A dapagliflozin-pioglitazone co-amorphous compound and its preparation method and application

Abstract

The present invention relates to a dapagliflozin-pioglitazone co-amorphous material and its preparation method and application. In the dapagliflozin-pioglitazone co-amorphous material, the molar ratio of dapagliflozin to pioglitazone is 1:2~2:1, and its X-ray powder diffraction pattern has no sharp diffraction peaks, indicating the formation of a co-amorphous state. The present invention obtains the co-amorphous form by utilizing a solvent-assisted grinding method and a rotary evaporation method. After experimental verification, the amorphous form has good solubility, dissolution, and bioavailability. After in vivo and in vitro hypoglycemic activity tests, it is verified that the activity is significantly better than that of dapagliflozin or pioglitazone alone. It can be seen that the co-amorphous supramolecular system of the present application has a synergistic hypoglycemic effect.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a dapagliflozin-pioglitazone co-amorphous compound and a preparation method and application thereof. Background Art

[0002] Pioglitazone, an insulin sensitizer and thiazolidinedione hypoglycemic drug used in the first-line clinical setting, is a PPAR-γ receptor agonist and an insulin-dependent drug. During the progression of type 2 diabetes mellitus (T2DM), its efficacy decreases with the decline in pancreatic β-cell function. At the same time, weight gain will further aggravate insulin resistance. The selective SGLT-2 inhibitor dapagliflozin is a new class of antidiabetic drugs that primarily lowers blood sugar in patients with T2DM by specifically inhibiting glucose reabsorption in the proximal convoluted tubule. Its glucose-lowering mechanism is independent of the state of insulin secretion and provides continuous and stable blood sugar control, which can prevent drug resistance and pancreatic islet dysfunction caused by excessive secretion of pancreatic β-cells and has the potential to protect β-cell function. However, long-term use of dapagliflozin carries the risk of inducing recurrent urinary tract infections. In summary, the dual-target combination of dapagliflozin and pioglitazone is clinically important for stable blood sugar control and reduced risk of side effects in the later stages of the disease.

[0003] The construction of drug-drug supramolecular systems combines supramolecular technology with combination therapy. Single-drug administration can achieve the purpose of combination therapy, improving the undesirable physical and chemical properties of each component. Crucially, the molecular-level synergistic effect of two-component supramolecular systems with different targets offers further advantages in enhancing efficacy and reducing toxicity, driving the advancement of combination therapy technology and theory. This invention proposes to explore new forms of combination therapy (technological innovation) by constructing a dual-target supramolecular system of dapagliflozin and pioglitazone, with the goal of improving the bioavailability of both drugs. Simultaneously, the synergistic mechanism of the two components in the supramolecular system is studied in depth at the molecular level (theoretical innovation), providing innovative clinical intervention strategies for the treatment of type 2 diabetes. Summary of the Invention

[0004] The purpose of the present invention is to provide a dapagliflozin-pioglitazone co-amorphous compound (DAP-PIO) having good solubility and bioavailability, and also to provide a preparation method and application thereof.

[0005] The present invention provides a dapagliflozin-pioglitazone co-amorphous compound, characterized in that the co-amorphous compound comprises dapagliflozin and pioglitazone, and the molar ratio of dapagliflozin to pioglitazone is 1:2 to 2:1.

[0006] As a further improvement of the present invention, the co-amorphous material is subjected to Cu-kα radiation, and the X-ray powder diffraction pattern expressed in 2θ shows a camel-shaped halo, and the crystalline diffraction peak disappears. This preliminarily indicates the formation of a co-amorphous state. TMDSC and TG analysis,Figure 2 As shown in the figure, an endothermic step appeared in the reversible heat flow signal curves of the samples of various ratios prepared by DAP, proving the formation of amorphous state in each DAP-PIO system.

[0007] As a further improvement of the present invention, the X-ray powder diffraction pattern is as follows Figure 1 -As shown in A.

[0008] As a further improvement of the present invention, the glass transition temperature of the co-amorphous material is 19.75-24.77°C.

