A daidzein drug co-crystal and its preparation method and application

Abstract

The present invention relates to the technical field of pharmaceutical co-crystals, and specifically discloses a daidzein pharmaceutical co-crystal, a preparation method thereof, and an application thereof. The daidzein pharmaceutical co-crystal comprises daidzein and betaine in a molar ratio of 1:2. The preparation method comprises the following steps: adding daidzein and betaine to a solvent, stirring and mixing uniformly, filtering, collecting powder, and drying to obtain a daidzein pharmaceutical co-crystal; wherein the solvent is a saturated solution of daidzein and betaine. The daidzein-betaine co-crystal provided by the present invention not only improves the in vitro solubility and oral bioavailability of the daidzein raw material, but also significantly enhances the hygroscopic stability of betaine, and daidzein and betaine synergistically can effectively enhance the efficacy of the co-crystal in preventing and treating obesity, and can be used to develop into a variety of preparations for use as a new drug for metabolic diseases such as obesity and diabetes, with high application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical co-crystals, and in particular to a daidzein pharmaceutical co-crystal and a preparation method and application thereof. Background Art

[0002] In recent years, obesity has become a widespread international health issue. The World Health Organization (WHO) estimates that by 2025, approximately one-fifth of the global adult population will be obese. The prevalence of obesity and overweight in people aged 3 to 17 and 18 years and older is 19% and 50.7%, respectively. Obesity is a chronic metabolic disease characterized by an imbalance between energy intake and expenditure and excessive fat accumulation. It is closely associated with type 2 diabetes mellitus (T2DM), non-alcoholic fatty liver disease (NAFLD), cardiovascular disease, and osteoarthritis. In animals, obesity is also one of the most common chronic metabolic diseases in dogs. Numerous studies have shown that obesity is a major cause of other canine diseases, such as diabetes, cancer, osteoarthritis, hypertension, and hyperlipidemia, ultimately leading to shortened lifespan and increased mortality, severely endangering their health and shortening their lifespan. Oral medication is currently a common treatment for obesity. In 1998, the European Medicines Agency (EMA) approved oritas as a long-term weight loss medication. However, its low oral bioavailability, first-pass metabolism, and severe side effects (steatorrhea, fecal incontinence, and liver and kidney damage) limit its use. Given the current high prevalence of obesity and limited treatment options, the development of safe, effective, and environmentally friendly weight loss medications is urgently needed.

[0003] Daidzein (DAI), whose chemical structure is shown in Formula (I), is the most abundant isoflavone found in plants such as soybeans, alfalfa, kudzu root, and red clover. Studies have shown that daidzein can act as a PPARγ and SCD1 inhibitor and UCP-1 activator, reducing high-fat diet-induced obesity in C57BL / 6J mice and rats. However, daidzein is a BSC IV drug with a solubility in water of only 5.3 μg / mL, which greatly limits its clinical application. Therefore, there is a need to improve the oral absorption and availability of daidzein to expand its clinical application.

[0004] Summary of the Invention

[0005] In view of the problems in the prior art such as poor solubility, low bioavailability and inability to fully exert the efficacy of daidzein, the present invention provides a daidzein drug co-crystal and a preparation method and application thereof.

[0006] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0007] In a first aspect, the present invention provides a daidzein drug co-crystal comprising daidzein and betaine in a molar ratio of 1:2.

[0008] The present invention provides a pharmaceutical co-crystal formed by combining two active pharmaceutical ingredients, daidzein and betaine, through hydrogen bonding forces. The co-crystal not only improves the in vitro solubility and oral bioavailability of the daidzein raw material, but also significantly enhances the hygroscopic stability of betaine. The daidzein-betaine co-crystal provided by the present invention not only retains the pharmacological properties of the two active ingredients themselves, but also improves the physicochemical properties such as drug stability and solubility, has more stable physicochemical properties, can improve bioavailability, and thus more effectively promotes drug absorption. In addition, compounding the two drugs with anti-obesity efficacy can produce a synergistic effect, significantly improving the preventive and therapeutic effects on obesity, and is a novel drug for preventing and treating obesity with high application prospects.

