM choline chloride, 2 mM KCl, 1 mM CaCl) without test substances was added to the SGLT1-overexpressing cell line treated with drugs for 24 h. 2 , 1 mM MgCl 2 , pH 7.4) and incubated for 1, 2, 4, 8, and 24 h to dissociate the drug bound to SGLT1.

[0149] One hour later, the preincubation buffer was removed and 10 μM [ 14 C]AMG (SGLT1 substrate) incubation buffer [10 mM HEPES, 5 mM Tris, 140 mM NaCl, 2 mM KCl, 1 mM CaCl 2, 1 mM MgCl 2 , pH 7.4], and reacted at 37°C in a thermomicromixer for two hours. After two hours, wash with 200 μL of ice-cold washing buffer [10 mM HEPES, 5 mM Tris, 140 mM choline chloride, 2 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 100 mM AMG, pH 7.4] and washed the cells twice.

[0150] After removing the culture medium, 40 μL of 10% SDS solution was added to lyse the cells, and then the sample was transferred to a measurement plate of a liquid scintillation counter and 150 μL of OptiPhase 500 as an isotope measurement mixture solution was added. The cells were incubated overnight to ensure that the cell solution was evenly mixed with the mixture solution. The [ 14 C]AMG, Figure 5 In cell lines overexpressing SGLT1, substrate [ 14 C] Intracellular uptake rate of AMG (percentage of control group). All data values ​​are expressed as the mean ± SD of four independent experiments.

[0151] like Figure 5 It was confirmed that when the CHO-SGLT1 cell line was treated with enagliflozin, M1, M2, dapagliflozin and empagliflozin for 24 hours, the activity of SGLT1 was reduced, and the ability to restore SGLT1 activity varied depending on the time of drug removal from the culture medium.

[0152] In other words, when cells were treated with enagliflozin, activity was not fully restored for up to two hours after washout, but was restored after four hours ( Figure 5 A).

[0153] In the case of M1 treatment, activity was partially restored for up to two hours after washout at low concentrations (50, 500, 5000 nM), but in the 20000 nM treatment group, activity recovery was incomplete even 24 hours after washout ( Figure 5 B).

[0154] In the case of M2 treatment, a very similar trend was observed as with enagliflozin. In other words, activity was not fully restored up to two hours after washout, but was restored after four hours ( Figure 5 C).

[0155] In the case of dapagliflozin and empagliflozin treatment, activity was fully restored two hours after washout, and it was confirmed that the recovery rate was faster than that of empagliflozin at the same concentration ( Figure 5 D and 5E).

[0156] From the above results, it can be confirmed that M1 dissociates from SGLT1 the slowest, enagliflozin and M2 dissociate faster, and dapagliflozin and empagliflozin dissociate faster. The above results mean that among the evaluated substances, M1 has the longest binding time to SGLT1.

[0157] (2) Comparative evaluation of the ability of enagliflozin and its metabolites to restore activity after clearance in cell lines overexpressing SGLT2

[0158] The evaluation method was the same as that for cells overexpressing SGLT1, except that a cell line overexpressing SGLT2 (CHO-SGLT2 stable cells) was used, and the concentrations of the evaluation substances used were as follows.

[0159] Enagagliflozin: 0.5, 5, 50, 500 nM

[0160] M1: 0.5, 5, 50, 500nM

[0161] M2: 0.5, 5, 50, 500nM

[0162] Dapagliflozin: 0.5, 5, 50, 500 nM

[0163] Empagliflozin: 0.5, 5, 50, 500 nM

[0164] like Figure 6 It was confirmed that when CHO-SGLT2 cells were treated with enagliflozin, M1, M2, dapagliflozin and empagliflozin for 24 hours, the activity of SGLT2 was reduced, and the ability to restore SGLT2 activity varied depending on the time of drug removal from the culture medium.

[0165] In the case of enagliflozin treatment, the recovery of activity over time after clearance showed different patterns, depending on the concentration of enagliflozin. When cells were treated with 0.5 nM, activity recovered after 2 hours, but when treated with 5 nM, recovery was shown after 24 hours. When cells were treated with 50 and 500 nM, the activity recovery was unstable even after 24 hours, with a recovery rate of less than 50%. The above results suggest that the activity recovery is slow ( ) due to the intracellular concentration of enagliflozin maintaining or delaying dissociation from SGLT2. Figure 6 A).

