A dapagliflozin- ezetimibe co-amorphous material and methods of making and using the same

Abstract

The present application relates to the technical field of pharmacy, and discloses a dapagliflozin- ezetimibe co-amorphous substance, a preparation method and application thereof. The dapagliflozin-ezetimibe co-amorphous substance is formed by combining dapagliflozin and ezetimibe; and the molar ratio of dapagliflozin and ezetimibe is 1:1. Through the above technical scheme, the problems of moisture absorption of DAP raw material, poor treatment of metabolic dysfunction related fatty liver disease and the like in the related art are solved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a dapagliflozin-ezetimibe co-amorphous compound, its preparation method, and its application. Background Technology

[0002] Metabolic dysfunction-associated fatty liver disease (MASLD) has a complex pathogenesis, and currently lacks effective clinical drugs. MASLD is closely related to hepatocyte autophagy inhibition, lipid metabolism disorders, and iron overload. Therefore, the key to treating MASLD lies in how to specifically enhance autophagy in hepatocytes, regulate lipid metabolism disorders, reduce excessive lipid deposition, and mitigate the toxic effects of iron overload on hepatocytes. Among these, autophagy activation is the core. Selective sodium-dependent glucose transporter 2 (SGLT2) inhibitors, dapagliflozin (DAP), and ezetimibe (EZE) enhance autophagy by activating the AMPK / mammalian target of rapamycin (mTOR) pathway, thereby improving hepatic steatosis. Meanwhile, the DAP structural analog empagliflozin can inhibit ferroptosis by activating the AMPK / mTOR pathway and alleviate mitochondrial dysfunction. However, there is limited research on whether DAP and EZE are involved in ferroptosis and reducing liver fat content in MASLD, and the synergistic effect of the two drugs combined needs further clarification. Recent studies show that lowering cholesterol levels may promote the expression of genes related to autophagy. EZE reduces cholesterol uptake by hepatocytes and intestinal epithelial cells by inhibiting Niemann-Pick C1-Like 1 (NPC1L1), altering intracellular cholesterol distribution, which may then activate endoplasmic reticulum stress and the AMPK pathway, ultimately inducing autophagy. Importantly, EZE activates autophagy only in hepatocytes and intestinal epithelial cells by inhibiting NPC1L1. This cell specificity allows for precise regulation of autophagy through cholesterol-autophagy linkages, providing the possibility for cell-specific autophagy activation. However, DAP has side effects such as increased low-density lipoprotein cholesterol and acute kidney injury. EZE can effectively reduce serum low-density lipoprotein cholesterol (LDL-C) levels, thereby significantly reducing the risk of cardiovascular disease by decreasing the formation and development of atherosclerotic plaques. Simultaneously, EZE reduces oxidative damage by inducing antioxidant enzymes, thus improving nephrotoxicity. The combined administration of DAP and EZE offers complementary mechanistic advantages and is significant for mitigating hepatocellular damage caused by lipid deposition and iron overload. Enterohepatic circulation plays a crucial role in enhancing the efficacy of certain drugs, and EZE itself possesses enterohepatic circulation properties. Whether the intermolecular interactions of supramolecular systems can enable EZE to influence the in vivo transport of DAP, inducing enterohepatic circulation of DAP and thus achieving synergistic effects warrants further investigation.

[0003] The construction of drug-drug supramolecular systems combines supramolecular technology with combination drug therapy, enabling single-drug administration to achieve the effects of combination therapy, improving the undesirable physicochemical properties of each component. Importantly, the synergistic effect at the molecular level of two-component supramolecular systems with different targets offers further advantages in enhancing efficacy and reducing toxicity, thus advancing the technology and theory of combination drug therapy. EZE is classified as a Biopharmaceutics Classification System (BCS) type II drug, exhibiting low solubility and high permeability. However, the low solubility of EZE limits its oral absorption; its dissolution rate in gastrointestinal fluids is extremely low and unstable, making bioavailability difficult to predict. To improve the therapeutic delivery efficiency and bioavailability of EZE, researchers have explored various strategies, such as ezetimibe-maleic acid cocrystals and nanoparticles. Similarly, to overcome the hygroscopicity problem of DAP active pharmaceutical ingredient, researchers prepared dapagliflozin-citric acid cocrystals. These studies demonstrate the promising application of supramolecular technology in improving the physicochemical properties of drugs such as DAP and EZE. For supramolecular binary compounds, whether their pharmacological activity originates from a simple mixture of components (PM) or from a unique structure formed by weak interactions between the components remains inconclusive. However, the differences in biological behavior between supramolecular systems and PM have been confirmed. Exploring how the forms in which these components exist in different systems affect in vivo and in vitro behavior and therapeutic efficacy is a key focus for future research. Proteomics provides comprehensive analysis of protein expression and modification, revealing the influence of supramolecular systems on protein synthesis, degradation, and functional regulation, thereby identifying target proteins and related signaling pathways. Metabolomics helps elucidate the metabolic mechanisms of interactions between components in cocrystallized systems. For example, cytotoxicity studies of oridonin-nicotinamide cocrystallization revealed differences between oridonin-PM and PM in glycerophospholipid metabolism and cysteine / methionine metabolism. Therefore, molecular regulatory networks constructed by integrating proteomics and metabolomics data can help to delve deeper into the biological effects of supramolecular systems, potentially providing insights into the synergistic mechanisms that distinguish supramolecular systems from simple combination drug administration. Summary of the Invention

[0004] This invention proposes a dapagliflozin-zemetab co-amorphous compound, its preparation method, and its application, which solves the problems of hygroscopicity, solubility, bioavailability, and poor treatment of fatty liver disease related to metabolic dysfunction in related technologies.

[0005] The technical solution of the present invention is as follows:

[0006] This invention proposes a dapagliflozin-ezetimibe co-amorphous compound, which is formed by combining dapagliflozin and ezetimibe.

[0007] Driven by proteomics and metabolomics data, this study explores the synergistic mechanism of a novel DAP-EZE supramolecular system (Co-amorphous, COA) in the treatment of MASLD. This system aims to comprehensively regulate autophagy, reduce lipid deposition, and inhibit ferroptosis through multi-target synergistic action. Specifically, it activates the cholesterol-autophagy axis and regulates enterohepatic circulation and the AMPK / Unc-51-like kinase 1 (ULK1) pathway, ultimately achieving a synergistic effect in enhancing the efficacy of MASLD treatment while reducing potential toxicity.

[0008] This invention constructs a dual-target supramolecular system of dapagliflozin and ezetimibe to improve the bioavailability of both drugs and explore a new form of combined drug use (technological innovation). At the same time, it conducts in-depth research on the synergistic mechanism of the two components in the supramolecular system at the molecular level, and utilizes the cholesterol-autophagy connection in the hepatic-gut axis circulation to achieve hepatocyte-specific autophagy activation and reduce lipid deposition in hepatocytes (theoretical innovation), providing an innovative clinical intervention strategy for the treatment of fatty liver disease related to metabolic dysfunction.

[0009] As a further technical solution, the molar ratio of dapagliflozin to ezetimibe is 1:1.

[0010] As a further technical solution, the dapagliflozin-ezetimibe co-amorphous compound is amorphous and, when subjected to Cu-kα radiation, exhibits an X-ray powder diffraction spectrum with 2θ showing no sharp diffraction peaks.

[0011] As a further technical solution, the dapagliflozin-ezetimibe co-amorphous compound is prepared by rotary evaporation.

[0012] As a further technical solution, the glass transition temperature of the dapagliflozin-ezetimibe co-amorphous compound is 61.56℃.

[0013] As a further technical solution, the dapagliflozin component in the dapagliflozin-ezetimibe co-amorphous compound has a solubility of 22.02 mg / mL and a dissolution rate of 77.43% in a medium with pH=1, a solubility of 26.31 mg / mL and a dissolution rate of 78.62% in a medium with pH=4, and a solubility of 21.28 mg / mL and a dissolution rate of 86.63% in a phosphate buffer solution with pH=6.8.

[0014] The ezetimibe component in the dapagliflozin-ezetimibe co-amorphous compound exhibits a solubility of 79.85 μg / mL and a dissolution rate of 19.83% in a medium with pH=1, a solubility of 483 μg / mL and a dissolution rate of 34.66% in a medium with pH=4, and a solubility of 387 μg / mL and a dissolution rate of 26.30% in a phosphate buffer solution with pH=6.8.

[0015] The present invention also proposes a method for preparing a dapagliflozin-ezetimibe co-amorphous compound, which includes the following steps: mixing dapagliflozin and ezetimibe in a solvent, evaporating the solvent, and drying to obtain the dapagliflozin-ezetimibe co-amorphous compound.

[0016] As a further technical solution, the total mass ratio of dapagliflozin and ezetimibe to the volume ratio of the solvent is 100 mg: 30~50 mL.

[0017] As a further technical solution, the total mass ratio of dapagliflozin and ezetimibe to the volume ratio of the solvent is 100 mg: 40 mL.

[0018] As a further technical solution, the solvent includes one or both of methanol and ethanol.

[0019] As a further technical solution, the mixing is ultrasonic mixing, and the ultrasonic mixing frequency is 30~50kHz, the power is 90~110W, and the time is 30~40min.

[0020] As a further technical solution, the frequency of the ultrasonic mixing is 40kHz, the power is 100W, and the time is 30min.

[0021] As a further technical solution, the temperature during solvent evaporation is 36~40℃, the rotation speed is 45~55rpm, and the time is 20~40min.

[0022] As a further technical solution, the temperature during solvent evaporation is 38°C, the rotation speed is 50 rpm, and the time is 30 min.

