Pharmaceutical compositions containing metabolites of inabogliflozin and their uses

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] This application relates to a pharmaceutical composition containing an inabogliflozin metabolite and its uses. [Background technology]

[0002] SGLT2 (sodium glucose cotransporter 2), along with SGLT1 (sodium glucose cotransporter 1), is a transporter responsible for glucose reabsorption in the kidneys, with SGLT2 playing the majority of the role. Therefore, when an SGLT2 inhibitor inhibits the SGLT2 transporter, the amount of glucose excreted in the urine increases, ultimately lowering blood glucose levels. Furthermore, the calories contained in blood glucose are excreted, resulting in weight loss.

[0003] One of the drugs developed as an SGLT2 inhibitor that can be useful as a treatment for type 2 diabetes due to these effects is inavogliflozin, represented by the following structural formula (chemical formula A), which 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] Inabogliflozin is a drug that treats type 2 diabetes by selectively inhibiting sodium-glucose cotransporter 2 (SGLT2), and exhibits equivalent or superior efficacy with only 0.3 mg, which is less than 1 / 30th the dose of existing SGLT2 inhibitors. Through a Phase 3 clinical trial conducted in patients with type 2 diabetes, it demonstrated superior efficacy in lowering glycated hemoglobin (HbA1c) and fasting blood glucose levels, as well as safety, compared to existing commercially available drugs, and received marketing approval.

[0006] According to Non-Patent Literature 1, inabogliflozin has been shown to produce five metabolites, M1, M2, M3, U1, and U2, in human hepatocytes. The formation of M1 and M2 from inabogliflozin was catalyzed by CYP3A4 and CYP2C19. M3 was produced by the 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 on drug metabolism in the liver by inabogliflozin and the elucidation of inabogliflozin metabolites and metabolic enzymes, but it does not clarify at all what kind of activity inabogliflozin metabolites exhibit. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Korean Published Patent 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. [Overview of the project] [Problem to be Solved by the Invention]

[0010] An object of the present invention is to provide a pharmaceutical use of an inabogliptin metabolite. [Means for Solving the Problem]

[0011] The present inventors found during the research on metabolites of inabogliptin that the inabogliptin M1 metabolite represented by chemical formula 1 acts as an SGLT1 / SGLT2 dual inhibitor and exhibits a pharmacological mechanism different from that of inabogliptin, which is a SGLT2-selective inhibitor, and thus completed the present invention.

[0012] [Chemical Formula]

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

[0014] Most of the conventionally developed SGLT2 inhibitors are drugs with high selective binding affinity for SGLT2, and inabogliptin developed by the present applicant is also a drug with extremely high selectivity for SGLT2 compared to SGLT1, and has recently been approved as a therapeutic agent for type 2 diabetes.

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

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

[0017] Hereinafter, the present invention will be described more specifically.

[0018] The present invention provides the use of inabogliflozin M1 metabolite or a pharmaceutically acceptable salt thereof as an SGLT1 / SGLT2 dual inhibitor, a pharmaceutical composition for the prevention or treatment of diabetes or heart failure comprising inabogliflozin M1 metabolite or a pharmaceutically acceptable salt thereof as an active ingredient, and a method for the prevention or treatment of diabetes or heart failure comprising administering an effective amount of inabogliflozin M1 metabolite or a pharmaceutically acceptable salt thereof to a subject in need.

[0019] The present invention provides a pharmaceutical composition for the prevention or treatment of diabetes or heart failure, comprising the inabogliflozin 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 the inabogliflozin M1 metabolite of chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0021] As can be seen in the examples below, the inabogliflozin M1 metabolite possesses binding affinity to SGLT2 while simultaneously exhibiting strong binding affinity to SGLT1. Therefore, it is a candidate drug that can act on both indications treatable as an SGLT2 inhibitor and indications treatable as an SGLT1 / SGLT2 dual inhibitor.

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

[0023] Typical indications for which SGLT2 inhibitors can be used for treatment include type 2 diabetes. In Example 3 below, the inabogliflozin M1 metabolite demonstrated excellent antidiabetic efficacy in a normal rat model through oral glucose tolerance tests (OGTT) and urinary glucose excretion (UGE).

