A nanocomposite preparation of sodium crocetinate with high bioavailability, preparation method, drug and application

Abstract

The present invention provides a nanocomposite preparation of crocetin sodium with high bioavailability, a preparation method, a drug and an application, relating to the technical field of biomedicine. The nanocomposite preparation comprises crocetin sodium and a nanomaterial, and the nanomaterial comprises a polymeric nanocarrier, a lipid nanocarrier or an inorganic nanoparticle carrier. In the present invention, nanoparticles of substances such as metal-organic frameworks, gold, copper silicate, chitosan or polyhydroxybutyric acid are used as drug carriers to load crocetin sodium (CRAT), and a nanocomposite preparation for treating heart failure and other cardiovascular diseases with high bioavailability can change the release behavior and state of CRAT in the intestine, accelerate the absorption of CRAT in the body and improve the bioavailability, can more effectively treat cardiovascular diseases, and at the same time improve various indexes of the heart, greatly expanding the clinical value and drugability of CRAT.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a nanocomposite preparation of sodium crocetinate with high bioavailability, a preparation method, a drug, and an application thereof. Background Art

[0002] As patients with cardiovascular underlying diseases age, especially those with ischemic heart disease and hypertensive heart disease, there is a high probability of developing heart failure syndrome. Chronic heart failure (CHF) refers to the gradual appearance of heart failure symptoms and signs on the basis of an original chronic heart disease. Currently, the heart failure treatment strategy that changes the biological properties of the failing heart has gradually shifted from short-term hemodynamic / pharmacological measures such as diuresis, cardiotonic, and vasodilation to long-term, reparative measures mainly based on neuroendocrine inhibitors. Traditional Chinese medicine has advantages in delaying the disease condition, improving cardiac function, and enhancing the quality of life, and has characteristics such as low cost, wide applicability, and few side effects, and is generally considered as an alternative strategy for treating heart failure (HF), especially CHF.

[0003] Myocardial ischemia refers to a pathological state in which the blood perfusion of the heart is reduced, resulting in a decrease in the oxygen supply to the heart, abnormal myocardial energy metabolism, and the inability to support the normal operation of the heart. The energy required for heart activity is almost entirely provided by aerobic metabolism. Therefore, even at rest, the myocardial oxygen uptake rate is very high (about 70%). Under normal circumstances, the body can adjust itself to make the blood supply and demand relatively constant to ensure the normal operation of the heart. When a certain cause leads to an imbalance between myocardial blood supply and demand, it constitutes true myocardial ischemia. Coronary heart disease is the main and most common cause of myocardial ischemia. In addition, myocardial injury refers to organic heart diseases, such as heart diseases. Clinically, coronary heart disease, angina pectoris, myocarditis, and myocardial infarction are common diseases of myocardial injury. Its symptoms mainly have three stages. The first stage shows myocardial ischemia, the second stage shows myocardial injury, and the third stage shows myocardial necrosis. Myocardial fibrosis, also known as myocardial calcification, is the result of persistent and (or) repeatedly aggravated myocardial ischemia and hypoxia caused by moderate to severe coronary atherosclerotic stenosis, leading to the gradual development of heart failure, that is, chronic ischemic heart disease.

[0004] Crocetin, also known as safranal or crocetin, is the main active ingredient of traditional Chinese medicines saffron and gardenia fruit, and is a natural carotenoid dicarboxylic acid; it has strong pharmacological activities such as anti-myocardial ischemia, antioxidant, and anti-myocardial hypertrophy. Crocetin significantly reduces cell damage in the I / R model through the SIRT3 / FOXO3a / SOD2 signaling pathway, highlighting its heart-protecting function, and then shows effects such as inhibiting myocardial infarction and cardiac hypertrophy, reducing blood pressure, and inhibiting platelet aggregation. CN108553469A discloses the use of crocetin or its derivatives in the preparation of drugs for treating heart failure. Crocetin or its derivatives can significantly improve the left ventricular cardiac function of rats with chronic heart failure caused by coronary artery ligation, inhibit the secretion of angiotensin II, neuroendocrine factor NT-proBNP, and heart failure regulatory factor IL-6 in the renin-angiotensin-aldosterone system (RAAS) that is over-activated during the process of heart failure, and inhibit myocardial pathological remodeling, and the effects of some indicators are better than Qili Qiangxin Capsule; in order to improve the stability of crocetin and its derivatives, CN114748461B discloses a crocetin preparation and its application. The bilayer membrane structure of the crocetin-loaded nano-liposome has good protection ability for crocetin molecules, which can not only protect crocetin molecules from digestion and decomposition in the harsh gastrointestinal environment, but also reduce the decomposition and destruction of crocetin under conditions such as light, but its effect is to improve the anti-fatigue ability of crocetin.

[0005] The combination of nano-drug carriers and modern medicine is an emerging discipline direction in modern drug innovation research. Compared with traditional small molecule drugs, nano-drugs play a unique advantage in improving the drugability of drugs: 1) improving the pharmaceutical properties of drugs, such as increasing the solubility of drugs, improving the stability of drugs, etc.; 2) changing the pharmacokinetic parameters of drugs in vivo, such as prolonging the in vivo half-life of drugs, improving the targeting of drugs; 3) increasing the therapeutic index of drugs, increasing the efficacy, and reducing adverse reactions; 4) improving the utilization rate of drugs, reducing the toxic and side effects of drugs on normal cells or tissues. Currently, nano-materials have been widely used in biological detection and in vitro diagnosis of diseases, and higher requirements are needed for the safety of nano-drugs directly applied to the human body. Since nano-drug preparations change the in vivo distribution behavior of the original drugs, new adverse reactions may be caused. All of these require comprehensive and in-depth toxicological studies on nano-drug preparations.