[0009] Betaine (BTN), also known as trimethylglycine or glycine betaine, has a structural formula as shown in Formula (II). It is a natural compound isolated from sugar beet molasses and has a wide range of pharmacological activities. It has high solubility and bioavailability. Studies have shown that betaine plays a positive role in regulating lipid metabolism in diseases such as obesity and diabetes. However, betaine is highly hygroscopic. At a relative humidity (RH) of 20%, anhydrous betaine will convert into monohydrate betaine, and at a RH of 54%, it will dissolve. The present invention forms a cocrystal of daidzein (DAI) and betaine (BTN), thereby simultaneously solving the problems of low solubility and poor bioavailability of daidzein and poor hygroscopic stability of betaine, thereby improving the bioavailability of both.

[0010] Furthermore, the daidzein drug co-crystal has characteristic diffraction peaks at 2θ=6.4°±0.2°, 9.4°±0.2°, 18°±0.2°, 21.5°±0.2°, and 22.1°±0.2°.

[0011] Furthermore, the daidzein drug co-crystal also has characteristic diffraction peaks at 2θ=7.9°±0.2°, 9.9°±0.2°, 10.7°±0.2°, 12.7°±0.2°, 12.9°±0.2°, 15.5°±0.2°, 15.9°±0.2°, 16.3°±0.2°, 16.9°±0.2°, 18.3°±0.2°, and 20.9°±0.2°.

[0012] Furthermore, the daidzein drug co-crystal is a monoclinic P21 / n space group with unit cell parameters of α=γ=90°, β=93.983(9)°.

[0013] In a second aspect, the present invention also provides a method for preparing a daidzein drug co-crystal, comprising the following steps: adding daidzein and betaine to a solvent, stirring and mixing evenly, filtering, collecting powder, and drying to obtain a daidzein drug co-crystal; wherein the solvent is a saturated solution of daidzein and betaine.

[0014] Pharmaceutical cocrystals are a new type of solid form, formed by two or more molecules (at least one of which is an active pharmaceutical ingredient, or API) bound together in the same crystal lattice through non-ionic and non-covalent bonds. This structure gives cocrystals unique physicochemical properties, which can often greatly improve the various physicochemical and pharmaceutical properties of drugs. However, not all drugs can form cocrystals. Appropriate intermolecular forces are required between the drug molecules and the coformer. The two substances need to be compatible and not cause problems with the chemical or physical stability of the two substances. Therefore, the selection of the coformer is crucial, and the preparation method of the cocrystal will also have a significant impact on the physicochemical and pharmaceutical properties of the cocrystal.

[0015] The preparation method of the daidzein-betaine cocrystal provided by the present invention is simple, has mild operating conditions, is not prone to forming hydrates and solvates, has low cost, causes little environmental pollution, is suitable for large-scale industrial production, and has high promotion value.

[0016] Furthermore, the molar ratio of daidzein to betaine is 1:1 to 1:3.

[0017] Furthermore, the mass volume ratio of the total mass of the daidzein and betaine to the solvent is 100 mg:1 mL to 10 mg:1 mL.

[0018] Furthermore, the solvent of the saturated solution of daidzein and betaine is at least one of an alcohol solvent, acetonitrile, ethyl acetate, water or acetone.

[0019] Specifically, the alcohol solvent is anhydrous ethanol, methanol or isopropanol.

[0020] As a specific embodiment of the present invention, the solvent of the saturated solution of daidzein and betaine is anhydrous ethanol.

[0021] Furthermore, the stirring speed is 200 r / min to 1000 r / min, the stirring temperature is 15° C. to 45° C., and the stirring time is 12 h to 48 h.

[0022] The preferred reaction conditions are conducive to the formation of daidzein-betaine co-crystals with high purity and good stability.

[0023] Furthermore, the drying temperature is 45°C to 55°C.

[0024] Optionally, the drying method is to place the solid powder obtained by filtration in an oven at 45° C. to 55° C. and dry it to a constant weight.

[0025] In a third aspect, the present invention further provides a pharmaceutical composition comprising any one of the above-mentioned daidzein drug co-crystals.

[0026] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0027] Pharmaceutically acceptable excipients include carriers, diluents, wetting agents, emulsifiers, binders, coating agents, fillers, glidants, lubricants, disintegrants or pH buffers known in the art that can be used in pharmaceutical preparations.

[0028] Furthermore, the pharmaceutical composition is in the form of tablets, capsules, pills, injection preparations, sustained-release preparations or controlled-release preparations.