[0024] Typical indications for which SGLT1 / SGLT2 dual inhibitors can be treated include type 1 diabetes and heart failure.

[0025] Inabogliflozin M1 metabolites can simultaneously block SGLT2 present in the kidney and SGLT1 present in the small intestine, making them effective in treating type 1 diabetes with impaired insulin secretion. Example 4 below demonstrates the antidiabetic efficacy of inabogliflozin M1 metabolites in a rat model of type 1 diabetes. Example 4 shows that inabogliflozin M1 metabolites may be the most effective drug for type 1 diabetes compared to inabogliflozin and sotagliflozin.

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

[0027] A pharmaceutical composition containing inabogliflozin M1 metabolite or a pharmaceutically acceptable salt thereof as an active ingredient may be used as a monotherapy or in combination therapy.

[0028] Inabogliflozin M1 metabolites are sufficient on their own to prevent or treat diabetes and / or heart failure. However, for various purposes, diabetes medications or heart failure medications are administered in combination, and these combinations can exhibit complementary or synergistic effects. Therefore, pharmaceutical compositions containing inabogliflozin M1 metabolites as an active ingredient can be administered in combination with other diabetes medications or heart failure medications.

[0029] For example, it may be administered in combination with known diabetes treatments such as baguanide drugs like metformin, DPP4 inhibitors, sulfonylurea insulin secretagogues, and / or insulin.

[0030] Another indication for treatment with an SGLT1 / SGLT2 dual inhibitor may be heart failure. The preventive or therapeutic effect of sotagliflozin as an SGLT1 / SGLT2 dual inhibitor on heart failure has been demonstrated by its approval by the U.S. FDA.

[0031] As used herein, the term “prevention” means any action that suppresses or delays the onset of diabetes or heart failure by administering a pharmaceutical composition according to the present invention.

[0032] Furthermore, as used herein, the term “treatment” means any action by which diabetes or heart failure is improved or beneficially altered by the administration of the pharmaceutical composition according to the present invention.

[0033] There are no particular restrictions on the dose of inalogliflozin M1 metabolites that can be used for the prevention or treatment of diabetes and / or heart failure in patients who have diabetes and / or heart failure, or who are at risk thereof, and may be appropriately adjusted depending on the severity of the patient's disease, weight, age, sex, and presence or absence of other complications.

[0034] Inabogliflozin M1 metabolites or pharmaceutically acceptable salts thereof may be administered orally or parenterally. The duration of administration may be adjusted as appropriate by the clinician based on the preventive or therapeutic effect on diabetes and / or heart failure in patients with or at risk of having 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 active ingredient inabogliflozin M1 metabolite of chemical formula 1 or a pharmaceutically acceptable salt thereof, and 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. Acceptable pharmaceutically acceptable carriers in compositions formulated as liquid solutions include those suitable for sterilization and biological use, such as physiological saline, sterile water, Ringer's solution, buffered physiological saline, albumin for injection, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components. Other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Diluents, dispersants, surfactants, binders, and lubricants may also be added to further formulations into injectable dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets.

[0037] In the present invention, the pharmaceutical composition may have a dosage form for oral administration, such as a tablet or a capsule. 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 excipients, disintegrants, and binders as additives.

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

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

[0042] Other examples of additives include lubricants and colorants.

[0043] Examples of the lubricants include stearic acid, stearate (e.g., magnesium stearate), hard anhydrous silicic acid, talc, corn starch, canauba wax, magnesium silicate, synthetic aluminum silicate, hydrogenated oil, tetrachloride, titanium dioxide, 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. However, it is not limited to this, but for example, a parenteral dosage form containing the composition of the present invention as an active ingredient can be formulated in an injectable form such as subcutaneous injection, intravenous injection, or intramuscular injection.

[0045] To formulate the composition of the present invention into an injectable dosage form, the composition can be mixed with a stabilizer or buffer in water to prepare a solution or suspension, which can then be formulated into ampoules or vials for unit administration.

