Anti-obesity combination therapy of enavogliflozin and low-dose liraglutide

Abstract

The present invention provides an obesity treatment therapy with reduced side effects through co-administration of enavogliflozin and low-dose liraglutide. The combination therapy of enavogliflozin and low dose liraglutide provides a synergistic effect in the treatment of obesity compared to the administration of each drug alone. In addition, the administration dose of liraglutide can be reduced compared to the administration of liraglutide alone, thereby reducing the liraglutide-associated direct cause of side effects related to the gastrointestinal and cardiovascular systems. In addition, the present invention has an advantage of additionally providing an effect of alleviating metabolic diseases and cardiovascular diseases related to blood glucose, blood lipids, blood pressure, fatty liver, and the like through the combination with enavogliflozin.

Description

Anti-obesity combination therapy with inavogliflozin and low-dose liraglutide

[0001] This is about the anti-obesity combination therapy of inabogliflozin and low-dose liraglutide.

[0002] GLP-1 receptor agonists (GLP-1RAs) are drugs that activate the GLP-1 receptor, reducing blood sugar and energy intake. GLP-1RAs mimic the action of GLP-1, an endogenous incretin hormone secreted by the intestines after meals. GLP-1RAs suppress glucagon secretion and stimulate insulin production, reducing hyperglycemia in patients with type 2 diabetes and reducing food intake, making them effective in treating obesity through weight loss. GLP-1RAs suppress glucagon secretion in proportion to glucose concentration. This means that glucagon secretion is suppressed at high blood sugar levels and not suppressed at low blood sugar levels, which has the advantage of not causing serious problems in hypoglycemic situations. Liraglutide, a GLP-1RA drug better known under the brand name Saxenda, was initially developed as a diabetes treatment. However, it has been shown to be effective in treating obesity at high doses, and is now widely used in the treatment of obesity. However, the obesity prescription dose of liraglutide is 3 to 5 times higher than the diabetes prescription dose, and unlike when administering the diabetes prescription dose, when administering the obesity prescription dose, it is known to have many side effects, including gastrointestinal side effects such as nausea and diarrhea due to high dose prescription, and cardiovascular side effects such as increased heart rate.

[0003] Meanwhile, SGLT-2 inhibitors are drugs with mechanisms of action that promote weight loss and lower blood pressure through osmotic diuresis and caloric loss due to glucose excretion. Enavogliflozin, an SGLT-2 inhibitor developed by the applicant, recently received marketing approval as an oral diabetes treatment. According to the applicant's preliminary research, enavogliflozin has been confirmed to have an excellent anti-obesity effect based on its ability to inhibit glucose reabsorption.

[0004] Accordingly, the inventors of the present invention hypothesized that the combined administration of inavogliflozin and low-dose liraglutide would synergistically enhance anti-obesity effects while reducing side effects compared to existing treatments, and conducted research based on this hypothesis. As a result, the combined administration of inavogliflozin and low-dose liraglutide reduced the side effects of liraglutide while demonstrating a synergistic effect on weight loss compared to when the two drugs were administered alone, thereby completing the present invention.

[0005] The present invention aims to provide a novel anti-obesity treatment method that improves anti-obesity effects while reducing side effects resulting from high-dose administration of liraglutide.

[0006] The present invention provides an anti-obesity pharmaceutical composition comprising inabogliflozin as an active ingredient and characterized by combined use with low-dose liraglutide.

[0007] In another specific embodiment, the present invention provides an anti-obesity pharmaceutical composition comprising low-dose liraglutide as an active ingredient and characterized by combined use with inavogliflozin.

[0008] In the pharmaceutical composition of the present invention, inabogliflozin or liraglutide includes all cases where it exists in the form of a pharmaceutically acceptable salt, a pharmaceutically acceptable amide, or a pharmaceutically acceptable ester.

[0009] Inavogliflozin is currently approved as a diabetes treatment drug and is sold under the product name Enblo Tablet. The dosage of inavogliflozin as a diabetes treatment drug is approved as 0.3 mg per day, but the dosage of inavogliflozin as an obesity treatment drug has not been determined. The present invention relates to the use of inavogliflozin for the treatment of obesity in combination with low-dose liraglutide. Based on the examples below, the dosage of inavogliflozin for combination with low-dose liraglutide may be 0.3 to 1.5 mg / day.

[0010] Here, low-dose liraglutide refers to a lower dosage compared to the dosage of liraglutide currently used to treat obesity. Liraglutide is currently approved for two uses: diabetes and obesity. The dosage for diabetes treatment is 0.6 to 1.8 mg per day, whereas the dosage for obesity treatment is 3.0 mg per day, which means that the approved dosage for obesity treatment is much higher than the approved dosage for diabetes treatment. The low-dose liraglutide intended in the present invention means a dosage less than 3.0 mg per day, which is the current approved dosage for obesity treatment, and is not limited thereto, but the dosage of liraglutide may be 1.2 to 2.4 mg / day. That is, when inavogliflozin and low-dose liraglutide are co-administered, in one specific example, the dosage of inavogliflozin may be 0.3 to 1.5 mg / day, and the dosage of liraglutide may be 1.2 to 2.4 mg / day. In another specific example, the dosage of inavogliflozin may be 0.6 to 1.2 mg / day, and the dosage of liraglutide may be 1.5 to 2 mg / day.

[0011] The term "combination" as used herein refers to co-administration of inavogliflozin and liraglutide to a subject in need thereof. There are no temporal restrictions regarding co-administration, and it includes administration separately, simultaneously, or sequentially. Furthermore, "combination" includes both cases where inavogliflozin and liraglutide are administered individually as separate formulations, and cases where inavogliflozin and liraglutide are administered as a combination formulation containing inavogliflozin and liraglutide.

