An IDH2-K272 lactoylation promoter based on enasidenib and a preparation method thereof

Abstract

This invention relates to the field of pharmaceutical technology and discloses an IDH2-K272 lactation promoter based on empagliflozin and its preparation method, comprising: empagliflozin, neutral phospholipids, cholesterol, sodium lactate, arginine, filler, disintegrant, and humectant. Empagliflozin regulates the activity of ACAT1 / HDAC1 modifying enzymes, and sodium lactate provides the IDH2-K272 modification substrate, synergistically enhancing the lactation level of IDH2-K272 in myocardial microvascular endothelial cells. This promotes competitive binding to the CSD domain of Cav1, dissociates the Cav1-eNOS inhibitory complex, upregulates eNOS activity, promotes NO release, drives angiogenesis in the infarct border zone of diabetic myocardial infarction, and reduces myocardial damage. The formulation can be made into granules or tablets, exhibiting good storage stability and rapid dissolution, overcoming the limitation of limited in vivo efficacy of free empagliflozin after oral administration.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to an IDH2-K272 lactation promoter based on empagliflozin and its preparation method. Background Technology

[0002] Patients with diabetes mellitus complicated by myocardial infarction (DMI) have a poor prognosis. The hyperglycemic and hypoxic microenvironment inhibits the proliferation and migration of myocardial microvascular endothelial cells (CMECs), impairs compensatory angiogenesis in the infarct border zone, further expands the myocardial infarction area, and aggravates myocardial fibrosis and ventricular remodeling. Current research has confirmed that lactation modification of lysine residue 272 (IDH2-K272) of isocitrate dehydrogenase 2 (IDH2) can competitively bind to the CSD domain of caveolin 1 (Cav1), weakening the inhibitory effect of Cav1 on eNOS, increasing eNOS activity, promoting NO synthesis, and ultimately promoting angiogenesis in CMECs; the SGLT-2 inhibitor empagliflozin can upregulate the lactation level of IDH2-K272 and exert a cardioprotective effect. Furthermore, the publicly disclosed experimental protocol directly uses free empagliflozin for oral gavage, a technique with significant drawbacks: oral administration results in gastrointestinal metabolism and first-pass effect in the liver, leading to low bioavailability; the drug is widely distributed throughout the body, but the concentration of the drug at the target site in myocardial microvascular endothelial cells is limited, requiring a higher dose to be effective; empagliflozin only promotes IDH2-K272 lactation by upregulating lactase ACAT1 and downregulating delactase HDAC1, but the insufficient supply of intracellular lactate substrate limits the extent of improvement in IDH2-K272 lactation modification; systemic administration can cause non-specific drug exposure in the kidneys and peripheral tissues, leading to potential side effects.

[0003] Therefore, developing an IDH2-K272 lactation promoter that combines enzyme regulation, substrate supply, synergistic endothelial protection, and excellent storage stability is of great practical significance. Summary of the Invention

[0004] In view of this, the present invention proposes an IDH2-K272 lactation promoter based on empagliflozin and its preparation method, aiming to solve the defects of the current technology, such as insufficient drug concentration in target cells when empagliflozin is administered orally, limited lactate substrate, poor storage stability of active ingredients, and lack of synergistic pharmacodynamic components.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention proposes an IDH2-K272 lactation accelerator based on empagliflozin, comprising the following components in parts by weight: Empagliflozin 1-10 parts, neutral phospholipids 15-40 parts, cholesterol 3-12 parts, sodium lactate 2-8 parts, arginine 1-5 parts, filler 30-70 parts, disintegrant 2-10 parts, wetting agent 0.5-3 parts.

[0006] Furthermore, the neutral phospholipid is selected from soybean lecithin and distearate.

[0007] Furthermore, the filler is selected from at least one of microcrystalline cellulose, lactose, mannitol, and dextrin.

[0008] Furthermore, the disintegrant is selected from at least one of crospovidone, sodium carboxymethyl starch, and low-substituted hydroxypropyl cellulose.