[0029] The daidzein-betaine co-crystals provided by the present invention can be prepared into a variety of dosage forms, such as tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, pellets, and dripping pills. They can also be prepared into sustained-release and controlled-release preparations. The preparation can be carried out according to conventional methods for dosage forms in the art, and the present invention does not impose any particular limitations thereon.

[0030] In a fifth aspect, the present invention further provides use of any of the aforementioned daidzein drug co-crystals or any of the aforementioned pharmaceutical compositions in the preparation of drugs for preventing and treating obesity.

[0031] The daidzein-betaine co-crystal provided by the present invention has high solubility, good stability, and high bioavailability. The daidzein and betaine synergistically can effectively enhance the efficacy of the co-crystal in preventing and treating obesity. The co-crystal can be used to develop into various preparations as a new drug for metabolic diseases such as obesity and diabetes, and has high application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 This is a powder X-ray diffraction (PXRD) pattern of the daidzein-betaine powder prepared in Comparative Example 1 of the present invention;

[0034] Figure 2This is a powder X-ray diffraction (PXRD) pattern of the daidzein-betaine powder prepared in Comparative Example 2 of the present invention;

[0035] Figure 3 The powder X-ray diffraction (PXRD) patterns of daidzein, melatonin, and the daidzein-melatonin mixture in Comparative Example 3 of the present invention are shown;

[0036] Figure 4 The powder X-ray diffraction (PXRD) patterns of betaine, genistein, and the betaine-genistein mixture in Comparative Example 4 of the present invention are as follows;

[0037] Figure 5 The powder X-ray diffraction (PXRD) patterns of daidzein, chlorogenic acid, and the daidzein-chlorogenic acid mixture in Comparative Example 5 of the present invention are shown;

[0038] Figure 6 This is the powder X-ray diffraction (PXRD) pattern of the daidzein-betaine co-crystal prepared in Example 1 of the present invention;

[0039] Figure 7 The single crystal structure diagram of the daidzein-betaine co-crystal prepared in Example 1 of the present invention, wherein (A) is the asymmetric unit, (B) is the hexameric structure diagram, (C) is the one-dimensional chain structure diagram, (D) is the two-dimensional structure diagram, and (E) is the three-dimensional structure diagram;

[0040] Figure 8 This is a thermogravimetric analysis (TG) of the daidzein-betaine co-crystal prepared in Example 1 of the present invention;

[0041] Figure 9 is a differential scanning calorimetry (DSC) diagram of the daidzein-betaine co-crystal prepared in Example 1 of the present invention;

[0042] Figure 10 This is a graph showing the intrinsic dissolution rate of the daidzein-betaine co-crystal prepared in Example 1 of the present invention;

[0043] Figure 11 This is a dynamic water vapor adsorption curve of the daidzein-betaine co-crystal prepared in Example 1 of the present invention;

[0044] Figure 12 This is a drug-time curve of the daidzein-betaine co-crystal prepared in Example 1 of the present invention;

[0045] Figure 13 This is a graph showing weekly weight changes of rats in the daidzein group, betaine group, daidzein-betaine physical mixture group, and daidzein-betaine cocrystal group in Test Example 1 of the present invention;

[0046] Figure 14This is a comparison chart of body weight gain of the daidzein group, betaine group, daidzein-betaine physical mixture group and daidzein-betaine cocrystal group in Test Example 1 of the present invention after 8 weeks of experiment. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0048] In order to better illustrate the present invention, further examples are given below.

[0049] Example 1

[0050] This embodiment provides a daidzein-betaine co-crystal, comprising daidzein and betaine in a molar ratio of 1:2.

[0051] The preparation method of the daidzein-betaine co-crystal comprises the following steps:

[0052] 20 mg of daidzein and 120 mg of betaine were added to 2 mL of anhydrous ethanol, sonicated for 10 min, and filtered to obtain a saturated solution of daidzein and betaine, which was recorded as the solvent;

[0053] Accurately weigh daidzein and betaine in a molar ratio of 1:1 and add them to the above solvent, wherein the ratio of the total mass of daidzein and betaine to the above solvent is 100 mg:1 mL. Stir continuously at 25°C and 600 r / min for 24 h, filter with filter paper, and dry the obtained solid in a 45°C oven to constant weight. Pass through an 80-mesh sieve to obtain daidzein-betaine cocrystal.

[0054] Example 2

[0055] This embodiment provides a daidzein-betaine co-crystal, comprising daidzein and betaine in a molar ratio of 1:2.