[0012] There are no specific restrictions on the dosage regimen for combining the two drugs. Although inavogliflozin was initially developed as an oral drug, its parenteral administration is not restricted. Liraglutide is currently administered parenterally for the treatment of obesity. Therefore, for example, inavogliflozin can be administered orally or parenterally, while liraglutide can be administered parenterally. Specific examples of parenteral administration suitable for the present invention include subcutaneous or intramuscular administration. Liraglutide for anti-obesity use is currently administered via subcutaneous injection once daily.

[0013] Although not limited thereto, in one specific embodiment of the present invention, inabogliflozin may be administered orally and liraglutide may be administered via subcutaneous injection.

[0014] In another embodiment, inavogliflozin and liraglutide may be administered via subcutaneous injection.

[0015] In the following examples, it was confirmed that when inavogliflozin was administered orally and liraglutide was administered subcutaneously, and when a depot composition was prepared as a combination preparation containing inavogliflozin and liraglutide and these drugs were administered together, an enhanced anti-obesity effect could be achieved even with a low dose of liraglutide.

[0016] That is, the difference in the administration route of each drug does not affect the achievement of the enhanced anti-obesity effect through the combined use of inavogliflozin and liraglutide.

[0017] Combination therapy with inavogliflozin and low-dose liraglutide offers a synergistic effect in the treatment of obesity compared to either drug alone. Furthermore, compared to liraglutide alone, the dose of liraglutide can be reduced, thereby reducing the direct cause of liraglutide's gastrointestinal and cardiovascular side effects. Furthermore, combination therapy with inavogliflozin offers the additional benefit of improving metabolic and cardiovascular conditions, including blood sugar, blood lipids, blood pressure, and fatty liver.

[0018] Figure 1 is a graph showing the weight loss effect of co-administration of inabogliflozin and liraglutide in a high-fat diet-induced obesity mouse model.

[0019] Figure 2 is a graph showing the change in body weight (%) according to the co-administration of inabogliflozin and liraglutide in a high-fat diet-induced obesity mouse model.

[0020] Figure 3 shows the results of evaluating the difference in weight loss efficacy by co-administration of inabogliflozin and liraglutide according to the degree of obesity in a high-fat diet-induced obese mouse model.

[0021] Figure 4 shows the results of body composition analysis according to the co-administration of inabogliflozin and liraglutide in a high-fat diet-induced obese mouse model.

[0022] Figure 5 shows the results of an oral glucose tolerance test performed after co-administration of inabogliflozin and liraglutide in a high-fat diet-induced obese mouse model.

[0023] Figure 6 is a graph showing the body weight loss rate in an obesity-induced rat model following administration of a depot composition according to the present invention.

[0024] Figure 7 is a photograph showing a decrease in the fat ratio within the tissue as a result of body composition analysis of an obesity-induced rat model following administration of a depot composition according to the present invention.

[0025] Figure 8 shows the PK analysis results of inabogliflozin according to administration of each test substance in test groups G1 to G9.

[0026] Figure 9 shows the PK analysis results of liraglutide according to administration of each test substance in test groups G1 to G9.

[0027] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms. These embodiments are provided solely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention. The present invention is defined solely by the scope of the claims.

[0028] For convenience, in the tables and drawings of the examples below, liraglutide is also expressed by the abbreviation LIRA, and inavogliflozin is also expressed by the abbreviation ENAVO.

[0029] [Experimental Example 1] Evaluation of the anti-obesity efficacy of combined administration of inavogliflozin and liraglutide in an obese animal model.

[0030] To determine whether the combined administration of inabogliflozin and liraglutide has a synergistic effect on anti-obesity efficacy, the following experiment was conducted.

[0031] Six-week-old male C57BL / 6J mice were acclimatized for one week and fed a high-fat diet for 15 weeks to prepare a diet-induced obese mouse model (C57BL / 6J 6w, male + HFD 15w, n=11~12). The diet-induced obese mouse model was divided into the following groups, and inavogliflozin (3 mg / kg or 10 mg / kg) was administered orally once daily for 4 weeks, and liraglutide (10 nmol / kg) was administered subcutaneously. Body weight was measured once every 4 days.

[0032] Group Group name Number of animals (n) Administered substance G1 Lean 6 Saline + vehicle G2 Vehicle 11 Saline + vehicle G3 Liraglutide 11 Liraglutide 10 nmol / kg + vehicle G4 Inavogliflozin 3 mg / kg 12 Saline + Inavogliflozin 3 mg / kg G5 Inavogliflozin 10 mg / kg 12 Saline + Inavogliflozin 10 mg / kg G6 Liraglutide + Inavogliflozin 3 mg / kg 12 Liraglutide 10 nmol / kg + Inavogliflozin 3 mg / kg G7 Liraglutide + Inavogliflozin 10 mg / kg 12 Liraglutide 10 nmol / kg + Inavogliflozin 10 mg / kg

[0033] After 4 weeks, non-fasting blood glucose, glycated hemoglobin, and oral glucose tolerance tests were performed, while body fat mass, organ weight, and blood biochemistry tests were performed. Figure 1 is a graph showing the weight loss effect of co-administration of inavogliflozin and liraglutide in a high-fat diet-induced obese mouse model.

[0034] As can be seen in Fig. 1, in the body weight measured on the 28th day of the experiment, compared to the obese mouse G2 that was administered the vehicle, G3, which was administered only 10 nmol / kg of liraglutide, showed a 5.33% weight loss, G4, which was administered 3 mg / kg of inavogliflozin, showed a 3.5% weight loss, G5, which was administered 10 mg / kg of inavogliflozin, showed an 8.16% weight loss, G6, which was administered 10 nmol / kg of liraglutide + 3 mg / kg of inavogliflozin, showed a 12.31% weight loss, and G7, which was administered 10 nmol / kg of liraglutide + 3 mg / kg of inavogliflozin, showed a 14.92% weight loss. It was confirmed that the combined administration of liraglutide and inavogliflozin showed a synergistic effect in weight loss compared to the single administration of each drug.