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

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

[0048] Based on the results of the clinical trials, the appropriate once-daily dose of inabogliflozin is 0.1 mg to 0.5 mg. Therefore, it is expected that the inabogliflozin M1 metabolite can also be administered at a similar dose of 0.1 mg to 0.5 mg.

[0049] When the pharmaceutical composition is formulated into a unit dose 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 once to three times a day, for example, once a day, but is not limited thereto. [Effects of the Invention]

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

[0052] [Figure 1] This graph shows the inhibitory effects of inabogliflozin, M1, M2, dapagliflozin, and empagliflozin on AMG transport into CHO-SGLT1 cells, thereby inhibiting SGLT1 activity. [Figure 2] This graph shows the inhibitory effects of inabogliflozin, M1, M2, dapagliflozin, and empagliflozin on AMG transport into CHO-SGLT2 cells, thereby inhibiting SGLT2 activity. [Figure 3] This graph shows the changes in the inhibitory effect of inabogliflozin, M1, M2, dapagliflozin, and empagliflozin on SGLT1 activity depending on the drug pretreatment time. [Figure 4] This graph shows the changes in the inhibitory effects of inabogliflozin, M1, M2, dapagliflozin, and empagliflozin on SGLT2 activity depending on the drug pretreatment time. [Figure 5] This paper compares the activity recovery capabilities of inabogliflozin, M1, M2, dapagliflozin, and empagliflozin for SGLT1. [Figure 6] This paper compares the activity recovery capabilities of inabogliflozin, M1, M2, dapagliflozin, and empagliflozin for SGLT2. [Figure 7] The results of comparing the binding affinity and dissociation of inabogliflozin, M1, M2, and dapagliflozin to SGLT1 are shown. [Figure 8] The results of comparing the binding affinity and dissociation of inabogliflozin, M1, M2, and dapagliflozin to SGLT2 are shown. [Figure 9] This graph shows the changes in blood glucose over time after administration of inabogliflozin, M1, or M2 in normal rats. [Figure 10] This graph shows the area under the blood glucose concentration-time curve after administration of inabogliflozin, M1, or M2 in normal rats. [Figure 11] This graph shows non-fasting blood glucose levels observed up to 72 hours after administration of the test substance in STZ-induced type 1 diabetic rats. [Figure 12] This graph shows the blood glucose levels obtained from an oral glucose tolerance test in STZ-induced type 1 diabetic rats. [Figure 13] This graph shows the area under the blood glucose concentration-time curve in STZ-induced type 1 diabetic rats after an oral glucose tolerance test. [Figure 14] This graph shows the changes in inflammatory response-related cytokine levels over time after administration of the test substance in STZ-induced type 1 diabetic rats. [Modes for carrying out the invention]

[0053] The advantages and features of the present invention, as well as methods for achieving them, will become apparent by referring in detail to the experimental and manufacturing examples described below. However, the present invention is not limited to the experimental and manufacturing examples disclosed below, and can be realized in a variety of different forms, provided merely to complete the disclosure of the present invention and to fully inform those who are ordinary skill in the art to which the present invention pertains.

[0054] [Examples] [Example 1] Evaluation of SGLT1 and SGLT2 inhibitory activity of inabogliflozin metabolites According to the applicant's prior research, inabogliflozin (also referred to as DWP16001 in the examples below) showed higher inhibitory activity against SGLT2 compared to SGLT1 compared to its competitors, dapagliflozin and ipragliflozin, and that its inhibitory activity remained unchanged even after pretreatment for up to 2 hours. Through Dixon plots and Replot results for inhibitory activity evaluation based on substrate concentration, the inhibition of SGLT2 showed a reversible competitive inhibitory mechanism. However, after 24 hours of culture, quantification of intracellular drug concentration revealed that the amount of inabogliflozin remaining in cells was significantly higher compared to dapagliflozin and ipragliflozin. Furthermore, in the case of inabogliflozin, a time-dependent delay phenomenon was observed in the recovery ability of intracellular SGLT2 activity, but this was not observed in the competitors, dapagliflozin and ipragliflozin. These results indicate that inabogliflozin has better sustained efficacy compared to dapagliflozin and ipragliflozin.