[0023] As a further technical solution, the drying includes vacuum drying, wherein the vacuum drying temperature is 40°C and the time is 24 hours.

[0024] The present invention also proposes the application of the dapagliflozin-ezetimibe co-amorphous compound or the dapagliflozin-ezetimibe co-amorphous compound prepared by the aforementioned method in the treatment of metabolic dysfunction-related fatty liver disease.

[0025] The working principle and beneficial effects of this invention are as follows:

[0026] 1. The amorphous compounds of dapagliflozin and ezetimibe of the present invention have good dissolution, solubility and bioavailability.

[0027] 2. The dapagliflozin-ezetimibe co-amorphous compound provided by this invention has been verified in in vitro and in vivo to be significantly more effective than either drug alone in treating metabolic disorders-related fatty liver disease (MASLD). This demonstrates that the co-amorphous compound of this invention can enhance the therapeutic effects of dapagliflozin and ezetimibe on MASLD. Simultaneously, a novel SGLT-2 / NPC1L1 dual-target supramolecular system was successfully constructed, and its significant advantages in optimizing the physicochemical properties of the active pharmaceutical ingredients and improving bioavailability were systematically verified. This invention is the first to utilize supramolecular technology to achieve enterohepatic circulation of one drug with another, thus achieving synergistic effects. Pharmacokinetic studies show that EZE can induce enterohepatic circulation of DAP in vivo. Proteomics and metabolomics analyses indicate that, based on enterohepatic circulation, COA exerts a synergistic therapeutic effect on MASLD through multi-target, multi-pathway regulatory mechanisms, including autophagy activation, ferroptosis inhibition, and cholesterol metabolism balance regulation. Specifically, at the cellular level, COA significantly improved lipid accumulation in the HepG2 cell fatty liver model by activating the AMPK / ULK1 signaling pathway, promoting autophagy, and inhibiting the ferroptosis signaling pathway. Furthermore, COA enhanced the oxidative stress resistance of HepG2 cells by upregulating SOD activity and inhibiting ROS production. Regarding cholesterol metabolism regulation, COA reduced the expression of the cholesterol-absorbing protein NPC1L1 in Caco-2 cells through cholesterol-autophagy linkages, effectively lowering blood lipid concentrations in the HepG2 cell fatty liver model. Further in vivo experiments confirmed that this system upregulates the phosphorylation level of the AMPK-ULK1 signaling pathway in the liver of MASLD mice, significantly improving hepatic steatosis and cholesterol metabolism disorders by regulating cholesterol-autophagy linkages. In summary, based on the physiological basis of enterohepatic circulation, DAP-EZECOA enhances autophagy, inhibits ferroptosis, and reduces lipid deposition and oxidative stress by activating the AMPK / ULK1 signaling pathway and regulating cholesterol-autophagy linkages, thereby reducing lipid deposition and oxidative stress and ultimately improving cholesterol metabolism disorders and insulin resistance, thus achieving synergistic treatment for MASLD. Attached Figure Description

[0028] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0029] Figure 1 This is the characterization spectrum of the dapagliflozin-ezetimibe amorphous compound obtained in Example 1 of this invention.

[0030] In the figure, A is the PXRD spectrum, B is the TMDSC spectrum, C-F are the TG spectra, G is the FT-IR spectrum, and H is... 1 H-NMR characterization diagram;

[0031] Figure 2The physicochemical properties and bioavailability of the dapagliflozin-ezetimibe amorphous compound obtained in Example 1 of this invention are as follows:

[0032] In the figure, A is the solubility diagram of DAP, B is the solubility diagram of EZE, C is the PXRD pattern of the powder after 72 hours of solubility, D~I are the dissolution diagrams, J is the PXRD pattern of the powder after 72 hours of dissolution, K is the hygroscopic diagram, L~M are the stability test diagrams, N~O are the drug-time curves in SD rats, P is the binding conformation of DAP and EZE molecules after molecular docking, Q is the binding conformation of DAP and NPC1L1 protein after molecular docking, R is the binding conformation of EZE and NPC1L1 protein after molecular docking, and S is the binding conformation of the DAP-EZE assembled molecule and NPC1L1 protein after molecular docking (red represents hydrogen bonds; cyan represents halogen bonds; purple represents hydrophobic interactions).

[0033] In the picture, * P <0.05, ** P <0.01, *** P <0.001;

[0034] Figure 3 This invention provides a cellular-level efficacy evaluation of dapagliflozin-ezetimibe co-amorphous compounds: cytotoxicity analysis and observation of intracellular lipid droplet accumulation in each group using Oil Red O staining and Bodipy staining methods.

[0035] In the figure, A represents the cell survival rate of fatty liver model cells with different concentrations of sodium palmitate / sodium oleate; B represents the cell survival rate of HepG2 cells with different concentrations of the drug; C represents the cell survival rate of Caco-2 cells with different concentrations of the drug; D represents Oil Red O staining; E represents Bodipy immunofluorescence staining; and F represents the average gray value of fluorescence corresponding to Bodipy immunofluorescence staining.

[0036] In the picture, * P <0.05, ** P <0.01, *** P <0.001;

[0037] Figure 4 This is a diagram showing the intracellular inhibition of ferroptosis in different groups of the HepG2 cell fatty liver model using dapagliflozin-ezetimibe co-amorphous compound, as described in this invention.

[0038] In the figure, A shows the Western blot results of p-AMPK and p-ULK1 in each treatment group; B shows the quantitative analysis of p-AMPK protein expression; C shows the quantitative analysis of p-ULK1 protein expression; D shows the CLSM (confocal microscopy) image of LC3; E shows the quantitative statistical analysis of LC3; F shows the CLSM image of P62; and G shows the quantitative statistical analysis graph of P62.

[0039] In the picture, * P <0.05, ** P <0.01, *** P <0.001;

[0040] Figure 5 This is a graph showing the detection of ferroptosis-related indicators in the HepG2 cell fatty liver model using dapagliflozin-ezetimibe co-amorphous compound, as described in this invention.

[0041] In the figure, A represents the iron ion content, B represents the CLSM image of ROS, C represents the quantitative statistical analysis of ROS, D represents the CLSM image of GSH, E represents the quantitative statistical analysis of GSH, F represents the CLSM image of GPX4, and G represents the quantitative statistical analysis of GPX4.

[0042] In the picture, * P <0.05, ** P <0.01, *** P <0.001;

[0043] Figure 6 This diagram illustrates the effect of the dapagliflozin-ezetimibe co-amorphous compound of the present invention on the regulation of NPC1L1 protein expression in SP / SO-induced Caco-2 cells and blood lipid levels in a HepG2 cell fatty liver model via cholesterol-autophagy linkage.

[0044] In the figure, A is the CLSM image of NPC1L1, B is the quantitative statistical analysis of NPC1L1, C is the WB result of NPC1L1, D is the statistical analysis of protein expression, E is triglycerides (TRIG), F is total cholesterol (TC), G is low-density lipoprotein cholesterol (LDL-C), and H is high-density lipoprotein cholesterol (HDL-C).

[0045] Figure 7 This is a graph showing the body weight and glucose metabolism levels of MASLD mice after COA intervention according to this invention.

[0046] In the diagram, A represents Body weight (n=6), B represents OGTT (n=3), C represents OGTT-AUC (n=3), D represents ITT (n=3), and E represents ITT-AUC (n=3).

[0047] In the figure, mean ± SD * P <0.05, ** P <0.01, *** P <0.001;

[0048] Figure 8 This is a graph showing the effects of COA intervention on blood lipid levels and liver function in MASLD mice.

[0049] In the diagram, A stands for TRIG, B for TC, C for LDL-C, D for HDL-C, E for ALT, and F for AST.

[0050] In the figure, mean ± SD, * P <0.05, ** P <0.01, *** P <0.001;

[0051] Figure 9 This invention improves hepatic steatosis in MASLD mice after COA intervention.

[0052] In the image, A represents the abdominal ultrasound image, and B represents the average liver / kidney grayscale ratio.

[0053] In the figure, mean ± SD * P <0.05, ** P <0.01, *** P <0.001;

[0054] Figure 10 This invention relates to liver weight and liver index in MASLD mice after COA intervention.

[0055] In the figure, A represents liver weight and B represents liver index (calculated as liver weight / body weight).

[0056] In the figure, mean ± SD * P <0.05, ** P <0.01, *** P <0.001;

[0057] Figure 11 This is a liver tissue section of MASLD mice after COA treatment according to the present invention.

[0058] In the figure, A shows the H&E staining results of mouse liver tissue in each group; B shows the Oil Red O staining results of mouse liver tissue in each group; C shows the PAS staining results of mouse liver tissue in each group (Scale bar=100µm).

[0059] Figure 12 This invention relates to the expression levels of p-AMPK / p-ULK1, LC3, and p62 in the livers of MASLD mice after COA intervention.

[0060] In the figure, A is the protein blot band map, B is the p-AMPK expression level map, C is the p-ULK1 expression level map, D is the LC3 expression level map, and E is the p62 expression level map.

[0061] In the figure, mean ± SD * P <0.05, ** P <0.01, *** P <0.001;

[0062] Figure 13 This invention relates to the levels of liver oxidative stress, lipid peroxidation, and ferroptosis in MASLD mice after COA intervention.

[0063] In the figure, A represents liver GSH content, B represents liver SOD activity, C represents liver MDA content, D represents liver GPX4 white matter blot pattern, and E represents liver GPX4 protein expression level.