[0006] Due to the very poor stability of sodium crocetin in solution, as well as its poor stability and absorption effect in vivo, its bioavailability is extremely low. Therefore, it is difficult to develop sodium crocetin into an oral preparation. Moreover, the production cost of crocetin is high. If the daily clinical dosage is large, it will inevitably affect the drugability. Therefore, it is imperative to develop a highly efficient anti-heart failure nano-preparation of crocetin or its derivatives with good stability, high bioavailability, and oral administration. Summary of the Invention

[0007] In view of the problems existing in the prior art, the present invention provides a sodium crocinate nano-complex preparation with high bioavailability, a preparation method, a drug and uses thereof. Using nanomaterials as drug carriers, efficient loading and effective release of CRAT are achieved, realizing oral delivery of small molecule drugs. This improved nano-preparation of CRAT can greatly improve the drug stability, bioavailability and absorption efficiency compared with the traditional ordinary CRAT, effectively improve the drug metabolism in vivo, can more effectively treat heart failure and other cardiovascular diseases, and at the same time improve various cardiac indexes, greatly expanding the clinical value and druggability of CRAT.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] The present invention provides a sodium crocinate nano-complex preparation with high bioavailability, wherein the complex preparation comprises sodium crocinate and nanomaterials, and the nanomaterials include polymer nano-carriers, lipid nano-carriers or inorganic nano-particle carriers.

[0010] Preferably, the polymer nano-carrier is selected from polylactic acid, polyglycolic acid, poly(lactic acid-co-glycolic acid), polycaprolactone, alkyl cyanoacrylate, polyhydroxybutyrate (PHB), polyorthoester, polyanhydride, hydroxypropyl methylcellulose phthalate, n-butyl cyanoacrylate, chitosan (CTB), cyclodextrin, sodium alginate, dextran or derivatives thereof.

[0011] Preferably, the lipid nano-carrier is selected from nano-lipid particles, nano-liposomes, nano-emulsions, nano-capsules or polymer micelles.

[0012] Preferably, the inorganic nano-particle carrier is selected from silica nano-particles (SiNPs), gold nano-particles (AuNPs), magnetic nano-particles (MNPs), metal-organic framework nano-particles (MOFs), copper nano-particles, iron oxide nano-particles, carbon nanotubes (CNTs), nano-diamonds (NDs), fullerenes, hydroxyapatite nano-particles or graphene oxide nano-particles.

[0013] Further preferably, the copper nano-particles are copper silicate (CuSiO3) nano-particles.

[0014] Further preferably, the metal-organic framework nano-particles are γ-cyclodextrin metal-organic framework (γ-CDMOFs) nano-particles.

[0015] Preferably, the sodium crocinate is loaded on the nanomaterials.

[0016] Preferably, the mass ratio of the γ-cyclodextrin metal-organic framework (γ-CDMOFs) nanoparticles to the sodium crocinate is 1-12:1.

[0017] The present invention also provides a method for preparing a sodium crocinate nano-complex preparation, comprising the following steps:

[0018] (1) γ-cyclodextrin and KOH are dissolved in water together, methanol is added according to the volume ratio of water to methanol of 1-5:1, heated to 60-80 °C until clarified and then stirred to obtain solution A;

[0019] (2) Polyethylene glycol 20000 is dissolved in methanol and added to solution A to obtain a mixture;

[0020] (3) The mixture is heated, allowed to stand and cool overnight to obtain a crude product of γ-cyclodextrin metal-organic framework;

[0021] (4) The crude product of γ-cyclodextrin metal-organic framework is washed with methanol, centrifuged, activated with dichloromethane and dried to obtain γ-cyclodextrin metal-organic framework nanoparticles.

[0022] (5) The methanol solution of sodium crocinate is rotary evaporated and dispersed in the γ-cyclodextrin metal-organic framework nanoparticles, ultrasonicated, stirred, and rotary evaporated to dryness to obtain the sodium crocinate cyclodextrin metal-organic framework complex preparation.

[0023] Preferably, in step (1), the heating temperature is 60-80 °C, stirring is carried out while heating, and the stirring time is 1-3 h.

[0024] Preferably, in step (3), the heating temperature is 60-80 °C, and the cooling temperature is room temperature.

[0025] Preferably, in step (4), the solvent used for washing is methanol, and the solvent used for activation is dichloromethane.

[0026] The present invention also provides a drug for treating coronary heart disease, acute and chronic heart failure, cardiomyopathy, congenital heart disease, myocarditis, pericarditis or myocardial infarction, characterized in that the drug comprises the above-mentioned sodium crocinate nano-complex preparation and pharmaceutically acceptable excipients.

[0027] Preferably, the drug is in an oral dosage form or an injection dosage form.

[0028] More preferably, the oral dosage form is selected from tablets or capsules.

[0029] Preferably, the excipients are one or more of diluents, disintegrants, binders, lubricants.

[0030] Further preferably, the diluent is selected from one or more of microcrystalline cellulose, lactose, starch, dextrin, and sucrose.

[0031] Further preferably, the disintegrant is selected from one or more of croscarmellose sodium, crospovidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, and dry starch.

[0032] Further preferably, the binder is selected from one or more of hypromellose, methylcellulose, ethylcellulose, povidone, and sodium carboxymethyl cellulose.

[0033] Further preferably, the lubricant is selected from one or more of magnesium stearate, calcium stearate, talc powder, colloidal silica, and silicon dioxide.

[0034] The present invention also relates to the use of the above-mentioned sodium crocinate nano-composite preparation in the preparation of drugs for treating heart diseases, and the heart diseases include coronary heart disease, acute and chronic heart failure, cardiomyopathy, myocarditis, pericarditis, or myocardial infarction.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) The present invention provides a sodium crocinate nano-composite preparation. The dissolution and release process of loading CRAT in the nanomaterial is slower than that of CRAT alone. In addition, the drug detection rate of the nano-formulation enteric-coated capsules changes little after 6 months under accelerated conditions, indicating that loading CRAT on the nanocarrier can significantly improve the stability of CRAT. In the dissolution experiment of the example, after 60 minutes, ordinary CRAT and CDMOFs@CRAT, AuNRs@CRAT, CuSiO3@CRAT, CTS@CRAT, and PHB@CRAT can all be completely dissolved, while when CRAT is loaded on the nanocarrier, the dissolution and release of the CRAT drug is more gentle, showing a sustained-release effect.

[0037] (2) Loading CRAT onto the nanomaterial can change the release and physicochemical properties of CRAT in the intestine and improve the bioavailability of CRAT absorbed in vivo. In the example, the relative oral bioavailability of CDMOFs@CRAT is 198% higher than that of CRAT. Among them, for the comparison of the drug exposure amounts obtained by the two dosing sequences, the highest relative bioavailability of CDMOFs@CRAT can be increased by 287.5% and 376.7%, indicating the ability of CDMOFs as a delivery carrier to significantly improve the bioavailability of CRAT drugs and improve the drug metabolism situation.

