Pharmaceutical compositions containing metabolites of inavogliflozin and uses thereof

Abstract

The present invention relates to a pharmaceutical composition containing an inavogliflozin metabolite and its use. The inavogliflozin M1 metabolite of Chemical Formula 1 is an SGLT1 / SGLT2 dual inhibitor, and exhibits a different pharmacological mechanism from inavogliflozin, which is a selective SGLT2 inhibitor. A pharmaceutical composition containing the inavogliflozin M1 metabolite as an active ingredient as an SGLT1 / SGLT2 dual inhibitor can be useful for the prevention or treatment of diabetes or heart failure.

Description

[Technical Field]

[0001] The present application relates to a pharmaceutical composition containing an inavogliflozin metabolite and uses thereof. [Background technology]

[0002] SGLT2 (sodium glucose cotransporter 2) is a transporter that, together with SGLT1 (sodium glucose cotransporter 1), is responsible for the reabsorption of glucose in the kidney, with SGLT2 playing most of the role. Therefore, when SGLT2 inhibitors inhibit the SGLT2 transporter, the amount of glucose excreted in the urine increases, ultimately lowering blood glucose levels and further excreting the calories contained in blood glucose, resulting in weight loss.

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

[0004] [ka] Compound name: (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

[0005] Inavogliflozin is a drug that treats type 2 diabetes by selectively inhibiting sodium-glucose cotransporter 2 (SGLT2), and is as effective as or better than existing SGLT2 inhibitors at just 0.3 mg, which is one-thirtieth of the dose. A Phase 3 clinical trial conducted on type 2 diabetes patients demonstrated superior efficacy and safety in lowering glycated hemoglobin (HbA1c) and fasting blood glucose levels compared to existing commercially available drugs, and was approved for sale.

[0006] According to Non-Patent Document 1, inavogliflozin has been confirmed to produce five metabolites, M1, M2, M3, U1, and U2, in human hepatocytes. The formation of M1 and M2 from inavogliflozin was catalyzed by CYP3A4 and CYP2C19. M3 was produced by hydroxylation of M1 and was catalyzed by CYP3A4. The formation of U1 was catalyzed by UGT2B7, while the formation of U2 was catalyzed by UGT1A4, UGT1A9, and UGT2B7.

[0007] Non-Patent Document 1 is a paper regarding the drug metabolism of inavogliflozin in the liver and the elucidation of the metabolites and metabolic enzymes of inavogliflozin, but does not clarify at all what activity the metabolites of inavogliflozin exhibit. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Korean Patent Publication No. 10-2014-0022086 [Non-patent literature]

[0009] [Non-Patent Document 1] Ju-Hyun Kim et al., Pharmaceutics. 2020 Sep 11;12(9):865. doi: 10.3390 / pharmaceutics12090865. Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide pharmaceutical uses of inavogliflozin metabolites. [Means for solving the problem]

[0011] In the course of studying the metabolites of inavogliflozin, the present inventors discovered that the inavogliflozin M1 metabolite of Chemical Formula 1 is an SGLT1 / SGLT2 dual inhibitor and exhibits a different pharmacological mechanism from that of inavogliflozin, an SGLT2 selective inhibitor, and thus completed the present invention.

[0012] [ka]

[0013] SGLT-2 inhibitors target SGLT2, which is responsible for the reabsorption of more than 90% of glucose filtered by the kidney, while SGLT1 inhibitors target SGLT1, which is responsible for the reabsorption of the remaining 10% of glucose in small intestinal epithelial cells.

[0014] Most of the SGLT2 inhibitors developed to date have been drugs with highly selective binding to SGLT2, but inavogliflozin, developed by the applicant, is also a drug with extremely high selectivity for SGLT2 over SGLT1, and has recently been approved as a treatment for type 2 diabetes.

[0015] Lexicon Pharmaceuticals' sotagliflozin, a dual SGLT1 / SGLT2 inhibitor, has been approved in the EU for type 1 diabetes and by the US FDA for heart failure (HF).

[0016] The present invention provides the use of the inavogliflozin M1 metabolite as an SGLT1 / SGLT2 dual inhibitor.

[0017] The present invention will be described in more detail below.

[0018] The present invention provides use of inavogliflozin M1 metabolite or a pharmaceutically acceptable salt thereof as an SGLT1 / SGLT2 dual inhibitor, a pharmaceutical composition for preventing or treating diabetes mellitus or heart failure, which comprises inavogliflozin M1 metabolite or a pharmaceutically acceptable salt thereof as an active ingredient, and a method for preventing or treating diabetes mellitus or heart failure, which comprises administering an effective amount of inavogliflozin M1 metabolite or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0019] The present invention provides a pharmaceutical composition for preventing or treating diabetes or heart failure, comprising the inavogliflozin M1 metabolite of Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0020] Furthermore, the present invention provides an SGLT1 / SGLT2 dual inhibitor comprising, as an active ingredient, the inavogliflozin M1 metabolite of Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[0021] As can be seen in the following examples, the inavogliflozin M1 metabolite has binding ability to SGLT2 and also exhibits strong binding ability to SGLT1. Therefore, it is a drug candidate that can treat both indications that can be treated as an SGLT2 inhibitor and as an SGLT1 / SGLT2 dual inhibitor.

[0022] In the present invention, the diabetes may be type 1 diabetes or type 2 diabetes.

[0023] A typical indication that can be treated with an SGLT2 inhibitor may be type 2 diabetes. In Example 3 below, the excellent antidiabetic efficacy of inavogliflozin M1 metabolite was confirmed through oral glucose tolerance test (OGTT) and urinary glucose excretion (UGE) in a normal rat model.

