Colchicine and ferulic acid co-amorphous substance, and preparation method and application thereof

Abstract

The present application relates to the medical pharmaceutical technical field, especially to a colchicine and ferulic acid co-amorphous substance and a preparation method and application thereof.The molar ratio of colchicine and ferulic acid in the colchicine and ferulic acid co-amorphous substance is 1-3:1.The colchicine and ferulic acid co-amorphous substance provided by the present application has good physical stability, significantly reduces the dissolution rate of colchicine while improving the solubility of colchicine, realizes the slow release and stable blood drug concentration of the drug, has the effect of "synergistic and reducing toxicity", and the glass transition temperature of the colchicine and ferulic acid co-amorphous substance of the present application is significantly lower than that of colchicine, which can promote transdermal absorption, provide a new technical path for developing colchicine external preparation, has wide application prospect, and effectively solves the problems of narrow treatment window, high toxicity and poor patient compliance of colchicine.

Description

Technical Field

[0001] This invention relates to the field of medical pharmaceutical technology, and in particular to an amorphous compound of colchicine and ferulic acid, its preparation method, and its application. Background Technology

[0002] Colchicine (COL) is derived from the European lily family plant autumn crocus (Colchella asiatica). Colchicum autumnale L. COL is a phenolic alkaloid extracted and isolated from the bulb of the plant. As a classic drug, COL remains a first-line treatment for gout both domestically and internationally, and is the only drug that can be used for acute gout attacks, acute gouty arthritis attacks, and prevention, making it irreplaceable. In recent years, COL has also been found to have some new clinical applications. In addition to being a specific drug for familial Mediterranean fever, it has been used in the fields of skin diseases (such as Behçet's disease and scleroderma), cardiovascular diseases (pericarditis and atrial fibrillation), and anti-fibrotic treatment.

[0003] COL is a BCS Class III drug, characterized by high solubility and low permeability. Although COL is widely used clinically with significant efficacy, its main challenges include high toxicity, significant side effects, and poor patient compliance. Firstly, COL has a narrow therapeutic window (blood concentration of 0.5-33 ng / mL), meaning the therapeutic dose and toxic dose are very close. Secondly, as an inhibitor of mitosis, COL has significant toxic side effects; during short-term treatment, the most common adverse reactions include gastrointestinal symptoms such as diarrhea, abdominal cramps, nausea, and vomiting. Recent studies have found that COL exhibits cardiotoxicity in certain populations at specific doses, posing an additional challenge to medication safety in gout patients with coexisting cardiovascular disease. Furthermore, existing conventional COL formulations require multiple doses, leading to poor patient compliance, large fluctuations in blood concentrations, and pronounced peak-and-trough phenomena, further increasing the risk of toxicity.

[0004] Therefore, it is hoped that an effective improvement solution can be provided to address the issues of narrow therapeutic window, high toxicity, and poor patient compliance in COL treatment. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a colchicine and ferulic acid co-amorphous compound, its preparation method, and its applications. The colchicine and ferulic acid co-amorphous compound provided by this invention exhibits good physical stability. While improving the solubility of colchicine, it significantly reduces its dissolution rate, achieving slow drug release and stable blood drug concentration, thus possessing a "synergistic effect and reduced toxicity" effect. Furthermore, the glass transition temperature of the colchicine and ferulic acid co-amorphous compound of this invention is significantly lower than that of colchicine, promoting transdermal absorption. This provides a new technical pathway for developing topical colchicine preparations, with broad application prospects, effectively solving the problems of colchicine's narrow therapeutic window, high toxicity, and poor patient compliance.

[0006] In a first aspect, the present invention provides a colchicine and ferulic acid co-amorphous compound, wherein the molar ratio of colchicine to ferulic acid in the colchicine and ferulic acid co-amorphous compound is 1-3:1, for example 1:1, 2:1, 3:1, etc.