[0166] In the case of M1 treatment, activity was restored after 2 hours at a low concentration (0.5 nM), but when cells were treated with 5 and 50 nM and then cleared, activity was restored over time for up to eight hours and was almost completely restored after 24 hours. When cells were treated with 500 nM, delayed activity recovery was confirmed ( Figure 6 B).

[0167] In the case of M2 treatment, it was confirmed that the difference according to the concentration was not significant, and the activity recovered over time. In other words, the activity recovered over time up to four hours after the washout, and recovered after eight hours ( Figure 6 C).

[0168] In the case of dapagliflozin treatment, a similar trend as M2 was shown. In other words, it was confirmed that the difference according to concentration was not significant and the activity recovered over time. The activity recovered over time up to four hours after washout and recovered after 8 hours ( Figure 6 D).

[0169] In the case of empagliflozin treatment, it was confirmed that the activity was fully restored two hours after the washout, and the recovery rate was faster than that of empagliflozin, M1, M2, and dapagliflozin at the same concentration ( Figure 6 E).

[0170] The above results show that enagliflozin dissociates the slowest from SGLT2, and at high concentrations, activity recovery is not complete even after 24 hours, with a recovery rate of less than 50%. For M1, it has been confirmed that activity recovery is delayed by concentration and clearance time. In M2 and dapagliflozin, activity recovery does not vary greatly by concentration and is delayed by clearance time, while enagliflozin has the fastest activity recovery and is not delayed by pretreatment concentration or time.

[0171] [Example 2] Evaluation of drug-target binding affinity and dissociation degree of enagliflozin metabolites

[0172] When the cell lines overexpressing SGLT1 and SGLT2 (CHO-SGLT1, CHO-SGLT2 stable cells) were cultured to 60% confluence, the cells were treated with 0.05% trypsin to detach from the bottom of the plate, suspended in 200 μL of culture medium, and used at room temperature.

[0173] To evaluate the target binding affinity and dissociation degree of enagliflozin, M1, M2 and dapagliflozin for SGLT1 and SGLT2, glucose currents were recorded using whole-cell patch clamp mode, and drug-target binding affinity and dissociation degree were confirmed by evaluating their binding time to the target and clearance.

[0174] Glucose reabsorption is mediated by sodium ions (Na + ) is induced by the sodium-glucose cotransporter 2 (SGLT-2), and glucose diffuses across the cell membrane into the blood via the glucose transporter 2 (GLUT-2), which enables glucose transport. Each glucose molecule transported by SGLT2 absorbs a sodium ion (Na +), and each glucose molecule transported by the sodium-glucose cotransporter 1 (SGLT1) absorbs two sodium ions (Na + Therefore, by changing the composition of the buffer, it is possible to record only the glucose current and exclude the sodium ions (Na + ) current.

[0175] The experiment was performed as follows.

[0176] 1) Using a Narishige needle puller, glass microelectrodes were made at two stages at 62.7°C and 57°C, filled with internal solution (145 mM cesium chloride, 5 mM sodium chloride, 10 mM HEPES, 11 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), pH 7.2), and then inserted into the Ag / AgCl 2 (silver chloride) electrode.

[0177] 2) SGLT1 or SGLT2 cells were seeded in a chamber pre-coated with poly-L-lysine and then flowed into an external solution (150 mM NaCl, 10 mM HEPES, 1 mM CaCl 2 , 1 mM MgCl 2 , pH 7.4) to ensure that the cells are fully immersed. The chamber temperature is maintained at 36.5±0.5°C using a temperature control device.

[0178] 3) Using a micromanipulator, attach a glass microelectrode to the cell membrane to form a gigaohm seal, which is then ruptured to isolate the cell membrane (whole-cell mode, 3 to 5 gigaohms).

[0179] 4) The cells were fixed at -60 mV (holding potential) and the current [I total Current (Na + / glucose current (pA)].