[0024] Representative indications treatable with SGLT1 / SGLT2 dual inhibitors may be type 1 diabetes and heart failure.

[0025] The inavogliflozin M1 metabolite can simultaneously block SGLT2 in the kidney and SGLT1 in the small intestine, and therefore can be effective in treating type 1 diabetes with impaired insulin secretion. In Example 4 below, the antidiabetic efficacy of the inavogliflozin M1 metabolite was demonstrated in a rat model of type 1 diabetes. Example 4 demonstrates that the inavogliflozin M1 metabolite may be the most effective drug for type 1 diabetes compared to inavogliflozin and sotagliflozin.

[0026] In one embodiment, the pharmaceutical composition may be administered to a patient with diabetes or a patient in the pre-diabetic stage.

[0027] A pharmaceutical composition comprising the inavogliflozin M1 metabolite or a pharmaceutically acceptable salt thereof as an active ingredient may be used in the form of single administration or combined administration.

[0028] Inavogliflozin M1 metabolite alone is sufficient to prevent or treat diabetes and / or heart failure. However, for various purposes, antidiabetic agents or antiheart failure agents are administered in combination, and complementary or synergistic effects can be exhibited when administered in combination. Therefore, a pharmaceutical composition containing inavogliflozin M1 metabolite as an active ingredient can be administered in combination with other antidiabetic agents or antiheart failure agents.

[0029] For example, it may be administered in combination with biguanide drugs such as metformin, which are known diabetes treatment agents, DPP4 inhibitors, sulfonylurea insulin secretagogues and / or insulin.

[0030] Another indication that can be treated with an SGLT1 / SGLT2 dual inhibitor is heart failure. The preventive or therapeutic effect of sotagliflozin as an SGLT1 / SGLT2 dual inhibitor for heart failure has been proven by its approval by the US FDA.

[0031] The term "prevention" as used herein means any action of inhibiting or delaying the onset of diabetes or heart failure by administering a pharmaceutical composition according to the present invention.

[0032] Additionally, the term "treatment" as used herein means any action that improves or beneficially alters diabetes or heart failure by administering a pharmaceutical composition according to the present invention.

[0033] The dose of inalogliflozin M1 metabolite that can be used for the prevention or treatment of diabetes and / or heart failure in patients with or at risk of diabetes and / or heart failure is not particularly limited and may be appropriately adjusted depending on the severity of the disease, body weight, age, sex, presence or absence of other complications, etc. of the patient.

[0034] Inavogliflozin M1 metabolite or a pharmaceutically acceptable salt thereof may be administered orally or parenterally. The administration period may be adjusted appropriately by a clinician based on the preventive or therapeutic effect of the administration on diabetes and / or heart failure in patients with or at risk of diabetes and / or heart failure.

[0035] The pharmaceutical composition according to the present invention may further contain a pharmaceutically acceptable carrier in addition to the inavogliflozin M1 metabolite of Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, or may be formulated together with the carrier.

[0036] In the present invention, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate living organisms and does not inhibit the biological activity and properties of the administered compound. Pharmaceutical carriers acceptable for compositions formulated into liquid solutions include sterile and biocompatible carriers, such as saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components. Other common additives, such as antioxidants, buffers, and bacteriostats, may also be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may also be added to formulate the compositions into injectable forms such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, and tablets.

[0037] In the present invention, the pharmaceutical composition may have a dosage form for oral administration such as a tablet, a capsule, etc. In one embodiment of the present invention, the pharmaceutical composition may have a dosage form of a tablet.

[0038] When the pharmaceutical composition of the present invention is in a dosage form for oral administration, it may contain additives such as an excipient, a disintegrant, and a binder.

[0039] Examples of excipients include lactose (including hydrates), dextrin, mannitol, sorbitol, starch, microcrystalline cellulose (e.g., Celphere™), silicified microcrystalline cellulose (e.g., Prosolv™), calcium phosphate hydrate, calcium phosphate anhydrous, calcium carbonate, sugars, or mixtures thereof. In an embodiment of the present invention, the preferred excipient is microcrystalline cellulose.

[0040] Examples of disintegrants include crospovidone, croscarmellose sodium, sodium starch glycolate, and low-substituted hydroxypropyl cellulose. In an embodiment of the present invention, the preferred excipient is croscarmellose sodium.

[0041] Examples of binders include polyvinylpyrrolidone, povidone, gelatin, starch, sucrose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl alkylcellulose (e.g., hydroxypropylmethylcellulose), and mixtures thereof. In an embodiment of the present invention, the preferred binder is hydroxypropylcellulose.

[0042] Examples of other additives include lubricants and colorants.

[0043] The lubricants include stearic acid, stearates (e.g., magnesium stearate), hard anhydrous silicic acid, talc, corn starch, canauba wax, magnesium silicate, synthetic aluminum silicate, hydrogenated oil, white lead, titanium oxide, microcrystalline cellulose, macrogol 4000 and 6000, isopropyl myristate, calcium hydrogen phosphate, and mixtures thereof.

[0044] In the present invention, the pharmaceutical composition may have a dosage form for parenteral administration. For example, but not limited to, the dosage form for parenteral administration containing the composition of the present invention as an active ingredient may be formulated into an injectable form such as a subcutaneous injection, an intravenous injection, or an intramuscular injection.

[0045] To prepare an injectable dosage form, the composition of the present invention is mixed with a stabilizer or buffer in water to prepare a solution or suspension, which can then be formulated into a unit dose in an ampule or vial.