[0038] (3) Sodium crocinate nano - complex formulation can significantly delay the pathological enlargement of the left ventricular cavity caused by HFrEF, improve EF, FS and cardiac ejection function, can significantly delay the development process of HFrEF, has a relatively obvious inhibitory effect on myocardial fibrosis, and improves the heart failure situation of rats.

[0039] (4) Sodium crocinate nano - complex formulation shows a stronger positive regulatory effect than CRAT in reducing the levels of NT - proBNP, CK, LDH and inflammatory - related indicators IL - 6, TNF - α, indicating that the 10 mg / kg dose of CDMOFs@CRAT has a stronger ability to slow down the heart failure process in multiple aspects than the single 30 mg / kg dose of CRAT. The activity of CDMOFs@CRAT is more than 3 times higher than that of single CRAT, and it can more effectively relieve the onset process of heart failure and related cardiovascular diseases and improve various cardiac indicators. Description of the Drawings

[0040] Figure 1 For the 1H - NMR spectra of CRAT, γ - CD, CDMOFs@CRAT in Example 3 1 1H - NMR spectra;

[0041] Figure 2 For the SEM images of CDMOFs and CDMOFs@CRAT in Example 3

[0042] Figure 3 For the representative SEM elemental mapping images of CDMOFs and CDMOFs@CRAT in Example 3

[0043] Figure 4 For the elemental spectra of CDMOFs and CDMOFs@CRAT in Example 3

[0044] Figure 5 For the XPS spectra of CDMOFs and CDMOFs@CRAT in Example 3

[0045] Figure 6 For the XRD spectra of γ - CD, CDMOFs, CRAT, CDMOFs@CRAT in Example 3

[0046] Figure 7 For the dissolution curves of CRAT, CDMOFs@CRAT, AuNRs@CRAT, CuSiO3@CRAT, CTS@CRAT, PHB@CRAT in Example 5

[0047] Figure 8For the characterization analysis of CDMOFs@CRAT in Example 7, where A is the state of CRAT and CDMOFs@CRAT at room temperature under different concentrations; B is the SEM image of CDMOFs after incubation in pH 6.8 solution for 30 minutes; C is the SEM image of CRAT after incubation in pH 6.8 solution for 30 minutes; D is the SEM image of CDMOFs@CRAT after incubation in pH 6.8 solution for 30 minutes;

[0048] Figure 9 Schematic illustration for the pharmacodynamic study of CRAT and CDMOFs@CRAT in the coronary artery ligation rat model of Example 9;

[0049] Figure 10 Representative results and scoring measurement analysis of heart failure-related biomarker levels in the rat serum of Example 9;

[0050] Figure 11 Results of the inhibitory effect on myocardial collagen fibers after administration of CRAT and CDMOFs@CRAT by Masson staining in Example 9. Detailed implementation manners

[0051] It should be noted that the raw materials used in the present invention are all ordinary commercially available products, and their sources are not specifically limited.

[0052] Explanation of the meanings of technical terms:

[0053] γ-CDMOFs, namely CDMOFs: γ-cyclodextrin metal-organic framework;

[0054] γ-CD: γ-cyclodextrin;

[0055] CRAT: crocin sodium;

[0056] CDMOFs@CRAT: crocin sodium γ-cyclodextrin metal-organic framework complex preparation;

[0057] AuNRs@CRAT: crocin sodium gold nanoparticle complex preparation;

[0058] CuSiO3@CRAT: crocin sodium copper silicate nanoparticle complex preparation;

[0059] CTS@CRAT: crocin sodium chitosan nano-complex preparation;

[0060] PHB@CRAT: crocin sodium polyhydroxybutyrate nano-complex preparation.

[0061] Example 1 Preparation of crocin sodium nano-complex preparation

[0062] (1) Sodium crocinate γ-cyclodextrin metal-organic framework complex preparation

[0063] 1 g of γ-cyclodextrin and 0.35 g of KOH were added to 31 mL of ultrapure water, and they were fully dissolved. Then 10 mL of methanol was added to obtain a cyclodextrin methanol solution. The cyclodextrin methanol solution was passed through a nylon 6 filter head (0.22 μm) and then stirred in a water bath at 60 °C for 1 hour. 0.62 g of PEG20000 (polyethylene glycol 20000) was dissolved in 3.1 mL of methanol to obtain a PEG20000 methanol solution, and then it was slowly added to the cyclodextrin methanol solution. The mixed solution continued to be stirred in a water bath at 60 °C for 10 minutes, and then it was allowed to stand and cool at room temperature for 12 h overnight to obtain a crude product of γ-CDMOFs. The crude product was washed with methanol. After centrifugation, it was soaked and activated with 10 mL of dichloromethane overnight. Then the solid was recovered by centrifugation and dried to obtain a γ-CDMOFs sample.

[0064] The dried γ-CDMOFs (8 g) were dispersed in 16 L (added in 4 batches for rotary evaporation, 4 L each time) of CRAT methanol (0.1% NaOH) solution (3.1 g, 4 L, 0.77 g / L), ultrasonicated for 5 min, and then stirred for 1 h. Then it was rotary evaporated to dryness at room temperature (25 °C). The solid was transferred to a centrifuge tube in batches, and the remaining material adhering to the glass bottle wall was transferred to the sample centrifuge tube in batches through 800 mL of methanol. The supernatant was removed by centrifugation (9500 rpm, 10 min, 4 °C), and the solid was washed 4 times in batches with 1000 mL of methanol. Then it was dried overnight (16 h) in a vacuum drying oven to obtain the final product, sodium crocinate γ-cyclodextrin metal-organic framework complex preparation CDMOFs@CRAT.

[0065] (2) Sodium crocinate gold nanoparticle complex preparation

[0066] 10 mL of an aqueous solution of cetyltrimethylammonium bromide with a concentration of 0.2 mol / L was placed in a constant temperature water bath at 25 °C, and 1 mL of an aqueous solution of chloroauric acid with a concentration of 5×10 -4 mol / L was added thereto. After stirring and mixing evenly, the solution turned yellowish-brown. Then 1 mL of an aqueous solution of ice-cold sodium borohydride with a concentration of 0.01 mol / L was added to the above solution, and it was rapidly stirred for 2 min and then left standing in the water bath at this temperature to obtain a gold nanoparticle seed solution.