[0055] The inventors of this invention sought to evaluate whether the same results would be obtained for the M1 and M2 metabolites of inabogliflozin (referred to as M1 and M2 respectively in this specification) through this study. They also sought to evaluate and compare dapagliflozin and empagliflozin, which have recently been the subject of active clinical research.

[0056] Example 1-1. Evaluation of the inhibitory effect of inabogliflozin metabolites on SGLT subtypes. (1) Evaluation of SGLT1 inhibitory activity Cell lines overexpressing SGLT1 (CHO-SGLT1 stable cells) and mock cells (CHO-mock cells) were cultured, and when 80% confluence was reached, 1 × 10⁶ cells were transferred to a 96-well plate. 5 Seeded using cells / well.

[0057] After 24 hours, the culture medium was removed, and 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, and the culture was incubated for 1 hour.

[0058] After 1 hour, remove the pretreatment buffer and add the SGLT1 substrate material, 10 μM [ 14 Incubation buffer containing C]AMG and inhibitory evaluation substances at different concentrations [10mM HEPES, 5mM Tris, 140mM NaCl, 2mM KCl, 1mM CaCl2, 1mM MgCl2, pH 7.4] was added, and the mixture was reacted for 2 hours in a 37°C thermomicromixer.

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

[0060] For reference, the M2 metabolite has the following compound name and structure (Chemical Formula 2).

[0061] [ka]

[0062] After a predetermined time, the samples 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 CaCl2, 1 mM MgCl2, 100 mM AMG, pH 7.4].

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

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

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

[0066] (2) Evaluation of SGLT2 inhibitory activity 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 substance was used as described below.

[0067] The concentrations of the inhibitory substances evaluated are as follows: Inabogliflozin: 0.01, 0.05, 0.2, 2, 10, 50, 100, 500 nM 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, 500 nM Empagliflozin: 0.01, 0.05, 0.2, 2, 10, 50, 100, 500 nM

[0068] (3) Evaluation of the inhibitory effects of inabogliflozin and M1 and M2 metabolites in SGLT1 and SGLT2 overexpressing cell lines The inhibitory activity of inabogliflozin, M1, M2, dapagliflozin, and empagliflozin against SGLT1 or SGLT2 was evaluated and is shown in Table 1 and Figures 1-2 below.

[0069] [Table 1]

[0070] The low-resolution values ​​calculated from the evaluation results were similar to those of previous studies. Compared to dapagliflozin and empagliflozin, inabogliflozin showed stronger inhibition of SGLT2, and its selectivity, as determined by the ratio of low-resolution values ​​for SGLT1 to SGLT2, was evaluated as superior. Furthermore, both inabogliflozin metabolites M1 and M2 showed inhibitory activity against SGLT2.

[0071] Unlike inabogliflozin, M2, dapagliflozin, and empagliflozin, which specifically and selectively inhibit SGLT2, the metabolite M1 also exhibits excellent SGLT1 activity inhibition (IC 50 (Value at 28.4nM level), SGLT1 / SGLT2 IC 50 The ratio was extremely low, confirming that it can act as a dual inhibitor against both SGLT1 and SGLT2.

[0072] Examples 1-2. Evaluation of the inhibitory mechanisms of inabogliflozin and its M1 and M2 metabolites on SGLT1 and SGLT2. (1) Comparative evaluation of changes in inhibitory activity in SGLT1 overexpressing cell lines based on drug exposure time. CHO-SGLT1 cells were pretreated with inabogliflozin, M1, M2, dapagliflozin, and empagliflozin at different concentrations in a sodium-negative pretreatment buffer for 1 or 2 hours. The concentration-dependent inhibitory effect of inabogliflozin on AMG transport into CHO-SGLT1 cells was then evaluated and compared to 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 evaluation results indicate that inabogliflozin, M2, dapagliflozin, and empagliflozin did not show increased or significantly changed inhibitory activity even with increasing exposure time (p>0.05). On the other hand, M1 showed a significant increase in low resolution with increasing exposure time (p<0.05).