[0046] Alternatively, the compositions of the present invention can be formulated into various forms for parenteral administration such as, for example, eye drops, microneedles, patches, depots, and the like.

[0047] The compositions of the present invention are administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease. The effective dose level can be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug, the administration time, administration route and excretion rate, the duration of treatment, and other factors well known in the medical field, including other factors including other drugs used concomitantly. The compositions of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, and in single or multiple administrations. That is, the total effective amount of the compositions of the present invention may be administered to a patient in a single dose or in a fractionated treatment protocol in which multiple doses are administered over a long period of time. Taking all of the above factors into consideration, it is important to administer an amount that can achieve maximum efficacy at the minimum dose without side effects, which can be easily determined by those skilled in the art.

[0048] The appropriate once-daily dose of inavogliflozin determined in the course of clinical trials is 0.1 mg to 0.5 mg, and based on the results of the Examples below, it is expected that the inavogliflozin M1 metabolite can also be administered at a dose of 0.1 mg to 0.5 mg, similar to that of inavogliflozin.

[0049] 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.

[0050] The pharmaceutical composition according to the present invention may be administered from 1 to 3 times a day, for example, but not limited to, once a day. [Effects of the Invention]

[0051] The pharmaceutical composition of the present invention containing the inavogliflozin M1 metabolite as an active ingredient can be useful for the prevention or treatment of diabetes or heart failure. [Brief explanation of the drawings]

[0052] [Figure 1] 1 is a graph showing the SGLT1 activity inhibitory effects of inavogliflozin, M1, M2, dapagliflozin, and empagliflozin on AMG transport into CHO-SGLT1 cells. [Figure 2] 1 is a graph showing the SGLT2 activity inhibitory effects of inavogliflozin, M1, M2, dapagliflozin, and empagliflozin on AMG transport into CHO-SGLT2 cells. [Figure 3] 1 is a graph showing changes in the inhibitory effects of inavogliflozin, M1, M2, dapagliflozin, and empagliflozin on SGLT1 activity depending on the drug pretreatment time. [Figure 4] 1 is a graph showing changes in the inhibitory effects of inavogliflozin, M1, M2, dapagliflozin, and empagliflozin on SGLT2 activity depending on the drug pretreatment time. [Figure 5] 1 shows the results of a comparison of the activity recovery abilities of inavogliflozin, M1, M2, dapagliflozin, and empagliflozin against SGLT1. [Figure 6] 1 shows the results of a comparison of the activity recovery abilities of inavogliflozin, M1, M2, dapagliflozin, and empagliflozin against SGLT2. [Figure 7] 1 shows the results of comparing the binding affinity and dissociation degree of inavogliflozin, M1, M2, and dapagliflozin to SGLT1. [Figure 8] 1 shows the results of comparing the binding affinity and dissociation degree of inavogliflozin, M1, M2, and dapagliflozin to SGLT2. [Figure 9] 1 is a graph showing blood glucose changes over time after administration of inavogliflozin, M1, or M2 in normal rats. [Figure 10] 1 is a graph showing the area under the blood glucose concentration-time curve after administration of inavogliflozin, M1, or M2 in normal rats. [Figure 11] 1 is a graph showing non-fasting blood glucose levels observed up to 72 hours after administration of test substances in STZ-induced type 1 diabetic rats. [Figure 12] 1 is a graph showing blood glucose levels in STZ-induced type 1 diabetic rats in an oral glucose tolerance test. [Figure 13] 1 is a graph showing the area under the blood glucose concentration-time curve in an oral glucose tolerance test in STZ-induced type 1 diabetic rats. [Figure 14] 1 is a graph showing changes in inflammatory response-related cytokine levels over time after administration of a test substance in STZ-induced type 1 diabetic rats. DETAILED DESCRIPTION OF THE INVENTION

[0053] The advantages and features of the present invention, as well as methods for achieving them, will become apparent from the following detailed experimental and manufacturing examples. However, the present invention is not limited to the experimental and manufacturing examples disclosed below, and may be realized in various different forms. The following examples are provided solely for the purpose of completing the disclosure of the present invention and fully conveying the scope of the present invention to those skilled in the art.

[0054] [Example] Example 1: Evaluation of SGLT1 and SGLT2 inhibitory activity of inavogliflozin metabolites Our preliminary research demonstrated that inavogliflozin (also referred to as DWP16001 in the Examples below) exhibited stronger inhibitory activity against SGLT2 than against SGLT1 compared with the competing drugs dapagliflozin and ipragliflozin, and that this inhibitory activity remained unchanged after up to 2 hours of pretreatment. Dixon plot and Replot results for the evaluation of inhibitory activity as a function of substrate concentration indicated a reversible competitive inhibition mechanism for SGLT2 inhibition. However, quantification of intracellular drug concentrations after 24 hours of incubation confirmed that significantly higher amounts of inavogliflozin remained intracellularly compared with dapagliflozin and ipragliflozin. Furthermore, inavogliflozin exhibited a time-dependent delay in the recovery of intracellular SGLT2 activity, whereas the competing drugs dapagliflozin and ipragliflozin did not. These results demonstrate that inavogliflozin has a longer duration of efficacy than dapagliflozin and ipragliflozin.

[0055] In this study, the inventors attempted to evaluate whether the same results would be obtained for the M1 and M2 metabolites of inavogliflozin (also referred to as M1 and M2, respectively, herein), and also to evaluate and compare them with dapagliflozin and empagliflozin, which have recently been actively studied in clinical trials.