[0035] Figure 2 is a graph showing the change in body weight (%) according to the co-administration of inabogliflozin and liraglutide in a high-fat diet-induced obesity mouse model.

[0036] Looking at the body weight change (%) of mice G1 to G7 in Fig. 2, as in Fig. 1, it was confirmed that the body weight change of G6, which was administered 10 nmol / kg of liraglutide + 3 mg / kg of inavogliflozin, and G7, which was administered 10 nmol / kg of liraglutide + 3 mg / kg of inavogliflozin, was significantly higher than that of liraglutide alone.

[0037] Figure 3 shows the results of evaluating the difference in weight loss efficacy by combined administration of inavogliflozin and liraglutide according to the degree of obesity in a high-fat diet-induced obese mouse model. The mice were divided into a group weighing less than 40 g and a group weighing 40 g or more, and the weight loss efficacy compared to the initial body weight (Day 0) was evaluated. As shown in Figure 3, the anti-obesity effects of G6, which received 10 nmol / kg of liraglutide + 3 mg / kg of inavogliflozin, and G7, which received 10 nmol / kg of liraglutide + 3 mg / kg of inavogliflozin, were confirmed to be significant. In particular, the weight loss efficacy was prominent in the subgroup with a relatively low degree of obesity (BW<40 mg).

[0038] Figure 4 shows the results of body composition analysis according to the co-administration of inavogliflozin and liraglutide in a high-fat diet-induced obese mouse model. As a result of observing the body composition after 4 weeks of co-administration, the body fat mass of the vehicle-administered group significantly increased compared to the Lean group, and as can be seen in Figure 4, all administration groups except G4, which received 3 mg / kg of inavogliflozin, showed a tendency for fat to decrease, and it was confirmed that lean tissue was significantly reduced in the co-administration group (liraglutide 10 nmol / kg + inavogliflozin 3 mg / kg, liraglutide 10 nmol / kg + inavogliflozin 10 mg / kg).

[0039] As a result of measuring metabolic parameters, as can be seen in Table 2, the inavogliflozin monotherapy group showed increased water and feed intake in a dose-dependent manner compared to the vehicle group, while the liraglutide monotherapy group showed a tendency to decrease. When inavogliflozin and liraglutide were co-administered, the water and feed intake tended to decrease compared to the inavogliflozin monotherapy group. It is thought that when inavogliflozin is administered, food intake increases due to compensatory psychology for sugar loss, but when liraglutide is co-administered, appetite is suppressed, showing a synergistic effect on weight loss in the co-administration group.

[0040] [Table 2]

[0041]

[0042] In addition, the non-fasting blood sugar level of the vehicle group significantly increased compared to the Lean group, and the blood sugar levels in the inavogliflozin 3, 10 mg / kg monotherapy group, liraglutide 10 nmol / kg + inavogliflozin 3 mg / kg, and liraglutide 10 nmol / kg + inavogliflozin 10 mg / kg administration groups were significantly reduced compared to the vehicle group. The glycated hemoglobin levels of all administration groups except for inavogliflozin 10 mg / kg were not significantly different from the vehicle group.

[0043] Figure 5 shows the results of an oral glucose tolerance test performed after co-administration of inavogliflozin and liraglutide in a high-fat diet-induced obese mouse model. As a result of the oral glucose tolerance test performed in the third week of co-administration, as shown in Figure 5a, significantly lower blood glucose levels were observed in all inavogliflozin-administered groups compared to the vehicle-administered group 15, 30, 60, and 120 minutes after glucose administration. As shown in Figure 5b, a comparison of the AUC (area under the curve) of the blood glucose graph versus time after glucose administration showed that glucose tolerance was significantly and significantly improved in a dose-dependent manner in all inavogliflozin-administered groups. Glucose tolerance was significantly improved in the co-administration group (liraglutide 10 nmol / kg + inavogliflozin 3, 10 mg / kg) compared to the liraglutide monotherapy group.

[0044] In addition, as shown in Table 3, the blood biochemistry test results showed that the 10 mg / kg inavogliflozin group and the liraglutide monotherapy group significantly reduced cholesterol (CHO) compared to the vehicle group, but no significant reduction effect was observed in the combination therapy. The inavogliflozin and liraglutide groups showed a tendency for ALT to decrease, but the significance was not observed. The inavogliflozin group (monotherapy, combination therapy) showed a tendency to increase AST, but the difference was not significant. Triglyceride (TG) significantly increased in the inavogliflozin 10 mg / kg group and the inavogliflozin 10 mg / kg + liraglutide 10 nmol / kg group compared to the vehicle group.

[0045] [Table 3]

[0046]

[0047] As a result of long-term weight measurement, as shown in Table 4, the liver weight of all administration groups except the 10 mg / kg inabogliflozin administration group was significantly lower than that of the vehicle group, and no effect on kidney weight was observed according to administration group.

[0048] [Table 4]

[0049]

[0050]

[0051] In summary, inavogliflozin exhibited significant weight loss efficacy compared to the vehicle group despite co-administration with liraglutide at a lower dose (10 nmol / kg / day = 3 mg / day in humans), compared to the standard dose of liraglutide (100 nmol / kg / day). Therefore, it is expected that superior weight loss efficacy can be achieved while reducing the dose of liraglutide, thereby reducing the side effects of liraglutide.

[0052]

[0053] Accordingly, the inventors of the present invention have devised a method for producing a combination formulation that can reduce the dosage of liraglutide while simultaneously administering it with inavogliflozin and improve the convenience of taking the medication for patients, and have produced a depot composition containing inavogliflozin and liraglutide as follows and evaluated its anti-obesity efficacy.