[0009] Furthermore, the wetting agent is selected from polyethylene glycol and stearyl alcohol.

[0010] A method for preparing an IDH2-K272 lactation accelerator based on empagliflozin includes the following steps: (1) Empagliflozin, sodium lactate and arginine were sieved separately; neutral phospholipids and cholesterol were mixed and heated to melt, resulting in a molten lipid mixture; (2) The sieved empagliflozin, sodium lactate, arginine, filler and disintegrant are mixed evenly to obtain powder material. Molten lipid mixture and wetting agent are sprayed into the powder material and wet granules are obtained after granulation. (3) The wet granules are dried under reduced pressure and then sieved and granulated to obtain a granular accelerator, or the granulated granules are further compressed to obtain a tablet accelerator.

[0011] Furthermore, the sieving in step (1) is sieve sieve 80-100 mesh; the heating and melting parameters are: temperature 55-70℃, time 5-15min.

[0012] Furthermore, the granulation temperature in step (2) is 45-55℃; the injection rate is 8-15mL / min.

[0013] Furthermore, the reduced pressure drying temperature in step (3) is 40-50℃; the sieving is done through a 20-40 mesh sieve.

[0014] The present invention also provides an application of the empagliflozin-based IDH2-K272 lactation promoter described in the above technical solution, specifically the application of the empagliflozin-based IDH2-K272 lactation promoter in the preparation of oral medications for treating diabetic myocardial infarction.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention employs a neutral phospholipid-cholesterol lipid complex structure to encapsulate empagliflozin and sodium lactate, and introduces arginine. On the one hand, this improves the storage stability of the formulation, and on the other hand, arginine acts as a NO precursor to synergize with the eNOS pathway. Combined with fillers, disintegrants, and humectants, granules or tablets are prepared, which dissolve rapidly after oral administration. Empagliflozin regulates the modifying enzyme, and sodium lactate provides lactation substrates, thus synergistically enhancing the lactation modification level of IDH2-K272, promoting angiogenesis after diabetic myocardial infarction, and improving cardiac damage.

[0016] This invention combines empagliflozin and sodium lactate, which both regulate lactation modification enzymes and supplement lactate modification substrates. Compared with empagliflozin alone, IDH2-K272 has a more significant effect on lactation modification. The formulation introduces arginine, which, in addition to stabilizing the formulation, also serves as a precursor for eNOS to catalyze the generation of NO, synergistically amplifying the effects of endothelial protection and angiogenesis.

[0017] In this invention, neutral phospholipids and cholesterol form a lipid complex structure to encapsulate the active components, reducing the degradation of empagliflozin due to environmental temperature and humidity; arginine regulates the pH of the powder microenvironment, further improving the storage stability of the active substances.

[0018] The present invention, when combined with fillers, disintegrants, and humectants, can be made into granules or tablets, with fast dissolution rate, mature preparation process, and easy industrial scale-up production. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0020] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] This invention proposes an IDH2-K272 lactation accelerator based on empagliflozin, comprising the following components in parts by weight: Empagliflozin 1-10 parts, neutral phospholipids 15-40 parts, cholesterol 3-12 parts, sodium lactate 2-8 parts, arginine 1-5 parts, filler 30-70 parts, disintegrant 2-10 parts, wetting agent 0.5-3 parts.

[0025] In this invention, the IDH2-K272 lactation accelerator is further preferably composed of the following components in parts by weight: 2-10 parts empagliflozin, 18-40 parts neutral phospholipids, 5-12 parts cholesterol, 3-8 parts sodium lactate, 2-5 parts arginine, 35-70 parts filler, 4-10 parts disintegrant, and 1-3 parts humectant.

[0026] In this invention, the neutral phospholipid is selected from soybean lecithin and distearate.

[0027] In this invention, the filler is selected from at least one of microcrystalline cellulose, lactose, mannitol, and dextrin.

[0028] In this invention, the disintegrant is selected from at least one of crospovidone, sodium carboxymethyl starch, and low-substituted hydroxypropyl cellulose.