[0009] As a further improvement of the present invention, the co-amorphous material is prepared by solvent-assisted grinding or rotary evaporation.

[0010] Another aspect of the present invention provides a method for preparing the above-mentioned dapagliflozin-pioglitazone co-amorphous compound.

[0011] Option 1: Solvent-assisted grinding method: grind and mix dapagliflozin and pioglitazone, add solvent to grind, and dry to obtain the product.

[0012] As a further improvement of the present invention, the solvent is selected from 100% ethanol, and the ratio of the solvent to the material (dapagliflozin and pioglitazone) is 0.3-1 mL:200 mg; the grinding time after adding the solvent is 30-40 min, and the grinding speed is 1200-1500 rpm.

[0013] As a further improvement of the present invention, the molar ratio of dapagliflozin to pioglitazone is 2:1.

[0014] Option 2: Using the rotary evaporation method: add dapagliflozin and pioglitazone to the solvent, ultrasonically extract the sample at a solid-liquid ratio of 5-8 mg / mL for 30-40 minutes, and obtain a co-amorphous form by rotary evaporation at 38±2°C.

[0015] As a further improvement of the present invention, the solvent is selected from methanol, ethanol, a mixture of methanol or ethanol and water, and the concentration of the mixture of ethanol and water is greater than or equal to 50%.

[0016] As a further improvement of the present invention, the molar ratios of dapagliflozin and pioglitazone are 1:1, 1:2, and 2:1, respectively.

[0017] In another aspect, the present invention provides the use of the above-mentioned dapagliflozin-pioglitazone co-amorphous compound in preparing dapagliflozin-pioglitazone amorphous compound in preparing drugs for treating diabetes and obesity.

[0018] The beneficial effects of adopting the above technical solution are:

[0019] The present invention uses dapagliflozin and pioglitazone as raw materials, and obtains a co-amorphous material through a solvent-assisted grinding method and an ultrasonic rotary evaporation method. Experimental verification shows that the co-amorphous material has good dissolution, solubility and bioavailability.

[0020] The co-amorphous compound provided by the present invention has been verified by in vitro and in vivo tests for the treatment of diabetes and obesity, and is significantly better than the activity of the two drugs used alone. This shows that the co-amorphous compound of the present application can enhance the hypoglycemic activity and weight loss of dapagliflozin and pioglitazone. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Characterization spectra of the dapagliflozin-pioglitazone co-amorphous compound obtained in Example 2 of the present invention: (A) XRD, (B) IR, and (C) Raman characterization patterns;

[0022] Figure 2 This is a TMDSC-TG characterization chart of the co-amorphous material obtained in Example 2 of the present invention;

[0023] Figure 3 These are the solubility diagrams of Experimental Example 2 of the present invention: (A) solubility of DAP, (B) solubility of PIO, and (C) powder PXRD after 72 h at pH = 1;

[0024] Note:*P<0.05(vs.DAP or PIO); **P<0.01(vs.DAP or PIO), ★ P<0.05(vs.PM)and ★★ P < 0.01 (vs. PM);

[0025] Figure 4 This is the dissolution curve of Test Example 3 of the present invention in 0.1 mol / L hydrochloric acid medium (38° C., n=3);

[0026] Figure 5 This is the drug-time curve of Experimental Example 4 of the present invention in SD rats (n=6);

[0027] Figure 6 Statistical graphs of glucose consumption rates of different drug concentrations in the cellular insulin resistance model of Experimental Example 5 of the present invention: H9C2 (Figures AC) and HepG2 (Figures DF);

[0028] Figure 7 Glucose uptake in the supramolecular system (DAP-PIO) of the H9C2 cell insulin resistance model in Experimental Example 6 of the present invention (Note: blue is DAPI; green is 2-NBDG);

[0029] Figure 8Effects of DAP-PIO co-amorphous on metabolic parameters and glucose homeostasis in HFD-induced diabetic mice in Experimental Example 7 of the present invention;

[0030] Figure 9 This is the effect of DAP-PIO co-amorphous in Experimental Example 7 of the present invention on lipid levels in the liver and heart of mice on HFD and HFD treatment. DETAILED DESCRIPTION

[0031] In order to make the objectives, technical solutions and advantages of the present invention more clear, the invention is clearly and completely described below in conjunction with specific embodiments.