[0054]

[0055] [Manufacturing Examples 1 and 2]

[0056] Depot compositions containing inabogliflozin and liraglutide were prepared according to the composition and manufacturing method of Table 5, and the depot compositions of Manufacturing Examples 1 and 2 were obtained.

[0057] Manufacturing Example Manufacturing Method Polymer Concentration / Type Additives and Features ENAVO Concentration (w / w%) LIRA Concentration (w / w%) Continuous Phase (CP) Conditions 1st Homogenization Condition (RPM) 2nd Homogenization Condition (RPM) CP Injection Speed / Injection Temperature (°C) Discontinuous Phase (DP) Injection Speed ​​Volatilization Conditions Temperature / Time / RPM Size D10 / D50 / D90 / D[4,3] 1 W / O / W 35% PLGARG 75 2 HNaAc 1% 16.67 27.17 0.25% PVA 3L+1% mannitol 4,000 RPM 3 min 4,000 RPM 3 min 1.5 L / min 20℃ 5ml / min 25℃ / 3 hours / 150 3.4 / 26.8 / 65.1 / 30.92 W / O / W 30% PLAR202HMg(OH)210%16.6727.170.50% PVA 5L+1% mannitol4,000 RPM3min4,000 RPM3min1.5 L / min20℃5ml / min30℃ / 3hrs / 1503.8 / 23.1 / 63.7 / 29.1

[0058] Liraglutide was sufficiently dissolved in distilled water for more than 30 minutes to prepare a first phase solution (W). Inavogliflozin was sufficiently dissolved in methanol for more than 30 minutes before use. The biodegradable polymer was dissolved in methylene chloride, an oil solvent. An oil phase solution (O) was prepared by mixing a solution of inavogliflozin dissolved in methanol and a solution of the biodegradable polymer dissolved in methylene chloride. The first phase solution (W) and the oil phase solution (O) were mixed and homogenized using a homogenizer to prepare a water-on-oil (W / O) emulsion. In addition, an aqueous solution containing polyvinyl alcohol (PVA) as a surfactant was prepared as a second phase solution. When the W / O emulsion was injected through a rubber pipe using a micro-injector, it was exposed to the surfactant aqueous solution and simultaneously mixed by the homo-mixer to form microspheres. The organic solvent present in the microparticles was removed by rotating the mixture of the water-soluble emulsion and the surfactant aqueous solution at a constant speed. To remove the organic solvent, the temperature of the preparation vessel was adjusted to 15-35℃ and maintained for 3 hours to remove the organic solvent. After the organic solvent was removed, the suspension was repeatedly washed with distilled water to remove the residual polyvinyl alcohol, and the recovered microparticles were freeze-dried.

[0059] [Experimental Example 2] Evaluation of the anti-obesity efficacy of Manufacturing Examples 1 and 2

[0060] To evaluate the anti-obesity efficacy of the depot compositions of Manufacturing Examples 1 and 2, obesity was induced in 5-week-old male SD rats and used as an animal model.

[0061] Obesity was induced in rats by feeding them a high-fat diet for 13 weeks. To minimize stress on the experimental animals due to subcutaneous administration of the test substance, all animals were administered saline subcutaneously for 2 weeks prior to administration of the test substance to provide prior adaptation training for subcutaneous administration.

[0062] The test groups were set up as follows: vehicle group (G1), inavogliflozin monotherapy group (G2), Saxenda™ (high-dose liraglutide) monotherapy group (G3), and groups administered a depot formulation containing inavogliflozin and low-dose liraglutide every 2 or 4 weeks (G4 to G6) (n=8 per group). All test groups were administered the test substance via subcutaneous (SC) injection of 2 mg / kg containing the test substance and vehicle for 4 weeks.

[0063] Group Group name Test substance and single dose Dosage G1 Vehicle Vehicle (PBS) Once a day G2 Inavogliflozin Inavogliflozin (0.30 mg / kg) Once a day G3 Saxenda™ Liraglutide (0.31 mg / kg) Once a day G4 Manufacturing example 1 (2W) ENAVO (5.32 mg / kg), LIRA (2.17 mg / kg) Once every 2 weeks G5 Manufacturing example 2 (2W) ENAVO (4.20 mg / kg), LIRA (2.17 mg / kg) Once every 2 weeks G6 Manufacturing example 2 (4W) ENAVO (8.40 mg / kg), LIRA (4.34 mg / kg) Once every 4 weeks

[0064] As a result of analyzing the weight loss rate by group, as can be seen in Figure 6, the weight loss effect of G2 was minimal compared to G1 (-3.39%), but the combination depot injection groups G4 (-8.85%) and G5 (-9.26%) combined with low-dose liraglutide administered at 2-week intervals showed a superior weight loss effect than the high-dose liraglutide alone group G3 (-7.09%). In addition, the combination depot injection group G6 combined with low-dose liraglutide administered at 4-week intervals showed a similar weight loss effect to the high-dose liraglutide alone group G3 (-7.09%). In the case of G6, it is a formulation that significantly improves patient medication convenience in that the weight loss effect was achieved with only one injection compared to G3, which requires 28 injections over 4 weeks.

[0065] In addition, as a result of analyzing the fat weight and the ratio of fat in the tissue through body composition analysis, as can be seen in Table 7 and Figure 7, it was confirmed that the G5 group administered depot containing inabogliflozin and low-dose liraglutide was reduced to a level similar to that of the G3 group administered high-dose liraglutide alone compared to the placebo group (the red part in Figure 7 represents fat).