[0064] In the figure, mean ± SD * P <0.05, ** P <0.01, *** P <0.001;

[0065] Figure 14 This is the gross Oil Red O staining (Scale bar=100µm) of the aorta of MASLD mice after COA intervention according to the present invention;

[0066] Figure 15 The following are the histopathological staining results of MASLD mice after COA intervention according to the present invention: In the figure, A is the result of HE staining of pancreas, B is the result of immunohistochemical staining of pancreas, and C is the result of HE staining of small intestine (Scale bar=100µm).

[0067] Figure 16 This refers to the protein expression level of NPC1L1 in the intestines of MASLD mice after COA intervention according to this invention.

[0068] In the figure, mean ± SD * P <0.05, ** P <0.01, *** P <0.001;

[0069] Figure 17 This invention relates to cholesterol levels in the livers of MASLD mice after COA intervention.

[0070] In the figure, mean ± SD * P <0.05, ** P <0.01, *** P <0.001;

[0071] Figure 18 This is a schematic diagram illustrating the pharmacodynamics and mechanism of the SGLT-2 / NPC1L1 dual-receptor supramolecular system for treating MASLD. Detailed Implementation

[0072] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0073] Example 1

[0074] The preparation method of dapagliflozin-ezetimibe co-amorphous form includes the following steps:

[0075] The rotary evaporation method was used: 100.00 mg of a physical mixture of dapagliflozin and ezetimibe (molar ratio of dapagliflozin to ezetimibe was 1:1) was placed in a 100 mL round-bottom flask, 40 mL of ethanol was added, and the mixture was ultrasonically mixed at 40 kHz and 100 W for 30 min at room temperature to ensure complete dissolution. The mixture was then rotated at 38 °C and 50 rpm for 20 min. The solvent was evaporated under the same conditions, and the mixture was vacuum dried at 40 °C for 24 h to obtain the dapagliflozin-ezetimibe co-amorphous compound.

[0076] Experimental Example 1: Structural Characterization of Co-amorphous Materials

[0077] 1. X-ray powder diffraction detection

[0078] Appropriate amounts of DAP (dapagliflozin), EZE (ezetimibe), and COA (DAP-EZE co-amorphous compound prepared in Example 1) were weighed onto the stage, and the X-ray diffraction patterns of each sample were measured. The scanning range was 5° to 40° (2θ), the step size was 0.02°, and the scanning speed was 10° / min. The measured PXRD patterns are shown below. Figure 1 As shown in Figure A.

[0079] As can be seen from the figure, crystalline EZE has obvious characteristic diffraction peaks, while the PXRD pattern of COA shows a camel-shaped halo without sharp characteristic peaks, and the crystal diffraction peaks disappear, which initially indicates the formation of amorphous morphology.

[0080] 2. DSC-TG detection

[0081] TMDSC analysis: 4 mg of DAP, EZE, and COA (DAP-EZE co-amorphous compound prepared in Example 1) powder were precisely weighed into aluminum crucibles, using an empty crucible as a reference, and the TMDSC spectra of each sample were measured. The test conditions were: temperature 0–250 °C, heating rate 2.000 K / min, period 60 seconds, and amplitude 0.5 K.

[0082] Thermogravimetric (TG) analysis: 4 mg of each of the following powders (DAP, EZE, PM, and COA – the DAP-EZE co-amorphous compound prepared in Example 1) were accurately weighed into an aluminum crucible. The test conditions were: flow rate 40 mL / min, temperature range 30–800 °C, and heating rate 20 °C / min.

[0083] The results are as follows Figure 1 As shown in Figure B, an endothermic step appeared in the reversible heat flow signal curve of the prepared COA, proving the formation of the DAP-EZE co-amorphous material. Figure 1 C~ Figure 1 As can be seen from the TG spectrum of the DAP-EZE co-amorphous compound, the DTG curve clearly shows that EZE has two thermal decomposition peaks, indicating that its decomposition process is a two-step decomposition, while DAP has four thermal decomposition peaks, indicating that its decomposition process is a four-step decomposition. The difference between PM and COA is significant, indicating that the physical mixture is just a mixture of samples, while the co-amorphous compound has undergone a fundamental change.

[0084] 3. FT-IR test

[0085] The FT-IR spectra of DAP, EZE, and COA (co-amorphous compounds) were measured using the KBr pellet method on a Spectrum Two FT-IR spectrometer (PerkinElmer Company, USA). 1 mg of DAP, EZE, and COA powder were weighed out, and 160 mg of KBr was weighed out. The powders were mixed in equal increments and thoroughly ground, then pelleted. A blank KBr pellet was used as a reference, and the resolution was 4 cm⁻¹. -1 The scanning range is 4000 to 400 cm. -1 The total number of scans was 40, and the signals were accumulated and averaged. The FT-IR spectrum is as follows: Figure 1 As shown in G.

[0086] In the figure, COA (DAP-EZE supramolecular system) is attributed to the stretching vibration of C=O in the ezetimibe structure, which originates at 1731 cm⁻¹. -1 Moved to 1726cm -1 CC stretching vibration 1155cm -1 Moved to 1157cm respectively -1 It is inferred that the C=O group in ezetimibe forms hydrogen bonds with certain groups in the dapagliflozin structure. The stretching vibration attributable to OH in the dapagliflozin structure is 3407 cm⁻¹. -1 Move to 3399cm -1 CO stretching vibration from 1244 cm -1 1178cm -1 Move to 1227cm -1 1176cm -1 This indicates that OH participates in the formation of hydrogen bonds between dapagliflozin and ezetimibe in the co-amorphous supramolecular system, and the dapagliflozin-ezetimibe supramolecular system was successfully constructed.

[0087] 4. 1 H-NMR test

[0088] To investigate the weak interaction between DAP and EZE, NMR spectra of DAP, EZE, PM, and COA were obtained at room temperature using an AVANCE NEO 600 NMR spectrometer in DMSO-d6 (deuterated dimethyl sulfoxide). 1 H-NMR spectra. All spectra were calibrated using a DMSO-d6 water peak signal with a δ of 2.50 ppm, a spectral width of 11905 Hz, a cumulative count of 12000, and a relaxation time of 1 s. Figure 1 As shown in H.

[0089] In the figure, the peak at 3.43 ppm is stronger in COA compared to PM, while the intensity and shift of the hydrogen atoms at 4.44 ppm and 4.33 ppm, attributed to the phenolic hydroxyl group of dapagliflozin, are slightly different. The hydrogen atoms at 4.44 ppm and 4.43 ppm in PM, attributed to the phenolic hydroxyl group of dapagliflozin, shift to 4.49 ppm and 4.45 ppm in COA, moving towards a higher field. This illustrates the difference between COA and PM. In the DAP raw material spectrum, the peak at 1.11 ppm is unique to dapagliflozin and disappears after the formation of a supramolecular system, but it is present in both DAP raw material and PM, indicating intermolecular interactions between DAP and EZE in solution.

[0090] Experimental Example 2: Determination of the solubility of dapagliflozin-ezetimibe co-amorphous compound

[0091] The equilibrium solubility of COA (dapagliflozin-ezetimibe co-amorphous compound prepared in Example 1) at pH=1, pH=4, and pH=6.8 was determined using the shake-flask method. Excess sample was added to shake flasks containing 10 mL of media at different pH values ​​(pH=1, pH=4, pH=6.8), placed on an air bath shaker, and the air bath shaker was set to a constant temperature of 37±0.5℃ and a rotation speed of 150 r·min. -1 Shake until the concentrations of dapagliflozin and ezetimibe in the solution no longer change; this is the saturated solution. After 72 hours of equilibrium, use a disposable syringe to draw up the suspension and filter it through a 0.22 μm microporous membrane. Utilization-performance liquid chromatography (HPLC) is used to determine the equilibrium solubility of DAP, EZE, PM, and COA. The solid phase is collected, dried, and PXRD is measured. All experiments are performed in triplicate. The equilibrium solubility of DAP, EZE, PM, and COA in media at pH (1, 4, 6.8) is shown in [reference needed]. Figure 2 From A to 2B, with increasing pH, the solubility of EZE in the COA supramolecular system generally increases, while the solubility of COA is significantly better than that of PM. After determining the equilibrium solubility in pH (1, 4, 6.8) media, solid samples were collected for PXRD analysis. Figure 2 As shown in Figure C, no new diffraction peaks appeared for COA, indicating that COA remains stable after repeated dissolution and precipitation in the solvent.

[0092] Experimental Example 3: Determination of dissolution rate of dapagliflozin-ezetimibe co-amorphous compound

[0093] The dissolution rates of DAP, EZE, PM, and COA (dapagliflozin-ezetimibe co-amorphous compound prepared in Example 1) were determined using the second method (paddle method) in Part IV of the Pharmacopoeia on an RC-808D dissolution apparatus (Tianjin Tianda Tianfa Co., Ltd.). Accurately weigh 50 mg of DAP, 50 mg of EZE, 100 mg of PM (containing 50 mg of both DAP and EZE), and 100 mg of COA (containing 50 mg of both DAP and EZE). Use 500 mL of dissolution medium at pH (1, 4, 6.8). Stir at 100 rpm at 37 ± 0.5 °C. Samples are taken at 1 min, 5 min, 10 min, 15 min, 25 min, 40 min, 60 min, 120 min, 240 min, and 360 min, respectively. Simultaneously, the same volume of dissolution medium at the same temperature is added. After filtration through a 0.22 μm microporous membrane, the samples are diluted and their concentrations are determined by high-performance liquid chromatography (HPLC). All experiments are performed in triplicate. The dissolution rates of DAP, EZE, PM, and COA in pH (1, 4, 6.8) media are shown in [reference needed]. Figure 2 D~ Figure 2 I.