[0074] [Table 2]

[0075] (2) Evaluation of inhibitory effect by substrate AMG concentration in SGLT1 overexpressing cell lines To determine the inhibitory mechanism and low resolution (Ki) of SGLT1, the concentration-dependent inhibitory effects of inabogliflozin, M1, M2, dapagliflozin, and empagliflozin were evaluated in the CHO-SGLT1 cell line by varying the substrate AMG concentration.

[0076] The absorption rate of AMG and the concentrations of inabogliflozin, M1, M2, dapagliflozin, and empagliflozin were shown in a Dixon plot, and the AMG concentration and Dixon slopes were shown graphically. The results were also shown in an LB plot, and the inhibitor concentrations and LB slopes were shown graphically (not shown). From this, the inhibition mechanism was confirmed, and the low resolution was calculated. The low resolution and inhibition 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 lowest among the test substances. A smaller Ki value indicates greater affinity, and this result suggests that M1 has a greater affinity for SGLT1 compared to the test substances, resulting in stronger inhibition of SGLT1.

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

[0080] As shown in Table 4 and Figure 4, the evaluation results indicate that for inabogliflozin, M1, M2, dapagliflozin, and empagliflozin, the inhibitory effect either increased or remained significantly unchanged even with increasing exposure time (p>0.05).

[0081] [Table 4]

[0082] (4) Evaluation of inhibitory effect of substrate AMG concentration in SGLT2 overexpressing cell lines To determine the inhibitory mechanism and low resolution (Ki) of SGLT2, the concentration-dependent inhibitory effects of inabogliflozin, M1, M2, dapagliflozin, and empagliflozin were evaluated in the CHO-SGLT2 cell line by varying the substrate AMG concentration.

[0083] The absorption rate of AMG and the concentrations of inabogli flozin, M1, M2, dapagliflozin and empagliflozin are represented by Dixon plot, and the AMG concentration and Dixon slopes are shown in a graph. In addition, the results are represented by an L-B plot, and the inhibitor concentration and L-B slopes are shown in a graph (not shown). Based on this, the inhibition mechanism was confirmed, and the low resolution was calculated. The low resolution calculated from the slope of the L-B plot and the inhibition mechanism are shown in Table 5.

[0084] [Table 5]

[0085] Examples 1-3. Comparative evaluation of the activity recovery ability of inabogliflozin and its metabolites after 24-hour pretreatment for SGLT1 and SGLT2. (1) Comparative evaluation of the activity recovery ability of inabogliflozin and its metabolites after washout in SGLT1 overexpressing cell lines A cell line overexpressing SGLT1 (CHO-SGLT1 stable cells) was cultured, and when it reached 80% confluence, 1×10 5 cells were seeded at cells / well. After 24 hours, the medium was removed, replaced with medium containing test substances at different concentrations, and cultured for another 24 hours.

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

[0087] Cell lines overexpressing SGLT1, treated with the drug for 24 hours, were given 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] that did not contain the evaluation substance. 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, remove the pretreatment buffer and add the SGLT1 substrate material, 10 μM [ 14 Incubation buffer containing C]AMG [10mM HEPES, 5mM Tris, 140mM NaCl, 2mM KCl, 1mM CaCl2, 1mM MgCl2, pH 7.4] was added and the mixture was reacted in a 37°C Thermo Micro Mixer for 2 hours. After 2 hours, the mixture was washed twice with 200 μL of ice-cold washing buffer [10mM HEPES, 5mM Tris, 140mM choline chloride, 2mM KCl, 1mM CaCl2, 1mM MgCl2, 100mM AMG, pH 7.4].

[0089] After removing the culture medium, 40 μL of 10% SDS solution was added to lyse the cells, then the sample was transferred to a liquid scintillation counter measurement plate, 150 μL of OptiPhase Supermix® (registered trademark) isotope measurement cocktail solution was added, and the sample was incubated overnight to ensure that the cell solution was homogeneously mixed with the cocktail solution. 14 After measuring C]AMG with a liquid scintillation counter, substrate drugs in cell lines overexpressing SGLT1 [ 14 The intracellular absorption rate (% of control) of [C]AMG is shown in Figure 5. All data values ​​are expressed as the mean ± SD of four independent experiments.