[0007] The colchicine and ferulic acid co-amorphous compound provided by this invention exhibits good physical stability. While improving the solubility of colchicine, it reduces its dissolution rate, enabling slow release of colchicine. Furthermore, the synergistic effect of colchicine and ferulic acid effectively enhances the therapeutic efficacy for diseases such as gout, achieving the goal of synergistic effect and reduced toxicity. In addition, the colchicine and ferulic acid co-amorphous compound effectively lowers the Tg of colchicine, promoting transdermal absorption. This provides a new technical pathway for developing topical colchicine formulations, expands the clinical application scenarios of colchicine, and has good industrial application prospects. Specifically: The colchicine and ferulic acid co-amorphous compound provided by this invention uses colchicine as the active pharmaceutical ingredient, and its chemical name is (S)- N -(5,6,7,9-tetraoxo-1,2,3,10-tetramethoxy-9-oxobenzo[α]-heptaden-7-yl)acetamide, with the molecular formula C 22 H 25 NO6, its structural formula is .

[0008] Simultaneously, ferulic acid is used as the drug ligand, with the molecular formula C. 10 H 10 O4, its structural formula is .

[0009] Ferulic acid (FEA) is a natural phenolic acid compound widely found in traditional Chinese medicinal herbs such as Angelica sinensis and Ligusticum chuanxiong, exhibiting high biocompatibility and multiple pharmacological activities. Ferulic acid can reduce uric acid production and promote uric acid excretion by inhibiting xanthine oxidase (XO) activity, thereby exerting an anti-gout effect. Simultaneously, ferulic acid possesses significant antioxidant, anti-inflammatory, and cardiovascular protective effects, including improving myocardial energy metabolism, and can effectively antagonize the potential cardiotoxicity of colchicine (COL). Therefore, the colchicine and ferulic acid co-amorphous compound provided by this invention not only achieves sustained-release delivery of colchicine through solid-state engineering, but also, through the synergistic effect of the two active ingredients, enhances the efficacy of anti-gout and other disease treatments while reducing the systemic toxicity of colchicine, achieving excellent therapeutic effects. Furthermore, the colchicine and ferulic acid co-amorphous compound of the present invention possesses unique physicochemical properties. Compared to the colchicine crystal form, this co-amorphous compound effectively reduces the glass transition temperature (Tg) of colchicine while maintaining or improving its equilibrium solubility. This characteristic can promote the transdermal absorption of the drug, providing a new technical route for the development of colchicine topical preparations, expanding the clinical application scenarios of colchicine, and showing good prospects for industrial transformation.

[0010] As a preferred embodiment of the present invention, the molar ratio of colchicine to ferulic acid in the colchicine-ferulic acid co-amorphous compound is 1:1.

[0011] As a preferred embodiment of the present invention, the equilibrium solubility of the colchicine and ferulic acid co-amorphous compound in water at 37°C is 17.9 mg / mL, calculated as colchicine.

[0012] As a preferred embodiment of the present invention, the inherent dissolution rate of the colchicine and ferulic acid co-amorphous compound in water at 37°C, calculated as colchicine, is 0.8 mg / (min·cm). 2 ).

[0013] The colchicine-ferulic acid co-amorphous compound provided by this invention has higher equilibrium solubility and lower inherent dissolution rate than colchicine, which can achieve slow release of colchicine and stable blood drug concentration, and has the effect of "enhancing efficacy and reducing toxicity".

[0014] In a second aspect, the present invention provides a method for preparing the colchicine and ferulic acid co-amorphous compound as described in the first aspect, the preparation method comprising: The prescribed amounts of colchicine and ferulic acid were soaked in the first solvent, ground until a gel-like consistency was obtained, and the solvent was allowed to evaporate completely to obtain the amorphous product of colchicine and ferulic acid. Alternatively, the preparation method includes: The prescribed amounts of colchicine and ferulic acid were dissolved in a second solvent and dried to obtain the colchicine and ferulic acid co-amorphous compound.

[0015] The colchicine and ferulic acid co-amorphous compound provided by this invention is prepared by grinding or solution crystallization. The preparation method is simple and has good prospects for industrial application.

[0016] As a preferred embodiment of the present invention, the ratio of the total mass of colchicine and ferulic acid to the volume of the first solvent is 59.36 mg:(1-2) mL, for example, 59.36 mg:1 mL, 59.36 mg:1.5 mL, 59.36 mg:2 mL, etc.