[0032] Example 1 Preparation of Dapagliflozin-Pioglitazone Co-amorphous Compound

[0033] Accurately weigh 60 mg of dapagliflozin (DAP) and 28.83 mg of pioglitazone (PIO) (molar ratio DAP / PIO 2:1) into a 2 ml grinding tube. Add two 5 mm zirconium oxide grinding balls and mix thoroughly. Add 0.2 mL of 100% ethanol (v / v) to maintain a slurry. Grind at 1200 rpm for 30 minutes, pausing every 5 minutes to cool the material. After grinding, dry in a 45°C vacuum oven overnight to obtain a dapagliflozin-pioglitazone co-amorphous compound.

[0034] Example 2 Preparation of Dapagliflozin-Pioglitazone Co-amorphous Form

[0035] Rotary evaporation method: 200.00 mg of each physical mixture containing dapagliflozin and pioglitazone (molar ratio of 1:2, 1:1, 2:1) was placed in a 100 mL round-bottom flask, and 40 mL of ethanol was added respectively. The mixture was ultrasonicated at room temperature for 30 min to completely dissolve, and the mixture was rotated at 38°C for 20 min (speed of 50 rpm). The solvent was evaporated under the same conditions, and the mixture was vacuum dried to obtain dapagliflozin-pioglitazone co-amorphous products with different molar ratios for later use.

[0036] Experimental Example 1 Structural Characterization of Co-amorphous Compounds

[0037] In this test example, the dapagliflozin-pioglitazone co-amorphous compounds with different molar ratios obtained in Example 2 were named COA-Ⅰ (DAP:PIO=1:1), COA-Ⅱ (DAP:PIO=2:1), and COA-Ⅲ (DAP:PIO=1:2), and their corresponding physical mixtures were PM-Ⅰ (DAP:PIO=1:1), PM-Ⅱ (DAP:PIO=2:1), and PM-Ⅲ (DAP:PIO=1:2).

[0038] (1) X-ray powder diffraction detection

[0039] Dapagliflozin, pioglitazone, and the co-amorphous compound prepared in different molar ratios in Example 2 were weighed and placed on a table. The X-ray diffraction patterns of the samples were measured. The scanning range was 5 to 40° (2θ), the step size was 0.02°, and the scanning speed was 10° / min. The PXRD patterns were as follows: Figure 1 As shown in A.

[0040] It can be seen from the figure that crystalline PIO has obvious characteristic diffraction peaks, while the PXRD patterns of the DAP-PIO supramolecular system at different ratios show a camel-shaped halo without sharp characteristic peaks, and the crystalline diffraction peak disappears, preliminarily indicating the formation of a co-amorphous state. The amorphous material prepared in Example 1 has the same pattern as the dapagliflozin-pioglitazone 2:1 prepared in Example 2.

[0041] (2) FT-IR and Raman detection

[0042] FT-IR spectra of DAP, PIO, and DAP-PIO co-amorphous were measured using the KBr pellet method on a Spectrum Two FT-IR (PerkinElmer Company, USA). Approximately 1 mg of each of DAP, PIO, and co-amorphous powder was weighed, along with approximately 160 mg of KBr. These were mixed in equal increments, thoroughly ground, and pressed into pellets. A blank KBr pellet was used as a reference, and the resolution was 4 cm. -1 , scanning range 4000 to 400cm -1 The total number of scans is 40, and the signals are accumulated and averaged.

[0043] The XploRA Plus Raman imaging spectrometer (Horiba France Sas, France) was used to measure the Raman spectra of DAP, PIO, and co-amorphous -1 Raman spectra within the range. The excitation wavelength was 638 nm; the RTD time was 1 s, and the scans were repeated three times with the average value accumulated.

