Daidzein pharmaceutical eutecticum and preparation method and application thereof

Abstract

The invention relates to the technical field of pharmaceutical co-crystals, and particularly discloses a daidzein pharmaceutical co-crystal as well as a preparation method and application thereof. The daidzein pharmaceutical eutecticum comprises daidzein and betaine in a molar ratio of 1: 2. The preparation method comprises the following steps: adding daidzein and betaine into a solvent, uniformly stirring and mixing, filtering, collecting powder, and drying to obtain the daidzein pharmaceutical eutecticum, wherein the solvent is a saturated solution of daidzein and betaine. The daidzein-betaine eutectic provided by the invention not only improves the in-vitro solubility and oral bioavailability of the daidzein bulk drug, but also significantly enhances the moisture absorption stability of betaine, and the daidzein and betaine cooperate to effectively enhance the efficacy of the eutectic for preventing and treating obesity. The compound can be developed into a plurality of preparations, can be used as a novel medicine for treating obesity, diabetes and other metabolic diseases, and has a relatively high application prospect.

Description

Technical Field

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

[0002] In recent years, obesity has become a widespread international health problem. According to the World Health Organization (WHO), by 2025, about one-fifth of the world's adult population will suffer from obesity, among which the prevalence of obesity and overweight in the population aged 3 to 17 and ≥18 years old is 19% and 50.7%, respectively. Obesity is a chronic metabolic disease characterized by imbalance in energy intake and consumption and excessive fat accumulation. It is closely related to 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. A large number of studies have shown that obesity is the main cause of other diseases in dogs, such as diabetes, tumors, osteoarthritis, hypertension, hyperlipidemia, etc., which ultimately leads to shortened lifespan and increased mortality in dogs, seriously endangering health and shortening the lifespan of dogs. Oral drug treatment is currently a common method for treating obesity. In 1998, the European Medicines Agency (EMA) approved oritas as a long-term weight loss drug. However, its low oral bioavailability, first-pass metabolism, and severe side effects (steatorrhea, fecal incontinence, liver and kidney damage) restrict its use. Given the current high prevalence of obesity and limited treatment options, safe, effective, and green weight loss drugs are in urgent need of development.

[0003] The chemical structure of daidzein (DAI) is shown in formula (I). It is the most abundant isoflavone component found in plants such as soybean, alfalfa, kudzu root and red clover. Studies have shown that daidzein can be used as a PPARγ, SCD1 inhibitor and UCP-1 activator to reduce high-fat diet-induced obesity in C57BL / 6J mice and rats. However, daidzein belongs to BSC IV class drugs and has a solubility in water of only 5.3 μg / mL, which greatly limits its clinical application. Therefore, it is necessary to improve the oral absorption and utilization 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 drug co-crystal formed by combining two active drug components of daidzein and betaine through hydrogen bonding forces, which not only improves the in vitro solubility and oral bioavailability of the daidzein raw material drug, 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 components themselves, but also can improve the physicochemical properties such as drug stability and solubility, has more stable physicochemical properties, can improve bioavailability, and thus more effectively promote drug absorption. In addition, compounding two drugs with anti-obesity efficacy can produce a synergistic effect, significantly improve its prevention and treatment effects on obesity, and is a novel drug for preventing and treating obesity diseases with high application prospects.

[0009] The structural formula of betaine (BTN) is shown in formula (II), which is also known as trimethylglycine or glycine betaine. It is a natural compound isolated from beet molasses with a wide range of pharmacological activities and has high solubility and bioavailability. Studies have shown that betaine has a positive effect on regulating lipid metabolism in diseases such as obesity and diabetes. However, betaine has strong hygroscopicity. Anhydrous betaine will be converted into monohydrate betaine when the relative humidity (RH) is 20%, and it will dissolve when the RH is 54%. The present invention forms a eutectic with soybean aglycone (DAI) and betaine (BTN), thereby solving the problems of low solubility and poor bioavailability of soybean aglycone 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 drug molecules and coformers. The two substances need to be compatible and will not cause problems with the stability of the chemical or physical properties of the two substances. Therefore, the selection of coformers is crucial, and the preparation method of cocrystals will also have an important impact on the physicochemical and pharmaceutical properties of cocrystals.