[0017] As a preferred embodiment of the present invention, the ratio of the total mass of colchicine and ferulic acid to the volume of the second solvent is 59.36 mg:(1-3) mL, for example, 59.36 mg:1 mL, 59.36 mg:2 mL, 59.36 mg:3 mL, etc.

[0018] As a preferred embodiment of the present invention, the first solvent and the second solvent each independently include any one or more of ethanol, methanol, acetone, and acetonitrile.

[0019] As a preferred embodiment of the present invention, when the prescribed amounts of colchicine and ferulic acid are dissolved in the second solvent, stirring is performed at a speed of 500-1000 rpm, such as 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, 1000 rpm, etc.; and the stirring time is 6-24 h, such as 6 h, 12 h, 16 h, 20 h, 24 h, etc.

[0020] As a preferred embodiment of the present invention, the drying is vacuum drying, and the temperature of the vacuum drying is 40-50℃, such as 40℃, 42℃, 44℃, 46℃, 48℃, 50℃, etc.; the vacuum drying time is 3-5 h, such as 3h, 3.5h, 4h, 4.5h, 5h, etc.

[0021] Thirdly, the present invention provides a pharmaceutical composition comprising the colchicine and ferulic acid amorphous compound as described in the first aspect or the colchicine and ferulic acid amorphous compound prepared by the preparation method described in the second aspect, and pharmaceutical excipients.

[0022] As a preferred embodiment of the present invention, the pharmaceutical excipients include any one or more of diluents, disintegrants, binders, lubricants, isotonic agents, and buffers.

[0023] As a preferred embodiment of the present invention, the dosage form of the pharmaceutical composition includes cream, patch, gel, capsule, injection, granule, and tablet.

[0024] Fourthly, the present invention provides the use of the colchicine and ferulic acid co-amorphous compound as described in the first aspect, or the colchicine and ferulic acid co-amorphous compound prepared by the preparation method described in the second aspect, or the pharmaceutical composition described in the third aspect, in the preparation of medicaments for treating gout, gouty arthritis, familial Mediterranean fever, skin diseases (such as Behçet's disease and scleroderma), cardiovascular diseases (pericarditis and atrial fibrillation), and antifibrotic drugs.

[0025] The technical solution provided by the embodiments of the present invention has the following advantages compared with the prior art: 1. The colchicine and ferulic acid co-amorphous compound provided by this invention has good physical stability. While improving the solubility of colchicine, it significantly reduces its dissolution rate, achieving slow drug release and stable blood drug concentration, thus having the effect of "enhancing efficacy and reducing toxicity". Moreover, the glass transition temperature of the colchicine and ferulic acid co-amorphous compound of this invention is significantly lower than that of colchicine, which can promote transdermal absorption. This provides a new technical path for the development of colchicine topical preparations, with broad application prospects, and effectively solves the problems of colchicine's narrow therapeutic window, high toxicity, and poor patient compliance.

[0026] 2. The colchicine and ferulic acid co-amorphous compound provided by the present invention is prepared by grinding or solution crystallization. The preparation method is simple and has good prospects for industrial application. Attached Figure Description