[0094] As shown in the figure, the overall dissolution rate of EZE increased after the formation of the co-amorphous form, and the dissolution rate of the co-amorphous COA was significantly better than that of the physical mixture PM. The dissolution rate of DAP also increased with increasing pH, and at pH=6.8, the solubility of the co-amorphous form was significantly better than that of the physical mixture. After the dissolution rate measurements at pH (1, 4, 6.8), solid samples were collected for PXRD, and the results are as follows... Figure 2 As shown in Figure J, no new diffraction peaks were observed in the co-amorphous COA, indicating that the properties of the co-amorphous COA remain stable after repeated dissolution and precipitation in the solvent.

[0095] Experimental Example 4: Determination of the hygroscopicity of dapagliflozin-ezetimibe co-amorphous compound

[0096] The hygroscopicity test was conducted according to the guidelines for hygroscopicity testing as specified in the 2020 edition of the Pharmacopoeia of the People's Republic of China. A dry, stoppered weighing bottle (50mm × 15mm) was placed in a suitable constant-temperature desiccator at 25℃ ± 1℃ (with a saturated ammonium chloride solution at the bottom) one day before the test, and the weight was accurately recorded as m1. Appropriate amounts of DAP, EZE, and COA (dapagliflozin-ezetimibe amorphous compound prepared in Example 1) were placed in the weighing bottle, approximately 1mm thick, and the weight was accurately recorded as m2. The weighing bottle was left open and placed under the above conditions with the cap. After 24 hours, the weight was accurately recorded as m3. The COA was placed at 25℃ and 80% relative humidity for 24 hours, and the percentage weight gain was calculated. The results are as follows: Figure 2 K. Wherein, the percentage weight gain = (m3-m2) / (m2-m1)×100%.

[0097] As can be seen from the figure, the COA system can interact with water and has a certain hygroscopicity, but it is not high, lower than that of DAP and PM, and is relatively stable. The experimental results show that: with the increase of the ezetimibe sample, the hygroscopicity of the co-amorphous form is improved, making the sample tend to be stable, and there is a statistical difference.

[0098] Experimental Example 5 Stability Experiment Determination of Dapagliflozin-Ezetimibe Co-Amorphous

[0099] Room temperature storage stability experiment: The COA (dapagliflozin-ezetimibe co-amorphous obtained in Example 1) sample was placed in a room temperature desiccator for 0 days, 90 days, and 180 days for the room temperature storage stability experiment (as Figure 2 shown in L). It can be seen from the figure that COA still maintains the amorphous state and no crystallization phenomenon occurs, indicating that the supramolecular system sample is stable and can be stored for a long time;

[0100] High temperature and high humidity experiment: It was determined according to the guiding principle of high temperature and high humidity test specified in the Pharmacopoeia of the People's Republic of China (2020 Edition). DAP and COA (dapagliflozin-ezetimibe co-amorphous obtained in Example 1) were exposed and placed in weighing bottles, and placed under the conditions of 45 °C and relative humidity (RH) 75%. Samples were taken and observed and determined on the 0th day, 3rd day, 7th day, and 14th day. The samples were placed at 40 °C and 75% for 0, 3, 7, 14 days for the high temperature and high humidity experiment (as Figure 2 shown in M). In the figure, DAP and COA still maintain the amorphous state and no crystallization phenomenon occurs, indicating that the samples are stable and the stability of the co-amorphous form is less affected by temperature and humidity.

[0101] Experimental Example 6 Pharmacokinetic Determination of Dapagliflozin-Ezetimibe Co-Amorphous

[0102] Twenty-four male SD rats with a body weight of about 250 ± 25 g were selected as the test animals (from the Hebei Experimental Animal Center, SPF, license number: SCXK (Hebei) 2022-001). They were fasted for 12 h before the experiment until the end of the experiment and were allowed free access to water. All animal facilities complied with the requirements of the International Association for Assessment and Accreditation of Laboratory Animal Care. The feeding environment of the experimental animals: temperature 22-24 °C, relative humidity 50 ± 5%, 12 h of light followed by 1 h of darkness.

[0103] Twenty-four SD rats were randomly divided into four groups of six each. Each group was orally administered DAP, EZE, PM, or COA (dapagliflozin-ezetimibe amorphous compound prepared in Example 1), respectively. The rats were housed in an environment with a temperature of 22–24°C and a relative humidity of 50 ± 5%. Before the experiment, the rats were weighed and the dosage was calculated (equivalent to 80 mg / kg dapagliflozin and 80 mg / kg ezetimibe). The rats were fasted for 12 hours before administration, but had free access to water. A single gavage administration was performed. At 0.083 h, 0.25 h, 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 5 h, 8 h, 10 h, 16 h, and 24 h, 0.3 mL of blood was collected from the orbital sinus into anticoagulant tubes containing dried heparin. The tubes were centrifuged at 3500 rpm for 10 min at 4°C, and the plasma supernatant was stored at -20°C. After processing, 10 μL of the supernatant from the plasma sample was accurately measured and injected into the liquid chromatograph, and the chromatogram was recorded. A drug-time curve was plotted with blood collection time on the x-axis and the concentrations of DAP and EZE in the plasma sample on the y-axis. Pharmacokinetic parameters such as AUC0-t and Cmax were calculated using DAS2.0 software, and statistical analysis of the pharmacokinetic parameters was performed using SPSS. The final results were set at a significance level of 0.05; a p-value < 0.05 was considered statistically significant. The concentration-time curves of DAP and EZE in rat plasma are shown below. Figure 2 N~ Figure 2 As shown in O.

[0104] In the figure, some drugs exhibit a second peak on the plasma concentration-time curve due to enterohepatic circulation, i.e., a bimodal phenomenon. For the amorphous DAP component, co-amorphous DAP showed increased Cmax and AUC0-t compared to PM and amorphous DAP; the results indicate that COA has higher bioavailability than single-drug DAP or PM of both. Dapagliflozin also exhibited enterohepatic circulation due to the influence of ezetimibe. For the amorphous EZE component, COA showed increased Cmax and AUC0-t compared to PM and EZE, and also exhibited a bimodal phenomenon, indicating that EZE also exhibits enterohepatic circulation after being prepared into COA. These experimental results validate the synergistic enterohepatic circulation of supramolecular systems, laying the foundation for hepatocyte-specific autophagy activation.

[0105] Experimental Example 7

[0106] The three-dimensional structures of DAP (CID: 9887712) and EZE (CID: 150311) were obtained from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ). The crystal structure of the NPC1L1 protein (PDBID: 3QNT) was obtained from the protein database (https: / / www.rcsb.org / ). Since no specific binding pocket was provided, global docking was performed. Hydrogenation was performed on both ligands and the protein receptor using AutoDockTools-1.5.7. Using AutodockVina 1.2.5 software, the binding conformations between DAP and EZE, DAP and protein, EZE and protein, and DAP-EZE assembled molecules and proteins were studied through molecular docking. The conformation with the highest score was selected for further analysis, and the binding affinity was calculated using AutovinaScore.

[0107] Intermolecular interactions are a crucial factor determining the effectiveness of supramolecular drug assembly. This study used... 1 1H-NMR spectroscopy confirmed the weak interaction between the two APIs (DAP and EZE) in the supramolecular solution. Previous pharmacokinetic results indicated that EZE itself possesses enterohepatic circulation characteristics. After preparation as a COA, DAP in the system was also affected by EZE, exhibiting enterohepatic circulation. EZE is the main driver of this circulation, with its direct target being the NPC1L1 protein. Enterohepatic circulation increases the chance of drug binding to the NPC1L1 receptor in the small intestine, allowing the reabsorbed COA to continuously inhibit NPC1L1 and prolong the drug's effect. However, the stability of the intermolecular forces in the COA during this process requires further investigation. To investigate the stability of intermolecular forces in the supramolecular system during enterohepatic circulation and to confirm the effect of these weak interactions on the target, molecular docking was used to analyze the interaction energy and stability between the molecular conformation and the target protein, emphasizing the role of hydrogen bonds and their influence on the NPC1L1 receptor. The binding mode is as follows: Figure 2 P~ Figure 2 As shown in S.

[0108] As shown in the figure, in the binding analysis of DAP and EZE, 3 pairs of hydrogen bonds and 2 pairs of π-π interactions were observed, with a binding affinity of -3.29 kcal / mol (e.g., Figure 2 P). In the docking results between DAP and proteins, no hydrogen bonds were formed, and the binding affinity was -6.13 kcal / mol (e.g., Figure 2 Q). During EZE docking with proteins, EZE forms hydrogen bonds with residues V191, R201, and D208, with a binding affinity of -6.19 kcal / mol (e.g., ...). Figure 2 R). For example, Figure 2As shown in Figure S, during the docking of the DAP-EZE assembly molecule with the protein, the assembly molecule formed two hydrogen bonds with residue T219, a halogen bond with N204, and a hydrophobic interaction with D208, resulting in a significantly increased binding affinity (-8.15 kcal / mol).

[0109] Docking results showed that the DAP-EZE assembled molecules bound to the protein more strongly than individual DAP or EZE molecules. The DAP-EZE assembled molecules formed a wider variety of interactions with the protein, resulting in more stable binding within the protein cavity. Binding affinity analysis further indicated that the DAP-EZE assembled molecules had a significantly higher affinity for the protein than individual DAP or EZE molecules. These results suggest that the DAP-EZE assembled molecules exhibit greater stability and stronger affinity when binding to the NPC1L1 protein.