[0015] The preparation method of the daidzein-betaine co-crystal provided by the present invention is simple, has mild operating conditions, is not easy to form hydrates and solvates, has low cost, has little pollution to the environment, 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 constant weight.

[0025] In a third aspect, the present invention further provides a pharmaceutical composition comprising any 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-crystal provided by the present invention can be made into a variety of dosage forms, such as tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, powders, pellets, dripping pills, etc., and can also be made into sustained-release preparations and controlled-release preparations. It can be prepared according to the conventional method of dosage forms in the field, and the present invention does not make special restrictions.

[0030] In a fifth aspect, the present invention further provides use of any of the above-mentioned daidzein drug co-crystals or any of the above-mentioned 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, and the daidzein and betaine can synergistically effectively enhance the efficacy of the co-crystal in preventing and treating obesity. The co-crystal can be used to develop a variety of preparations as a new drug for metabolic diseases such as obesity and diabetes, and has a high application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

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

[0034] Figure 2The figure 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 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;

[0037] Figure 5 It is the powder X-ray diffraction (PXRD) pattern of daidzein, chlorogenic acid and daidzein-chlorogenic acid mixture in Comparative Example 5 of the present invention;

[0038] Figure 6 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 soybean aglycone-betaine co-crystal prepared in Example 1 of the present invention, wherein (A) is an asymmetric unit, (B) is a hexameric structure diagram, (C) is a one-dimensional chain structure diagram, (D) is a two-dimensional structure diagram, and (E) is a three-dimensional structure diagram;

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

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

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

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

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

[0045] Fig.13 This is a graph showing weekly changes in body weight 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] Fig.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 testing. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solution 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 used 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, ultrasonicated 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, 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, continue stirring at 25°C and 600 r / min for 24 h, filter with filter paper, dry the obtained solid in an oven at 45°C to constant weight, and pass through an 80-mesh sieve to obtain daidzein-betaine co-crystals.

[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, ultrasonicated 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, 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, continue stirring at 15°C and 200 r / min for 48 h, filter with filter paper, dry the obtained solid in an oven at 50°C to constant weight, and pass through an 80-mesh sieve to obtain daidzein-betaine co-crystals.

[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, ultrasonicated 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, 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, continue stirring at 45°C and 1000 r / min for 12 h, filter with filter paper, dry the obtained solid in an oven at 55°C to constant weight, and pass through an 80-mesh sieve to obtain daidzein-betaine co-crystals.

[0064] Comparative Example 1

[0065] Precisely weigh daidzein and betaine in a molar ratio of 1:2, with a total mass of 60 mg, and put them into a centrifuge tube. Add zirconium oxide balls and 20 μL of methanol into the tube, put it into a ball mill, and vibrate at a frequency of 20 Hz 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-crystals were obtained.

[0066] Comparative Example 2

[0067] Precisely weigh daidzein and betaine in a molar ratio of 1:2, with a total mass of 120 mg, and put them into 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 the obtained solid powder is dried, pass it through an 80-mesh sieve, and measure the PXRD of the solid powder. Figure 2 As shown, the results demonstrated that no daidzein-betaine co-crystals were obtained.

[0068] Comparative Example 3

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

[0070] Precisely weigh daidzein and melatonin in a molar ratio of 1:1, add to the above solvent, wherein the ratio of the total mass of daidzein and melatonin to the above solvent is 100 mg:1 mL, continue stirring at 25°C and 600 r / min for 24 h, filter with filter paper, dry the obtained solid in an oven at 45°C to constant weight, pass through an 80-mesh sieve, and measure the PXRD of the solid powder, such as 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, 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, and there is 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, ultrasonicated 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, 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, continue stirring at 25°C and 600 r / min for 24 h, filter with filter paper, dry the obtained solid in an oven at 45°C to constant weight, pass through an 80-mesh sieve, and measure the PXRD of the solid powder, such as 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, with 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 structure, and 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 testing light source was Mo Kα ray The test temperature was 114.4K; cell refinement and data reduction were performed using the CrysAlisPRO program; structure analysis and refinement were performed using the SHELXTL tool within the Olex-2 software. Optimization was performed on F2 using the full matrix least squares method, and all non-hydrogen atoms were refined using anisotropic displacement parameters. All hydrogen atoms were placed in calculated positions with fixed isotropic thermal parameters. Finally, the crystal structure was drawn using Mercury 4.2.0 software.