[0027] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0029] Figure 1 PXRD patterns of the amorphous product of colchicine and ferulic acid from Example 1, colchicine, ferulic acid, and the mixture of colchicine and ferulic acid from Comparative Example 1. Figure 2 The following is a DSC chromatogram of the colchicine and ferulic acid co-amorphous compound, colchicine, and ferulic acid from Example 1; Figure 3Infrared images of the amorphous product of colchicine and ferulic acid in Example 1, colchicine, ferulic acid, and the mixture of colchicine and ferulic acid in Comparative Example 1; Figure 4 PXRD patterns of the amorphous compound of colchicine and ferulic acid after 0, 5, and 10 days of storage at high temperature; Figure 5 PXRD images of the colchicine and ferulic acid co-amorphous compound after 0, 5, and 10 days of storage under high humidity conditions; Figure 6 PXRD images of the amorphous compound of colchicine and ferulic acid after 0, 5, and 10 days of storage under light conditions; Figure 7 PXRD patterns of colchicine and ferulic acid co-amorphous compound after 0 days, 1 month, and 3 months of storage in a desiccator at 25°C and low relative humidity; Figure 8 The equilibrium solubility comparison chart shows the amorphous product of colchicine and ferulic acid in Example 1, colchicine, and the mixture of colchicine and ferulic acid in Comparative Example 1. Figure 9 Serum IL-1β levels in rats of different groups after administration of the drug to rats with gout; Figure 10 Serum IL-6 levels in rats of different groups after administration of the drug to gout rats; Figure 11 Serum TNF-α levels in rats of different groups after administration of the drug to gout rats; Figure 12 The staining results show the generation of reactive oxygen species in different groups after different drugs stimulate cardiomyocytes. Figure 13 The effects of different drugs on the mitochondrial membrane potential of cardiomyocytes. Detailed Implementation

[0030] To better understand the above-mentioned objectives, features, and advantages of the present invention, the solutions of the present invention will be further described below. It should be noted that, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

[0031] Many specific details are set forth in the following description in order to provide a full understanding of the invention, but the invention may also be practiced in other ways different from those described herein; obviously, the embodiments in the specification are only some embodiments of the invention, and not all embodiments.

[0032] Example 1 This embodiment provides a colchicine and ferulic acid co-amorphous compound and its preparation method, the preparation method comprising the following steps: Accurately weigh 39.94 mg of colchicine and 19.42 mg of ferulic acid and place them in a mortar. Add 2 mL of ethanol and grind until a gel-like consistency is reached. After the solvent has completely evaporated, an amorphous compound of colchicine and ferulic acid is obtained.

[0033] Example 2 This embodiment provides a colchicine and ferulic acid co-amorphous compound and its preparation method, the preparation method comprising the following steps: Accurately weigh 79.88 mg of colchicine and 19.42 mg of ferulic acid and place them in a mortar. Add 1.7 mL of methanol and grind until a gel-like consistency is reached. After the solvent has completely evaporated, an amorphous compound of colchicine and ferulic acid is obtained.

[0034] Example 3 This embodiment provides a colchicine and ferulic acid co-amorphous compound and its preparation method, the preparation method comprising the following steps: Accurately weigh 119.82 mg of colchicine and 19.42 mg of ferulic acid and place them in a mortar. Add 4.5 mL of acetone and grind until a gel-like consistency is reached. After the solvent has completely evaporated, an amorphous compound of colchicine and ferulic acid is obtained.

[0035] Example 4 This embodiment provides a colchicine and ferulic acid co-amorphous compound and its preparation method, the preparation method comprising the following steps: Accurately weigh 39.94 mg of colchicine and 19.42 mg of ferulic acid into a vial, mix them evenly, add 2 mL of ethanol, place the vial on a magnetic stirrer, and stir continuously at 800 rpm for 12 h until the above solution is basically evaporated. Then place the vial in a vacuum drying oven and dry at 40 °C for 3 h to obtain a co-amorphous product of colchicine and ferulic acid. Place the product in a desiccator and store it at room temperature for later use.

[0036] Example 5 This embodiment provides a colchicine and ferulic acid co-amorphous compound and its preparation method, the preparation method comprising the following steps: Accurately weigh 79.88 mg of colchicine and 19.42 mg of ferulic acid into a vial, mix them evenly, add 1.7 mL of acetonitrile, place the vial on a magnetic stirrer, and stir continuously at 500 rpm for 24 h until the above solution is basically evaporated. Then place the vial in a vacuum drying oven and dry at 50 °C for 3 h to obtain a co-amorphous product of colchicine and ferulic acid. Place the product in a desiccator and store it at room temperature for later use.

[0037] Example 6 This embodiment provides a colchicine and ferulic acid co-amorphous compound and its preparation method, the preparation method comprising the following steps: Accurately weigh 119.82 mg of colchicine and 19.42 mg of ferulic acid into a vial, mix them evenly, add 7 mL of methanol, place the vial on a magnetic stirrer, and stir continuously at 1000 rpm for 6 h until the above solution is basically evaporated. Then place the vial in a vacuum drying oven and dry at 45 °C for 4 h to obtain a co-amorphous product of colchicine and ferulic acid. Place the product in a desiccator and store it at room temperature for later use.