[0056] The preparation method of the daidzein-betaine co-crystal comprises the following steps:

[0057] 20 mg of daidzein and 120 mg of betaine were added to 2 mL of anhydrous ethanol, sonicated for 10 min, and filtered to obtain a saturated solution of daidzein and betaine, which was recorded as the solvent;

[0058] Accurately weigh daidzein and betaine in a molar ratio of 1:2 and add them to the above solvent, wherein the ratio of the total mass of daidzein and betaine to the above solvent is 100 mg:5 mL. Stir continuously at 15°C and 200 r / min for 48 h, filter with filter paper, and dry the obtained solid in a 50°C oven to constant weight. Pass through an 80-mesh sieve to obtain daidzein-betaine cocrystal.

[0059] Example 3

[0060] This embodiment provides a daidzein-betaine co-crystal, comprising daidzein and betaine in a molar ratio of 1:2.

[0061] The preparation method of the daidzein-betaine co-crystal comprises the following steps:

[0062] 10 mg of daidzein and 60 mg of betaine were added to 1 mL of anhydrous ethanol, sonicated for 15 min, and filtered to obtain a saturated solution of daidzein and betaine, which was recorded as the solvent;

[0063] Accurately weigh daidzein and betaine in a molar ratio of 1:3 and add them to the above solvent, wherein the ratio of the total mass of daidzein and betaine to the above solvent is 100 mg:10 mL. Stir continuously at 45°C and 1000 r / min for 12 h, filter with filter paper, and dry the obtained solid in a 55°C oven to constant weight. Pass through an 80-mesh sieve to obtain daidzein-betaine cocrystal.

[0064] Comparative Example 1

[0065] Accurately weigh daidzein and betaine in a molar ratio of 1:2, with a total mass of 60 mg, and place them in a centrifuge tube. Add zirconia balls and 20 μL of methanol to the tube, place it in a ball mill, vibrate at a frequency of 20 Hz, and ball mill for 30 min. The obtained solid powder is naturally dried and passed through an 80-mesh sieve. The PXRD of the solid powder is measured, as shown in FIG. Figure 1 As shown, the results demonstrated that no daidzein-betaine co-crystal was obtained.

[0066] Comparative Example 2

[0067] Accurately weigh daidzein and betaine in a molar ratio of 1:2, with a total mass of 120 mg, and place them in a round-bottom flask. Add 25 mL of anhydrous ethanol to the flask, place it on a magnetic stirrer, stir at a speed of 600 rpm, and heat at 60°C until the drug is completely dissolved. Use a rotary evaporator to evaporate the solvent at 45°C. After drying, the obtained solid powder is passed through an 80-mesh sieve, and the PXRD of the solid powder is measured. Figure 2 As shown, the results demonstrated that no daidzein-betaine co-crystal was obtained.

[0068] Comparative Example 3

[0069] 20 mg of daidzein and 280 mg of melatonin were added to 2 mL of anhydrous ethanol, sonicated for 10 min, and filtered to obtain a saturated solution of daidzein and melatonin, which was recorded as the solvent;

[0070] Accurately weigh daidzein and melatonin in a molar ratio of 1:1 and add them to the above solvent, wherein the ratio of the total mass of daidzein and melatonin to the above solvent is 100 mg:1 mL. Stir continuously at 25°C and 600 r / min for 24 h, filter with filter paper, and dry the obtained solid in a 45°C oven to constant weight. Pass through an 80-mesh sieve, and measure the PXRD of the solid powder. Figure 3 shown.

[0071] As can be seen from the figure, the peaks of the solid product obtained in the experiment are superimposed peaks of daidzein and melatonin, with neither the appearance of new peaks nor the disappearance of old peaks. Therefore, the obtained product is a physical mixture of daidzein and melatonin.

[0072] Comparative Example 4

[0073] 80 mg of genistein and 120 mg of betaine were added to 2 mL of anhydrous ethanol, ultrasonicated for 10 min, and filtered to obtain a saturated solution of genistein and betaine, which was recorded as the solvent;

[0074] Accurately weigh genistein and betaine in a molar ratio of 1:1, add them to the above solvent, wherein the ratio of the total mass of genistein and betaine to the above solvent is 100 mg:1 mL, and continue stirring at 25 ° C and 600 r / min for 24 h. Filter with filter paper, dry the obtained solid in a 45 ° C oven to constant weight, pass through an 80 mesh sieve, and measure the PXRD of the solid powder. Figure 4 shown.