[0110] 1. In vitro cellular level efficacy evaluation:

[0111] 1.1 Cell Culture and Treatment

[0112] Human HepG2 and Caco2 cells were provided by the School of Pharmacy, Hebei Medical University (Shijiazhuang, Hebei, China) and cultured in DMEM medium 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 maintained overnight in DMEM containing normal glucose after 60% confluence. A HepG2 fatty liver in vitro cell model induced by sodium palmitate / sodium oleate (2:1) was designed and established. The optimal ratio of dapagliflozin-ezetimibe co-amorphous agents for the treatment of metabolic-related fatty liver disease was screened at the cellular level. Protein expression of the NPC1L1 receptor in Caco-2 and HepG2 cells, as well as cholesterol uptake in HepG2 cells, were evaluated. The study investigated whether the supramolecular system could activate the AMPK pathway, increase ULK1 phosphorylation, promote autophagy (as evidenced by increased LC3B protein levels and decreased p62 levels), reduce intracellular lipid accumulation, and improve ferroptosis. The effects of ferroptosis-related markers on the levels of MDA, GSH, and antioxidant enzymes in HepG2 liver cells were investigated. Cells induced by sodium palmitate / sodium oleate (2:1) were treated in the absence or presence of different concentrations of DAP, EZE, PM, and COA (dapagliflozin-ezetimibe co-amorphous compound prepared in Example 1).

[0113] 1.2 Cytotoxicity analysis and observation of intracellular lipid droplet accumulation in each group using Oil Red O staining and Bodipy staining:

[0114] MTT assay for cell viability: Cells were inoculated at a concentration of 6 × 10⁶ cells / year. 3 / wells were seeded in 96-well plates and incubated at 37°C and 5% CO2 for 24 h. Then, sodium palmitate / sodium oleate (2:1) was used to induce lipid accumulation for 24 h. Different concentrations of DAP, EZE, physical mixture (PM), and DAP-EZE COA were added, and the effects on cell viability of each group were investigated by MTT assay.

[0115] Oil Red O staining: HepG2 cells in logarithmic growth phase were seeded into 12-well plates containing cell spreaders and cultured for 24 h. Lipid accumulation was induced for 24 h with sodium palmitate / sodium oleate (2:1). Cells were divided into a control group (CON) receiving no treatment, and a simple fatty liver cell model group (MOD), DAP, EZE, physical mixture (PM), and COA group. Lipid accumulation was induced with sodium palmitate / sodium oleate (2:1) before drug administration, followed by 24 h of incubation. The cells were fixed with 4% paraformaldehyde every other day, incubated with Oil Red O solution at 37°C, destained with 75% ethanol, stained with hematoxylin, destained, mounted, and observed under an upright microscope for lipid droplet accumulation.

[0116] Bodipy staining: HepG2 cells in logarithmic growth phase were seeded into 12-well plates containing cell spreaders and cultured for 24 hours. Lipid accumulation was induced for 24 hours with sodium palmitate / sodium oleate (2:1). Cells were then grouped and treated (grouping was the same as for Oil Red O staining). The 12-well plates were removed and the culture medium discarded the next day. The cells were washed once with PBS. Then, the cells were fixed with 4% paraformaldehyde for 20 minutes, the paraformaldehyde was discarded, and the cells were washed three times with PBS. Under light-protected conditions, 2 mL of Bodipy working solution (Bodipy stock solution:PBS = 1:1000) was added to each well for staining for 30 minutes. The cells were then washed three times with PBS, followed by nucleus staining with DAPI for 10 minutes, and then washed five times with PBS. The cells were mounted with an anti-fluorescence quencher and observed under an upright microscope.

[0117] The MTT assay was used to determine the toxic effects of different concentrations and systems of the supramolecular system COA on HepG2 and Caco2 cell models of fatty liver induced by sodium palmitate / sodium oleate (PA / OA). Results are as follows: Figure 3 As shown in A~3F, for HepG2 cells, sodium palmitate / sodium oleate (300μM:150μM) was ultimately selected for subsequent experimental studies (e.g., ...). Figure 3 A). Through screening different concentrations of COA in the supramolecular system, COA (DAP:EZE=10μM:10μM) was finally determined to be the optimal therapeutic concentration. Figure 3 BC). Oil Red O staining results showed that the number of orange-red lipid droplets in HepG2 cells of the MOD group was significantly increased compared with that of the CON group; compared with the MOD and PM groups, the number of orange-red lipid droplets in HepG2 cells of the COA (supramolecular system group) was significantly reduced, indicating that the supramolecular system had significant efficacy. Figure 3D). Therefore, this invention used Bodipy C-11 to verify the advantages of the supramolecular system COA in improving lipid peroxidation. Compared with the model groups MOD and PM, the supramolecular system significantly improved the level of palmitic acid / oleic acid-induced lipid peroxidation and alleviated intracellular lipid accumulation (D). Figure 3 E- Figure 3 F).

[0118] 1.3 Detection of autophagy-related proteins in HepG2 cells:

[0119] AMPK / ULK1 protein expression level detection

[0120] This study investigated whether DAP-EZE COA activates autophagy through the AMPK / ULK1 signaling pathway, thereby reducing lipid deposition and improving MASLD, using Western blotting (WB). HepG2 cells were treated with SP / SO (2:1) to induce lipid accumulation for 24 h. After incubation with the selected drug for 24 h, the cells were washed with PBS and then lysed using cell lysis buffer containing PMSF and a phosphatase inhibitor to extract total protein. Protein samples were mixed with loading buffer, denatured, and then the protein expression levels of p-AMPK and p-ULK1 in each group of cells were detected by WB.

[0121] To verify the effect of COA on the autophagy pathway in a HepG2 cell fatty liver model, the protein expression levels of p-AMPK and p-ULK1 were detected by Western blotting. The results showed that compared with the CON group, the protein expression levels of p-AMPK and p-ULK1 in the HepG2 cell fatty liver model were significantly reduced in the MOD group. After drug intervention, the expression of p-AMPK and p-ULK1 proteins in HepG2 cells significantly increased in the DAP, EZE, PM, and COA treatment groups, with a more significant increase in the COA group (P<0.05). The supramolecular system upregulated the expression of autophagy-related p-AMPK and p-ULK1 proteins in the HepG2 cell fatty liver model. Figure 4 A- Figure 4 C).

[0122] Immunofluorescence staining for LC3 and p62 proteins

[0123] HepG2 cells at 2×10 4Cells were seeded at a density of 1 / 2 well in 24-well plates covered with coverslips and cultured at 37°C and 5% CO2 for 24 h. Subsequently, cells were treated with SP / SO (2:1) to induce lipid accumulation for 24 h, followed by the addition of different groups of drugs. After 24 h of incubation, cells were incubated overnight with primary antibodies against autophagy-related proteins (LC3, p62), followed by incubation with secondary antibodies. Cells were then washed with PBS, fixed with 4% paraformaldehyde, stained with DAPI, mounted, and observed using CLSM.

[0124] The effects of different sample groups on the expression of autophagy-related LC3 and p62 proteins in the HepG2 cell fatty liver model are shown in the figure. Figure 4 D- Figure 4 (G). As shown in the figure, in HepG2 cells of the MOD group, the expression level of LC3 protein was significantly lower than that of the CON group, while the expression level of P62 protein was significantly higher, indicating an inhibition of autophagy. After treatment with the supramolecular system, the expression level of LC3 protein was significantly upregulated, while the expression level of P62 protein was significantly downregulated. The results indicate that the supramolecular system may effectively reduce intracellular lipid accumulation by activating the intracellular autophagy pathway, promoting the clearance of damaged organelles and proteins.

[0125] 1.4 Observation of intracellular ferroptosis inhibition in HepG2 cell fatty liver model:

[0126] Iron content detection

[0127] HepG2 cells were used at a rate of 1×10 7 Cells were seeded at a density of / wells in culture dishes and cultured at 37°C and 5% CO2 for 24 h. Lipid accumulation was induced for 24 h with SP / SO (2:1). Groups receiving the drug (CON, MOD, DAP, EZE, PM, and COA) were incubated for 24 h. Cells were digested and centrifuged to collect the cell pellet. Cells were sonicated to disrupt the cell structure, and the supernatant was collected and stored on ice for analysis. Quantitative analysis of iron content was performed using an iron content assay kit with the ferroazine colorimetric method, measuring absorbance.

[0128] Intracellular iron content in HepG2 cells was detected using an iron ion assay kit. Results are shown below. Figure 5 A. The results showed that the iron content in the cells of the MOD group was significantly increased compared to the CON group. After treatment with different intervention groups (DAP, EZE, PM, and COA), the intracellular iron content in each intervention group was significantly lower than that in the MOD group, with the COA group showing a more significant reduction in iron content (P<0.05). These results indicate that the SP / SO effect leads to an increase in intracellular iron content in HepG2 cells, while the supramolecular system can effectively alleviate intracellular iron overload.

[0129] ROS detection

[0130] The effects of different drugs on the regulation of ROS production in SP / SO-induced HepG2 cells were investigated. HepG2 cells were grown at a rate of 2 × 10⁶ cells / year. 4 Cells were seeded at a density of / wells in 24-well plates and cultured overnight under suitable conditions. After inducing lipid accumulation for 24 h with SP / SO (2:1), cells were treated with different concentrations of DAP, EZE, PM, and COA, either in the absence of these drugs or in the presence of these drugs, followed by incubation for 24 h with different drug groups. Cells in normal glucose DMEM were considered the CON group. After incubation, cells were washed with PBS, stained with the DCFH-DA probe for 20 min, fixed with 4% paraformaldehyde, stained with DAPI, mounted, and observed and imaged using CLSM.