[0088] The single crystal structure analysis of daidzein-betaine drug cocrystal showed that the cocrystal belonged to the monoclinic 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 bonding. The two asymmetric units are connected by C18-H18A··O3 and C9-H9··O5 Bonds form hexamer ( Figure 7 B) Hexamer through 2 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). About 5-10 mg of daidzein-betaine cocrystal sample was placed in an aluminum pan 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 25°C to 500°C at a rate of 10°C / min. The obtained thermogravimetric analysis is 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 calorimeter (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 increased from 40°C to 250°C at a rate of 10°C / min. The differential scanning calorimetry analysis diagram was as follows: Fig. 9 shown.

[0096] Depend on Fig. 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 crystalline phase.

[0097] 1.5 Determination of intrinsic dissolution rate

[0098] The intrinsic dissolution rate of daidzein and daidzein-betaine co-crystal prepared in Example 1 was measured.

[0099] Weigh 300 mg of sifted daidzein and daidzein-betaine co-crystal prepared in Example 1, respectively, and press into tablets with a diameter of 1.3 cm at a pressure of 2t for 30s. Place each tablet in a mold and seal one side with wax. The test method for intrinsic dissolution is the paddle method, the dissolution medium is 900 mL of anhydrous ethanol-water (v:v, 50 / 50), the temperature is 37°C, and the speed is 100 rpm; take 1 mL of solution at 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, and 40 min, and add 1 mL of blank solution; filter the solution with a 0.22 μm filter, dilute to an appropriate multiple, and analyze the concentration of daidzein in the solution by high performance liquid chromatography.

[0100] The results are shown in Table 1 and Fig.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 of betaine and daidzein-betaine drug cocrystal with a particle size of 75-150 μm were weighed and placed on a sample plate; the test conditions were: nitrogen flow rate of 200 sccm, temperature of 25°C, relative humidity gradient of 10%, and 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 Fig.11 As shown in Table 2. The results show that compared with betaine API, daidzein-betaine cocrystal has good stability. The adsorption of betaine to water vapor increases sharply at RH of 40% to 90%, and the weight changes from 15.3% to 230%, while the adsorption of daidzein-betaine cocrystal to water vapor increases slowly at 0% to 60%, and increases rapidly at 60% to 90%, and the weight changes from 9.5% to 172%. In the range of 0% to 95%, the mass change of daidzein-betaine cocrystal is less than that of betaine, which proves that the cocrystal has good hygroscopic stability. During the desorption process, obvious mass loss can be observed in the curves of betaine and daidzein-betaine cocrystal.

[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 daidzein raw material was compared with that of the daidzein raw material.

[0112] Select 7-8 week old SD rats, male, a total of 12, body weight 250-270g, after adaptive feeding for 1 week, randomly divided into 2 groups, i.e. daidzein group and daidzein-betaine drug cocrystal group, fasting for 12h before the test, free drinking water. Daidaidzein and daidzein-betaine cocrystal prepared in Example 1 were suspended in a 0.5% sodium carboxymethylcellulose (CMC-Na) solution, and given to rats by gavage at a dosage of 100mg / kg. Then, 400μL of blood was collected from the retroorbital venous plexus of rats at 0.16h, 0.25h, 0.42h, 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, and 12h, respectively, and placed in a centrifuge tube containing sodium heparin, centrifuged at 6000rpm for 5min, and -20°C for standby use.

[0113] Take 100 μL of plasma sample, add 900 μL of methanol, vortex for 3 minutes, centrifuge at 12000 rpm for 10 minutes, take the supernatant, blow with nitrogen, blow dry, add 200 μL of methanol to reconstitute, vortex for 3 minutes, centrifuge at 12000 rpm for 3 minutes, filter with 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 (4.6×250mm, 5μm); mobile phase is methanol: water = 60:40; flow rate is 1.0mL / min; column temperature is 37°C; injection volume is 20μL; detection wavelength is 249.5nm.