[0067] 10 mL of an aqueous solution of cetyltrimethylammonium bromide with a concentration of 0.2 mol / L was placed in a constant temperature water bath at 25 °C, 1 mL of a silver nitrate solution with a concentration of 4×10 -3 mol / L was added, and after stirring evenly, 1×10 -31 mL of chloroauric acid aqueous solution with a concentration of mol / L was stirred evenly, and the solution changed from colorless to brownish-yellow. Then, ascorbic acid aqueous solution with a concentration of 0.0788 mol / L was added to the mixed solution, and it was quickly stirred until the mixed solution became colorless, which was called the growth solution.

[0068] 30 mL of seed solution was added to 10 mL of growth solution. After mixing evenly, it was placed in a water bath at 30 °C. After about 5 minutes, the solution changed from colorless to dark red. It was then continuously placed in a water bath pot under this condition for 12 h to obtain an aqueous solution of gold nanorods. 4 mg of CRAT was ultrasonically dissolved in a methanol (0.1% NaOH) solution. Methanol was removed by rotary evaporation under reduced pressure to form a transparent film in a round-bottom flask. 4 mL of the aqueous solution of gold nanorods was added, and hydration was continued by rotation. After complete hydration, it was ultrasonically dissolved again and sonicated 40 times in an ice bath using an ultrasonic cell disruptor. It was centrifuged at 8000 r / min for 10 min, the precipitate was discarded, vortexed for 1 min, and reacted on a shaker at room temperature for 12 h to obtain sodium crocinate gold nanoparticles AuNRs@CRAT.

[0069] (3) Sodium crocinate copper silicate nanoparticle complex preparation

[0070] Cetyltrimethylammonium bromide (0.18 g) and ammonium fluoride (0.3 g) were dissolved in 45 mL of deionized water. After magnetic stirring at 80 °C for 1 h, a mixed solution of tetraethyl orthosilicate (1 mL) and ethanol (4 mL) was slowly added dropwise to the above solution. After magnetic stirring at 80 °C for 4 h, the synthesized nanospheres were collected by centrifugation (9000 rpm, 5 min) and washed three times with ethanol. After freeze-drying the product for 48 h, mesoporous silica nanospheres with a size of about 80 - 120 nm could be obtained. Mesoporous silica (10 mg) was added to 10 mL of deionized water and ultrasonically dispersed to form a white emulsion; copper acetate (10 mg) and disodium maleate (100 mg) were dissolved in 10 mL of deionized water, and the two solutions were mixed evenly and ultrasonically stirred for 30 min. The mixed emulsion was transferred to a stainless-steel autoclave with a polytetrafluoroethylene lining and kept at 140 °C for 12 h. After cooling to room temperature, it was centrifuged (9000 rpm, 5 min) and then washed 3 times with deionized water to obtain the product CuSiO3. 8 mg of the prepared copper silicate nanoparticles was added to ethanol (5 mL) and ultrasonically treated for 10 min to form a uniform dispersion. Then, 2 mg of CRAT and 1 mg of distearoylphosphatidylethanolamine-polyethylene glycol-amine conjugate DSPE-PEG-NH2 were added to ethanol (5 mL). The above two solutions were mixed evenly and magnetically stirred at room temperature for 6 h. After centrifugation (9000 rpm, 5 min), it was washed three times with ethanol and water to obtain CuSiO3@CRAT nanoparticles.

[0071] (4) Sodium crocinate chitosan nano complex preparation

[0072] 8 g of chitosan (CTS) was added to 40 mL of 1% acetic acid solution and stirred for 1 hour. After the chitosan was completely dissolved, the solution was filtered through a 0.22 μm filter paper. 5 g of sodium tripolyphosphate was added to 40 mL of deionized water and stirred for 30 minutes. After the sodium tripolyphosphate was completely dissolved, the solution was filtered through a 0.22 μm filter paper. 2 g of CRAT was ultrasonically dissolved in methanol (0.1% NaOH) solution, and then dropped into the chitosan solution. Under continuous magnetic stirring, the sodium tripolyphosphate solution was slowly dropped into the solution and stirred for 30 minutes to form chitosan nanoparticles loaded with CRAT. Subsequently, centrifugation was carried out at a speed of 12,000 rpm, and the centrifuged product was freeze-dried in a freeze dryer to obtain chitosan nanoparticle powder CTS@CRAT loaded with CRAT.

[0073] (5) Sodium crocinate polyhydroxybutyrate nano complex preparation

[0074] 3 g of polyhydroxybutyrate (PHB) was weighed and dissolved in 100 mL of methanol. It was magnetically stirred at room temperature until completely dissolved, 0.3 g of CRAT was added, and magnetic stirring was continued for 10 minutes until evenly dissolved to form an organic phase. 30 g of polyvinyl alcohol was weighed and dissolved in 1000 mL of water. It was dissolved at high temperature for 1 hour until completely dissolved and cooled to room temperature to form an aqueous phase. 2 mL of the above organic phase and 200 mL of the aqueous phase were taken and placed in a dry beaker, and ultrasonic treatment was carried out at 100 W for 10 minutes to form an emulsion. Immediately, the emulsion was poured into a rotary evaporator to remove methanol. After 1 hour, high-speed centrifugation and resuspension of the nanoparticles could obtain sodium crocinate polyhydroxybutyrate nanoparticles PHB@CRAT.

[0075] Example 2 Determination of particle size, encapsulation efficiency and yield

[0076] The drug encapsulation efficiency and loading amount were calculated by the solution concentration (HPLC) before the reaction and weighing. The particle size of the nano preparation was measured by a laser nano particle size analyzer.

[0077] Calculation of drug loading amount and yield: 5 mg of the active substance powders of CDMOFs@CRAT, AuNRs@CRAT, CuSiO3@CRAT, CTS@CRAT and PHB@CRAT prepared in Example 1 were weighed respectively and a 50 μg / mL solution was prepared. The loading amount of CRAT in the γ-CDMOFs carrier was quantified by HPLC and the loading efficiency was calculated. Taking CDMOFs as an example, corresponding replacements were made for other nano preparations, and the drug encapsulation efficiency and yield were calculated by the following formulas.