[0066]

[0067] Body fat mass and muscle mass of SD rats fed 60% HFD administered test substances during the experimental period Group Value (g) BMD (g / cm 2 )Bone Area(cm 2 )Bone Volume(cm 3 )FAT In Tissue (%)Total MassBMCFATLEANG1VehicleNMean856.1619.4574358.8684477.83790.244679.569411.768642.68938SD65.281.006658.857028.74930.00953.46830.60884.3588G2InavogliflozinNMean820.0318.8277311.1294490.07050.242377.726311.387738.62818SD73.051.077752.643730.36520.00594.45650.65183.6568G3Saxenda ™NMean793.2419.1914293.3549480.69090.242579.075411.607737.706 68SD65.201.623847.818826.41590.00924.80240.98213.5796G4 Manufacturing Example 1 ( 2W)NMean778.9118.5477304.1375456.22130.235078.946811.218439. 76807SD79.421.430854.677332.01080.00745.73220.86543.4135G5 Manufacturing example 2(2W)NMean775.8118.6810289.3078467.82310.239178.114411.29903 7.93768SD72.930.911154.330829.17600.00463.04890.55104.2511G6 Joye2(4W)NMean794.1218.4799297.5711478.07340.2340*79.058811.17 7438.19648SD55.280.580246.407426.70880.00953.44530.35094.0630

[0068] BMC: bone mineral content; BMD: bone mineral density. Significant difference from G1 HFD vehicle control group by One-way ANOVA followed by the Dunnett's test: * p<0.05, ** p<0.01.

[0069] In addition, as shown in Table 8, in the serological analysis, the LDL-C reduction results were significantly reduced in the combination depot group G4 containing inavogliflozin and low-dose liraglutide compared to the high-dose liraglutide monotherapy group G3. In addition, the TG levels in the combination depot groups G5 and G6 containing inavogliflozin and low-dose liraglutide also significantly decreased compared to the G1 group.

[0070] Blood chemistry test results of SD rats fed 60% HFD administered test substances during the experimental period Group Blood chemistry test AST (U / L) ALT (U / L) TG (mg / dL) T-Chol (mg / dL) LDL-C (mg / dL) HDL-C (mg / dL) G1 Vehicle NMean 339.63 198.1379.25 96.75 6.88 22.638 SD30 6.04 180.52 21.83 21.17 1.55 4.84 G2 Inavogliflozin NMean 226.50 123.38 61.1386.385.1324.008 SD15 1.1996.09 18.70 18.57 0.64 3.25 G3 Saxenda ® NMean184.25105.5040.88**80.506.1322.888SD84.1299.4710.2615.720.994.64G4 Manufacturing Example 1 (2W) NMean167.2972.8652.7178.864.71*22.577SD57.8338.3712.2024.410.956.05G5 Joye2(2W)NMean157.7573.6349.63*74.385.0022.258SD79.2843.2310.9316.071.413.96G 6 Manufacturing example 2 (4W)NMean135.8855.7551.50*90.756.7521.888SD42.8327.143.8912.161.832.17

[0071] Significant difference from G1 vehicle control group by One-way ANOVA followed by the Dunnett's test: * p<0.05, ** p<0.01.AST: aspartate aminotransferase, ALT: alanine aminotransferase, TG: triglyceride T-Chol: total cholesterol, LDL-C: low density lipoprotein-cholesterol, HDL-C: high density lipoprotein-cholesterol.

[0072] In addition, as can be seen in Table 9, which compares the internal organ weights of SD rats through autopsy after the end of the test, the absolute organ weight of the liver was significantly reduced in the combination depot injection groups G4, G5, and G6 containing inavogliflozin and low-dose liraglutide compared to the high-dose liraglutide monotherapy group G3. In addition, the relative organ weight in the combination depot injection group G6 containing inavogliflozin and low-dose liraglutide also significantly decreased compared to the G1 group.

[0073] Absolute organ weight (g) and relative organ weight (organ weight / body weight ratio) at the time of sacrifice Group Body weight (g) Absolute organ weight (g) Relative organ weight Hepatorenal fat Hepatorenal fat G1 Vehicle NMean 811.13 19.50 1526.14 350.02 400.03 218 SD6 0.05 3.09 7 24.44 36 0.00 300.00 44 G2 Inavogliflozin NMean 777.17 18.68 1723.29 77 0.02 410.02 95 8 SD71.98 2.82 277.09 75 0.00 320.00 65 G3 Saxenda ® NMean744.0917.163221.52370.02310.02878SD62.642.37004.58150.00270.0041G4 Manufacturing Example 1 (2W )NMean729.4215.7746*21.62600.02180.02967SD75.521.47513.90170.00250.0042G5 Manufacturing Example 2 ( 2W)NMean735.4615.2485*20.64180.02060.02818SD69.092.75443.21070.00240.0041G6 Manufacturing example 2(4W)NMean751.3414.5738**23.48280.0194**0.03118SD52.721.11086.10030.00130.0071

[0074] Significant difference from G1 HFD vehicle control group by One-way ANOVA followed by the Dunnett's test: * p<0.05, ** p<0.01.

[0075] In conclusion, in a rat model in which obesity was induced by feeding a high-fat diet for 13 weeks, we confirmed that the combination depot injections G4 and G5 containing inavogliflozin and low-dose liraglutide administered once every two weeks had a superior weight loss effect than the group (G3) administered Saxenda™ (high-dose liraglutide), an existing obesity treatment. In addition, the combination depot injection G6 administered once every four weeks showed a similar weight loss effect to G3. In addition, the results of fat weight, serological analysis, and absolute and relative liver tissue weights were combined to show that the combination depot injection containing inavogliflozin and low-dose liraglutide has additional benefits related to improving obesity metabolism that cannot be confirmed with high-dose liraglutide administration, while at the same time reducing side effects, showing a similar weight loss effect, and further improving patient medication convenience. This was confirmed through an anti-obesity efficacy evaluation in SD rats.

[0076] Accordingly, the following study was conducted to conduct further research on the sustained drug release of the depot composition.