[0029] In this invention, the wetting agent is selected from polyethylene glycol and stearyl alcohol.

[0030] A method for preparing an IDH2-K272 lactation accelerator based on empagliflozin includes the following steps: (1) Empagliflozin, sodium lactate and arginine were sieved separately; neutral phospholipids and cholesterol were mixed and heated to melt, resulting in a molten lipid mixture; (2) The sieved empagliflozin, sodium lactate, arginine, filler and disintegrant are mixed evenly to obtain powder material. Molten lipid mixture and wetting agent are sprayed into the powder material and wet granules are obtained after granulation. (3) The wet granules are dried under reduced pressure and then sieved and granulated to obtain a granular accelerator, or the granulated granules are further compressed to obtain a tablet accelerator.

[0031] In this invention, the sieving in step (1) is preferably sieve 80-100 mesh, and more preferably sieve 90-100 mesh; the heating and melting parameters are preferably: temperature 55-70℃, time 5-15min, and more preferably temperature 58-70℃, time 5-12min.

[0032] In this invention, the granulation temperature in step (2) is preferably 45-55℃, more preferably 48-55℃; the injection rate is preferably 8-15mL / min, more preferably 10-15mL / min.

[0033] In this invention, the vacuum drying temperature in step (3) is preferably 40-50℃, more preferably 42-50℃; the sieving is preferably through a 20-40 mesh sieve, more preferably through a 20-30 mesh sieve.

[0034] The present invention also provides an application of the empagliflozin-based IDH2-K272 lactation promoter described in the above technical solution, specifically the application of the empagliflozin-based IDH2-K272 lactation promoter in the preparation of oral medications for treating diabetic myocardial infarction.

[0035] In this invention, the neutral phospholipids in the IDH2-K272 lactation promoter form a lipid complex with cholesterol to encapsulate empagliflozin and sodium lactate. Arginine not only improves the stability of the formulation system but also acts as a precursor for nitric oxide synthesis, synergistically enhancing endothelial protection. After oral administration, the formulation rapidly dissolves and releases the active components. Empagliflozin regulates the activity of ACAT1 / HDAC1 modifying enzymes, while sodium lactate provides the substrate for IDH2-K272 lactation modification. Together, they synergistically enhance the lactation modification level of IDH2-K272 in myocardial microvascular endothelial cells, prompting the IDH2-K272 lactated protein to competitively bind to the CSD domain of Cav1, dissociating the Cav1-eNOS inhibitory complex, upregulating eNOS activity, promoting NO release, promoting angiogenesis in the infarct margin zone of diabetic myocardial infarction, and reducing myocardial damage.

[0036] In this invention, unless otherwise specified, all raw materials required for preparation are commercially available products well known to those skilled in the art.

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1 Ingredients: 2 parts empagliflozin, 18 parts soy lecithin, 5 parts cholesterol, 3 parts sodium lactate, 2 parts arginine, 35 parts mannitol, 4 parts crospovidone, 1 part polyethylene glycol; Preparation method: (1) Empagliflozin, sodium lactate and arginine were passed through a 90-mesh sieve; neutral phospholipids and cholesterol were mixed and heated to melt at 58°C for 12 min to obtain a molten lipid mixture; (2) The sieved empagliflozin, sodium lactate, arginine, mannitol, and cross-linked polyvinylpyrrolidone were put into a fluidized bed and mixed thoroughly to obtain powder material. The fluidized bed inlet air temperature was set to 48℃, the material temperature was controlled at 38℃, and the spray rate was 10mL / min. Molten lipid mixture and polyethylene glycol were sprayed into the powder material, and wet granules were obtained after granulation. (3) The wet granules are dried under reduced pressure at 42°C and then granulated through a 20-mesh sieve to obtain the granule morphology promoter.