[0131] Increased iron ion levels facilitate the Fenton reaction, leading to elevated intracellular ROS levels. The DCFH-DA fluorescent probe exhibits green fluorescence after interaction with ROS. The ROS levels in different experimental groups after SP / SO induction treatment of HepG2 cells are shown below. Figure 5 As shown in Figure BC, the HepG2 cell fatty liver model exhibited a significant green fluorescence signal, indicating that intracellular iron overload induced the Fenton reaction, ultimately leading to the accumulation of intracellular ROS. After intervention in the DAP, EZE, PM, and COA groups, the intracellular ROS levels in HepG2 cells were significantly lower than those in the MOD group, with the COA group showing the best intervention effect (P<0.05). These results indicate that SP / SO can induce the accumulation of intracellular ROS in HepG2 cells, while the supramolecular system can effectively alleviate intracellular ROS accumulation.

[0132] GSH level

[0133] GSH fluorescence imaging of HepG2 cells was performed using Cell Tracker Green CMFDA. HepG2 cells were stored at a concentration of 2 × 10⁶ cells / cells. 4 Cells were seeded in 24-well plates with coverslips and cultured at 37°C and 5% CO2 for 24 h. Lipid accumulation was induced by SP / SO (2:1) for 24 h, with grouped administration and incubation for 24 h. Cell samples were fixed with 4% paraformaldehyde for 20 min, then 1 μg / mL CMFDA staining agent (1 μL stock solution added to 1 mL culture medium) was added, and the cells were incubated at 37°C for 30 min. After washing three times with PBS, CLSM observation was performed.

[0134] GSH plays a crucial role in scavenging lipid peroxides. As a key substrate for GPX4 to exert its catalytic activity, it can efficiently inhibit iron-dependent cell death. Figure 5DE represents the GSH expression level in a HepG2 cell fatty liver model after drug treatment. As shown in the figure, the GSH content in the MOD group was significantly lower than that in the CON group in HepG2 cells. Compared with the MOD group, the drug treatment group showed enhanced GSH activity in HepG2 cells, and the increase in GSH activity was more significant after supramolecular system treatment (P<0.05). The results indicate that SP / SO treatment can lead to a decrease in intracellular GSH content in HepG2 cells, while the supramolecular system can effectively increase intracellular GSH levels, thereby inhibiting ferroptosis.

[0135] GPX4 generation

[0136] HepG2 cells at 2×10 4 / wells were seeded into 24-well plates with coverslips and incubated at 37°C and 5% CO2 for 24 h. After 24 h of lipid accumulation induced by SP / SO (2:1), different groups of drugs were added. After 24 h of incubation, the cells were stained with GPX4 probe, washed with PBS, fixed with 4% paraformaldehyde, stained with DAPI nuclei, mounted, and observed by CLSM.

[0137] GSH depletion can inhibit GPX4 activity, and this inhibition further promotes the accumulation of lipid peroxides in cells, ultimately leading to ferroptosis. Figure 5 Figure FG shows the GPX4 expression level in a HepG2 cell fatty liver model after drug treatment. As shown in the figure, in the HepG2 cell fatty liver model, intracellular ferroptosis inhibition in each group was observed using GPX4 staining. It was found that ferroptosis was closely related to the decrease in GPX4 levels induced by intracellular iron overload. The results indicate that the supramolecular system can promote the expression of the ferroptosis-related protein GPX4 and inhibit ferroptosis in HepG2 cells induced by SP / SO.

[0138] 1.5 Effects of cholesterol-autophagy linkage regulation on cholesterol metabolism in Caco-2 and HepG2 cells:

[0139] NPC1L1 protein expression level in Caco-2 cells

[0140] To observe the effects of the drug on cells, Caco-2 cells were cultured at a density of 2 × 10⁻⁶. 4 Cells were seeded in 24-well plates (with or without coverslips) and incubated at 37°C and 5% CO2 for 24 h. After inducing lipid accumulation with SP / SO (2:1) for 24 h, different groups of drugs were added and incubated for another 24 h. Subsequently, NPC1L1 immunostaining, 4% paraformaldehyde fixation, DAPI staining of cell nuclei, mounting, and observation of cells using CLSM were performed.

[0141] HepG2 cell intracellular lipid level measurement

[0142] HepG2 cells were incubated at 37°C and 5% CO2 for 24 h (1×10⁻⁶ cells / year). 7 / well). After cell adhesion, SP / SO (2:1) induced lipid accumulation for 24 h. Groups were set up with CON, MOD, DAP, EZE, PM, and COA, and incubated for 24 h. 5 × 10⁶ cells were collected. 6 Cells were transferred to centrifuge tubes, the supernatant was discarded, and the cells were sonicated with anhydrous ethanol. The cells were then centrifuged at 4°C, and the supernatant was collected and placed on ice for analysis. Blood lipid levels were assessed using kits for measuring intracellular triglycerides, cholesterol, HDL-C, and LDL-C.

[0143] Immunofluorescence staining and Western blotting were used to investigate the regulation of NPC1L1 protein expression in a Caco-2 cell fatty liver model by cholesterol-autophagy linkages. Results are shown in [Figure number missing]. Figure 6 AD. Simultaneously, the impact on blood lipid levels in the HepG2 cell fatty liver model was assessed, and the results are shown in […]. Figure 6 EH. As shown in the figure, in the intestinal Caco-2 cell model, the supramolecular system significantly inhibited the expression of NPC1L1 protein, and its efficacy was superior to PM. In addition, the experiment also observed a decrease in blood lipid levels in HepG2 cells, indicating that the supramolecular system can not only reduce intestinal cholesterol absorption, but also inhibit lipid deposition in the HepG2 cell fatty liver model, thereby improving lipid metabolism abnormalities.

[0144] 2. In vivo pharmacodynamic evaluation

[0145] Male C57BL / 6J mice [SPF (Beijing) Biotechnology Co., Ltd., license number: SCXK (Beijing) 2024-0001] at 4 weeks of age were used as test animals and were adaptively fed for 1 week. All animal experiments were strictly carried out in accordance with relevant regulations and were approved by the Animal Ethics Committee of Hebei Medical University (IACUC-HEBMU-2024002). The control group was fed a standard diet, and the model group was fed a high-fat diet [High-fat diet, HFD, 60% fat (SFD020)]. The feed was stored at -4°C to -15°C to maintain its freshness and hardness. Feeding the high-fat diet for 3 months induced MASLD, and the success of the model was judged by abdominal ultrasound of mice and measuring serum cholesterol levels. After that, the successfully modeled MASLD model mice were selected and continued to be fed the high-fat diet, while the control group (CON) was fed the standard diet. In vivo pharmacodynamic evaluations were performed on mice in the normal diet group (CON), HFD-induced MASLD mice, and treatment group mice. DAP, EZE, PM, and COA drugs were orally administered by gavage every day. The experimental groups were divided into a control group (CON), a model group (MOD), DAP, EZE, PM, and DAP-EZECOA groups. Before the experiment, the body weights of the mice were weighed and the dosing doses were calculated (equivalent to DAP 2 mg / kg, EZE 2 mg / kg), and the drug administration continued for 5 weeks (n = 6 for each group).

[0146] Investigation of blood glucose and insulin sensitivity

[0147] Determination of oral glucose tolerance test (Oral glucose tolerance test, OGTT) and insulin tolerance test (Insulin tolerance test, ITT): In the final stage of the experiment, the fasting blood glucose of the mice was measured using a Roche blood glucose meter. In the OGTT and ITT experiments, the mice were fasted overnight and then orally administered glucose (1 g / kg) or intraperitoneally injected with insulin (0.75 U / kg). The blood glucose levels were monitored at different time points (0 min, 15 min, 30 min, 60 min, 90 min, and 120 min), and the total area (AUC) was calculated. The mice were sacrificed, and plasma and tissue samples were collected. The body weight, liver, and heart weights were recorded, and the corresponding indices were calculated.

[0148] After treatment with drug intervention, the body weights of the mice in the EZE, DAP, PM, and COA groups were significantly lower than those in the MOD group ( Figure 7 A). The results of the OGTT and ITT experiments are as Figure 7 , and the results showed that COA could significantly improve the whole-body glucose metabolism function of MASLD mice ( Figure 7 B~E). Figure 7 B and D show the time-dependent change trends of MASLD mice during the treatment process, while Figure 7 C and E present the corresponding area under the curve (AUC) data. Compared with the CON group, the MOD group mice showed elevated blood glucose levels. After treatment with EZE, DAP, PM, and COA, the blood glucose levels of MASLD mice significantly decreased. Furthermore, compared with the PM group, the supramolecular system demonstrated superior overall glucose tolerance improvement in OGTT and ITT tests, specifically reflected in a lower AUC value (…). Figure 7 C and E; P<0.05).

[0149] Liver function and blood lipid tests: After drug administration, blood was randomly collected from the orbits of three mice in each group, and serum was separated by centrifugation. Serum lipid metabolism indicators (including TC, TRIG, HDL-C, LDL-C) and liver function enzymes (ALT, AST) were measured using commercially available kits (purchased from Changchun Huili Biotechnology Co., Ltd.). Serum biochemical results are as follows: Figure 8 As shown.

[0150] As shown in the figure, compared with the PM group, the COA group mice had significantly lower levels of TC, TRIG, and LDL-C, while their HDL-C levels were significantly higher; all these differences were statistically significant. To further evaluate the effects of different treatment regimens on liver function, ALT and AST levels, which reflect liver function status, were measured. Figure 8 (E and F). The results showed that, compared with the CON group, the serum ALT and AST levels in the MOD group mice were significantly increased, confirming the liver function impairment in MASLD mice. Notably, all interventions could reduce serum ALT and AST levels to some extent. Among them, the reduction in the COA group was similar to that in the CON group, indicating that the COA group has a potential hepatoprotective effect.