[0044] FT-IR spectrum such as Figure 1 As shown in B: The stretching vibration of C=O in the DAP-PIO supramolecular system is attributed to the 1744 cm -1 Move to 1750cm -1 , by 1694cm -1 Moved to 1696cm respectively -1 , 1698cm -1 , moving to the high field. And the NH stretching vibration is shifted from 3084cm -1 Move to 3036cm respectively -1 , 3034cm -1, moving downfield. It is inferred that the C=O and NH in pioglitazone form hydrogen bonds with certain groups in the dapagliflozin structure. The stretching vibration of OH in the dapagliflozin structure is at 3378cm -1 Move to 3386cm respectively -1 , 3388cm -1 , 3422cm -1 The results indicate that OH participates in the formation of hydrogen bonds between dapagliflozin and pioglitazone in the co-amorphous supramolecule.

[0045] like Figure 1 As shown in the Raman spectrum of C, the C=O of PIO raw material is 1610 cm -1 , the -OH of DAP is 873 cm -1 In the Raman spectrum of the physical mixture, overlapping peaks of DAP and PIO can be observed. In contrast, the intensity, width and position of the peaks of the co-amorphous compound have changed. -1 、3067cm -1 The peak at 827 cm-1 is obviously broadened, indicating the formation of disordered structure. Meanwhile, the -OH vibration of DAP-PIO supramolecular merges and shifts to 827 cm-1. -1 These obvious changes and mergers also suggest that the co-amorphous structure may be formed through intermolecular hydrogen bonding between the -OH group of DAP and the C=O group of PIO.

[0046] (2)DSC-TG detection

[0047] TMDSC analysis: Approximately 4 mg of DAP, PIO, and DAP-PIO co-amorphous powders at varying molar ratios were accurately weighed into an aluminum crucible. Using an empty crucible as a reference, TMDSC spectra were measured for each sample. Temperature: 0-250°C, heating rate: 2.000 K / min, cycle: 60 seconds, amplitude: 0.5 K.

[0048] Thermogravimetric (TG) analysis: Approximately 4 mg of DAP, PIO, and DAP-PIO co-amorphous powder were accurately weighed and placed in an aluminum crucible. The N2 flow rate was set at 40 mL / min, the temperature range was 40-900°C, and the heating rate was 10 K / min.

[0049] like Figure 2 A and Figure 2 As shown in Figure B, the reversible heat flow curves for all DAP-PIO samples show an endothermic step, confirming the formation of an amorphous phase in each DAP-PIO system. Furthermore, the Tg of the co-amorphous COA-II is higher than that of the amorphous DAP alone. Comparing these Tg values preliminarily suggests that the formation of the co-amorphous phase enhances the stability of DAP.

[0050] Depend on Figure 2 As can be seen from C-2F, in the TG spectrum of the co-amorphous form, the DTG curve clearly shows that pioglitazone has only one thermal decomposition peak, indicating that its decomposition process is a one-step decomposition, and dapagliflozin has two thermal decomposition peaks, indicating that its decomposition process is a two-step decomposition. There are differences between the physical mixture and the co-amorphous form, indicating that the physical mixture is just a separate mixture of samples, while the co-amorphous form has undergone essential changes.

[0051] Test Example 2 Determination of the solubility of dapagliflozin-pioglitazone

[0052] The equilibrium solubility of dapagliflozin-pioglitazone co-amorphous compound at pH = 1, pH = 1.2, pH = 4, and pH = 6.8 was determined by the shake flask method. An excess of the sample to be tested was added to a shake flask containing 10 mL of the medium and placed on an air bath shaker. The air bath shaker was set to a constant temperature of 37 ± 0.5 ° C and the shaker was rotated at 150 r·min. -1 The solution was shaken until the concentrations of dapagliflozin and pioglitazone remained constant, indicating a saturated solution. After 72 hours of equilibrium, the suspension was aspirated with a disposable syringe and filtered through a 0.22 μm microporous membrane. The equilibrium solubilities of pioglitazone hydrochloride, dapagliflozin, and their co-amorphous form at varying molar ratios (1:1, 2:1, and 1:2) were determined by HPLC. The solid phase was collected, dried, and analyzed by PXRD. All experiments were repeated three times.