[0039] Example 2 Ingredients: 5 parts empagliflozin, 25 parts soy lecithin, 8 parts cholesterol, 5 parts sodium lactate, 4 parts arginine, 50 parts microcrystalline cellulose, 7 parts crospovidone, 2 parts polyethylene glycol; Preparation method: (1) Empagliflozin, sodium lactate and arginine were passed through a 90-mesh sieve; soybean lecithin and cholesterol were mixed and heated at 65°C for 10 min to obtain a molten lipid mixture; (2) The sieved empagliflozin, sodium lactate, arginine, microcrystalline cellulose, and cross-linked polyvinylpyrrolidone were put into a fluidized bed and mixed thoroughly to obtain powder material. The fluidized bed inlet air temperature was set to 50℃, the material temperature was controlled at 38℃, and the spray rate was 12mL / min. Molten lipid mixture and polyethylene glycol were sprayed into the powder material, and wet granules were obtained after granulation. (3) The wet granules are dried under reduced pressure at 45°C and then granulated through a 20-mesh sieve to obtain the granule morphology promoter.

[0040] Example 3 Ingredients: 10 parts empagliflozin, 40 parts distearate, 12 parts cholesterol, 8 parts sodium lactate, 5 parts arginine, 70 parts lactose, 10 parts crospovidone, 3 parts polyethylene glycol; Preparation method: (1) Empagliflozin, sodium lactate and arginine were passed through a 100-mesh sieve; distearylcholine and cholesterol were mixed and heated to melt at 70°C for 5 min to obtain a molten lipid mixture; (2) The sieved empagliflozin, sodium lactate, arginine, lactose, and cross-linked polyvinylpyrrolidone were put into a fluidized bed and mixed thoroughly to obtain powder material. The fluidized bed inlet air temperature was set at 55℃, the material temperature was controlled at 40℃, and the spray rate was 15mL / min. Molten lipid mixture and polyethylene glycol were sprayed into the powder material, and wet granules were obtained after granulation. (3) The wet granules are dried under reduced pressure at 50°C, then granulated by passing them through a 30-mesh sieve. The granulated granules are further compressed into tablets to obtain the tablet form accelerator.

[0041] Example 4 Ingredients: 1 part empagliflozin, 15 parts distearate, 3 parts cholesterol, 2 parts sodium lactate, 1 part arginine, 30 parts mannitol, 2 parts sodium carboxymethyl starch, 0.5 parts stearyl alcohol; Preparation method: (1) Empagliflozin, sodium lactate and arginine were passed through an 80-mesh sieve; distearylcholine and cholesterol were mixed and heated to melt at 55°C for 15 min to obtain a molten lipid mixture; (2) The sieved empagliflozin, sodium lactate, arginine, mannitol, and sodium carboxymethyl starch were put into a fluidized bed and mixed thoroughly to obtain powder material. The fluidized bed inlet air temperature was set to 45℃, the material temperature was controlled at 35℃, and the spray rate was 8mL / min. Molten lipid mixture and wetting agent stearyl alcohol were sprayed into the powder material and wet granules were obtained after granulation. (3) The wet granules are dried under reduced pressure at 40°C, then granulated by passing them through a 20-mesh sieve. The granulated granules are further compressed into tablets to obtain the tablet form of the accelerator.

[0042] Comparative Example 1 Sodium lactate was removed from the raw materials, and microcrystalline cellulose was used to make up the total mass of the formula: 5 parts empagliflozin, 25 parts soybean lecithin, 8 parts cholesterol, 4 parts arginine, 55 parts microcrystalline cellulose, 7 parts crospovidone, and 2 parts polyethylene glycol. Preparation method: (1) Empagliflozin and arginine were passed through a 90-mesh sieve; soybean lecithin and cholesterol were mixed and heated at 65°C for 10 min to obtain a molten lipid mixture; (2) The sieved empagliflozin, arginine, microcrystalline cellulose, and cross-linked polyvinylpyrrolidone were put into a fluidized bed and mixed thoroughly to obtain powder material. The fluidized bed inlet air temperature was set to 50℃, the material temperature was controlled at 38℃, and the spray rate was 12mL / min. Molten lipid mixture and polyethylene glycol were sprayed into the powder material, and wet granules were obtained after granulation. (3) The wet granules are dried under reduced pressure at 45°C and then granulated through a 20-mesh sieve to obtain the granule morphology promoter.