[0151] In vivo liver tissue lipid analysis

[0152] Near the end of the treatment period, abdominal ultrasound was used to observe the fatty liver condition in mice treated with DAP-EZECOA. The liver-to-kidney ratio was used to determine whether the fatty liver condition in HFD mice had improved after supramolecular system treatment. Mouse weight was recorded weekly. At the end of the intervention, mouse livers were harvested, liver weight was recorded, and liver index was calculated.

[0153] Long-term HFD-induced mice exhibited significant liver pathological changes, specifically hepatocyte swelling, disordered arrangement, and intracellular lipid accumulation. These pathological features included microvesicular and macrovesicular steatosis, ultimately leading to enlarged liver volume. To assess the effects of COA on MASLD mice, abdominal ultrasound was performed on the mice at the end of the dosing period. The results are shown in [Figure 1]. Figure 9 .

[0154] The results showed that the liver-to-kidney ratio was significantly reduced in the COA treatment group, indicating that supramolecular system intervention can effectively alleviate hepatic steatosis. At the end of the experiment, mice were sacrificed and liver tissue was collected. Body weight and liver weight were recorded, and the liver index was calculated. The results are shown below. Figure 10 Compared to the MOD group, the liver weight of mice in the COA treatment group ( Figure 10 A) and liver index ( Figure 10 B) A significant decrease, a result that strongly demonstrates that COA has a protective effect against HFD-induced liver enlargement.

[0155] 3. Histopathological staining

[0156] Liver, heart, pancreas, spleen, small intestine, and kidney tissues were collected from MASLD mice, fixed in 4% paraformaldehyde, and embedded in paraffin. After sectioning, hematoxylin and eosin (H&E) staining was performed on each tissue, and Oil Red O staining was applied to the liver and aorta to observe the morphological structure of the liver and visualize intracellular fat. Glycogen accumulation in the liver was observed using iodic acid-Schiff (PAS) staining. Insulin immunoperoxidase staining was used to detect insulin localization and expression, and images were acquired using an optical microscope.

[0157] To investigate the effects of coagulase on liver tissue structure, histological analysis was performed using H&E staining. The results are as follows: Figure 11 As shown in Figure A. The results indicated that the liver structure of the normal group mice was intact, without obvious fatty degeneration or inflammatory cell infiltration, and the hepatocytes were arranged in a single layer radially around the central vein. In contrast, the liver tissue of the MOD group mice showed a disordered structure, with irregular hepatocyte arrangement, accompanied by fatty infiltration and intracellular lipid droplet accumulation. After drug intervention, the liver tissue structure and morphology of both the PM and COA groups were significantly improved compared to the MOD group, with the COA group showing a more significant improvement. Simultaneously, liver Oil Red O staining (… Figure 11 The results of examination B) further support this conclusion. In addition, the PAS staining results ( Figure 11 C) indicates that COA can effectively reverse the decrease in liver glycogen synthesis caused by long-term HFD feeding. In conclusion, COA has a protective effect on the liver tissue of MASLD mice, and compared with the PM group, the COA group plays an important role in the regulation of glucose and lipid metabolism in MASLD mice.

[0158] 4. Western blot analysis showed that DAP-EZECOA enhanced autophagy in liver cells and reduced the expression of proteins related to excessive lipid deposition in vivo.

[0159] After liver tissue processing, total protein was extracted by lysis in RIPA lysis buffer. The lysate samples were separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane, where they were incubated with primary antibodies p-AMPK, p-ULK1, LC3, and p62. Protein bands were analyzed using software and probed with a specific secondary antibody.

[0160] Mechanism study of COA in improving MASLD

[0161] Autophagy activation is a key factor in the treatment of MASLD. However, given the complexity and dual effects of autophagy, achieving specific activation of hepatic autophagy remains a challenge. Cholesterol depletion can activate autophagy, and there is a close link between cholesterol homeostasis and autophagy regulation. Notably, enterohepatic circulation plays a crucial role in enhancing the efficacy of certain drugs. Previous studies have confirmed that EZE can affect the in vivo transport of DAP, thereby inducing enterohepatic circulation. Based on this, further research was conducted on how COA utilizes the enterohepatic circulation mechanism to specifically activate hepatic autophagy through multiple synergistic effects, thereby improving the pathological state of MASLD.

[0162] COA activates autophagy through the AMPK-ULK1 pathway, improving lipid accumulation in MASLD mice.

[0163] To further explore the potential molecular mechanism by which COA reduces lipid metabolism disorders in MASLD mice, the protein expression levels of the AMPK / ULK1, LC3, and P62 autophagy pathways related to lipid metabolism were detected by Western blotting. The results are as follows: Figure 12 .

[0164] AMPK, a key intracellular energy sensor, plays a central role in regulating cellular lipid metabolism, glucose metabolism, and autophagy. When AMPK is activated, it phosphorylates its downstream target protein ULK1, thereby initiating autophagy. As shown in the figure, the protein expression of p-AMPK, p-ULK1, and LC3 in the liver of mice in the MOD group was significantly lower than that in the CON group, while the expression level of the autophagy-related substrate P62 protein was significantly increased, indicating that autophagy was inhibited. This suggests that HFD induction may inhibit the AMPK-ULK1 autophagy pathway, leading to impaired autophagy function in liver cells. Compared with the MOD group, all treatment groups improved the expression of autophagy-related proteins, with the COA group showing particularly significant effects. The expression of p-AMPK, p-ULK1, and LC3 was significantly increased (P<0.05), while the expression of P62 was significantly decreased (P<0.05), indicating that COA can activate the AMPK-ULK1 autophagy pathway, thereby promoting autophagy. In summary, COA effectively promotes autophagy in hepatocytes by activating the AMPK-ULK1 autophagy pathway, thereby clearing abnormally accumulated lipid substances and playing a crucial role in preventing and improving hepatic steatosis caused by HFD.

[0165] 5. Detection of iron-related indicators in animal liver tissue

[0166] GSH level

[0167] Weigh 0.1g of liver tissue and add it to 1mL of pre-chilled Extraction Buffer. Homogenize on ice. Centrifuge the homogenate at 4°C and collect the supernatant. Analyze on ice. Use the CheKine™ GSH assay kit (micro-method) for detection. The experimental groups were divided into CON, MOD, DAP, EZE, PM, and COA groups.

[0168] SOD activity

[0169] 0.1 g of liver tissue was homogenized with 1 mL of pre-cooled Lysis Buffer. The homogenized sample was centrifuged at 4°C for 5 min, and the supernatant was collected for analysis using a SOD activity assay kit. The experimental groups were divided into CON, MOD, DAP, EZE, PM, and COA groups.

[0170] MDA content

[0171] A 0.1g liver sample was placed in 1mL of pre-chilled Extraction Buffer and homogenized on ice. The sample was then centrifuged at 4°C for 10 min, and the supernatant was used for subsequent analysis. MDA activity assay kit was used for detection. The experimental groups were CON, MOD, DAP, EZE, PM, and COA groups.

[0172] GPX4 protein expression level

[0173] After liver tissue processing, total protein was extracted by lysis in RIPA lysis buffer. The lysate samples were separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane, where they were incubated with the primary antibody GPX4. The membrane was probed with a specific secondary antibody, and protein bands were analyzed using software.

[0174] Intracellular iron metabolism imbalance, the occurrence of the Fenton reaction, inhibition of systemic Xc- function, and decreased levels of GPX4 and GSH have all been confirmed to be closely related to ferroptosis. Systemic Xc- (SLC7A11 / SLC3A2), as a key transporter on the cell membrane, is responsible for mediating cysteine ​​uptake, thereby promoting GSH synthesis. GPX4, as an important lipid repair enzyme, can convert harmful lipid oxides into harmless lipid alcohols with the participation of GSH, thus effectively inhibiting lipid peroxidation-induced cell damage. Therefore, increasing GPX4 expression is considered an effective strategy to mitigate ferroptosis-induced cell damage. Ferroptosis is closely related to the imbalance of intracellular oxidative stress, its main characteristic being the significant generation of ROS. To resist ROS-induced cell damage, cells activate a series of antioxidant defense mechanisms, among which SOD plays a crucial role in mitigating oxidative stress by catalyzing the disproportionation reaction of superoxide anions. Furthermore, ROS-induced lipid oxidation is also a crucial step in the ferroptosis signaling pathway, generating large amounts of MDA, which serves as a bioindicator of lipid oxidation. The level of MDA directly reflects the degree of cell damage. In summary, MDA accumulation, SOD activity, and the functional state of the GSH / GPX4 system all play important roles in the fine regulation of ferroptosis. To further investigate the effect of COA on hepatocyte ferroptosis, this invention used MASLD mice as a model to systematically detect ferroptosis-related indicators in liver tissue. The results are shown in [Figure number missing]. Figure 13 .

[0175] As shown in the figure, compared with the CON group, the MDA content in the liver tissue of the MOD group mice was significantly increased, while the GSH level, SOD activity, and GPX4 protein expression level were significantly decreased, indicating that ferroptosis occurred in the liver of the MOD group mice. Compared with the MOD group, all treatment groups showed improvement in ferroptosis-related indicators, especially the COA group, which showed the most significant improvement, with a significant decrease in MDA content and a substantial increase in GSH level, SOD activity, and GPX4 protein expression level (P<0.05). These results indicate that the COA supramolecular system effectively inhibits ferroptosis through multiple pathways, including regulating GSH level, upregulating GPX4 expression, scavenging ROS, and inhibiting lipid peroxidation.