[0053] The equilibrium solubility of DAP, PIO and DAP-PIO amorphous compound in pH (1, 1.2, 4, 6.8) media is shown in Figure 3 A and 3B, with the increase of pH value, the drug solubility shows an overall downward trend, but compared with the physical mixture, the solubility of the co-amorphous is better than that of the physical mixture, with a significant difference. After the pH = 1 equilibrium solubility determination, solid samples were collected for PXRD, such as Figure 3 As shown in Figure 1-C, the co-amorphous COA-II exhibited no new diffraction peaks, indicating that the co-amorphous COA-II remained stable after repeated dissolution and precipitation in the solvent, with no precipitation of pioglitazone free base, thus preventing the disproportionation of pioglitazone hydrochloride. At a pH of 1, after 72 hours of reaction, the physical mixture of dapagliflozin and pioglitazone formed a co-amorphous form, increasing solubility and facilitating drug absorption.

[0054] Test Example 3: Dissolution Determination of Dapagliflozin-Pioglitazone

[0055] The dissolution rates of dapagliflozin, pioglitazone, physical mixture, and co-amorphous were determined by the second method (paddle method) in the fourth part of the pharmacopoeia on an RC-808D dissolution tester (Tianjin Tianda Tianfa Co., Ltd.). Accurately weigh 30 mg of DAP, 28.83 mg of PIO, 58.83 mg of PM-I (containing 30 mg of DAP and 28.83 mg of PIO), and 58.83 mg of COA-I (containing 30 mg of DAP and 28.83 mg of PIO) respectively. Using 500 mL of 0.1 mol / L hydrochloric acid solution as the dissolution medium, stir at 100 rpm at 37 ± 0.5 °C. Sampling was carried out at 5, 10, 15, 20, 25, 30, 45, 60, 90, 120, 180, 240, 300, and 360 min respectively, and the same volume of dissolution medium at the same temperature was supplemented at the same time. After the samples were filtered through a 0.22 μm microporous filter membrane, they were appropriately diluted and then the concentration was determined by high performance liquid chromatography. All experiments were carried out in parallel three times.

[0056] It can be seen from Figure 4 that after the formation of co-amorphous, the overall dissolution rate of the drug has increased. Especially after the formation of co-amorphous COA-I and co-amorphous COA-III, the dissolution of the drug has increased significantly, which helps to improve the bioavailability of the drug in vivo. It can be seen from the figure that the dissolution of the physical mixture has increased, which may be due to the interaction between DAP and PIO in the 0.1 mol / L hydrochloric acid medium, increasing their dissolution with each other.

[0057] Experimental Example 4 Pharmacokinetic Determination of Dapagliflozin-Pioglitazone

[0058] Twenty-four male SD rats weighing about 250 ± 25 g were selected as the test animals (Hebei Experimental Animal Center, SPF, license number: SCXK (Ji) 2022-001). They were fasted from 12 h before the experiment until the end of the experiment and allowed free access to water. All animal facilities complied with the requirements of the International Association for Assessment and Accreditation of Laboratory Animal Care. Experimental animal feeding environment: temperature 22 - 24 °C, relative humidity 50 ± 5%, 12 h of light followed by 12 h of darkness.

[0059] Twenty-four SD rats were randomly divided into four groups of six rats each and orally administered with DAP, PIO, PM-II, or COA-II. The rats were housed at a temperature of 22-24°C and a relative humidity of 50 ± 5%. Rats were weighed and the dose calculated (equivalent to 83 mg / kg of dapagliflozin and 80 mg / kg of pioglitazone) before administration. Rats were fasted for 12 hours with free access to water. A single oral gavage was administered. Blood (0.3 mL) was collected from the orbital cavity into a tube containing dry heparin at 5, 15, 30 minutes, and at 1, 1.5, 2, 3, 5, 8, 12, 16, and 24 hours. The blood was centrifuged at 3500 rpm for 10 minutes at 4°C, and the plasma supernatant was collected and stored at -20°C. After processing, 10 μL of the supernatant was accurately measured and injected into a liquid chromatograph, and the chromatogram was recorded. Drug-dose curves were plotted using the time of blood collection as the horizontal axis and the concentrations of DAP and PIO in plasma samples as the vertical axis. Pharmacokinetic parameters such as AUC0-t and Cmax were calculated using DAS2.0 software. Pharmacokinetic parameters were statistically analyzed using SPSS. The final results were tested at a p-value of 0.05, and statistically significant differences were considered when p < 0.05.