[0043] Comparative Example 2 Arginine was removed from the raw materials, and microcrystalline cellulose was used to make up the total mass of the formula: 5 parts empagliflozin, 25 parts soybean lecithin, 8 parts cholesterol, 5 parts sodium lactate, 54 parts microcrystalline cellulose, 7 parts crospovidone, and 2 parts polyethylene glycol. Except for the addition of arginine, the other steps in the preparation method are the same as in Example 2.

[0044] Comparative Example 3 Raw materials: Same as in Example 2; Preparation method: (1) All solid raw materials are passed through a 90-mesh sieve, and all powders are added to the fluidized bed and mixed evenly; (2) Using purified water as a binder, the fluidized bed inlet air temperature was set to 50°C, the material temperature to 38°C, and the spray rate to 12 mL / min. Pure water was sprayed in for wet granulation to obtain wet granules. (3) The wet granules are dried under reduced pressure at 45°C and then granulated through a 20-mesh sieve to obtain granules.

[0045] Comparative Example 4 Ingredients: Empagliflozin 5 parts, microcrystalline cellulose 73 parts, crospovidone 7 parts, polyethylene glycol 2 parts; Preparation method: (1) The raw materials are passed through a 90-mesh sieve, and the powder is added to the fluidized bed for mixing; (2) Wet granulation using purified water as a binder; (3) Dry under reduced pressure at 45℃, and granulate by passing through a 20-mesh sieve to obtain granules.

[0046] Performance testing: I. Physicochemical Properties Testing of the Formulation (1) Appearance: Under natural indoor light conditions, observe the color and shape of the sample directly with the naked eye and record the appearance of the preparation; observe the surface and color of the tablet sample, and observe the color and uniformity of the granules, and whether there are lumps, discoloration or foreign matter.

[0047] (2) Particle size D50: A laser particle size analyzer was used; granules were directly sampled for testing; tablet samples were first lightly crushed and then passed through a 10-mesh sieve to remove large fragments. Pure water was used as the dispersion medium, and the particles were ultrasonically dispersed for 30 seconds with the light-blocking ratio controlled at 10%-20%. The particle size was then measured and the median diameter D50 was output.

[0048] (3) Moisture content: Karl Fischer volumetric method was used. Approximately 0.2 g of the sample to be tested was accurately weighed and placed into the reaction cell of the Karl Fischer moisture analyzer. The sample was titrated to the endpoint using Karl Fischer titrant, and the volume of titrant consumed was recorded. The water content of the sample was then calculated.

[0049] (4) In vitro dissolution at 30 min: Dissolution and release were determined by paddle method. The dissolution medium was pH 6.8 phosphate buffer, with a volume of 900 mL, a temperature of 37 °C, and a stirring speed of 50 r / min. The formulation sample (equivalent to about 10 mg of empagliflozin) was accurately weighed and placed into the dissolution vessel. The sample was taken at 30 min, filtered through a 0.45 μm filter membrane, and the concentration of empagliflozin in the filtrate was determined by HPLC. The percentage of empagliflozin dissolved at 30 min was calculated.

[0050] (5) Empagliflozin encapsulation efficiency: The lipid complex-encapsulated drug and free drug were separated by gel filtration. A quantitative amount of the formulation suspension was loaded onto a pre-equilibrated gel column, and the lipid complex component was collected by elution. An equal amount of the formulation was taken, and methanol was added to break the membrane to obtain the total empagliflozin in the formulation. The empagliflozin content of both samples was determined by HPLC, and the encapsulation efficiency (%) = mass of empagliflozin in the lipid complex ÷ total mass of empagliflozin in the formulation sample × 100%.