[0078]

[0079]

[0080]

[0081] The results are shown in Table 1.

[0082] Table 1 Particle size, encapsulation efficiency and yield data of sodium crocinate complex preparation

[0083]

[0084] Example 3 Characterization of sodium crocinate γ-cyclodextrin metal-organic framework complex preparation

[0085] After mixing γ-cyclodextrin and potassium ions in a round-bottom flask by hydrothermal method, the formation of γ-CDMOFs crystal nuclei was controlled by adding PEG20000 as an activator to limit the size of crystal particles. The characterization of sodium crocinate γ-cyclodextrin metal-organic framework complex preparation CDMOFs@CRAT is as Figure 1 shown. In the 1 1H-NMR spectrum of γ-cyclodextrin ( Figure 1 ), the characteristic signals of this substance are at 5.12 ppm and 3.5 - 4 ppm respectively, where 4.79 ppm is the heavy water solvent peak. Through analysis, the characteristic peak at 5.12 ppm is the hydrogen atom at position 1 on the five-membered ring. Due to the influence of two electron-withdrawing oxygen atom groups, the electron cloud density around the hydrogen atom decreases, so it is the H with the largest numerical value in the chemical shift. The hydrogen atoms at other positions 2, 3, 4, 5, and 6 are mainly distributed in the range of 3.5 - 4 ppm. By comparing the 1H-NMR spectra of γ-cyclodextrin and γ-CDMOFs, it can be found that the characteristic absorption peaks of the two are basically the same, indicating the successful synthesis of γ-CDMOFs. In addition, since the H on the OH functional group is an inactive hydrogen, no absorption peak appears in the spectrum, probably due to the exchange with the deuterated hydrogen in the solvent D2O. In the 1H-NMR spectrum of CDMOFs@CRAT, the characteristic absorption belonging to γ-CDMOFs can also be clearly observed in the present invention. In addition, the characteristic absorption of CRAT is at 6 - 7 ppm, indicating the successful loading of CRAT. 1 The measurement conditions of 1H-NMR spectrum are: solvent (D2O, 1% NaOH), 25 °C.

[0086] The present invention also uses SEM surface electron microscopy scanning and surface element localization to confirm the changes in the morphology, size, dimensions, etc. of γ-CDMOFs and CDMOFs@CRAT. It can be analyzed from the characterization result diagram of SEM ( Figure 2), the γ-CDMOFs crystal particles have a regular cubic morphology with a smooth surface and a size of less than about 10 μm. The uneven crystal size may be due to the influence of solvents and temperature on the formation and rate of crystal nucleus growth. Energy spectrum data proves that the γ-CDMOFs crystal contains C, O, and K elements, indicating the successful synthesis and purity of γ-CDMOFs. The SEM-EDS result analysis of CDMOFs@CRAT shows no obvious difference in structure from the γ-CDMOFs crystal in terms of size. After drug loading, it can be observed that the surface of γ-CDMOFs is no longer smooth, and Na element can also be clearly observed in the EDS result ( Figure 3 、 Figure 4 ). Since SEM is a detection method for the surface of a substance, a large part of the adsorption of CRAT may be adsorbed into the internal pores of γ-CDMOFs. According to this result, the loading amount of CRAT cannot be judged in this invention. However, the loading behavior has no obvious effect on the crystal size of the γ-CDMOFs carrier, which confirms the feasibility of this loading scheme. Meanwhile, the experimental results of XPS further confirm that C and O are the main components of γ-CDMOFs. In the elemental composition of CRAT@CDMOFs, the appearance of the Na element of CRAT is clearly observed, which confirms the successful loading of CRAT. It is worth noting that the reason why the K element is not shown in the XPS spectrum is that the K element is in the pores of γ-CDMOFs and its detection intensity is relatively low ( Figure 5 ). As Figure 6 shows, γ-CDMOFs and CDMOFs@CRAT have obvious crystal diffraction peaks in the range of 5°-30°. From the results, the characteristic peaks of the X-ray powder diffraction data of γ-CDMOFs and the characteristic diffraction peaks of CDMOFs@CRAT are basically the same in peak shape, indicating that the drug loading behavior has no effect on the crystallinity of the crystal carrier. The characteristic diffraction peaks of CRAT can be observed in the diffraction results of CDMOFs@CRAT, which effectively confirms the successful synthesis of CRAT@CDMOFs.

[0087] Example 4 Preparation of Enteric Capsule Preparation

[0088] CRAT enteric capsule preparation: Weigh 40 mg of CRAT and 214.4 mg of lactose into a PE bag, mix for 5 min and then pass through a 60-mesh sieve twice. Then add 40 mg of pregelatinized starch, 16 mg of low-substituted hydroxypropyl cellulose, and 6.4 mg of silicon dioxide, mix for 5 min and then pass through a 60-mesh sieve. Finally, add 3.2 mg of magnesium stearate and mix for 3 min to obtain the total mixture, which is filled into CRAT enteric capsules.

[0089] CDMOFs@CRAT Enteric Capsule Preparation: Weigh 66.67 mg of CDMOFs@CRAT and 142.93 g of lactose into a PE bag. After mixing for 5 minutes, pass through a 60-mesh sieve twice. Then add 84.8 mg of pregelatinized starch, 16 mg of low-substituted hydroxypropyl cellulose, and 6.4 mg of silicon dioxide, mix for 5 minutes and then pass through a 60-mesh sieve. Finally, add 3.2 mg of magnesium stearate and mix for 3 minutes to obtain the total mixture, which is filled into CDMOFs@CRAT enteric capsules.

[0090] The formulation tables of CRAT and CDMOFs@CRAT enteric capsule preparations are shown in Table 2. The preparation methods of AuNRs@CRAT, CuSiO3@CRAT, CTS@CRAT, and PHB@CRAT enteric capsules refer to the preparation process of the above-mentioned CDMOFs@CRAT enteric capsules.