[0056] Example 1-1. Evaluation of the inhibitory activity of inavogliflozin metabolites against SGLT subtypes (1) Evaluation of SGLT1 inhibition SGLT1-overexpressing cell lines (CHO-SGLT1 stable cells) and mock cells (CHO-mock cells) were cultured. When they reached 80% confluence, 1 × 10 cells were plated in a 96-well plate. 5 cells / well.

[0057] After 24 hours, the medium was removed, and a pre-incubation buffer [10 mM HEPES, 5 mM Tris, 140 mM choline chloride, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2 pH 7.4] was added, followed by incubation for 1 hour.

[0058] After 1 hour, the pretreatment buffer was removed and 10 μM [ 14 C] AMG and inhibitory evaluation substances were added to incubation buffer [10 mM HEPES, 5 mM Tris, 140 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, pH 7.4] at various concentrations, and the mixture was incubated at 37°C in a thermomicromixer for 2 hours.

[0059] The concentrations of the inhibitory evaluation substances used in the experiment are as follows: Inavogliflozin: 1, 10, 100, 500, 1000, 5000, 20000, 50000nM M1:1, 10, 100, 500, 1000, 5000, 20000, 50000nM M2:1, 10, 100, 500, 1000, 5000, 20000, 50000nM Dapagliflozin: 1, 10, 100, 500, 1000, 5000, 20000, 50000nM Empagliflozin: 1, 10, 100, 500, 1000, 5000, 20000, 50000nM

[0060] For reference, M2 metabolites have the following compound names and structures:

[0061] [ka]

[0062] After a predetermined time, the plate was washed twice with 200 μL of ice-cold washing buffer [10 mM HEPES, 5 mM Tris, 140 mM choline chloride, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 100 mM AMG, pH 7.4].

[0063] After removing the medium, 40 μL of 10% SDS solution was added to lyse the cells, and the sample was then transferred to a liquid scintillation counter plate. 150 μL of OptiPhase Supermix, a cocktail for isotope measurement, was added and the mixture was incubated overnight to ensure that the cell lysate was uniformly mixed into the cocktail.

[0064] In the sample 14 C]AMG was measured using a liquid scintillation counter.

[0065] In SGLT1-overexpressing cell lines, 14 The intracellular influx rate of [C]AMG was compared with that in CHO-mock cells, and the substrate drug [ 14 The IC50 (half maximal inhibitory concentration) was calculated from the intracellular uptake rate (% of control) of [C] AMG and the concentration of the inhibitory evaluation substance added. 50 Values ​​were calculated using the inhibitory effect Sigmoid Emax model with the WinNonlin program (ver. 2.0). All data values ​​are expressed as the mean ± SD of three independent experiments.

[0066] (2) Evaluation of SGLT2 inhibition The evaluation was performed in the same manner as the SGLT1 inhibitory evaluation method, except that a cell line overexpressing SGLT2 (CHO-SGLT2 stable cells) was used and the inhibitory evaluation substances were used as follows.

[0067] The concentrations of the inhibitory evaluation substances are as follows: Inavogliflozin: 0.01, 0.05, 0.2, 2, 10, 50, 100, 500nM M1:0.01, 0.05, 0.2, 2, 10, 50, 100, 500nM M2:0.01, 0.05, 0.2, 2, 10, 50, 100, 500nM Dapagliflozin: 0.01, 0.05, 0.2, 2, 10, 50, 100, 500nM Empagliflozin: 0.01, 0.05, 0.2, 2, 10, 50, 100, 500nM

[0068] (3) Evaluation of the inhibitory effects of inavogliflozin and its M1 and M2 metabolites in SGLT1 and SGLT2 overexpressing cell lines The inhibitory activities of inavogliflozin, M1, M2, dapagliflozin, and empagliflozin against SGLT1 or SGLT2 were evaluated and are shown in Table 1 below and Figures 1 and 2.

[0069] [Table 1]

[0070] The low-resolution values ​​calculated from the evaluation results were similar to those of previous studies, and compared with dapagliflozin and empagliflozin, inavogliflozin had stronger inhibitory activity against SGLT2, and its selectivity, calculated as the ratio of low-resolution values ​​between SGLT1 and SGLT2, was evaluated to be superior. Furthermore, both inavogliflozin's metabolites M1 and M2 exhibited inhibitory activity against SGLT2.

[0071] Unlike inavogliflozin, M2, dapagliflozin, and empagliflozin, which selectively inhibit SGLT2, metabolite M1 also has a superior inhibitory effect on SGLT1 activity (IC 50 value of 28.4 nM level), and IC of SGLT1 / SGLT2 50 The ratio was very low, confirming that it can act as a dual inhibitor of SGLT1 and SGLT2.

[0072] Example 1-2. Evaluation of the inhibitory mechanism of inavogliflozin and its M1 and M2 metabolites on SGLT1 and SGLT2 (1) Comparative evaluation of inhibitory effects of drug exposure time on SGLT1-overexpressing cell lines CHO-SGLT1 cells were pretreated with inavogliflozin, M1, M2, dapagliflozin, and empagliflozin in a sodium-free pretreatment buffer for 1 or 2 hours, and the concentration-dependent inhibitory effect of inavogliflozin on AMG transport into CHO-SGLT1 cells was evaluated and compared with the untreated group. All data are expressed as the mean ± SD of four independent experiments.

[0073] As shown in Table 2 and Figure 3, the inhibitory activity of inavogliflozin, M2, dapagliflozin, and empagliflozin did not increase or change significantly with increasing exposure time (p>0.05). On the other hand, the low resolution of M1 increased significantly with increasing exposure time (p<0.05).