[0060] The concentration-time curves of DAP and PIO in rat plasma are shown in Figure 5 AUC was calculated using DAS2.0 software. 0-t 、C max , t 1 / 2 and T max See Table 1 and Table 2. For DAP, the co-amorphous compound is compared with PM-II and amorphous DAP, C max The AUC increased by approximately 1.52 times and 3.09 times, respectively. 0-t The AUC was improved by 1.33 times and 3.06 times respectively. 0-∞ The results were improved by 1.28 times and 3.19 times respectively. max The results showed that DAP-PIO co-amorphous had higher bioavailability than single-drug DAP or PM of both. For PIO, the co-amorphous had higher bioavailability than PM and PIO. max No significant change, AUC 0-t , AUC 0-∞ Slightly decreased, but T max It was advanced by about 1.17h and 0.58h, t 1 / 2α The distribution half-life was advanced by approximately 0.94h and 0.87h. 1 / 2β The elimination half-life was delayed by about 5.88h and 2.33h, indicating that the distribution of the drug in the body became faster and the elimination of the drug in the body slowed down after PIO was prepared into amorphous form.

[0061] Table 1 Average pharmacokinetic parameters of DAP in rats after administration

[0062] Table 4 Mean pharmacokinetic parameters of DAPin rats after administration

[0063]

[0064] Note:ns=no significance(vs.DAP or PM),*P<0.05(vs.DAP); **P<0.01(vs.DAP), ★ P<0.05(vs.PM)and ★★ P<0.01 (vs. PM)

[0065] Table 2 Average pharmacokinetic parameters of PIO in rats after administration

[0066] Table 5 Mean pharmacokinetic parameters of PIO in rats after administration

[0067]

[0068] Note:ns=no significance(vs.PIO or PM),*P<0.05(vs.PIO); **P<0.01(vs.PIO), ★ P<0.05(vs.PM)and ★★ P<0.01 (vs. PM)

[0069] Experimental Example 5: Evaluation of drug efficacy at the in vitro cell level

[0070] Cell culture and treatment:

[0071] Human HepG2 cells and H9c2 rat cardiac myoblasts were provided by the School of Pharmacy, Hebei Medical University (Shijiazhuang, Hebei, China) and cultured in DMEM medium containing 25 mM glucose, supplemented with 10% FBS and 1% penicillin / streptomycin, at 37°C and 5% CO2. Cells were passaged using trypsin-EDTA. Cells were seeded into 24-well plates and, after 60% confluence, maintained overnight in DMEM containing normal glucose. Cells incubated with 25 mM DMEM were considered the control group. 30 mM glucose plus 5 μM insulin (HGHI) for 28 hours was used to simulate T2DM in vitro. Cells under HGHI conditions were treated in the absence or presence of different concentrations of DAP, PIO, PM, and COA. For mechanistic studies, the PI3K inhibitor LY294002 and the AMPK inhibitor compound C were pretreated for 2 hours before HGHI incubation.