[0090] As can be seen in Figure 5, the SGLT1 activity that was reduced after 24-hour treatment of the CHO-SGLT1 cell line with inabogliflozin, M1, M2, dapagliflozin, or empagliflozin differed in the ability to restore SGLT1 activity depending on the time the drug was removed from the culture medium.

[0091] In other words, when treated with inabogliflozin, activity was only partially restored up to 2 hours after washout, but fully restored after 4 hours (Figure 5A).

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

[0093] When treated with M2, it showed a trend very similar to that of inabogliflozin. Specifically, activity was incompletely restored up to 2 hours after washout, but fully restored after 4 hours (Figure 5C).

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

[0095] The results above confirm that M1 dissociates from SGLT1 the slowest, while inalogliflozin and M2 dissociate quickly, and dapagliflozin and empagliflozin dissociate quickly. These results indicate that M1 binds to SGLT1 for the longest period among the evaluated substances.

[0096] (2) Comparative evaluation of the activity recovery ability of inabogliflozin and its metabolites after washout in SGLT2 overexpressing cell lines The evaluation method is the same as for SGLT1 overexpressing cells, except that a cell line overexpressing SGLT2 (CHO-SGLT2 stable cells) was used and the concentration of the evaluation substance was set as follows. Inabogliflozin: 0.5, 5, 50, 500 nM M1: 0.5, 5, 50, 500 nM M2: 0.5, 5, 50, 500nM Dapagliflozin: 0.5, 5, 50, 500 nM Empagliflozin: 0.5, 5, 50, 500 nM

[0097] As can be seen in Figure 6, the SGLT2 activity that was reduced after 24-hour treatment of the CHO-SGLT2 cell line with inabogliflozin, M1, M2, dapagliflozin, or empagliflozin differed in the ability to restore SGLT2 activity depending on the time the drug was removed from the culture medium.

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

[0099] When treated with M1, activity recovered after 2 hours at low concentrations (0.5 nM). However, after washing out following 5 and 50 nM treatments, recovery progressed over time up to 8 hours, and almost complete recovery was observed after 24 hours. A delay in activity recovery was observed with 500 nM treatment (Figure 6B).

[0100] When treated with M2, the difference with concentration was not significant, and it was confirmed that the activity recovered over time. In other words, the activity recovered over time up to 4 hours after washout, and after 8 hours, it recovered (Figure 6C).

[0101] When treated with dapagliflozin, the results showed a similar trend to M2. That is, the difference with concentration was not large, and it was confirmed that the activity recovered over time. The results showed that the activity recovered over time up to 4 hours after washout, and 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 at the same concentration than with inabogliflozin, M1, M2, and dapagliflozin (Figure 6E).

[0103] The results above show that inabogliflozin had the slowest dissociation of SGLT2, and at high concentrations, activity recovery was incomplete even after 24 hours, showing less than 50% recovery. In the case of M1, it was confirmed that activity recovery was delayed depending on the concentration and washout time. In M2, dapagliflozin did not show a large difference in activity recovery with concentration, but showed a phenomenon of delay with washout time. Empagliflozin showed no delay phenomenon depending on the concentration or time of pretreatment, and was confirmed to have the fastest activity recovery.

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

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

[0106] Glucose reabsorption occurs in the kidneys via SGLT2. +Induced by an electrochemical potential gradient, glucose diffuses across the cell membrane into the bloodstream via glucose transporter 2 (GLUT-2), which facilitates glucose transport. One Na+ molecule per glucose molecule is transported via GLUT-2. + While one molecule is absorbed, SGLT1 absorbs two Na molecules per glucose molecule. + Ions are absorbed. Therefore, the composition of the buffer is changed, and Na + Only the glucose current, excluding the electrical current, was recorded.

[0107] The experiment was conducted using the following method.