[0075] As can be seen from the figure, the peaks of the solid product obtained in the experiment are superimposed peaks of genistein and betaine, with neither the appearance of new peaks nor the disappearance of old peaks. Therefore, the obtained product is a physical mixture of genistein and betaine.

[0076] Comparative Example 5

[0077] 20 mg of daidzein and 500 mg of chlorogenic acid were added to 2 mL of anhydrous ethanol, sonicated for 10 min, and filtered to obtain a saturated solution of daidzein and chlorogenic acid, which was recorded as the solvent;

[0078] Accurately weigh daidzein and chlorogenic acid in a molar ratio of 1:1 and add them to the above solvent, wherein the ratio of the total mass of daidzein and chlorogenic acid to the above solvent is 100 mg:1 mL. Stir continuously at 25°C and 600 r / min for 24 h, filter with filter paper, and dry the obtained solid in a 45°C oven to constant weight. Pass through an 80-mesh sieve, and measure the PXRD of the solid powder. Figure 5 shown.

[0079] As can be seen from the figure, the peaks of the solid product obtained in the experiment are superimposed peaks of daidzein and chlorogenic acid. There is neither the appearance of new peaks nor the disappearance of old peaks. Therefore, the obtained product is a physical mixture of daidzein and chlorogenic acid.

[0080] Test example

[0081] 1. Sample Determination and Structural Characterization

[0082] The daidzein-betaine co-crystal prepared in Example 1 was measured and characterized in terms of structure. The specific method is as follows:

[0083] 1.1 PXRD analysis

[0084] Powder X-ray diffraction (PXRD) was performed using a German Bruker D8 Advance X-ray diffractometer. Measurement conditions: The light source was Cu Kα The tube voltage was 30 kV, the tube current was 20 mA, the test step was set to 0.02°, the test speed was 0.01 s per step, the scanning range was 5° to 35°, and the test temperature was room temperature.

[0085] The powder X-ray diffraction pattern of the daidzein-betaine co-crystal obtained in Example 1 is as follows: Figure 6 The X-ray powder diffraction of daidzein-betaine co-crystal has characteristic diffraction peaks at diffraction angles 2θ = 6.4°±0.2°, 7.9°±0.2°, 9.4°±0.2°, 9.9°±0.2°, 10.7°±0.2°, 12.7°±0.2°, 12.9°±0.2°, 15.5°±0.2°, 15.9°±0.2°, 16.3°±0.2°, 16.9°±0.2°, 18°±0.2°, 18.3°±0.2°, 20.9°±0.2°, 21.5°±0.2°, and 22.1°±0.2°.

[0086] 1.2 Single crystal X-ray diffraction analysis (SCXRD)

[0087] The daidzein-betaine co-crystal sample prepared in Example 1 was placed on a sample testing device; the test light source was Mo Kα ray The testing temperature was 114.4 K. Cellular refinement and data reduction were performed using the CrysAlisPRO program. Structure solution and refinement were performed using the SHELXTL tool within Olex-2 software. Refinement was performed using full-matrix least-squares on F2, and all non-hydrogen atoms were refined using anisotropic displacement parameters. All hydrogen atoms were placed at calculated positions with fixed isotropic thermal parameters. The crystal structure was finally drawn using Mercury 4.2.0 software.

[0088] The single crystal structure analysis of daidzein-betaine drug cocrystal showed that the cocrystal belongs to the monoclinic system P21 / n space group (Z=4); in the asymmetric unit ( Figure 7 A) contains 1 daidzein molecule and 2 betaine molecules. Daidzein and betaine are linked by O1-H1···O5 and O3-H3···O7 Ionic hydrogen bond interaction. The two asymmetric units are connected by C18-H18A··O3 and C9-H9··O5 Bonds form hexamer ( Figure 7 B) Hexamer through two C24-H24A···O3 The bonds are connected along the c-axis to form a one-dimensional chain ( Figure 7 C). The one-dimensional chains further stack to form two-dimensional chains and the final three-dimensional structure ( Figure 7 D. Figure 7 E).

[0089] 1.3 Thermogravimetric analysis (TG)

[0090] Thermogravimetric analysis (TG) was performed on the daidzein-betaine co-crystal prepared in Example 1.