[0077]

[0078] [Manufacturing Examples 3 to 10]

[0079] In order to ensure sustained drug release of inavogliflozin and liraglutide, a depot composition containing inavogliflozin and liraglutide was manufactured through various attempts (S / O / W formulation method, spray-drying formulation method, and freeze-drying recovery and post-mixing formulation method after separating inavogliflozin and liraglutide and encapsulating them into microspheres). The specific manufacturing method is as follows.

[0080] In Manufacturing Examples 3 to 10, inabogliflozin and liraglutide acetate were used as active ingredients. They were manufactured using the S / O / W manufacturing method, the spray drying manufacturing method, or a method in which each active ingredient was separated, encapsulated in microspheres, and then mixed.

[0081] First, to prepare liraglutide acetate using hydrophobic ion pairing, 10 g of liraglutide and 4.2 g of sodium acetate were dissolved in 400 ml of distilled water and mixed for 30 minutes. 10 ml of acetic acid (Glacial, Sigma) or 10 ml of 1 N HCl was added to the mixture to lower the pH, thereby precipitating liraglutide acetate, which was then further reacted for 4 hours. After repeated washing with distilled water and methanol, and filtering through a 635 mesh sieve, the recovered liraglutide acetate was lyophilized and recovered.

[0082] In Manufacturing Example 3, the PLA polymer of R202H was dissolved in methylene chloride at a weight ratio of 30 w / w% and used, and inabogliflozin was completely dissolved in methanol at a weight ratio of 16.7 w / w%. Then, the polymer solution and solid liraglutide acetate were mixed using a homomixer at 10,000 RPM for 3 minutes to prepare a first mixture (S / O emulsion). After that, the first mixture (S / O emulsion) was put into a 5 L water tank containing a surfactant (PVA) at a weight ratio of 0.5%, and at the same time, a homomixer was applied at 4500 RPM for 3 minutes to prepare a second mixture (S / O / W microspheres). For the remaining volatilization process, the water tank temperature was adjusted to 30℃ and stirring was performed at 150 RPM using an overhead stirrer for 3 hours. Afterwards, the microspheres were recovered through centrifugation at 1500~3000 RPM for 3 minutes, and washed three times with distilled water to wash away the unencapsulated active ingredient and surfactant. Afterwards, the microsphere powder was recovered by freeze-drying at -40℃~5℃ for 48 hours. The recovered microspheres were finally secured by passing them through a 200 mesh sieve.

[0083] In Manufacturing Example 4, the PLA polymer of R203H was dissolved in methylene chloride at a weight ratio of 23 w / w% and used, and inabogliflozin was completely dissolved in methanol at a weight ratio of 16.7 w / w%. Then, the polymer solution and solid liraglutide acetate were mixed using a homomixer at 10,000 RPM for 3 minutes to prepare a first mixture (S / O emulsion). After that, the first mixture (S / O emulsion) was put into a 1 L water tank containing a surfactant (PVA, Gohsenol EG-40PW) at a weight ratio of 1.0 w / w%, and at the same time, a homomixer was applied at 3500 RPM for 3 minutes to prepare the second mixture (S / O / W microspheres). The remaining volatilization process was performed by adjusting the water tank temperature to 30℃ and stirring was performed at 150 RPM using an overhead stirrer for 3 hours. After that, the microspheres were recovered through centrifugation at 1500 to 3000 RPM for 3 minutes, and the washing was repeated 3 times using primary distilled water to wash away the unencapsulated active ingredient and surfactant. After that, the microsphere powder was recovered by freeze-drying at -40℃ to 5℃ for 48 hours. The recovered microspheres were finally obtained by passing through a 200 Mesh sieve.

[0084] In Manufacturing Example 5, the PLA polymer of R203H was dissolved in methylene chloride at a weight ratio of 15 w / w% and used, and inabogliflozin was completely dissolved in methanol at a weight ratio of 16.7 w / w%. Then, the polymer solution and solid liraglutide acetate were mixed using a homomixer at 15,000 RPM for 3 minutes to create a mixture (S / O emulsion), and then microspheres were prepared using a spray dryer (B”U”CHI, Mini B-290). Here, the mixture was injected at a speed of 1.0 ml / min using an injection pump, the inlet temperature was set to 55 to 75°C, the outlet temperature was set to 36 to 42°C, and the mixture was sprayed at a pressure of 20 to 50 psi to create microspheres and then recovered.

[0085] In Manufacturing Example 6, the PLA polymer of R203H was dissolved in methylene chloride at a weight ratio of 23 w / w% and used, and inabogliflozin was completely dissolved in methanol at a weight ratio of 16.7 w / w%. Then, the polymer solution and solid liraglutide acetate were mixed using a homomixer at 10,000 RPM for 3 minutes to prepare a first mixture (S / O emulsion). After that, the first mixture (S / O emulsion) was put into a 1 L water tank containing a surfactant (PVA, Merck 05P) at a weight ratio of 1.0%, and at the same time, a homomixer was applied at 3500 RPM for 3 minutes to prepare the second mixture (S / O / W microspheres). The remaining volatilization process was performed at 150 RPM for 3 hours using an overhead stirrer while adjusting the water tank temperature to 35℃. After that, the microspheres were recovered through centrifugation at 1500-3000 RPM for 3 minutes, and the washing was repeated 3 times using primary distilled water to wash away the unencapsulated active ingredient and surfactant. After that, the microsphere powder was recovered by freeze-drying at -40℃ to 5℃ for 48 hours. The recovered microspheres were finally secured by passing through a 200 Mesh sieve.