[0051] (6) Accelerated stability test: The formulation sample was sealed and placed in a constant temperature and humidity test chamber. The conditions were set as follows: temperature 40℃, relative humidity 75%, and placed continuously for 30 days. The empagliflozin content in the sample at the initial time and after 30 days of placement was measured. The empagliflozin residue (%) was calculated as: empagliflozin content measured after 30 days of accelerated testing ÷ initial empagliflozin content × 100%.

[0052] The accelerators prepared in Examples 1-4 were subjected to formulation physicochemical property testing, and the results are shown in Table 1 below. Table 1. Results of physicochemical properties of the accelerators prepared in Examples 1-4

[0053] Physicochemical performance analysis: The in vitro dissolution performance of the granules in Examples 1 and 2 was superior to that of the tablets in Examples 3 and 4. Thanks to the phospholipid-cholesterol lipid complex encapsulation, the retention rate of empagliflozin in all four samples was greater than 91% after 30 days of accelerated storage, indicating good storage stability. Among them, the formulation in Example 2 exhibited the best overall physicochemical performance in terms of dissolution rate, encapsulation efficiency, and accelerated stability. In Comparative Example 3, the encapsulation efficiency decreased significantly, accelerated stability deteriorated markedly, and in vitro efficacy was significantly reduced. Although the chemical composition was completely identical, the lack of a lipid complex encapsulation structure formed by phospholipid-cholesterol melt spraying meant that the active drug was not protected and was difficult for myocardial microvascular endothelial cells to take up.

[0054] II. In vitro cell pharmacodynamic monitoring One- to three-day-old suckling mice (C57BL / 6, purchased from the Animal Center of Jiangsu University, publicly available material) were euthanized with excessive carbon dioxide. After disinfection with alcohol, they were transferred to a sterile environment for further procedures: The suckling mouse was placed in the left hand, its sides held in place with the thumb and forefinger, and the chest skin taut. Along the left midclavicular line, using curved scissors, the ribs were cut deep into the fourth to fifth intercostal space, close to the inner side of the chest wall. The scissors were used to press down on both sides of the incision, and the heart was squeezed out using the left thumb and forefinger. The heart was then picked up with curved forceps, washed three times in pre-cooled PBS solution to remove fat and connective tissue, and then minced into pieces approximately 1 mm in size. 3 After rinsing the myocardial tissue blocks, add a solution containing 0.2% type II collagenase in DMEM medium, approximately 8-10 times the volume of the myocardial tissue blocks. Digest for 1 hour on a 37°C shaker at 120 rpm. Then, add DMEM medium containing 10% serum to neutralize (the added serum-containing DMEM medium is equal in volume to the type II collagenase solution, used to stop enzyme digestion). After resuspending in the solution, filter the cell suspension (i.e., the cell fluid separated after tissue digestion) through a 200-mesh sieve using a sterile Pasteur pipette, transfer it to a 15 mL centrifuge tube, and centrifuge at 900 rpm for 10 minutes at room temperature, discarding the supernatant. Resuspend the cells in DMEM medium containing 10% serum and culture at 37°C in a 5% CO2 incubator for 1 hour using differential culture. During this process, myocardial fibroblasts are the first to adhere to the culture vessel. Non-adherent cells were collected, centrifuged again, resuspended in ECM medium (ECM purchased from Sciencell, USA), and transferred to gelatin-coated culture dishes for further culture. The next day, non-adherent cells were washed away, and new DMEM medium containing 10% serum was used for continued culture.

[0055] Cell passage: When cells reach 70%-80% confluence in the culture dish, passage is performed. After washing twice with sterile PBS, 1 mL of 0.25% trypsin is added for digestion. When the cell morphology changes from tightly adherent to translucent round cells under a microscope, the trypsin solution is discarded, and 2 mL of ECM medium is added for neutralization. The cells are gently pipetted and mixed, then transferred to a sterile centrifuge tube and centrifuged at 900 rpm for 5 minutes at room temperature. The cells are then resuspended in ECM medium to obtain primary mouse myocardial microvascular endothelial cells (CMECs). Subsequently, the cells are seeded into culture plates according to experimental requirements for the next round of culture or experimental procedures.