[0115] The pharmacokinetic data of the test were calculated and analyzed using DAS2.0 software AUC 0～12 , C max , peak time (T max ), half-life (t 1 / 2 ) and other pharmacokinetic parameters, and SPSS23.0 software was used to perform significance analysis on the data.

[0116] The drug-time curve and main pharmacokinetic parameters drawn are as follows Fig.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 cocrystals was 1271.82 ng / mL, which is 4.6 times that of daidzein API. 0～12 The AUC of daidzein-betaine cocrystal was 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: 60 mice were randomly divided into 2 groups, blank control group and modeling group. The blank control group was fed with 3.65kcal / g ordinary feed at 10% of their body weight (n=10), and the model group was fed with overfeeding, and the food intake was weighed weekly (n=50). At the end of 5 weeks, the blood lipid levels of overfed mice were measured and the obesity-tolerant mice were eliminated. 30 model group mice were selected and divided into 5 groups (n=6): model group: overfeeding; daidzein group: overfeeding + daidzein (100mg / kg); betaine group: overfeeding + betaine (64.7mg / kg); daidzein-betaine physical mixture group: overfeeding + daidzein-betaine physical mixture (164.7mg / kg); daidzein-betaine eutectic group: overfeeding + daidzein-betaine eutectic (164.7mg / kg), and 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 Fig.13 As shown in Table 4, at the beginning of the treatment experiment, there was no significant difference in body weight among the groups except the control group (P>0.05), while at the end of the treatment, the body weight of the mice in the daidzein 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) was significantly lower than that in the model group (56.89±0.67g). In addition, compared with the model group, the body weight of the daidzein-betaine cocrystal group showed a significant decrease 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 means no significant difference (P>0.05), * means significant difference (P<0.05), ** means extremely significant difference (P<0.01), and *** means extremely significant difference (P<0.001).

[0127] The weight of mice in each group was obtained as follows Fig.14As shown, the average weight gain of the model group was 7.43±0.42g, while the average weight gain of the intervention groups in the daidzein group, betaine group, daidzein-betaine physical mixture group, and daidzein-betaine cocrystal group was 5.42±0.25g, 5.22±0.45g, 4.62±0.25g, and 3.780.33g, respectively. It is worth noting that the effect of treatment on body weight in the daidzein-betaine cocrystal group was more significant than that in 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 drug, but also significantly enhances the hygroscopic stability of betaine. In addition, compounding the two drugs with anti-obesity efficacy can produce a synergistic effect, significantly improving the prevention and treatment effects of obesity, and is a new drug for preventing and treating obesity with high application prospects.

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

Claims

1. A daidzein drug co-crystal, characterized in that: The daidzein drug co-crystal comprises daidzein and betaine in a molar ratio of 1:

2.

2. The daidzein drug co-crystal according to claim 1, characterized in that: The daidzein drug co-crystal has characteristic diffraction peaks at 2θ=62θ=6.4°±0.2°, 9.4°±0.2°, 18°±0.2°, 21.5°±0.2°, and 22.1°±0.2°.

3. The daidzein drug co-crystal according to claim 2, characterized in that: 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°.

4. The daidzein drug co-crystal according to claim 1, characterized in that: The daidzein drug co-crystal is a monoclinic P21 / n space group with unit cell parameters of α=γ=90°, β=93.983(9)°.

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

6. The method for preparing daidzein drug co-crystal according to claim 5, characterized in that: 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 solvent of the saturated solution of daidzein and betaine is at least one of an alcohol solvent, acetonitrile, ethyl acetate, water or acetone; 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.

7. A pharmaceutical composition, characterized in that The invention comprises the daidzein drug co-crystal according to any one of claims 1 to 4.

8. The pharmaceutical composition according to claim 7, characterized in that Pharmaceutically acceptable excipients are also included.

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

10. Use of the daidzein drug co-crystal according to any one of claims 1 to 4 or the pharmaceutical composition according to any one of claims 7 to 9 in the preparation of drugs for preventing and treating obesity.

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