[0091] Table 2 Formulation Table of CRAT and CDMOFs@CRAT Enteric Capsules

[0092]

[0093] Example 5 Dissolution Experiment

[0094] Determination was carried out with reference to the dissolution and release determination method (Method 2, General Principles 0931, Volume IV, Chinese Pharmacopoeia 2020 Edition): Take 5 capsules each of CRAT enteric gel and CDMOFs@CRAT, AuNRs@CRAT, CuSiO3@CRAT, CTS@CRAT, and PHB@CRAT nano-enteric capsules prepared in Example 4. Using 900 mL of 0.1 mol / L hydrochloric acid solution as the dissolution medium, the paddle speed is 50 revolutions per minute. After 120 minutes, discard the acid solution in each dissolution cup, immediately add 900 mL of pH 7.8 phosphate buffer solution at 37°C ± 0.5°C, keep the speed unchanged, and take 10 mL of the dissolution solution at 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, and 120 minutes respectively, and promptly supplement 10 mL of the dissolution medium at 37°C ± 0.5°C. Filter with a 0.45 μm aqueous filter head, discard the initial filtrate of 5 mL, accurately measure 1 mL of the subsequent filtrate and place it in a 20 mL volumetric flask, dilute to the scale with pH 7.8 phosphate buffer solution, shake well, measure the absorbance at a wavelength of 421 nm, and calculate the dissolution amount per capsule.

[0095] Calculation formula:

[0096]

[0097]

[0098] Note: When t = 1, the cumulative dissolution degree (t)% is equal to the dissolution degree (t)%.

[0099] In the formula:

[0100] A t — Absorbance of the test sample solution at time point t; A 对 — Absorbance of the reference substance solution;

[0101] f — Correction factor; — Average correction factor;

[0102] m 对 — Sampling weight of the reference substance, mg; V 对 — Dilution volume of the reference substance, mL;

[0103] P — Content of the reference substance; V0 — Initial volume of the dissolution medium, mL;

[0104] V1 — Sampling volume, mL; V2 — Supplementary liquid volume, mL;

[0105] n — Number of sampling time points; t — The t-th sampling point;

[0106] M r — Labeled specification of the test sample, mg.

[0107] In the present invention, the dissolution of CRAT alone and nano-formulations of CDMOFs@CRAT, AuNRs@CRAT, CuSiO3@CRAT, CTS@CRAT, and PHB@CRAT was studied in a simulated small intestine physiological environment (pH 7.8 PBS solution). It can be observed from Figure 7 that the dissolution and release process of CRAT loaded in CDMOFs, AuNRs, CuSiO3, CTS, and PHB nanoparticles is slower than that of CRAT alone. Almost 100% of CRAT alone is dissolved after 30 minutes, but only 50 - 70% of CDMOFs@CRAT and other nano-formulations are dissolved at the same time. It is not until nearly 60 minutes later that the CRAT drug in CDMOFs@CRAT, AuNRs@CRAT, CuSiO3@CRAT, CTS@CRAT, and PHB@CRAT nano-formulations is completely dissolved. It can be seen that the dissolution and release of the CRAT drug loaded in nanoparticles CDMOFs, AuNRs, CuSiO3, CTS, and PHB is more gentle, and the nano-formulations have a certain drug sustained-release effect.

[0108] Example 6 Accelerated Stability Experiment

[0109] Accelerated stability tests were conducted on the CDMOFs@CRAT, AuNRs@CRAT, CuSiO3@CRAT, CTS@CRAT, and PHB@CRAT nanoenteric capsules prepared in Example 4. The above nanoformulations were taken and placed in a stability chamber at a temperature of 30°C ± 2°C and a relative humidity of 75% ± 5% for six months. Samples were taken at 1, 2, 3, and 6 months of acceleration to detect the content of CRAT. The detection method was high-performance liquid chromatography. An Agilent TC-C18 chromatographic column (4.6 mm × 200 mm, 5 μm) was used, the mobile phase was methanol:water:glacial acetic acid (75:24.5:0.5), the flow rate was 1.0 mL / min, the column temperature was 30°C, the detection wavelength was 423 nm, and the area normalization method was used to calculate the content of CRAT. The results are shown in Table 3.

[0110] The results of the accelerated stability test showed that the enteric-coated capsules of the CRAT nanoformulations prepared in the present invention had good stability under accelerated conditions. By detecting their content by HPLC, it was shown that there was no obvious degradation phenomenon after six months of acceleration. Compared with ordinary CRAT enteric-coated capsules, the drug stability of the nanoformulations CDMOFs@CRAT, AuNRs@CRAT, CuSiO3@CRAT, CTS@CRAT, and PHB@CRAT was good.

[0111] Table 3 Accelerated stability experiment data

[0112]

[0113] Characteristic analysis of CDMOFs@CRAT in Example 7

[0114] The characteristic analysis of CDMOFs@CRAT is as Figure 8 shown, where A shows the states of CRAT and CDMOFs@CRAT at room temperature at different concentrations. In addition, in order to verify the stability of CRAT in the physiological environment of the duodenum after being loaded, the present invention incubated γ-CDMOFs, CRAT, and CRAT@CDMOFs in an acidic environment at pH 6.8 and observed the morphological changes by SEM. Figure 8 B in it shows that although there is a certain collapse on the crystal surface of γ-CDMOFs in the acid, the overall morphology of the crystal still maintains its original appearance; while Figure 8 C in it shows that CRAT alone has completely agglomerated and formed flakes in the slightly acidic environment; Figure 8In D, it was observed that in an acidic environment, although the CRAT drug in CDMOFs@CRAT had been completely released and crystallized, it still surrounded the surface of the γ-CDMOFs carrier instead of forming a flaky structure. This morphological change after loading could significantly increase the specific surface area of CRAT, which was beneficial to the intestinal drug absorption of CRAT and the metabolism of the drug in the body. Loading CRAT into γ-CDMOFs to form CDMOFs@CRAT not only had a certain drug sustained-release effect, but also had a favorable impact on the intestinal absorption and oral bioavailability of CRAT.