[0056] Construction of cell model: Primary mouse myocardial microvascular endothelial cells (CMECs) were cultured for 2-3 more generations before being used for cell experiments. The cells were cultured in a high glucose medium with a glucose concentration of 25 mmol. In order to simulate the environment of CMECs in the infarct edge area, the cells were placed in an incubator at 37°C, 5% CO2, and 95% N2 to achieve oxygen deprivation.

[0057] (1) Relative expression level of IDH2-K272la: Western blotting was used to measure protein expression. Completed CMECs were collected, total cell protein was extracted using RIPA lysis buffer, and protein was quantified using a BCA kit. Equal amounts of protein were loaded onto SDS-PAGE gel electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% skim milk powder and incubated with IDH2-K272la primary antibody and internal control Tubulin primary antibody. After washing, the membranes were incubated with HRP-labeled secondary antibody. Chemiluminescence imaging was performed using ECL. ImageJ software was used to quantify the protein band gray values. The Tubulin gray value was used to correct the loading amount. The corrected value of the blank control group was set to 1.00, and the relative expression level of IDH2-K272la in each group was calculated.

[0058] (2) p-eNOS / eNOS relative ratio: Western blotting was used to collect processed CMECs, and total cell protein was extracted with RIPA lysis buffer and quantified using a BCA kit. p-eNOS antibody and total eNOS antibody were incubated separately and chemiluminescent imaging was performed. ImageJ software was used to read the gray levels of p-eNOS and total eNOS bands and calculate the p-eNOS / eNOS gray level ratio. The ratio was normalized to 1.00 with the blank control group ratio to obtain the p-eNOS / eNOS relative ratio for each group.

[0059] (3) Relative total length of lumen formation: Pre-cooled Matrigel was laid on 96-well plates and incubated at 37°C to solidify; CMECs cell suspension was seeded, and the cells were treated in groups and incubated at 37°C for 6 hours. Images of lumen formation were acquired under an inverted microscope, and the total length of lumen formation in each well was calculated using ImageJ software; the total length of lumen formation in the blank control group was normalized to 1.00, and the relative total length of lumen formation in each group was obtained.

[0060] The promoters prepared in Examples 1-4 were subjected to in vitro cell pharmacodynamic testing. A blank control group was established using CMECs complete culture medium under high glucose and hypoxia conditions without the addition of any test drugs. The test results were normalized using the blank control group data. The performance test results are shown in Table 2 below. Table 2. In vitro cellular pharmacodynamic assay results of the promoters prepared in Examples 1-4

[0061] In vitro cellular pharmacodynamic analysis: Compared to Example 2, the physicochemical indicators in Comparative Example 1 were similar to those in Example 2, but the cellular pharmacodynamics were significantly reduced. Lacking sodium lactate substrate, relying solely on empagliflozin to regulate the modifying enzyme was insufficient to efficiently enhance the lactation modification level of IDH2-K272, demonstrating that sodium lactate provides the lactate modification substrate and is an essential component for achieving synergistic effects. In Comparative Example 2, the accelerated stability was significantly worse, and the p-eNOS / eNOS ratio and lumen-forming ability also decreased. Arginine was confirmed to have a dual function: on the one hand, it regulates the micro pH of the powder and improves the storage stability of the active components; on the other hand, it acts as a precursor for NO synthesis to synergistically amplify the efficacy of endothelial protection. All formulations in the embodiments of this invention can significantly improve the lactation modification level of IDH2-K272 in CMECs cells, upregulate the phosphorylation level of eNOS, and promote the formation of myocardial microvascular endothelial cells lumen. The efficacy of Example 2 was the best. Example 3 is a high-dose tablet, and its efficacy is second only to Example 2. Example 4 is a low-dose formulation, and its efficacy index is the lowest among the four groups, but it is still significantly higher than that of the blank control group.