[0108] 1) Using a Narishige puller, glass micropipettes were prepared in two stages at 62.7°C and 57°C. After filling them with the internal solution (145 mM CsCl, 5 mM NaCl, 10 mM HEPES, 11 mM EGTA, pH 7.2), they were fitted onto the Ag / AgCl2 electrode of a perch-clamp head stage.

[0109] 2) SGLT1 or SGLT2 cells were spread into 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 flowed through to ensure the cells were fully immersed. A temperature control device was used to maintain the chamber temperature at 36.5 ± 0.5°C.

[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 detach the cell membrane (Whole-cell mode, 3~5MΩ).

[0111] 4) Fix the cells at -60mV (holding potential) and apply current [I] in gap-free data acquisition mode. totalThe current (Na+ / Glucose current (pA)) was measured.

[0112] 5) Continuously flow the external solution into the chamber containing the cells and apply an electric current (I total After recording for approximately 30 seconds to 1 minute (stabilization), add glucose to the external solution (Na + (Glucose external) (150mM NaCl, 10mM HEPES, 1mM CaCl2, 1mM MgCl2, 100mM Glucose, pH 7.4) is applied, and a glucose current (I glucose The following was recorded: Then, cells were exposed for 2 minutes to an external solution containing glucose at one concentration of the compound, and the glucose current suppressed by the compound was recorded.

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

[0114] 7) After confirming the restoration of current, use a sodium-free external solution (Na + - Replace with (free external) (150mM Choline-Cl, 1mM CaCl2, 1mM MgCl2, pH 7.4) and Na + The current was recorded. After stabilizing the current, it was replaced with an external solution, and the current in a resting state where the cells were not stimulated was recorded to ensure the reliability of the experimental system.

[0115] For statistical analysis of the data, we used either pCLAMP10.4 (Molecular Devices, LLC, San Jose, CA, USA) or Origin6.0 (OriginLab, Northampton, MA, USA).

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

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

[0118] Half-time of recovery (T) 1 / 2,off The dissociation time for M1 was 34.9 seconds, confirming that M2 (23.5 seconds) and dapagliflozin (30.5 seconds) dissociated at similar rates. On the other hand, the dissociation time for M1 was 34.9 seconds. 1 / 2,off The dissociation time 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 is more effective than inalogliflozin or its competitor dapagliflozin in binding to and maintaining the SGLT1 protein, thus preserving its efficacy.

[0120] [Table 6]

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

[0122] Half-time of recovery (T) 1 / 2,offM1 (369.9 seconds) and M2 (540.4 seconds) had a dissociation rate of 745.5 seconds, the slowest among the substances evaluated. This was approximately 2.8 times slower than the competing substance dapagliflozin (265.2 seconds), while M1 (369.9 seconds) and M2 (540.4 seconds) showed dissociation rates that were twice and 1.38 times slower, respectively, compared to inabogliflozin.

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

[0124] In conclusion, while M1 exhibited a dual inhibitory effect on SGLT-1 / SGLT-2, the recovery of inhibitory activity after washout from the SGLT-1 target showed a slower dissociation time than M2. Furthermore, M2 was shown to maintain 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 inabogliflozin metabolites Inabogliflozin, M1, or M2 were administered intravenously as a single dose to normal rats, and their antidiabetic efficacy was evaluated.

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

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

[0129] As shown in Figures 9 and 10, inabogliflozin, M1, and M2 were confirmed to improve glucose tolerance when administered intravenously to normal rats at a dose of 1 mg / kg. Metabolite M1 showed superior glucose tolerance to inabogliflozin when administered intravenously, while M2 showed a similar pattern to inabogliflozin.

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

[0131] On the other hand, when urinary glucose excretion (UGE) was examined, UGE was observed for metabolites M1 and M2 from 6 hours after intravenous administration of 1 mg / kg. The amount and pattern of glucose excreted were similar for M1 and inabogliflozin, but in the case of M2, less glucose was excreted than inabogliflozin and M1 up to 24 hours.

[0132] In summary, these results suggest that intravenous administration of the test drug exhibits in vivo activity through the action of SGLT1 and SGLT2 in the kidneys.