[0115] Example 8 Drug Metabolism Analysis of CDMOFs@CRAT

[0116] Animal source, animal feeding and ethics number: 6 ordinary-grade female Beagle dogs weighing 12 - 16 kg, which were reserve dogs from Boji Pharmaceutical Drug Evaluation Center (Experimental Animal Use License Number: SYXK(Yue)2022 - 0279), and the experimental animal quality certificate numbers were: No.110334210100040471, No.370825220100019471. 6 ordinary-grade female Beagle dogs weighing 12 - 16 kg were used. They were sorted by body weight and randomly divided into 2 groups, with 3 dogs in each of Group A and Group B (numbered A1, A2, A3, B1, B2, B3 respectively). The drugs were administered after meals by oral administration. A 7-day double-cycle cross-over dosing was carried out, and the ordinary CRAT enteric-coated capsules and CDMOFs@CRAT enteric-coated capsules prepared in Example 4 were given respectively; the drugs were administered once per cycle, 1 capsule each time. The cross-over dosing situation is shown in the following table:

[0117] Table 4 Double-Cycle Cross-Over Dosing Design

[0118]

[0119] Blood samples were collected from the two groups of animals before dosing and at 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, 18 h, 24 h after dosing. Approximately 2 mL of whole blood was collected from the cephalic vein of the forelimb into a heparinized blood collection tube, centrifuged at 4500 rpm for 10 min to separate plasma, and stored in an ultra-low temperature refrigerator for later measurement. The concentration of crocin in plasma was determined by a validated UPLC method. The blood drug concentration data were processed using DAS 3.3.0, the main pharmacokinetic parameters were calculated using a non-compartmental model, and the relative bioavailability of different formulations of sodium crocinate (CRAT) was compared.

[0120] The specific results are shown in Tables 5 - 7. It can be seen from the data in the tables that the half-life t 1 / 2 From CRAT t 1 / 2 of 2.466 ± 0.517 h and CDMOFs@CRAT t1 / 2 was equivalent to 2.436 ± 0.98 h; the time to peak drug concentration Tmax was prolonged from 2.37 h of CRAT to 3.33 h of CDMOFs@CRAT, an increase of 40%, with a significant effect; the peak drug concentration Cmax of CDMOFs@CRAT increased significantly, being 1260.302 μg / L for CRAT and 2188.522 μg / L for CDMOFs@CRAT, and CDMOFs@CRAT was 1.74 times that of CRAT. It can be seen that on the basis of the same concentration of active drug, CDMOFs@CRAT has better absorption effect and longer action time in vivo. It can be seen that whether CRAT or CDMOFs@CRAT is orally administered first, the plasma exposure AUC of CDMOFs@CRAT, 134.13 μg / L*h, was significantly higher than that of CRAT, 7303.27 μg / L*h, and CDMOFs@CRAT was 1.66 times that of CRAT; in addition, comparing the drug exposure AUC obtained by the two dosing sequences, the highest relative bioavailability of CDMOFs@CRAT in the two groups of animals could be increased by 287.5% and 376.7% respectively, and the relative oral bioavailability of CDMOFs@CRAT was 198% higher than that of CRAT. The above experimental results confirmed that compared with CRAT, both the peak concentration and peak time of CDMOFs@CRAT were improved, significantly increasing the in vivo bioavailability of crocetin sodium.

[0121] Table 5 Pharmacokinetic parameters of CRAT enteric-coated capsules

[0122]

[0123] Table 6 Pharmacokinetic parameters of CDMOFs@CRAT enteric-coated capsules

[0124]

[0125] Table 7 Relative oral bioavailability of CRAT and CDMOFs@CRAT enteric-coated capsules

[0126]

[0127] Example 9 Pharmacodynamic analysis of CDMOFs@CRAT

[0128] Animal source, animal feeding and ethical number: 170.6 - 258.1 g SD rats, SPF grade, 105 rats, 5 - 7 weeks old, male. Surplus animals will be euthanized after the experiment. Purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd., the experimental animal production license number is: SCXK(Yue)2022 - 0063, and the experimental animal quality certificate number is No.44829700004834.

[0129] SD rats were subjected to coronary artery ligation to induce chronic heart failure with reduced ejection fraction (HFrEF) for modeling, in order to investigate the therapeutic effects of the ordinary CRAT enteric capsules (low and high doses) and CDMOFs@CRAT enteric capsules prepared in Example 4 on HFrEF. Except for the 8 sham operation groups, the remaining rats were subjected to coronary artery ligation. The sham operation groups only had the thread passed through without ligation, and were routinely raised for 8 weeks after the operation. Subsequently, the animals with ejection fraction (EF) < 50% were randomly divided into a model control group, a CRAT low-dose group, a CRAT high-dose group, and a CDMOFs@CRAT group. Gastric gavage administration was started, and the specific administration is shown in Table 8, once a day for 5 consecutive weeks. During the administration treatment period, there was no significant difference in the body weight of the animals among the groups. Echocardiogram and blood biochemical tests were performed in the 5th week of administration, including serum N-terminal pro-brain natriuretic peptide (NT-proBNP), creatine kinase (CK), and lactate dehydrogenase (LDH), and the heart was taken for HE and Masson staining. The specific results are shown in Figures 9 - 11 。

[0130] Table 8 Administration conditions of different experimental groups

[0131]

[0132] To evaluate the ability of CRAT and CDMOFs@CRAT to regulate the cardiac function of HFrEF rats, the cardiac-related indexes in the echocardiograms of each group were compared in the 5th week of administration. From Figure 9Comparative analysis showed that, compared with the model control group, the ejection fraction (EF) and fractional shortening (FS) in the low-dose CRAT group showed an increasing trend, while the left ventricular end diastolic dimension (LVDd) and left ventricular end systolic dimension (LVDs) showed a decreasing trend; in the high-dose CRAT group, EF and FS also showed an increasing trend, and LVDd and LVDs showed a decreasing trend. In the CDMOFs@CRAT group, LVDs decreased significantly (p<0.05), EF and FS increased significantly (p<0.05), and LVDd showed a decreasing trend. At the same time, the research results showed that the EF and FS of CDMOFs@CRAT were higher than those of the low- and high-dose CRAT groups, and the LVDs and LVDd were lower than those of the low- and high-dose CRAT groups. The above results indicate that CRAT can significantly delay the pathological enlargement of the left ventricular cavity caused by HFrEF, improve EF and FS to a certain extent, and enhance the cardiac ejection function. Among them, CDMOFs@CRAT has a more significant improvement effect on various cardiac function indicators than the low- and high-dose CRAT groups. Moreover, the 10 mg / kg dose of CDMOFs@CRAT can more effectively relieve the disease progression of heart failure and improve various indicators of heart failure than the single 30 mg / kg dose of CRAT. The activity of CDMOFs@CRAT is more than 3 times higher than that of single CRAT, achieving unexpected technical effects.