[0091] Thermogravimetric analysis was performed using a thermogravimetric analyzer (SDT Q600, TA, USA). Approximately 5-10 mg of daidzein-betaine cocrystal sample was placed in an aluminum pan, covered with an aluminum lid, and heated from 25°C to 500°C at a rate of 10°C / min under nitrogen protection at a nitrogen flow rate of 50 mL / min. The obtained thermogravimetric analysis results are as follows: Figure 8 shown.

[0092] Figure 8 The TG graph showed that daidzein-betaine had no weight loss before the melting point, which was consistent with the DSC results.

[0093] 1.4 Differential Scanning Calorimetry (DSC)

[0094] The daidzein-betaine co-crystal prepared in Example 1 was subjected to differential scanning calorimetry (DSC).

[0095] The differential scanning calorimetry (SDT Q600, TA, USA) was used for the measurement. 3-5 mg of the sample was placed in an alumina crucible and covered with an aluminum lid. Under the protection of nitrogen, the nitrogen flow rate was 50 mL / min, and the temperature was raised from 40°C to 250°C at a rate of 10°C / min. The differential scanning calorimetry analysis graph was as follows: Figure 9 shown.

[0096] Depend on Figure 9 The results showed that the daidzein-betaine co-crystal had a sharp endothermic peak at 241°C, indicating that its melting point was 241°C, and further indicating that this substance was a single crystal phase.

[0097] 1.5 Determination of intrinsic dissolution rate

[0098] The intrinsic dissolution rates of daidzein and the daidzein-betaine co-crystal prepared in Example 1 were measured.

[0099] 300 mg each of sieved daidzein and the daidzein-betaine cocrystal prepared in Example 1 were weighed and compressed at a pressure of 2 t for 30 s into tablets with a diameter of 1.3 cm. Each tablet was placed in a mold and sealed on one side with wax. The intrinsic dissolution test method was a paddle method using 900 mL of anhydrous ethanol-water (50 / 50 v / v) at 37°C and 100 rpm. At intervals of 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, and 40 min, 1 mL of the solution was sampled and supplemented with 1 mL of blank solution. The resulting solution was filtered through a 0.22 μm filter, diluted to the appropriate dilution ratio, and analyzed for daidzein concentration by high-performance liquid chromatography.

[0100] The results are shown in Table 1 and Figure 10 The results showed that the daidzein-betaine co-crystal had a better dissolution behavior than the daidzein raw material, and the IDR value of daidzein was 102.83 μg·cm -2 min -1 The IDR value of daidzein drug cocrystal is 446.72 μg·cm -2 min -1 , the IDR of the cocrystal is about 4.34 times that of the API.

[0101] Table 1

[0102]

[0103] 1.6 Dynamic water vapor adsorption test

[0104] The daidzein-betaine drug cocrystal and betaine bulk drug prepared in Example 1 were subjected to dynamic water vapor adsorption tests to investigate the drug stability.

[0105] 30 μg each of betaine and daidzein-betaine drug cocrystals with particle sizes of 75-150 μm were weighed and placed on a sample tray. Testing conditions included a nitrogen flow rate of 200 sccm, a temperature of 25°C, a relative humidity gradient of 10%, and a relative humidity gradient cycle of 0% → 95% → 0% RH. Data processing software was Isotherm (ISO) analysis suite.

[0106] The water vapor adsorption and desorption results of betaine and daidzein-betaine drug co-crystals are shown in Figure 2. Figure 11 The results are shown in Table 2. The results indicate that the daidzein-betaine cocrystal exhibits superior stability compared to the betaine API. Betaine exhibits a sharp increase in water vapor adsorption at a relative humidity of 40% to 90%, with a weight change ranging from 15.3% to 230%. In contrast, the daidzein-betaine cocrystal exhibits a slow increase in water vapor adsorption from 0% to 60% and a rapid increase from 60% to 90%, with a weight change ranging from 9.5% to 172%. Within the 0% to 95% range, the mass change of the daidzein-betaine cocrystal is less than that of the betaine, demonstrating the cocrystal's superior hygroscopic stability. Significant mass loss is observed in the desorption curves of both betaine and daidzein-betaine cocrystals.

[0107] Table 2 Hygroscopic stability

[0108]

[0109]

[0110] 1.7 Pharmacokinetic studies

[0111] The pharmacokinetic test of the daidzein-betaine co-crystal prepared in Example 1 was carried out, and the in vivo metabolic behavior of the co-crystal was compared with that of the daidzein bulk drug.