[0074] [Table 2]

[0075] (2) Inhibitory effect of the substrate AMG on SGLT1-overexpressing cell lines To determine the inhibitory mechanism and Ki of SGLT1, the concentration-dependent inhibitory activity of inavogliflozin, M1, M2, dapagliflozin, and empagliflozin was evaluated in CHO-SGLT1 cell lines using varying concentrations of the substrate AMG.

[0076] The AMG absorption rate and the concentrations of inavogliflozin, M1, M2, dapagliflozin, and empagliflozin were plotted using a Dixon plot, and the AMG concentration and Dixon slopes were plotted. The results were also plotted using an LB plot, and the inhibitor concentrations and LB slopes were plotted (not shown). From this, the inhibitory mechanism was confirmed and the low resolution was calculated. The low resolution and inhibitory mechanism calculated from the slope of the LB plot are shown in Table 3.

[0077] [Table 3]

[0078] As can be seen from Table 3, the Ki value of the M1 metabolite for SGLT1 activity was 84.8 nM, the smallest value among the tested substances. A smaller Ki value indicates a higher affinity, and such results indicate that M1 has a higher affinity for SGLT1 than the test substances, and therefore has a stronger inhibitory effect on SGLT1.

[0079] (3) Comparative evaluation of inhibitory effects depending on drug exposure time in SGLT2-overexpressing cell lines CHO-SGLT2 cells were pretreated with inavogliflozin, M1, M2, dapagliflozin, and empagliflozin at different concentrations in a sodium-free pretreatment buffer for 1 or 2 hours, and the concentration-dependent inhibitory effect of inavogliflozin on AMG transport into CHO-SGLT2 cells was evaluated and compared with the untreated group. All data values ​​are expressed as the mean ± SD of four independent experiments.

[0080] As shown in Table 4 and FIG. 4, the evaluation results showed that for inavogliflozin, M1, M2, dapagliflozin, and empagliflozin, the inhibitory activity either increased or did not change significantly with increasing exposure time (p>0.05).

[0081] [Table 4]

[0082] (4) Evaluation of inhibitory effects of the substrate AMG on SGLT2 overexpressing cell lines To determine the inhibitory mechanism and Ki of SGLT2, the concentration-dependent inhibitory activity of inavogliflozin, M1, M2, dapagliflozin, and empagliflozin was evaluated in CHO-SGLT2 cell lines using varying concentrations of the substrate AMG.

[0083] The AMG absorption rate and the concentrations of inavogliflozin, M1, M2, dapagliflozin, and empagliflozin were plotted using a Dixon plot, and the AMG concentration and Dixon slopes were plotted. The results were also plotted using an LB plot, and the inhibitor concentrations and LB slopes were plotted (not shown). From this, the inhibitory mechanism was confirmed and the low resolution was calculated. The low resolution and inhibitory mechanism calculated from the slope of the LB plot are shown in Table 5.

[0084] [Table 5]

[0085] Example 1-3. Comparative evaluation of the activity recovery ability of SGLT1 and SGLT2 after 24-hour pretreatment with inavogliflozin and metabolites (1) Comparative evaluation of the recovery of activity after washout of inavogliflozin and its metabolites in SGLT1-overexpressing cell lines SGLT1-overexpressing cell lines (CHO-SGLT1 stable cells) were cultured until they reached 80% confluence, at which point 1 × 10 cells were plated onto a 96-well plate. 5 After 24 hours, the medium was removed and replaced with medium containing the test substance at each concentration, followed by another 24 hours of culture.

[0086] The concentrations of the evaluation substances used in the experiment are as follows: Inavogliflozin: 50, 500, 5000, 20000nM M1: 50, 500, 5000, 20000nM M2: 50, 500, 5000, 20000nM Dapagliflozin: 50, 500, 5000, 20000nM Empagliflozin: 50, 500, 5000, 20000nM

[0087] SGLT1-overexpressing cell lines were treated with drugs for 24 hours, and then a pretreatment buffer (10 mM HEPES, 5 mM Tris, 140 mM choline chloride, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2 pH 7.4) containing no substance to be evaluated was added. The cells were then cultured for 1, 2, 4, 8, and 24 hours to allow the drug bound to SGLT1 to dissociate.

[0088] After 1 hour, the pretreatment buffer was removed and 10 μM [ 14 The cells were incubated with incubation buffer (10 mM HEPES, 5 mM Tris, 140 mM NaCl, 2 mM KCl, 1 mM CaCl, 1 mM MgCl, pH 7.4) containing [C] AMG for 2 hours at 37°C in a Thermo Micromixer. After 2 hours, the cells were washed twice with 200 μL of ice-cold washing buffer (10 mM HEPES, 5 mM Tris, 140 mM choline chloride, 2 mM KCl, 1 mM CaCl, 1 mM MgCl, 100 mM AMG, pH 7.4).

[0089] After removing the medium, 40 μL of 10% SDS solution was added to lyse the cells, and the sample was transferred to a plate for liquid scintillation counter measurement. 150 μL of the cocktail solution for isotope measurement, OptiPhase Supermix®, was added and incubated overnight to ensure that the cell solution was homogeneously mixed with the cocktail solution. 14 C]AMG was measured by liquid scintillation counter, and then the substrate drug [ 14 The intracellular uptake rate of [C]AMG (% of control) is shown in Figure 5. All data values ​​are expressed as the mean ± SD of four independent experiments.

[0090] As can be seen from Figure 5, the activity of SGLT1 was reduced when the CHO-SGLT1 cell line was treated with inavogliflozin, M1, M2, dapagliflozin, or empagliflozin for 24 hours, and the ability to recover SGLT1 activity varied depending on the time the drug was removed from the culture medium.