[0086] In the case of Manufacturing Example 7, inabogliflozin and liraglutide acetate were prepared separately without mixing. In the case of biodegradable polymers, PLA polymer of R203H was dissolved in methylene chloride at a weight ratio of 23% and used, and after inabogliflozin was completely dissolved in methanol at a weight ratio of 16.7%, the polymer solution was mixed using a homomixer at 10,000 RPM for 3 minutes to create a first mixture (S / O emulsion), and the subsequent process was to put it into a 1 L water tank containing a surfactant (PVA, Merck 05P) at a weight ratio of 1.0% and simultaneously apply a homomixer at 3500 RPM for 3 minutes to create a second mixture (S / O / W microspheres). The remaining volatilization process was performed using an overhead stirrer at 150 RPM for 3 hours by adjusting the water tank temperature to 30℃. Afterwards, the microspheres were recovered through centrifugation at 1500~3000 RPM for 3 minutes, and washed three times using distilled water to wash away the unencapsulated active ingredient and surfactant. Afterwards, the microsphere powder was recovered by freeze-drying at -40℃~5℃ for 48 hours. The recovered microspheres were finally secured by passing through a 200 mesh sieve. In addition, liraglutide acetate was used by dissolving the PLA polymer of R203H in methylene chloride at a weight ratio of 23%, and the other manufacturing processes were manufactured in the same conditions as the preparation of microspheres in which inavogliflozin was separated and encapsulated.

[0087] In the case of Manufacturing Example 8, the PLA polymer of R202H was dissolved in methylene chloride at a weight ratio of 30% and used, and after inabogliflozin was completely dissolved in methanol at a weight ratio of 16.7%, the polymer solution and solid liraglutide acetate were mixed using a homomixer at 15,000 RPM for 3 minutes to create a first mixture (S / O emulsion), and then the subsequent process was to put it into a 1 L water bath containing a surfactant (PVA, Merck 05P) at a weight ratio of 1.0% and simultaneously apply a homomixer at 3500 RPM for 3 minutes to create a second mixture (S / O / W microspheres). The remaining volatilization process was performed using an overhead stirrer at 150 RPM for 3 hours while adjusting the water bath temperature to 30℃. Afterwards, the microspheres were recovered through centrifugation at 1500~3000 RPM for 3 minutes, and washed once with PBST (Tween 80, 0.05%) to wash away the unencapsulated active ingredient and surfactant, and then washed three times with distilled water. Afterwards, the microsphere powder was recovered by freeze-drying at -40℃~5℃ for 48 hours. The recovered microspheres were finally secured by passing through a 200 mesh sieve.

[0088] The PLA polymer of R203H of Manufacturing Example 9 was used by dissolving it in methylene chloride at a weight ratio of 23%, and after completely dissolving inavogliflozin in methanol at a weight ratio of 16.7%, 1N HCl was used instead of acetic acid to precipitate the polymer solution and solid liraglutide acetate, and using a homomixer, mixing at 15,000 RPM for 2.5 minutes to make the first mixture (S / O emulsion), and the subsequent process was to put it into a 1 L water bath containing a surfactant (PVA, Merck 05P) at a weight ratio of 1.0% and apply the homomixer at 3500 RPM for 3 minutes to make the second mixture (S / O / W microspheres), and for the other volatilization process, the water bath temperature was adjusted to 30℃ and an overhead stirrer was used at 150 RPM for 3 hours. The microspheres were recovered by centrifugation at 1500-3000 RPM for 3 minutes, and then washed once with PBST (Tween 80, 0.05%) to wash away the unencapsulated active ingredient and surfactant, and then washed three times with distilled water. Afterwards, the microsphere powder was recovered by freeze-drying at -40℃ to 5℃ for 48 hours. The recovered microspheres were finally secured by passing through a 200 mesh sieve.

[0089] In the case of Manufacturing Example 10, the PLA polymer of R203H was dissolved in methylene chloride at a weight ratio of 23% and used, and inabogliflozin was completely dissolved in methanol at a weight ratio of 16.7%, and then the polymer solution and solid liraglutide acetate were precipitated and recovered, and then washed repeatedly with distilled water for the first time and then washed with methanol for the final time to secure the precipitation, and then mixed for 3 minutes at 10,000 RPM using a homomixer to make the first mixed solution (S / O emulsion), and then the subsequent process was to put it into a 1 L water bath containing a surfactant (PVA, Merck 05P) at a weight ratio of 1.0% and apply the homomixer at 3500 RPM for 3 minutes to make the second mixed solution (S / O / W microspheres), and for the other volatilization processes, the water bath temperature was adjusted to 30℃ and an overhead stirrer was used for 3 hours. The mixture was centrifuged at 150 RPM. Afterwards, the microspheres were recovered through centrifugation at 1500-3000 RPM for 3 minutes, and washed once with PBST (Tween 80, 0.05%) to wash away the unencapsulated active ingredient and surfactant, and then washed three times with distilled water. Afterwards, the microsphere powder was recovered by freeze-drying at -40℃ to 5℃ for 48 hours. The recovered microspheres were finally secured by passing through a 200 mesh sieve.

[0090] To confirm the diameter and particle size distribution of the composite microspheres containing inabogliflozin and liraglutide acetate, analysis was conducted using a Malvern particle size analyzer.

[0091] Specifically, the depot compositions of Manufacturing Examples 3 to 10 were obtained according to the composition and manufacturing method of Table 10.