[0038] Comparative Example 1 This comparative example provides a mixture of colchicine and ferulic acid, wherein the amount of colchicine is 39.94 mg and the amount of ferulic acid is 19.42 mg. The two are directly stirred evenly to obtain the mixture of colchicine and ferulic acid.

[0039] Performance Test 1 Powder X-ray diffraction tests were performed on the amorphous co-form of colchicine and ferulic acid prepared in Example 1, colchicine, ferulic acid, and the mixture of colchicine and ferulic acid in Comparative Example 1.

[0040] Testing instrument: Rigaku D / max-2550 X-ray powder diffractometer Measurement conditions and methods: Cu / Ka l pha 1, 40KV-200 mA, I(max)=2244, 2θ=4-40°, λ=1.54056.

[0041] Example 1: PXRD patterns of the amorphous product of colchicine and ferulic acid, colchicine, ferulic acid, and the mixture of colchicine and ferulic acid in Comparative Example 1 are shown below. Figure 1 As shown. By Figure 1 It can be observed that colchicine and ferulic acid exhibit strong and clear characteristic diffraction peaks in the 2θ range of 3-40° (colchicine: 4.40°, 5.94°, 10.67°, 14.89°, 16.52°, 16.88°, 17.63°, 17.75°, 19.96°, 21.44°, 23.91°, 24.15°; ferulic acid: 9.01°, 10.47°, 12.8°, 15.62°, 17.4°, 26.45°). In contrast, the mixture of colchicine and ferulic acid in Comparative Example 1 shows a simple superposition of sharp diffraction peaks from colchicine and ferulic acid. In comparison, the PXRD pattern of the amorphous colchicine and ferulic acid co-formation in the embodiments of this invention shows typical "bun peaks," indicating the absence of an ordered structure, and successful co-amorphization of colchicine and ferulic acid.

[0042] Performance Test 2 The colchicine and ferulic acid co-amorphous compound, colchicine, and ferulic acid prepared in Example 1 were subjected to DSC analysis.

[0043] Testing instrument: Mettler Toledo DSC3 / 700 / 200 differential scanning calorimeter.

[0044] Determination conditions and methods: Approximately 3 mg of colchicine, ferulic acid, and the amorphous colchicine and ferulic acid compound prepared in Example 1 were weighed separately and placed in aluminum crucibles. Differential scanning calorimetry (DSC) was used for determination. Nitrogen flow rate was 50 mL / min, and the temperature was increased from 30 °C to 220 °C at a rate of 5 °C / min. The Tg value was determined based on the midpoint of the step change in the DSC curve.

[0045] Example 1: DSC chromatograms of the amorphous compound of colchicine and ferulic acid, colchicine, and ferulic acid are shown below. Figure 2 As shown in the figure, the DSC curve of crystalline colchicine shows a strong endothermic peak at 151.18°C, corresponding to the melting point of colchicine. The melting point of crystalline ferulic acid is 174.17°C, consistent with the literature report (173.9°C). Unlike the above crystalline raw materials, the DSC curve of the colchicine and ferulic acid co-amorphous compound prepared in Example 1 only shows a glass transition temperature (Tg) at 64.11°C, without a significant melting peak, indicating the formation of a uniform amorphous single-phase system. This demonstrates the successful preparation of the colchicine and ferulic acid co-amorphous compound of this invention.

[0046] Performance Test 3 Infrared spectroscopy was performed on the amorphous co-form of colchicine and ferulic acid prepared in Example 1, colchicine, ferulic acid, and the mixture of colchicine and ferulic acid in Comparative Example 1.

[0047] Testing instrument: Perkin Elmer Spectrum 400 infrared spectrometer.

[0048] Determination conditions and methods: Colchicine, ferulic acid, the amorphous co-form of colchicine and ferulic acid prepared in Example 1, and the mixture of colchicine and ferulic acid in Comparative Example 1 were analyzed and detected by infrared absorption spectroscopy in the range of 4000~400 cm⁻¹. -1 Scan within the range and record the infrared absorption spectrum.