[0112] Choose 7~8 week age SD rats, male, totally 12, body weight 250~270g, adaptability is raised after 1 week, is randomly divided into 2 groups, i.e. daidzein group and daidzein-betaine medicine eutectic group, fasting 12h before the test, can freely drink water.Daidzein-betaine eutectic that daidzein and embodiment 1 are prepared is suspended in concentration respectively and is in 0.5% sodium carboxymethyl cellulose (CMC-Na) solution, is gavaged with the dosage of 100mg / kg and is given to rat.Then respectively at 0.16h, 0.25h, 0.42h, 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, 12h time in the retroorbital venous plexus of rat place blood sampling 400 μ L, place the centrifuge tube that contains heparin sodium, 6000rpm centrifugal 5min,-20 ℃ standby.

[0113] Take 100 μL of plasma sample and add 900 μL of methanol, vortex for 3 minutes, centrifuge at 12000 rpm for 10 minutes, take the supernatant, blow with nitrogen, dry it, add 200 μL of methanol to dissolve it, vortex for 3 minutes, centrifuge at 12000 rpm for 3 minutes, filter it with a 0.22 μm filter membrane, and use high performance liquid chromatography to determine the concentration of daidzein in the plasma sample.

[0114] HPLC chromatographic conditions: chromatographic column is C 18 Column (4.6×250 mm, 5 μm); mobile phase: methanol: water = 60:40; flow rate: 1.0 mL / min; column temperature: 37°C; injection volume: 20 μL; detection wavelength: 249.5 nm.

[0115] The pharmacokinetic data were analyzed using DAS2.0 software to calculate the AUC 0～12 、C max , peak time (T max ), half-life (t 1 / 2 ) and other pharmacokinetic parameters, and the data were analyzed for significance using SPSS23.0 software.

[0116] The drug-time curve and main pharmacokinetic parameters drawn are as follows Figure 12 As shown in Table 3, the C max The C of daidzein-betaine drug cocrystal is 276.28 ng / mL. max The AUC of daidzein and daidzein-betaine cocrystal was 1271.82 ng / mL, which is 4.6 times that of daidzein API. 0～12 The AUC of daidzein-betaine cocrystal were 1233.68 ng / mL·min and 4599.46 ng / mL·min, respectively. 0～12 It is 3.73 times that of daidzein raw material. max is 1.79h, while the T max It is 0.42h.

[0117] Table 3 Pharmacokinetic parameters

[0118] Daidzein Daidzein-betaine cocrystal <![CDATA[t 1 / 2 (h)]]> 2.80±1.23** 9.06±3.24** <![CDATA[T max (h)]]> 1.79±2.06* 0.42±0.00* <![CDATA[C max (ng / mL)]]> 276.28±57.92** 1271.82±174.03** <![CDATA[AUC 0～12 (ng / mL min)]]> 1233.68±335.68 4599.46±719.94

[0119] Note: The same group without shoulder marks indicates no significant difference (P>0.05), * indicates significant difference (P<0.05), and ** indicates extremely significant difference (P<0.01).

[0120] 1.8 Pharmacodynamic studies

[0121] Pharmacodynamic tests were conducted on daidzein, betaine, a daidzein-betaine physical mixture (the molar ratio of daidzein to betaine was 1:2) and the daidzein-betaine co-crystal prepared in Example 1.

[0122] Animal Grouping and Treatment: Sixty mice were randomly divided into two groups: a blank control group and a modeling group. The blank control group was fed a 3.65 kcal / g normal diet at 10% of their body weight (n=10), while the model group was overfed, and food intake was measured weekly (n=50). At the end of 5 weeks, lipid levels in the overfed mice were measured, and obesity-tolerant mice were eliminated. Thirty modeling mice were equally divided into five groups (n=6): model group: overfed; daidzein group: overfed + daidzein (100 mg / kg); betaine group: overfed + betaine (64.7 mg / kg); daidzein-betaine physical mixture group: overfed + daidzein-betaine physical mixture (164.7 mg / kg); and daidzein-betaine cocrystal group: overfed + daidzein-betaine cocrystal (164.7 mg / kg). Body weight and diet were measured weekly. At week 8, blood, feces, and epididymal adipose tissue samples were collected and stored at −80°C for further analysis.