[0180] 5) The external solution is continuously flowed into the chamber containing the cells, and the current (I total ) for about 30 seconds to 1 minute (stabilization period), and then the external solution (Na + / glucose external solution) (150 mM NaCl, 10 mM HEPES, 1 mM CaCl 2 , 1 mM MgCl 2 , 100 mM glucose, pH 7.4) to record glucose current (I glucose ). The cells were then exposed to an external solution containing one concentration of the compound supplemented with glucose for two minutes, and the inhibition of the glucose current by the compound was recorded.

[0181] 6) After confirming the inhibition of the current, the external solution was replaced with a new glucose-containing solution without the compound, and the current change was confirmed during the washout process and recorded for a minimum of 3 minutes and a maximum of 10 minutes (when the reaction lasted longer and the seal could be maintained, the current was recorded for up to 15 minutes).

[0182] 7) After confirming the current recovery, the solution was replaced with a sodium-free external solution (150 mM choline chloride, 1 mM CaCl 2 , 1 mM MgCl 2 , pH 7.4), and record the sodium ion current. After the current stabilizes, the solution is replaced with an external solution to record the current in the resting state when the cell is not stimulated, thereby ensuring the reliability of the experimental system.

[0183] For statistical analysis of the data, pCLAMP10.4 (Molecular Devices, LLC, San Jose, CA, USA) or Origin6.0 (OriginLab, Northampton, MA, USA) was used.

[0184] Test results are presented as mean ± SD of the mean. Nonparametric Kruskal-Wallis test was performed using Statistical Package for Social Sciences (SPSS) for Windows (version 26.0, IBM Corporation, Armonk, New York, USA) to verify statistical significance. The significance level was set at p < 0.05.

[0185] The binding affinity and dissociation results of enagliflozin, M1, M2 and dapagliflozin to SGLT1 are shown in Figure 2 Figure 7 and as shown in Table 6.

[0186] The recovery half-life (T 1 / 2,off ) was 34.9 seconds, and M2 (23.5 seconds) and dapagliflozin (30.5 seconds) dissociated at similar rates. On the other hand, the recovery half-life (T 1 / 2,off ) was 59.8 seconds, indicating that the dissociation rate of M1 was 1.7 times slower than that of enagliflozin and 1.96 times slower than that of dapagliflozin.

[0187] These results suggest that M1 affects binding to the SGLT1 protein and maintains this binding for a longer period of time than enagliflozin and its competitor dapagliflozin, and that efficacy may be maintained.

[0188] [Table 6] Comparison of the binding affinity and dissociation degree of enagliflozin, M1, M2 and dapagliflozin to SGLT1

[0189]

[0190]

[0191] Each data point represents mean ± SD (n = 4-5); t 1 / 2，on : Binding rate half-life; t 1 / 2，off : Dissociation rate half-life; *: p < 0.05 compared with M2, +: p < 0.05 compared with dapagliflozin, by post hoc analysis and significant nonparametric Kruskal-Wallis test.

[0192] In addition, the evaluation results of the binding affinity and dissociation degree of enagliflozin, M1, M2 and dapagliflozin to SGLT2 are as follows Figure 8 And as shown in Table 7.

[0193] The half-life of SGLT2 recovery rate (T 1 / 2,off ) was 745.5 seconds, showing the slowest dissociation rate among the evaluated substances. It was about 2.8 times slower than the competing substance dapagliflozin (265.2 seconds), and the dissociation rates of M1 (369.9 seconds) and M2 (540.4 seconds) were 2 times and 1.38 times slower than those of enagliflozin, respectively.

[0194] These results suggest that enagliflozin binds to the SGLT2 protein the longest and that its inhibitory effect may be maintained because the binding is preserved. In addition, M1 and M2 may also maintain their efficacy because they bind to the SGLT2 protein and dissociate more slowly than their competitor dapagliflozin.

[0195] In conclusion, similar to the dual SGLT-1 / SGLT-2 inhibition of M1, the recovery of inhibitory activity after elimination from the SGLT-1 target showed a slower dissociation time than M2. In addition, it was found that M2 maintained its binding to the SGLT-2 target longer than M1.