[0091] That is, when treated with inavogliflozin, the activity was incompletely restored up to 2 hours after washout, but recovered after 4 hours (Figure 5A).

[0092] When M1 was treated, the activity was partially restored at low concentrations (50, 500, and 5000 nM) up to 2 hours after washout, but in the 20000 nM treatment group, the activity recovery was incomplete up to 24 hours after washout (Fig. 5B).

[0093] Treatment with M2 showed a trend very similar to that of inavogliflozin: activity was incompletely restored up to 2 hours after washout, but recovered by 4 hours (Figure 5C).

[0094] When treated with dapagliflozin or empagliflozin, the activity was fully recovered 2 hours after washout, and it was confirmed that the recovery was faster than that in the case of inavogliflozin at the same concentration (Figures 5D and 5E).

[0095] The above results confirmed that M1 dissociated from SGLT1 the slowest, inalogliflozin and M2 dissociated quickly, and dapagliflozin and empagliflozin dissociated quickly. These results indicate that among the evaluated substances, M1 bound to SGLT1 for the longest period of time.

[0096] (2) Comparative evaluation of the recovery of activity after washout of inavogliflozin and its metabolites in SGLT2-overexpressing cell lines The evaluation method was the same as for SGLT1-overexpressing cells, except that a cell line overexpressing SGLT2 (CHO-SGLT2 stable cells) was used and the concentrations of the evaluation substances were as follows: Inavogliflozin: 0.5, 5, 50, 500nM M1: 0.5, 5, 50, 500nM M2: 0.5, 5, 50, 500nM Dapagliflozin: 0.5, 5, 50, 500nM Empagliflozin: 0.5, 5, 50, 500nM

[0097] As can be seen from Figure 6, the activity of SGLT2 was reduced when the CHO-SGLT2 cell line was treated with inavogliflozin, M1, M2, dapagliflozin, or empagliflozin for 24 hours, and the ability to recover SGLT2 activity varied depending on the time the drug was removed from the culture medium.

[0098] When treated with inavogliflozin, the recovery of activity over time after washout varied depending on the inavogliflozin concentration. With 0.5 nM treatment, activity recovered after 2 hours, but with 5 nM treatment, recovery occurred after 24 hours. With 50 and 500 nM treatment, activity recovery was unstable even after 24 hours, with recovery of less than 50%. These results suggest that the intracellular concentration of inavogliflozin was maintained and its dissociation from SGLT2 was delayed, resulting in a delayed recovery of activity (Figure 6A).

[0099] When treated with M1 at a low concentration (0.5 nM), activity recovered after 2 hours, but when treated with 5 or 50 nM and washed out, activity recovered over time up to 8 hours and was almost fully recovered by 24 hours. Treatment with 500 nM confirmed a delayed recovery of activity (Figure 6B).

[0100] When M2 was treated, the activity recovered over time, with no significant difference depending on the concentration. That is, the activity recovered over time up to 4 hours after washout, and recovered again after 8 hours (Figure 6C).

[0101] When treated with dapagliflozin, the same trend as M2 was observed. That is, there was little difference in activity depending on the concentration, and it was confirmed that activity recovered over time. The results show that activity recovered over time up to 4 hours after washout, and recovered again after 8 hours (Figure 6D).

[0102] When treated with empagliflozin, activity was fully restored 2 hours after washout, and it was confirmed that the recovery was faster than that of inavogliflozin, M1, M2, and dapagliflozin at the same concentration (Figure 6E).

[0103] The results show that inavogliflozin dissociates most slowly from SGLT2, and at high concentrations, activity recovery is incomplete even after 24 hours, with recovery of less than 50%. Even in the case of M1, activity recovery is delayed depending on the concentration and washout time. M2 and dapagliflozin showed little difference in activity recovery depending on the concentration, but showed a delay depending on the washout time. Empagliflozin showed the fastest activity recovery, with no delay due to the pretreatment concentration or time.

[0104] [Example 2] Evaluation of drug-target binding affinity and dissociation degree of inavogliflozin metabolites Cell lines overexpressing SGLT1 and SGLT2 (CHO-SGLT1, CHO-SGLT2 stable cells) were cultured until they reached 60% confluence. The cells were then treated with 0.05% trypsin to detach them from the plate bottom, and then suspended in 200 μL of culture medium for use at room temperature.

[0105] To evaluate the target binding affinity and dissociation of inavogliflozin, M1, M2, and dapagliflozin to SGLT1 and SGLT2, glucose currents were recorded using whole-cell patch clamp, and the duration of target binding was assessed through washout to confirm the drug-target binding affinity and dissociation.

[0106] Glucose reabsorption occurs in the kidney via SGLT2 and Na +Driven by an electrochemical potential gradient, glucose diffuses across the cell membrane into the blood via glucose transporter 2 (GLUT-2), which allows facilitated glucose movement. One Na per glucose molecule is transported via SGLT2. + is absorbed, whereas SGLT1 absorbs two Na per glucose molecule. + Therefore, changing the composition of the buffer can increase the Na + Only glucose currents were recorded, excluding currents.

[0107] The experiment was carried out in the following way:

[0108] 1) Using a Narishige puller, glass micropipettes were prepared at two temperatures, 62.7°C and 57°C, and then filled with an internal solution (145 mM CsCl, 5 mM NaCl, 10 mM HEPES, 11 mM EGTA, pH 7.2) and fitted onto the Ag / AgCl2 electrode of the perch clamp headstage.

[0109] 2) SGLT1 or SGLT2 cells were seeded onto a chamber pre-coated with poly-L-lysine, and then an external solution (150 mM NaCl, 10 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, pH 7.4) was poured into the chamber to fully immerse the cells. The temperature of the chamber was maintained at 36.5 ± 0.5°C using a temperature control device.