[0133] The present invention analyzed the level of the heart failure-related marker N-terminal pro-brain natriuretic peptide (NT-proBNP) in rat serum. From Figure 10Data analysis shows that, compared with the sham operation group, the model control group showed an increasing trend. Compared with the model control group, the NT-proBNP levels in the high-dose CRAT group and the CDMOFs@CRAT group both showed a significant decreasing trend, and the NT-proBNP level of CDMOFs@CRAT was significantly lower than that of the low-dose CRAT group and the high-dose CRAT group, almost reaching the level of the normal model group. At the same time, the present invention also detected the levels of CK and LDH, representative substances of myocardial injury, to explore the protective effects of CRAT and CDMOFs@CRAT on myocardial injury. Compared with the sham operation group, the levels of CK and LDH in the serum of the model control group were significantly increased; compared with the model control group, significant decreases were observed in the serum CK and LDH levels of the low-dose CRAT group, the high-dose CRAT group, and the CDMOFs@CRAT group, and the serum CK and LDH levels of the CDMOFs@CRAT group were significantly lower than those of the low-dose and high-dose CRAT groups, and even lower than those of the sham operation control group. The serum CK and LDH values of CDMOFs@CRAT at a dose of 10 mg / kg were not only significantly lower than those of CRAT at a dose of 10 mg / kg, but also significantly lower than those of CRAT at a dose of 30 mg / kg. The research results indicate that CDMOFs@CRAT at a dose of 10 mg / kg has a stronger effect in improving heart failure and treating myocardial injury than CRAT at a dose of 30 mg / kg.

[0134] The present invention also observed the inhibitory effect on myocardial collagen fibers after administration of CRAT and CDMOFs@CRAT by Masson staining. The results are as Figure 11 shown. Compared with the sham operation control group, the positive area of Masson staining in the model control group, the CRAT group, and the CDMOFs@CRAT group increased significantly, presenting an obvious state of myocardial fibrosis. At the same time, each administration group of CRAT could reduce the positive area of Masson staining, indicating that the degree of myocardial fibrosis was effectively improved; among them, the positive area of the CDMOFs@CRAT group was smaller than that of the low-dose and high-dose CRAT groups, and there was a significant improvement compared with the model control group, indicating that CRAT has a relatively obvious inhibitory effect on myocardial fibrosis, especially that CDMOFs@CRAT has a stronger inhibitory effect on myocardial fibrosis than the conventional CRAT enteric capsule.

[0135] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than a limitation on the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art does not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A sodium crocetin nanocomplex preparation with high bioavailability, characterized in that: The sodium crocetin nano complex preparation comprises sodium crocetin and a nano material, wherein the nano material is a γ-cyclodextrin metal organic framework nano particle; the sodium crocetin is loaded on the nano material; the sodium crocetin nano complex preparation is prepared by the following steps: (1) γ-cyclodextrin and KOH are dissolved in water, methanol is added in a volume ratio of water to methanol of 1-5:1, the mixture is heated to 60-80°C until it becomes clear and stirred to obtain solution A; (2) Dissolve polyethylene glycol 20000 in methanol and add solution A to obtain a mixed solution; (3) The mixed solution is heated and allowed to cool overnight to obtain a crude γ-cyclodextrin metal organic framework product; (4) The crude product of γ-cyclodextrin metal organic framework is washed with methanol, centrifuged, activated with dichloromethane, and dried to obtain γ-cyclodextrin metal organic framework nanoparticles; (5) The methanol solution of sodium crocetin is dispersed in the γ-cyclodextrin metal organic framework nanoparticles by rotary evaporation, and then ultrasonicated, stirred, and rotary evaporated to dry.

2. The sodium crocetin nanocomplex preparation according to claim 1, characterized in that: The mass ratio of the γ-cyclodextrin metal organic framework nanoparticles to the sodium crocetin is 1-12:

1.

3. A method for preparing the sodium crocetin nanocomplex preparation according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: (1) γ-cyclodextrin and KOH are dissolved in water, methanol is added in a volume ratio of water to methanol of 1-5:1, the mixture is heated to 60-80°C until it becomes clear and stirred to obtain solution A; (2) Dissolve polyethylene glycol 20000 in methanol and add solution A to obtain a mixed solution; (3) The mixed solution is heated and allowed to cool overnight to obtain a crude γ-cyclodextrin metal organic framework product; (4) The crude product of γ-cyclodextrin metal organic framework is washed with methanol, centrifuged, activated with dichloromethane, and dried to obtain γ-cyclodextrin metal organic framework nanoparticles; (5) The methanol solution of sodium crocetin is dispersed in the γ-cyclodextrin metal organic framework nanoparticles by rotary evaporation, and then ultrasonicated, stirred, and rotary evaporated to obtain a sodium crocetin cyclodextrin metal organic framework complex preparation.

4. A drug for treating heart disease, characterized in that: The drug comprises the sodium crocetin nanocomplex preparation according to any one of claims 1 or 2 and pharmaceutically acceptable excipients, and the heart disease includes coronary heart disease, chronic heart failure, cardiomyopathy or myocardial infarction.

5. The drug according to claim 4, characterized in that The dosage form of the drug is an oral dosage form or an injection dosage form.

6. The drug according to claim 5, characterized in that The oral dosage form is selected from tablets or capsules.

7. The drug according to claim 4, characterized in that The auxiliary material is selected from one or more of a diluent, a disintegrant, a binder, and a lubricant; The diluent is selected from one or more of microcrystalline cellulose, lactose, starch, dextrin, and sucrose; The disintegrant is selected from one or more of cross-linked sodium carboxymethyl cellulose, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, and dry starch; The binder is selected from one or more of hydroxypropyl methylcellulose, methyl cellulose, ethyl cellulose, povidone, and sodium carboxymethyl cellulose; The lubricant is one or more of magnesium stearate, calcium stearate, talcum powder, micro-powder silica gel and silicon dioxide.

8. Use of the sodium crocetin nanocomplex preparation according to any one of claims 1-2 or the sodium crocetin nanocomplex preparation obtained by the preparation method according to claim 3 or the drug according to any one of claims 4-7 in the preparation of drugs for treating heart diseases, wherein the heart diseases include coronary heart disease, chronic heart failure, cardiomyopathy or myocardial infarction.

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