[0072] Glucose consumption rate determination:

[0073] The condition of 30 mM glucose plus 5 μM insulin (HGHI) for 28 hours was used to simulate T2DM in vitro. Cells under HGHI conditions were treated for 24 hours in the absence or presence of different concentrations of DAP, PIO, PM and COA. At the 24-hour time point, the glucose concentration of the culture medium of each group was measured by glucose determination reagent (Nanjing Jiancheng, Nanjing, China). The glucose consumption rate was calculated by (glucose concentration of each group - glucose concentration of the control group) / glucose concentration of the control group * 100%. Figure 6 It can be seen that for H9C2 and HepG2 cells, the co-amorphous drug-administered group had a better hypoglycemic effect than PM, and the glucose consumption in the cells increased, indicating that the glucose utilization rate of the cells increased, preliminarily confirming the synergistic hypoglycemic effect of the co-amorphous drug. However, as the drug concentration increased to a certain level, the glucose consumption actually decreased, indicating that the drug produced side effects as the drug concentration increased. Therefore, choosing the appropriate drug concentration can achieve the best therapeutic effect. For the co-amorphous systems COA-Ⅱ and COA-Ⅲ, the best effect was achieved when the concentration ratio was (10μM:5μM). For the co-amorphous system COA-Ⅰ, the corresponding (10μM:10μM) was selected for subsequent hypoglycemic effect studies.

[0074] Experimental Example 6: In vitro cell-level efficacy evaluation: glucose uptake test in insulin-resistant HepG2 and H9c2 cells

[0075] Glucose uptake assay in H9C2 cell insulin resistance model

[0076] The cells were cultured in DMEM medium containing 25mM glucose, supplemented with 10% FBS and 1% penicillin / streptomycin. Trypsin-EDTA was used for cell passaging. The cells were seeded in 6-well plates, and the medium was replaced after 60% confluence. 30mM glucose plus 5μM insulin (HGHI) was used to culture for 28 hours to simulate T2DM in vitro. Cells under HGHI conditions were cultured for 24 hours in the absence or presence of different drugs for glucose uptake assay experiments (n=3). 2-NBDG (fluorescently labeled glucose analog, used to detect glucose uptake in cells). After the cells were treated and washed, 500μL PBS containing 60μM 2-NBDG was added to each well and cultured for another 30 minutes. After culture, each well was washed twice with ice-cold PBS to stop glucose uptake and ensure that no 2-NBDG remained. The fluorescence intensity of 2-NBDG was measured by confocal microscopy at 488nm excitation and 550nm emission wavelengths. By Figure 7 It can be seen that the glucose uptake of the DAP-PIO co-amorphous supramolecular system is higher than that of the physical mixture and the single drug, indicating that the DAP-PIO co-amorphous supramolecular system improves the insulin resistance of cells.

[0077] Experimental Example 7 In vivo improvement of the dapagliflozin-pioglitazone supramolecular system on hyperglycemia and insulin sensitivity in HFD-induced diabetic mice

[0078] Four-week-old male C57BL / 6J mice were obtained from Spayfor Biotechnology Co., Ltd. and housed in a room at 25°C, 50% relative humidity, and a 12-hour light / 12-hour dark cycle with free access to food and water. The normal control group was fed a standard diet, and the diabetic model group was fed a high-fat diet (HFD, Research diet, D12492, 60% of calories from fat). The establishment of T2DM and IR began with two months of HFD feeding. Mice with high body weight and high blood sugar (fasting blood sugar ≥11.1 mmol / L) were classified as diabetic.

[0079] After T2DM was established, DAP, PIO, PM and COA-Ⅰ were orally administered daily by gavage for 2 weeks (n=6 per group). The diabetic model group and treated diabetic mice were fed with HFD, while the normal control group was fed with a normal diet.

[0080] Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT):

[0081] At the end of the experiment, the fasting blood glucose level of mice was measured by Accu-Chek Performa blood glucose meter (Roche, Switzerland). For oral glucose tolerance test (OGTT) and insulin tolerance test (ITT), mice were fasted overnight in the last two weeks, then oral 1g / kg glucose or intraperitoneal injection of 0.75U / kg insulin. Subsequently, glucose baseline levels were measured at 0, 15, 30, 60, 90, 120min, and calculated by area under the curve (AUC). After putting mice to death, their plasma samples and liver tissue were collected. The liver and cardiac index of record body weight, liver weight and heart weight were recorded.