[0110] 3) Using a micromanipulator, a glass micropipette was attached to the cell membrane to create a giga-ohm seal, which was then ruptured to separate the cell membrane (whole-cell mode, 3-5 MΩ).

[0111] 4) The cells were clamped at -60 mV (holding potential) and the current [I totalThe Na+ / Glucose current (pA) was measured.

[0112] 5) The external solution is continuously passed through the chamber containing the cells, and a current (I total ) was recorded for about 30 seconds to 1 minute (stabilization), and then the external solution (Na + / Glucose external) (150 mM NaCl, 10 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, 100 mM Glucose, pH 7.4) was applied to induce glucose current (I glucose The cells were then exposed to an external solution containing glucose containing one of the compounds at different concentrations for 2 minutes, and the glucose currents inhibited by the compounds were recorded.

[0113] 6) After confirming that the current was suppressed, the solution was replaced with a new solution containing glucose but not the compound, and the change in current was confirmed while washing out. The current was recorded for a minimum of 3 minutes and a maximum of 10 minutes (if the reaction continued and the seal could be maintained, it was recorded for up to 15 minutes).

[0114] 7) After confirming the recovery of the current, the sodium-free external solution (Na + -free external) (150 mM Choline-Cl, 1 mM CaCl2, 1 mM MgCl2, pH 7.4) + After the current stabilized, the external solution was replaced and the current was recorded in a resting state where the cells were not stimulated, ensuring the reliability of the experimental system.

[0115] Statistical analysis of the data was performed using pCLAMP10.4 (Molecular Devices, LLC, San Jose, CA, USA) or Origin6.0 (OriginLab, Northampton, MA, USA).

[0116] Test results were expressed as mean ± standard deviation (SD). Statistical significance was determined using the non-parametric Kruskal-Wallis test using SPSS for Windows (version 26.0, IBM Corp., Armonk, NY, USA). The significance level was set at p<0.05.

[0117] The evaluation results of the binding affinity and dissociation degree of inavogliflozin, M1, M2, and dapagliflozin to SGLT1 are shown in Figure 7 and Table 6.

[0118] Half-time of recovery (T) of inavogliflozin on SGLT1 1 / 2,off The dissociation rate of M2 (23.5 seconds) and dapagliflozin (30.5 seconds) was 34.9 seconds, confirming that they dissociated at similar rates. 1 / 2,off ) was 59.8 seconds, confirming that it dissociated 1.7 times slower than inalogliflozin and 1.96 times slower than dapagliflozin.

[0119] These results suggest that M1 binds and maintains SGLT1 protein better than inalogliflozin and its competitor dapagliflozin, maintaining efficacy.

[0120] [Table 6]

[0121] Furthermore, the evaluation results of the binding affinity and dissociation degree of inavogliflozin, M1, M2, and dapagliflozin to SGLT2 are shown in Figure 8 and Table 7.

[0122] Half-time of recovery (T) of inavogliflozin on SGLT2 1 / 2,off)) showed the slowest dissociation rate of the substances evaluated, at 745.5 seconds, approximately 2.8 times slower than the competitor dapagliflozin (265.2 seconds), while M1 (369.9 seconds) and M2 (540.4 seconds) showed dissociation rates that were 2 and 1.38 times slower than inavogliflozin, respectively.

[0123] These results indicate that inavogliflozin binds to the SGLT2 protein for the longest time, and that maintaining this binding maintains its inhibitory activity. Furthermore, M1 and M2 also bind to and dissociate from the SGLT2 protein more slowly than the competitor dapagliflozin, suggesting that their efficacy may be maintained.

[0124] In conclusion, M1 exhibited dual SGLT-1 / SGLT-2 inhibitory effects, but also showed a slower dissociation time than M2 upon washout from the SGLT-1 target, and M2 maintained binding to the SGLT-2 target for a longer period than M1.

[0125] [Table 7]

[0126] [Example 3] Evaluation of antidiabetic efficacy by intravenous administration of inavogliflozin metabolites Inavogliflozin, M1, or M2 was administered intravenously as a single dose to normal rats, and the antidiabetic efficacy was evaluated.

[0127] To evaluate oral glucose tolerance tests (OGTTs) and urinary glucose excretion (UGE), 8-week-old male normal rats were intravenously administered 1 mg / kg of inavogliflozin, M1, or M2, followed by a glucose solution (2 g / kg). Blood glucose levels were measured before and 5, 10, 15, 20, 30, 40, 60, 90, and 120 minutes after glucose administration to evaluate oral glucose tolerance. Urinary glucose excretion was also measured 6, 24, 48, and 72 hours after glucose administration to evaluate urinary glucose excretion (UGE).

[0128] Figure 9 is a graph showing the change in blood glucose level over time after intravenous administration of each test substance at a dose of 1 mg / kg to normal rats, and Figure 10 is a graph showing the area under the blood glucose concentration-time curve for 120 minutes after administration of the test substance.

[0129] As shown in Figures 9 and 10, inavogliflozin, M1, and M2 were confirmed to improve glucose tolerance when administered intravenously to normal rats at a dose of 1 mg / kg. When administered intravenously, metabolite M1 improved glucose tolerance better than inavogliflozin, and a similar pattern to that of inavogliflozin was observed for metabolite M2.

[0130] When comparing blood glucose AUC, M1 showed the most significant decrease in blood glucose AUC, while inavogliflozin and M2 showed a lower decrease than M1. Compared to vehicle, the glucose tolerance suppression rate was improved by 27% for inavogliflozin, 49% for M1, and 23% for M2 (Figure 10).