[0133] IC of metabolite M1 against SGLT2 50 Although its efficacy is higher than that of inabogliflozin, it exhibits a more potent drug effect against SGLT1. Therefore, metabolite M1 generally functions as a dual inhibitor against both SGLT1 and SGLT2, and as a result, M1 is thought to exhibit similar antidiabetic efficacy to inabogliflozin.

[0134] M2 shows effects for up to 24 hours and exhibits weaker efficacy than M1, but this is presumed to be because 1) M2 has a higher IC50 for SGLT1 than M1, resulting in weaker efficacy, and 2) rapid elimination occurred. 1 / 2 (Confirmed to be =0.8h).

[0135] [Example 4] Evaluation of the antidiabetic efficacy of inabogliflozin metabolites in a rat model of type 1 diabetes. In a rat model of type 1 diabetes induced by STZ, experiments were conducted to evaluate the antidiabetic efficacy of inabogliflozin metabolites, with test groups set up as shown in Table 8.

[0136] [Table 8]

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

[0138] (1) Non-fasting blood glucose Figure 11 shows the results of measuring non-fasting blood glucose levels at 6, 24, 48, and 72 hours after the first dose of the test drug in a rat model of type 1 diabetes induced with STZ.

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

[0140] Compared to Group G7, which received 1 mg / kg of sotagliflozin approved for type 1 diabetes, Groups G5 and G6, at the same dose, showed superior blood glucose-lowering effects.

[0141] (2) Oral glucose tolerance Blood glucose levels and AUC were calculated before glucose administration (0 minutes) and at 15, 30, 60, and 120 minutes after glucose administration, and are shown in Figures 12 and 13.

[0142] As shown in Figure 12, blood glucose levels decreased in all three groups: G5, G6, and G7, all administered 1 mg / kg of inabogliflozin M1 metabolite; G5, all administered 1 mg / kg of inabogliflozin; and G7, all administered 1 mg / kg of sotagliflozin. In the case of the M1 metabolite, a dose-dependent improvement in glucose tolerance was confirmed. In particular, the G5 group, administered 1 mg / kg of inabogliflozin M1 metabolite, showed the fastest and strongest blood glucose-lowering effect, and this effect was far superior to that of the G7 group, which was administered 1 mg / kg of sotagliflozin.

[0143] As shown in Figure 13, the G5 group, administered 1 mg / kg of inabogliflozin M1 metabolite, showed the best improvement in oral glucose tolerance in terms of AUC value. G5 showed an AUC value at a similar level to G1, the control group administered Vehicle, confirming the excellent blood glucose regulating effect of inabogliflozin M1 metabolite in oral glucose tolerance experiments.

[0144] (3) Assessment of inflammatory response Figure 14 shows the results of measuring cytokine levels in a rat model of type 1 diabetes induced with STZ after administration of the test drug.

[0145] As shown in Figure 14, interleukin-6 (IL-6), a cytokine that modulates inflammatory responses, increased in the vehicle group at all time points (6, 24, 48, and 72 hours) and decreased with M1 administration. Compared to the slight decreases observed with inabogliflozin and sotagliflozin, M1 significantly decreased in all dose groups (0.01, 0.1, and 1 mg / kg), with particularly significant decreases observed at 6 hours in the 0.01 mg / kg group and at 72 hours in the 0.1 mg / kg and 1 mg / kg groups.

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

Claims

1. A pharmaceutical composition for the prevention or treatment of diabetes or heart failure as an SGLT1 / SGLT2 dual inhibitor, comprising the inabogliflozin M1 metabolite of the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. 【Chemistry 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 it is for oral or parenteral administration.

5. The pharmaceutical composition according to claim 1, characterized in that the once-daily dose of inabogliflozin 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, characterized in that it is administered once a day.

7. An SGLT1 / SGLT2 dual inhibitor containing the inabogliflozin M1 metabolite of the chemical formula shown below (Chemical Formula 1) or a pharmaceutically acceptable salt thereof as an active ingredient. 【Chemistry 2】

Images