[0176] Histopathological staining

[0177] Pancreatic and small intestinal tissues were collected from MASLD mice, fixed in 4% paraformaldehyde, and embedded in paraffin. After sectioning, hematoxylin and eosin (H&E) staining was performed on each tissue, pancreatic immunohistochemistry was performed, and Oil Red O staining was performed on the aorta to observe the morphological structure of each organ and to visualize the fat in the aorta.

[0178] To comprehensively evaluate the impact of insulin resistance and cholesterol metabolism disorders on metabolic disorders such as dyslipidemia, atherosclerosis, and MASLD, this invention employed H&E staining and gross Oil Red O staining techniques to conduct detailed observations of the aorta, pancreas, and small intestine tissues of mice in different treatment groups to assess pathological changes at the tissue level. The results are shown in [Figure number missing]. Figure 14 and 15 .

[0179] The results showed that the aorta of mice in the MOD group exhibited significant red lipid deposition in Oil Red O staining, indicating atherosclerotic changes. Furthermore, the MOD group mice showed larger red staining areas at the aortic arch and branches, suggesting a higher degree of lipid deposition. In the PM group, scattered red spots or cord-like deposits were observed proximal to the heart, indicating mild lipid accumulation. In contrast, the COA group showed significantly reduced aortic lipid deposition, indicating that COA intervention can significantly improve aortic lipid deposition in MASLD mice, thereby slowing the progression of atherosclerosis.

[0180] Pancreatic H&E staining results as follows Figure 15 As shown in Figure A, the islet structure of mice in the CON group was intact, with regular and abundant β-cell distribution and normal acinar cell morphology. In contrast, the islets of mice in the MOD group showed atrophy, a significant decrease in the number of β-cells, acinar cell infiltration into the islets, vacuolation, and abnormal acinar structure. After 5 weeks of treatment, islet pathology and β-cell counts were significantly improved in all treatment groups, with the COA group showing better recovery than the PM group. Furthermore, the acinar cell morphology in the COA-treated group also showed significant improvement. Similarly, immunohistochemical results of pancreatic tissue further supported these findings. Figure 15 B) indicates that COA intervention can significantly improve the exocrine and endocrine functions of the pancreas and effectively enhance insulin sensitivity. H&E staining analysis of mouse small intestinal tissue showed ( Figure 15 (C) In the MOD group, the villi of the small intestine were more swollen and irregularly arranged compared to the CON group. A small number of lymphocytes were infiltrated in the lamina propria, and some intestinal epithelial cells showed vacuolation, indicating a mild inflammatory response and lipid metabolism disorder in the small intestine of these mice. The histopathological characteristics of the small intestine in all treatment groups were improved compared to the MOD group, showing a gradual reduction in intestinal epithelial cell vacuolation, with the COA treatment group showing the most significant improvement. These results indicate that the supramolecular system can improve the histopathological changes in the small intestine induced by a high-fat diet.

[0181] Intestinal NPC1L1 levels and liver cholesterol content

[0182] After processing the small intestinal tissue, the expression level of intestinal NPC1L1 protein was determined according to the aforementioned Western blot (WB) method. Simultaneously, the total cholesterol (TC) content in the liver was measured. 0.1 g of liver sample was homogenized with 1 mL of anhydrous ethanol on ice, then centrifuged at 4°C for 10 min. The supernatant was extracted and analyzed using a total cholesterol activity assay kit. The experimental groups were CON, MOD, DAP, EZE, PM, and COA groups.

[0183] Existing research indicates that insulin resistance and cholesterol metabolism disorders synergistically promote the occurrence and progression of various metabolic disorders, including dyslipidemia, atherosclerosis, and MASLD. Therefore, synergistic regulation of insulin resistance and cholesterol metabolism has become a key strategy for the prevention and treatment of these diseases. Notably, cells can sense changes in cholesterol levels through cholesterol-autophagy connections and activate autophagy, thereby regulating cholesterol metabolism and transport to maintain cholesterol homeostasis. Previous pharmacokinetic studies have preliminarily confirmed that in supramolecular systems, EZE significantly alters the in vivo transport of DAP and induces enterohepatic circulation. Based on this finding, to further verify the synergistic effect of supramolecular systems and enterohepatic circulation on enhanced drug efficacy, this invention utilizes the cholesterol-autophagy connection mechanism to deeply analyze the potential mechanism of action of supramolecular systems in improving abnormal cholesterol metabolism and insulin resistance.

[0184] To investigate the effects of supramolecular systems on cholesterol metabolism, this invention used Western blotting to detect the expression level of the key cholesterol transporter NPC1L1 in mouse small intestinal tissue. The results are shown in [Figure number missing]. Figure 16 .

[0185] NPC1L1 is a cholesterol transporter located on the brush border membrane of small intestinal epithelial cells and plays a crucial role in intestinal cholesterol absorption. EZE, a commonly used cholesterol absorption inhibitor in clinical practice, works by specifically targeting and inhibiting NPC1L1 function, thereby reducing intestinal cholesterol absorption. As shown in the figure, compared with the MOD group, all treatment groups inhibited the expression level of NPC1L1 protein in the small intestine to varying degrees, and the inhibitory effect of the COA group was superior to that of the PM group (P<0.05). This result indicates that the supramolecular system may effectively reduce the efficiency of intestinal cholesterol absorption by regulating NPC1L1 expression. Furthermore, the cholesterol levels in the liver of mice in all treatment groups were significantly lower than those in the MOD group (P<0.05), with the COA group showing the most significant reduction (e.g., ...). Figure 17This change not only reflects the optimization of cholesterol metabolism in the liver but also demonstrates a significant improvement in the liver's ability to process cholesterol. Comprehensive analysis of changes in small intestinal NPC1L1 protein expression and mouse liver cholesterol levels revealed that COA regulates hepatic cholesterol metabolism by reducing intestinal cholesterol absorption and improving insulin resistance, ultimately improving abnormal hepatic cholesterol metabolism. These findings provide new evidence for supramolecular systems regulating cholesterol metabolism and reveal their potential application value in lowering blood lipids and protecting liver function.

[0186] A schematic diagram illustrating the pharmacodynamics and mechanism of action of the SGLT-2 / NPC1L1 dual-receptor supramolecular system for treating MASLD of the present invention, as shown below. Figure 18 As shown.

[0187] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A dapagliflozin-ezetimibe amorphous compound, characterized in that, It is formed by the combination of dapagliflozin and ezetimibe; The molar ratio of dapagliflozin to ezetimibe is 1:1; The method for preparing the dapagliflozin-ezetimibe co-amorphous compound includes the following steps: adding dapagliflozin and ezetimibe to a solvent and mixing them ultrasonically, then evaporating the solvent and drying the mixture to obtain the dapagliflozin-ezetimibe co-amorphous compound.

2. The dapagliflozin-ezetimibe amorphous compound according to claim 1, characterized in that, The dapagliflozin-ezetimibe co-amorphous compound exhibits an amorphous form, and its X-ray powder diffraction spectrum, expressed as 2θ using Cu-kα radiation, shows no sharp diffraction peaks.

3. The dapagliflozin-ezetimibe amorphous compound according to claim 1, characterized in that, The glass transition temperature of the dapagliflozin-ezetimibe co-amorphous compound is 61.56 °C.

4. The dapagliflozin-ezetimibe amorphous compound according to claim 1, characterized in that, The dapagliflozin component in the dapagliflozin-ezetimibe co-amorphous compound exhibits a solubility of 22.02 mg / mL and a dissolution rate of 77.43% in a medium at pH=1, a solubility of 26.31 mg / mL and a dissolution rate of 78.62% in a medium at pH=4, and a solubility of 21.28 mg / mL and a dissolution rate of 86.63% in a phosphate buffer solution at pH=6.

8. The ezetimibe component in the dapagliflozin-ezetimibe co-amorphous compound exhibits a solubility of 79.85 μg / mL and a dissolution rate of 19.83% in a medium with pH=1, a solubility of 483 μg / mL and a dissolution rate of 34.66% in a medium with pH=4, and a solubility of 387 μg / mL and a dissolution rate of 26.30% in a phosphate buffer solution with pH=6.

8.

5. A method for preparing a dapagliflozin-ezetimibe amorphous compound, used to prepare the dapagliflozin-ezetimibe amorphous compound according to any one of claims 1 to 4, characterized in that, The process includes the following steps: adding dapagliflozin and ezetimibe to a solvent and mixing them ultrasonically, then evaporating the solvent and drying the mixture to obtain the dapagliflozin-ezetimibe co-amorphous compound.

6. The method for preparing a dapagliflozin-ezetimibe co-amorphous compound according to claim 5, characterized in that, The total mass ratio of dapagliflozin and ezetimibe to the volume ratio of solvent is 100 mg: 30-50 mL.

7. The method for preparing a dapagliflozin-ezetimibe co-amorphous compound according to claim 5, characterized in that, The ultrasonic mixing frequency is 30~50kHz, the power is 90~110W, and the time is 30~40min; The solvent is evaporated at a temperature of 36-40°C, a rotation speed of 45-55 rpm, and a time of 20-40 min.

8. The use of a dapagliflozin-ezetimibe amorphous compound according to any one of claims 1 to 4, or a dapagliflozin-ezetimibe amorphous compound prepared by the preparation method according to any one of claims 5 to 7, in the preparation of a drug for treating metabolic dysfunction-related fatty liver disease.

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