[0123] The results of pharmacodynamic tests showed that Figure 13 As shown in Table 4, at the beginning of the treatment trial, there was no significant difference in body weight among the groups except the control group (P>0.05). However, at the end of treatment, the body weights of the mice in the daidzein-betaine physical mixture treatment group (54.75±0.53g), betaine treatment group (54.43±0.5g), daidzein-betaine physical mixture treatment group (54.3±0.63g), and daidzein-betaine cocrystal treatment group (53.32±0.74g) were significantly lower than those in the model group (56.89±0.67g). In addition, the body weight of the daidzein-betaine cocrystal group was significantly lower than that of the model group in the first week of treatment (P<0.001).

[0124] Table 4 Body weight changes of mice in each group

[0125]

[0126] Note: For comparison in the same column, no shoulder mark indicates no significant difference (P>0.05), * indicates significant difference (P<0.05), ** indicates extremely significant difference (P<0.01), and *** indicates extremely significant difference (P<0.001).

[0127] The weight gain of mice in each group was as follows Figure 14As shown, the average weight gain in the model group was 7.43±0.42g, while the average weight gain in the intervention groups was 5.42±0.25g, 5.22±0.45g, 4.62±0.25g, and 3.780.33g, respectively, in the daidzein group, the betaine group, the daidzein-betaine physical mixture group, and the daidzein-betaine cocrystal group. It is noteworthy that the daidzein-betaine cocrystal group had a more significant effect on body weight compared with the API or physical mixture group.

[0128] In summary, the daidzein-betaine co-crystal provided by the present invention not only improves the in vitro solubility and oral bioavailability of the daidzein raw material, but also significantly enhances the hygroscopic stability of betaine. Furthermore, the combination of the two anti-obesity drugs can produce a synergistic effect, significantly improving their effectiveness in preventing and treating obesity. This represents a novel drug with promising application prospects for preventing and treating obesity.

[0129] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A daidzein drug co-crystal, characterized in that: The daidzein drug co-crystal is daidzein and betaine in a molar ratio of 1:2; the daidzein drug co-crystal has characteristic diffraction peaks at 2θ=6.4°±0.2°, 9.4°±0.2°, 18°±0.2°, 21.5°±0.2°, and 22.1°±0.2°.

2. The daidzein drug co-crystal according to claim 1, wherein The daidzein drug co-crystal also has characteristic diffraction peaks at 2θ=7.9°±0.2°, 9.9°±0.2°, 10.7°±0.2°, 12.7°±0.2°, 12.9°±0.2°, 15.5°±0.2°, 15.9°±0.2°, 16.3°±0.2°, 16.9°±0.2°, 18.3°±0.2°, and 20.9°±0.2°.

3. The daidzein drug co-crystal according to claim 1, wherein The daidzein drug cocrystal is a monoclinic P21 / n space group with unit cell parameters of a=6.0301(8) Å, b= 17.799(6) Å, c=22.318(2) Å, α=γ=90°, β=93.983(9)°.

4. The method for preparing the daidzein drug co-crystal according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: adding daidzein and betaine into a solvent, stirring and mixing uniformly, filtering, collecting powder, and drying to obtain daidzein drug co-crystal; wherein the solvent is a saturated solution of daidzein and betaine; and the solvent of the saturated solution of daidzein and betaine is anhydrous ethanol.

5. The method for preparing daidzein drug co-crystal according to claim 4, wherein: The molar ratio of daidzein to betaine is 1:1 to 1:3; and / or The mass volume ratio of the total mass of the daidzein and betaine to the solvent is 100 mg:1 mL to 10 mg:1 mL; and / or The stirring speed is 200 r / min to 1000 r / min, the stirring temperature is 15° C. to 45° C., and the stirring time is 12 h to 48 h; and / or The drying temperature is 45°C to 55°C.

6. A pharmaceutical composition, characterized in that The invention comprises the daidzein drug co-crystal according to any one of claims 1 to 3.

7. The pharmaceutical composition according to claim 6, wherein Pharmaceutically acceptable excipients are also included.

8. The pharmaceutical composition according to claim 6 or 7, characterized in that The dosage form of the pharmaceutical composition is tablet, capsule, pill, injection preparation, sustained-release preparation or controlled-release preparation.

9. Use of the daidzein drug co-crystal according to any one of claims 1 to 3 or the pharmaceutical composition according to any one of claims 6 to 8 in the preparation of a drug for preventing and treating obesity.

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