[0049] In Example 1, the infrared spectra of the amorphous product of colchicine and ferulic acid, colchicine, ferulic acid, and the mixture of colchicine and ferulic acid in Comparative Example 1 are shown below. Figure 3 As shown. It can be observed that in the infrared spectrum of the amorphous compound of colchicine and ferulic acid in Example 1, the crystalline colchicine is at 3314 cm⁻¹. - The sharp absorption peak of ν(NH) disappears, and ferulic acid at 3437 cm⁻¹... - The sharp absorption peak of ¹ disappears, while at 3435 cm⁻¹...- The formation of a broad peak at position ¹ indicates that the NH group in colchicine may interact intermolecularly with the carboxyl group of ferulic acid. These changes in the characteristic infrared absorption peaks suggest that the hydroxyl and carbonyl groups of ferulic acid interact with the amino and carbonyl groups of colchicine through hydrogen bonding.

[0050] Performance Test 4 The physical stability (short-term and long-term stability) of the colchicine and ferulic acid co-amorphous compound prepared in Example 1 was tested.

[0051] Testing instruments: HPP260 constant temperature and humidity incubator and Rigaku D / max-2550 X-ray powder diffractometer.

[0052] 1. High temperature test Take a small amount of sample and place it in a 50℃ constant temperature drying oven. After 0, 5 and 10 days, take the sample and perform PXRD test.

[0053] The results are as follows Figure 4 As shown, Figure 4 The images show the PXRD patterns of the colchicine and ferulic acid co-amorphous compound after 0, 5, and 10 days of high-temperature storage. It can be seen that after 10 days of high-temperature storage, the PXRD pattern of the colchicine and ferulic acid co-amorphous compound remains basically unchanged, indicating that it still retains its amorphous characteristics.

[0054] 2. High humidity test Take a small amount of sample and place it in a constant temperature and humidity incubator. Set the temperature to 25℃ and the relative humidity to 80%. After 0, 5 and 10 days, take the sample and perform PXRD test.

[0055] The results are as follows Figure 5 As shown, Figure 5 The images show the PXRD patterns of the colchicine and ferulic acid co-amorphous compound after 0, 5, and 10 days of storage under high humidity conditions. It can be seen that after 10 days of storage under high humidity conditions of 80%, the PXRD pattern of the colchicine and ferulic acid co-amorphous compound remains basically unchanged, indicating that it still retains its amorphous characteristics.

[0056] 3. Light test Take a small amount of sample and place it under a light source with an illuminance of 4500±500 lx; ​​take the sample after 0, 5 and 10 days respectively and perform PXRD test.

[0057] The results are as follows Figure 6 As shown, Figure 6The images show the PXRD patterns of the colchicine and ferulic acid co-amorphous compound after 0, 5, and 10 days of storage under light. It can be seen that after 10 days of storage under light, the PXRD pattern of the colchicine and ferulic acid co-amorphous compound remains basically unchanged, indicating that it still retains its amorphous characteristics.

[0058] 4. Long-term stability test Take an appropriate amount of sample and place it in a desiccator with low relative humidity containing color-changing silica gel. Place it at room temperature for 3 months. After 0, 1 and 3 months, take the above sample and perform PXRD test.

[0059] The results are as follows Figure 7 As shown, Figure 7 The PXRD images show the amorphous co-forms of colchicine and ferulic acid after 0 days, 1 month, and 3 months of storage in a desiccator at 25°C and low relative humidity. It can be seen that the amorphous co-forms of colchicine and ferulic acid remain amorphous after 3 months of storage.

[0060] High temperature, high humidity, light exposure, and long-term stability tests show that the colchicine and ferulic acid co-amorphous compound prepared in this invention has excellent stability.

[0061] Performance Test 5 The equilibrium solubility of the colchicine and ferulic acid co-amorphous compound prepared in Example 1, colchicine, and the mixture of colchicine and ferulic acid in Comparative Example 1 was determined.