[0082] The fasting blood glucose (FBG) levels of HFD mice increased significantly (≥11.1mmol / L) after 2 months of HFD feeding, indicating the successful establishment of T2DM. The fasting blood glucose (FBG) levels of HFD mice increased significantly (≥11.1mmol / L) after 2 months of HFD feeding, indicating the successful establishment of T2DM. Compared with the control group, HFD mice treated with the dapagliflozin-pioglitazone supramolecular system had improved whole-body glucose tolerance and insulin sensitivity ( Figure 8 The dapagliflozin-pioglitazone supramolecular system improved impaired glucose tolerance and whole-body IR in HFD-induced diabetic mice, indicating a beneficial effect on glycemic control.

[0083] HFD caused hepatocyte swelling, cellular disorganization, lipid accumulation, and microvesicular and macrovesicular lipid deposition in the liver, leading to an increase in liver volume. However, the increase in liver weight and liver index was avoided in the dapagliflozin-pioglitazone supramolecular system treatment group ( Figure 9 A, Figure 9 B) Hearts of HFD mice are prone to diabetes-induced myocardial hypertrophy, resulting in a significant increase in cardiac index, cardiomyocyte size, and left ventricular mass. Treatment with the dapagliflozin-pioglitazone supramolecular system significantly reduced the cardiac index of diabetic hearts ( Figure 9 C, Figure 9 D).

[0084] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A dapagliflozin-pioglitazone co-amorphous supramolecular system, characterized in that: The co-amorphous supramolecular system comprises dapagliflozin and pioglitazone, and the molar ratio of dapagliflozin to pioglitazone is 1:2 to 2:

1.

2. The dapagliflozin-pioglitazone co-amorphous supramolecular system according to claim 1, characterized in that: The co-amorphous supramolecular system has no sharp diffraction peaks in its X-ray powder diffraction spectrum expressed in 2θ using Cu-kα radiation.

3. The dapagliflozin-pioglitazone co-amorphous supramolecular system according to claim 1, characterized in that: The glass transition temperature of the co-amorphous supramolecular system is 19.75-24.77°C.

4. The dapagliflozin-pioglitazone co-amorphous supramolecular system according to claim 1, characterized in that: The co-amorphous supramolecular system is prepared by a solvent-assisted grinding method or a rotary evaporation method.

5. A method for preparing the dapagliflozin-pioglitazone co-amorphous supramolecular system according to any one of claims 1 to 4, characterized in that: Solvent-assisted grinding method: dapagliflozin and pioglitazone are ground and mixed, solvent is added to grind, and dried to obtain the product; The solvent is selected from 100% ethanol, and the ratio of the solvent to the materials dapagliflozin and pioglitazone is 0.3~1mL:200mg; the grinding time after adding the solvent is 30~40min, and the grinding speed is 1200~1500rpm.

6. The method for preparing the dapagliflozin-pioglitazone co-amorphous supramolecular system according to claim 5, characterized in that: The molar ratio of dapagliflozin to pioglitazone is 2:

1.

7. A method for preparing the dapagliflozin-pioglitazone co-amorphous supramolecular system according to any one of claims 1 to 4, characterized in that: The rotary evaporation method was used: dapagliflozin and pioglitazone were added to the solvent, the sample was ultrasonically extracted at a solid-liquid ratio of 5-8 mg / mL for 30-40 min, and the co-amorphous form was obtained by rotary evaporation at 38±2°C.

8. The method for preparing the dapagliflozin-pioglitazone co-amorphous supramolecular system according to claim 7, characterized in that: The solvent is selected from methanol, ethanol, a mixture of methanol or ethanol and water, and the concentration of the mixture of ethanol and water is greater than or equal to 50%.

9. The method for preparing the dapagliflozin-pioglitazone co-amorphous supramolecular system according to claim 7, wherein: The molar ratios of dapagliflozin and pioglitazone are 1:1, 1:2, and 2:1, respectively.

10. Use of the dapagliflozin-pioglitazone co-amorphous supramolecular system according to any one of claims 1 to 4 in the preparation of drugs for treating diabetes and obesity.

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