[0196] [Table 7] Comparison of the binding affinity and dissociation degree of enagliflozin, M1, M2 and dapagliflozin to SGLT2

[0197]

[0198]

[0199] Each data point represents mean ± SD (n = 3-6); t 1 / 2，on : Binding rate half-life; t 1 / 2，off : Dissociation rate half-life; *: p < 0.05 compared with enagliflozin, +: p < 0.05 compared with dapagliflozin, #: p < 0.05 compared with empagliflozin, by post hoc analysis and significant nonparametric Kruskal-Wallis test.

[0200] [Example 3] Evaluation of anti-diabetic efficacy by intravenous administration of enagliflozin metabolites

[0201] Enagagliflozin, M1, or M2 was intravenously administered once to normal rats, and its antidiabetic efficacy was evaluated.

[0202] In order to confirm oral glucose tolerance test (OGTT) and urine glucose excretion (UGE), enagliflozin, M1 or M2 were intravenously administered to male normal rats of 8 weeks old at a dosage of 1 mg / kg, and then glucose solution (2 g / kg) was applied to load blood glucose. Blood glucose levels were measured before and after administration of glucose solution at 5, 10, 15, 20, 30, 40, 60, 90 and 120 minutes to evaluate oral glucose tolerance. In addition, urine glucose excretion was evaluated by measuring the amount of glucose discharged in urine at 6, 24, 48 and 72 hours after administration of glucose solution.

[0203] Fig. 9 Shown is a graph showing changes in blood sugar levels over time after intravenous administration of each test substance at a dose of 1 mg / kg to normal rats. Fig.10 A graph showing analysis of the area under the blood glucose concentration-time curve (AUC) within 120 minutes after administration of the test substance is shown.

[0204] like Fig. 9 and Fig.10 As shown in Figure 2, it was confirmed that intravenous administration of enagliflozin, M1, and M2 at a dose of 1 mg / kg to normal rats improved glucose tolerance. When administered intravenously, metabolite M1 showed better glucose tolerance than enagliflozin, and M2 showed a similar pattern to enagliflozin.

[0205] In terms of the area under the blood glucose curve (AUC), M1 showed the most significant decrease in blood glucose AUC, and the decrease rates of enagliflozin and M2 were lower than that of M1. Compared with the solvent, the glucose tolerance inhibition rate of enagliflozin increased by 27%, M1 increased by 49%, and M2 increased by 23% ( Fig.10 ).

[0206] Meanwhile, when UGE was confirmed, UGE of metabolites M1 and M2 was observed 6 hours after intravenous administration of 1 mg / kg. The amount and pattern of glucose excretion were similar between M1 and enagliflozin, but in the case of M2, the amount of glucose excreted was less than that of enagliflozin and M1 for up to 24 hours.

[0207] In summary, it was judged that intravenous administration of the test drug exhibited in vivo activity by acting on SGLT1 and SGLT2 in the kidney.

[0208] Since the IC of metabolite M1 to SGLT250 It is higher than that of enagliflozin, but metabolite M1 shows a stronger effect on SGLT1, so it is judged that metabolite M1 acts as a dual inhibitor of SGLT1 and SGLT2. Ultimately, M1 shows anti-diabetic efficacy similar to that of enagliflozin.

[0209] The effect of M2 can last up to 24 hours and is weaker than that of M1. The reasons are: 1) M2 has an IC of SGLT1. 50 Higher than M1; 2) quickly dissipated (by T 1 / 2 = 0.8 hours confirmed).

[0210] [Example 4] Evaluation of the anti-diabetic efficacy of enagliflozin metabolites in a type 1 diabetes rat model

[0211] In order to evaluate the anti-diabetic efficacy of enagliflozin metabolites in the streptozotocin (STZ)-induced type 1 diabetes rat model, the test groups shown in Table 8 were set up and the experiments were performed.

[0212] [Table 8]

[0213]

[0214] STZ was administered to Sprague Dawley (SD) male 5-week-old rats on day 0 (G2 to G7, excluding G1), and non-fasting blood glucose levels were measured on day 3 to determine whether type 1 diabetes was induced. On day 7, the experimental substances were administered to each group, and blood and urine samples were collected at 6h, 24h, 48h, and 72h to measure non-fasting blood glucose levels. On day 14 of the experiment, the experimental animals fasted for 16h, and on day 15, 2g / kg of glucose solution was orally administered after the administration of the experimental substances. Thereafter, blood glucose levels were measured at 15, 30, 60, 90, and 120min after glucose administration. After blood collection, serum was separated, and the interleukin-6 (IL-6) enzyme-linked immunosorbent assay (ELISA) kit was used to evaluate the IL-6 inflammatory response.