[0092]

[0093] Manufacturing Example Manufacturing Method Polymer Concentration and Type Additives and Features ENAVO Concentration w / w % Continuous Phase Conditions 1st Homogenization Conditions (RPM) 2nd Homogenization Conditions (RPM) Volatilization Conditions Temperature / Time / RPM Size D10 / D50 / D90 / D[4,3]3 S / O / W 30% PLAR20 2 HLIRA-Acetate 16.7 0.50% PVA 5L Gohsenol EG-40 PW 10,000 3 min 4,500 3 min 30°C / 3 / 150 2.3 / 13.0 / 37.4 / 16.94 S / O / W 23% PLAR20 3 HLIRA-Acetate 16.7 1.0%, PVA 1L Gohsenol EG-40 PW 10,000 3 min 3,500 3 min 30°C / 3 / 1501.9 / 13.0 / 34.2 / 15.75SprayDrying15% PLAR203HLIRA-Acetate16.71.0%, PVA 1LMerckPVA05P15,0003minN.AN.A2.7 / 17.6 / 44.2 / 21.66S / O / W23% PLAR203HLIRA-Acetate16.71.0%, PVA 1LMerckPVA05P10,0003min3,5003min35°C / 3 / 1502.0 / 15.6 / 36.4 / 17.57O / W23% PLAR202HENAVOSeparating agent16.71.0%, PVA 1LMerckPVA05P10,0003min3,5003min30°C / 3 / 1502.0 / 14.6 / 33.3 / 18.1S / O / W23% PLAR203HLIRA-AcetateN.A1.0%, PVA 1LMerckPVA05P10,0003 minutes3,5003 minutes30°C / 3 / 1502.0 / 16.6 / 37.6 / 22.28S / O / W30% PLAR202HLIRA-Acetate / PBST0.05%washing16.71.0%, PVA 1LMerckPVA05P15,0003 minutes3,5003 minutes30°C / 3 / 1508.0 / 31.2 / 64.0 / 34.09S / O / W23% PLAR203HLIRA-Acetate(HCl)16.71.0%, PVA 1LMerckPVA05P15,0002.5min3,5003min30°C / 3 / 1505.2 / 24.4 / 51.2 / 26.910S / O / W30% PLAR202HLIRA-Acetate / MeOH Washing16.71.0%, PVA 1LMerckPVA05P10,0003 minutes3,5003 minutes30°C / 3 / 1507.4 / 28.8 / 65.1 / 32.1.

[0094] [Experimental Example 3] PK analysis according to administration of the depot compositions of Manufacturing Examples 3 to 10

[0095] For PK analysis according to administration of the depot compositions of Manufacturing Examples 3 to 10, 5-week-old male SD rats were used as an animal model.

[0096] The test groups were set as the inabogliflozin + liraglutide combination administration group (G1) and the depot formulation administration group of Manufacturing Examples 3 to 10 containing inabogliflozin and liraglutide (G2 to G9), as shown in Table 11 below (G1: n=3, G2 to G9: n=2 per group).

[0097] Test substance Dosage Dose (mg / kg) Volume (mL / kg) G1 Inavogliflozin + liraglutide SC Once a day Inavogliflozin 0.093 + liraglutide 0.186 Inavogliflozin 0.93 + liraglutide 1.86 G2 Manufacturing example 3 SC Single Inavogliflozin 1.87 + liraglutide 2.60 G3 Manufacturing example 4 Inavogliflozin 2.86 + liraglutide 2.60 G4 Manufacturing example 5 Inavogliflozin 2.77 + liraglutide 2.60 G5 Manufacturing example 6 Inavogliflozin 3.00 + liraglutide 2.60 G6 Manufacturing example 7 Inavogliflozin 2.60 + liraglutide 2.60 G7 Manufacturing example 8 Inavogliflozin 2.30 + Liraglutide 2.60G8 Manufacturing Example 9 Nabogliflozin 1.68 + Liraglutide 2.60G9 Manufacturing Example 10 Nabogliflozin 1.86 + Liraglutide 2.60

[0098] Plasma samples were collected at specified times and stored at -70°C until analysis using an LC-MS / MS system. Protein precipitation was performed on 20 μL of plasma samples using 180 μL of acetonitrile containing internal standards. After mixing and centrifugation, the supernatant (150 μL) was analyzed using an LC-MS / MS system with MRM mode. LC / MS / MS data were analyzed using Analyst 1.6.3. PK parameters were calculated using noncompartmental analysis using WinNonlin software. Figure 8 shows the PK analysis results of inavogliflozin according to the administration of each test substance.

[0099] As a result of evaluating the level of sustained drug release through blood drug concentration measured 4 weeks (total 672 h) after administration of the test substance, the drug was detected at 2 weeks (336 h point) in G2 / G4 / G7 / G9 compared to the ENAVO control drug administration group (G1), and the drug was detected up to 4 weeks (672 h point) in G7 and G9 compared to the ENAVO control drug administration group (G1), confirming that the drug was continuously released.

[0100] Figure 9 shows the PK analysis results of liraglutide according to administration of each test substance.

[0101] The sustained drug release level was evaluated through the blood drug concentration measured 4 weeks (total 672 h) after administration of the test substance. As a result, the drug was detected at 2 weeks (336 h point) in G2 / G7 / G9 compared to the LIRA control drug administration group (G1), confirming that the drug was continuously released.

[0102] In summary, it was confirmed that in the case of the depot compositions of Manufacturing Examples 3, 8, and 10, both inagliflozin and liraglutide continued to release the drugs for more than two weeks.

[0103] More preferably, it was confirmed that the liraglutide drug release of the depot composition of Manufacturing Example 10 was maintained at a relatively higher level than other Manufacturing Examples at 2 weeks (336 h point).

Claims

1. An anti-obesity pharmaceutical composition comprising inabogliflozin as an active ingredient and characterized by combined use of low-dose liraglutide.

2. In paragraph 1, the low-dose liraglutide is an anti-obesity pharmaceutical composition having an administered dose of less than 3 mg / day of liraglutide.

3. An anti-obesity pharmaceutical composition in paragraph 1, wherein the dosage of inabogliflozin is 0.3 to 1.5 mg / day, and the dosage of liraglutide is 1.2 to 2.4 mg / day.

4. An anti-obesity pharmaceutical composition in the first paragraph, wherein the dosage of inabogliflozin is 0.6 to 1.2 mg / day, and the dosage of liraglutide is 1.5 to 2 mg / day.

5. An anti-obesity pharmaceutical composition according to claim 1, wherein inabogliflozin is administered orally and liraglutide is administered via subcutaneous injection.

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