[0131] Meanwhile, urinary glucose excretion (UGE) was confirmed, and metabolites M1 and M2 showed UGE from 6 hours after intravenous administration of 1 mg / kg. The amount and pattern of glucose excreted by M1 and inavogliflozin were similar, but M2 excreted less glucose than inavogliflozin and M1 up to 24 hours.

[0132] Taking these results together, it is believed that intravenous administration of the test drug exerts in vivo activity through the action of SGLT1 and SGLT2 in the kidney.

[0133] IC of metabolite M1 on SGLT2 50 Although its activity is higher than that of inavogliflozin, it exhibits stronger efficacy against SGLT1. Therefore, it is thought that metabolite M1 functions as a dual inhibitor of SGLT1 and SGLT2, and as a result, M1 exhibits similar antidiabetic efficacy to inavogliflozin.

[0134] M2 showed efficacy up to 24 hours and showed weaker efficacy than M1, which is presumably due to 1) the IC50 of M2 against SGLT1 being higher than that of M1, resulting in weaker efficacy, and 2) rapid elimination (t 1 / 2 =0.8h).

[0135] Example 4: Evaluation of antidiabetic efficacy of inavogliflozin metabolites in a type 1 diabetes rat model To evaluate the antidiabetic efficacy of inavogliflozin metabolites in an STZ-induced type 1 diabetes rat model, the test groups were set up as shown in Table 8 and experiments were conducted.

[0136] [Table 8]

[0137] Five-week-old male SD rats were administered STZ on day 0 (G2–G7, excluding G1) and non-fasting blood glucose was measured on day 3 to confirm the induction of type 1 diabetes. On day 7, each group was administered the experimental substance. Blood and urine samples were collected at 6, 24, 48, and 72 hours, and non-fasting blood glucose was measured. On day 14, the experimental animals were fasted for 16 hours. On day 15, after administration of the experimental substance, a 2 g / kg glucose solution was orally administered. Blood glucose levels were then measured at 15, 30, 60, 90, and 120 minutes after glucose administration. After blood collection, serum was separated and used to evaluate the inflammatory response of IL-6 using an interleukin-6 (IL-6) ELISA kit.

[0138] (1) Non-fasting blood glucose Non-fasting blood glucose levels were measured 6, 24, 48, and 72 hours after the first administration of the test drug in an STZ-induced type 1 diabetic rat model, and the results are shown in FIG.

[0139] As shown in Figure 11, blood glucose levels were maintained low in G5 (inavogliflozin M1 metabolite) and G6 (inavogliflozin) at the same dose of 1 mg / kg, and this phenomenon confirmed that a statistically significant blood glucose lowering effect was maintained compared to the vehicle group up to 48 hours after administration. In particular, G5 showed the lowest blood glucose level 48 hours after administration, demonstrating a sustained blood glucose lowering effect.

[0140] Compared with G7, which is a 1 mg / kg sotagliflozin group approved for type 1 diabetes, G5 and G6, which had the same dose, showed superior blood glucose lowering effects.

[0141] (2) Oral glucose load The blood glucose levels measured before glucose administration (0 min) and 15, 30, 60 and 120 min after glucose administration and the AUC were calculated and shown in FIGS.

[0142] As shown in Figure 12, blood glucose reduction was observed in G5, which was administered 1 mg / kg of inavogliflozin M1 metabolite, G6, which was administered 1 mg / kg of inavogliflozin, and G7, which was administered 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, which was administered 1 mg / kg of inavogliflozin M1 metabolite, showed the fastest and strongest blood glucose reduction effect, which was far superior to the G7 group, which was administered 1 mg / kg of sotagliflozin.

[0143] As shown in Figure 13, the G5 group, which was administered 1 mg / kg of the inavogliflozin M1 metabolite, also showed the most excellent effect in improving oral glucose tolerance in terms of AUC value, and G5 showed an AUC value at the same level as G1, the control group administered with the vehicle, confirming the excellent blood glucose regulating effect of the inavogliflozin M1 metabolite in the oral glucose tolerance experiment.

[0144] (3) Inflammatory response evaluation The test drug was administered to an STZ-induced type 1 diabetes rat model, and cytokines were measured. The results are shown in FIG.

[0145] As shown in Figure 14, interleukin-6 (IL-6), a cytokine that regulates inflammatory responses, increased at all time points (6, 24, 48, and 72 hours) in the vehicle group and was confirmed to decrease with M1 administration. Compared to the slight decreases with inavogliflozin and sotagliflozin, M1 significantly decreased at all doses (0.01, 0.1, and 1 mg / kg), with a tendency for a more significant decrease especially in the 0.01 mg / kg group at 6 hours and in the 0.1 mg / kg and 1 mg / kg groups at 72 hours.

[0146] These results imply that M1 can regulate the inflammatory response associated with type 1 diabetes.

Claims

1. A pharmaceutical composition for preventing or treating diabetes or heart failure, comprising, as an active ingredient, an inavogliflozin M1 metabolite represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: 【Chemical 1】

2. The pharmaceutical composition according to claim 1, characterized in that the diabetes is type 1 diabetes.

3. The pharmaceutical composition according to claim 1, characterized in that the diabetes is type 2 diabetes.

4. The pharmaceutical composition according to claim 1, characterized in that the pharmaceutical composition is for oral or parenteral administration.

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

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

7. An SGLT1 / SGLT2 dual inhibitor comprising, as an active ingredient, an inavogliflozin M1 metabolite represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: 【Chemistry 2】

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