[0215] (1) Non-fasting blood glucose level

[0216] Fig.11 Shown are non-fasting blood glucose levels measured 6, 24, 48, and 72 hours after the first administration of the test drug to the STZ-induced type 1 diabetes rat model.

[0217] like Fig.11As shown in the results, at the same dose of 1 mg / kg, the blood glucose levels of G5 (enagagliflozin M1 metabolite) and G6 (enagagliflozin) were observed to remain at a low level, and it was confirmed that they had a statistically significant hypoglycemic effect up to 48 hours after administration compared with the vehicle group. In particular, G5 showed the lowest blood glucose level 48 hours after administration, indicating a sustained hypoglycemic effect.

[0218] Compared with the G7 group administered with 1 mg / kg sotagliflozin (approved for type 1 diabetes), the G5 and G6 groups at the same dose showed better blood sugar-lowering effects.

[0219] (2) Oral glucose tolerance

[0220] Fig.12 and Fig.13 Blood glucose levels measured before (0 min) and 15, 30, 60 and 120 min after glucose administration are shown along with the calculated area under the curve (AUC).

[0221] like Fig.12 As shown, a decrease in blood glucose level was observed in the G5 group administered with 1 mg / kg of enagliflozin M1 metabolite, the G6 group administered with 1 mg / kg of enagliflozin, and the G7 group administered with 1 mg / kg of sotagliflozin, and in the case of the M1 metabolite, a dose-dependent improvement in glucose tolerance was confirmed. In particular, the G5 group administered with 1 mg / kg of enagliflozin M1 metabolite showed the fastest and strongest hypoglycemic effect, and the effect was much better than that of the G7 group administered with 1 mg / kg of sotagliflozin.

[0222] like Fig.13 As shown, the G5 group administered with 1 mg / kg of enagliflozin M1 metabolite showed the best oral glucose tolerance improvement effect in terms of AUC value, and the AUC value of G5 was similar to that of the control group G1 administered with the vehicle, confirming that enagliflozin M1 metabolite has superior blood glucose control effect in the oral glucose tolerance test (OGTT).

[0223] (3) Evaluation of inflammatory response

[0224] Fig.14 Shown are the results of measuring cytokines after administration of the test drugs to the STZ-induced type 1 diabetes rat model.

[0225] like Fig.14As shown, interleukin-6 (IL-6), a cytokine that has been confirmed to regulate inflammatory responses, increased at all time points (6, 24, 48, and 72 hours) in the vehicle group, while it decreased after M1 administration. In contrast, interleukin-6 (IL-6) slightly decreased in the enagliflozin and sotagliflozin administration groups, and M1 decreased significantly in all dose groups (0.01, 0.1, and 1 mg / kg), especially in the 0.01 mg / kg group after 6 hours, showing a more significant downward trend, and the same is true in the 0.1 mg / kg and 1 mg / kg groups after 72 hours.

[0226] These results suggest that M1 can modulate the inflammatory response associated with type 1 diabetes.

Claims

1. A pharmaceutical composition for preventing or treating diabetes or heart failure, comprising an enagliflozin M1 metabolite represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, [Chemical formula 1] 2. The pharmaceutical composition according to claim 1, wherein The diabetes is type 1 diabetes.

3. The pharmaceutical composition according to claim 1, wherein The diabetes is type 2 diabetes.

4. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition is for oral or parenteral administration.

5. The pharmaceutical composition according to claim 1, wherein The daily dosage of the enagliflozin M1 metabolite of Chemical Formula 1 or a pharmaceutically acceptable salt thereof is 0.1 to 0.5 mg.

6. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition is administered once a day.

7. A sodium-glucose cotransporter 1 (SGLT1) / sodium-glucose cotransporter 2 (SGLT2) dual inhibitor, comprising an enagliflozin M1 metabolite represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, [Chemical formula 1]

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