[0062] In summary, the IDH2-K272 lactation promoter prepared using the formulation described in this invention utilizes a lipid complex structure formed by neutral phospholipids and cholesterol to encapsulate empagliflozin and sodium lactate. Arginine not only improves the stability of the formulation system but also acts as a precursor for nitric oxide synthesis, synergistically enhancing endothelial protection. After oral administration, the formulation rapidly dissolves and releases the active components. Empagliflozin regulates the activity of ACAT1 / HDAC1 modifying enzymes, while sodium lactate provides the substrate for IDH2-K272 lactation modification. Together, they synergistically enhance the lactation modification level of IDH2-K272 in myocardial microvascular endothelial cells, prompting the competitive binding of IDH2-K272 lactated protein to the CSD domain of Cav1, dissociating the Cav1-eNOS inhibitory complex, upregulating eNOS activity, promoting NO release, promoting angiogenesis in the infarct border zone of diabetic myocardial infarction, and reducing myocardial damage. The formulation of this invention has simple components and can be prepared into granules or tablets. It has good storage stability, fast dissolution rate, and is suitable for oral administration, thus overcoming the limitation of the limited efficacy of free empagliflozin in vivo when taken orally.

[0063] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. The scope of protection of the present invention is determined by the appended claims.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A lactation accelerator based on empagliflozin, characterized in that, The components comprise the following parts by weight: Empagliflozin 1-10 parts, neutral phospholipids 15-40 parts, cholesterol 3-12 parts, sodium lactate 2-8 parts, arginine 1-5 parts, filler 30-70 parts, disintegrant 2-10 parts, wetting agent 0.5-3 parts.

2. The IDH2-K272 lactation accelerator based on empagliflozin according to claim 1, characterized in that, The neutral phospholipid is selected from soybean lecithin and distearate.

3. The IDH2-K272 lactation accelerator based on empagliflozin according to claim 1, characterized in that, The filler is selected from at least one of microcrystalline cellulose, lactose, mannitol, and dextrin.

4. The IDH2-K272 lactation accelerator based on empagliflozin according to claim 1, characterized in that, The disintegrant is selected from at least one of crospovidone, sodium carboxymethyl starch, and low-substituted hydroxypropyl cellulose.

5. The IDH2-K272 lactation accelerator based on empagliflozin according to claim 1, characterized in that, The wetting agent is selected from polyethylene glycol and stearyl alcohol.

6. A method for preparing an IDH2-K272 lactation accelerator based on empagliflozin as described in any one of claims 1-5, characterized in that, Includes the following steps: (1) Empagliflozin, sodium lactate and arginine were sieved separately; neutral phospholipids and cholesterol were mixed and heated to melt, resulting in a molten lipid mixture; (2) The sieved empagliflozin, sodium lactate, arginine, filler and disintegrant are mixed evenly to obtain powder material. Molten lipid mixture and wetting agent are sprayed into the powder material and wet granules are obtained after granulation. (3) The wet granules are dried under reduced pressure and then sieved and granulated to obtain a granular accelerator, or the granulated granules are further compressed to obtain a tablet accelerator.

7. The method for preparing an IDH2-K272 lactation accelerator based on empagliflozin according to claim 6, characterized in that, The sieving in step (1) is sieve 80-100 mesh; the heating and melting parameters are: temperature 55-70℃, time 5-15min.

8. The method for preparing an IDH2-K272 lactation accelerator based on empagliflozin according to claim 6, characterized in that, The granulation temperature in step (2) is 45-55℃; the injection rate is 8-15mL / min.

9. The method for preparing an IDH2-K272 lactation accelerator based on empagliflozin according to claim 6, characterized in that, The vacuum drying temperature in step (3) is 40-50℃; the sieving is through a 20-40 mesh sieve.

10. The application of the IDH2-K272 lactation accelerator based on empagliflozin as described in any one of claims 1-5, characterized in that, The specific application is the use of the empagliflozin-based IDH2-K272 lactation promoter in the preparation of oral medications for treating diabetic myocardial infarction.

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