[0062] Test method: Accurately weigh an excess of the amorphous co-form of colchicine and ferulic acid from Example 1, colchicine, and the mixture of colchicine and ferulic acid from Comparative Example 1, and place them in stoppered centrifuge tubes. Add an appropriate amount of pure water medium and conduct an equilibrium solubility test by shaking flask method at a temperature of 37±2°C and a rotation speed of 120 rpm.

[0063] Samples were taken within 0-72 h, filtered through a 0.22 μm microporous membrane, and detected by high performance liquid chromatography at an absorption wavelength of 254 nm. The content was calculated using the external standard method.

[0064] Plot the solubility curves of colchicine in water with time (h) on the x-axis and colchicine dissolution (mg / ml) on the y-axis.

[0065] Test results are as follows Figure 8 As shown, Figure 8 This is a comparison of the equilibrium solubility of the amorphous product of colchicine and ferulic acid from Example 1, colchicine, and the mixture of colchicine and ferulic acid from Comparative Example 1. Figure 8It can be observed that, unlike the mixture of colchicine and ferulic acid, and colchicine itself, the amorphous co-form of colchicine and ferulic acid exhibits a slow release trend. Within 0-30 h, at each sampling point, the solubility of the amorphous co-form of colchicine and ferulic acid, calculated as colchicine, was lower than that of colchicine. Equilibrium was reached within 70-80 h, with a final equilibrium solubility of 17.9 mg / mL, which is 1.44 times that of colchicine. This indicates that the amorphous co-form of colchicine and ferulic acid of this invention improves the equilibrium solubility of colchicine while simultaneously achieving a slow release of colchicine.

[0066] Performance Test 6 The intrinsic dissolution rate of colchicine and ferulic acid co-amorphous compound prepared in Example 1 and colchicine were determined.

[0067] Test Method: Approximately 10 mg of colchicine and colchicine-ferulic acid amorphous compound were placed separately in a Mini-IDR tableting module and compressed at 100 psi for 1.5 min to form discs with a fixed surface area of ​​0.0707 cm². Each disc was mounted in a stir-mold assembly and immersed in a flat-bottomed glass vial containing 15 mL of purified water at 37°C. Stirring was immediately started at a paddle speed of 150 rpm; drug concentration was monitored every 10 seconds using an in-situ UV probe for 10 min. All experiments were repeated three times, and the IDR value (intrinsic dissolution rate) was calculated using the following formula.

[0068]

[0069] In the formula, V represents the volume of the medium; dc / dt represents the linear slope of the dissolution curve; and A represents the fixed surface area of ​​the disk.

[0070] The IDR of colchicine was determined to be 5.5 mg / (min·cm). 2) The IDR of the amorphous compound of colchicine and ferulic acid was 0.8 mg / (min·cm). 2) The IDR of the colchicine-ferulic acid co-amorphous compound decreased to 15% of that of colchicine. This indicates that co-amorphization significantly slowed the dissolution rate of colchicine, achieving slow drug release and stable blood drug concentration, thus resulting in better safety.

[0071] Performance Test 7 The therapeutic effect of the colchicine and ferulic acid co-amorphous compound prepared in Example 1 on gout rats was investigated.

[0072] Experimental Methods: Fifty male SD rats were randomly divided into five groups: blank control group, model group, colchicine group, comparative example 1 group, and example 1 group. After one week of acclimatization, the rats were labeled and administered the drugs. Except for the blank control group, all other groups were anesthetized by intraperitoneal injection of 2% pentobarbital, followed by intra-articular injection of 0.1 mL of sodium urate crystal suspension (concentration of 20 mg / mL). The blank control group rats were injected with the same volume of physiological saline into the right ankle joint and intraperitoneal cavity, respectively. The injection standard was swelling of the contralateral joint capsule. Colchicine, the mixture of colchicine and ferulic acid from comparative example 1, and the amorphous product of colchicine and ferulic acid from example 1 were dissolved in physiological saline to form suspensions with a concentration of 0.2 mg / mL (all calculated based on colchicine). Rats were administered colchicine at a dose of 0.6 mg / kg to the colchicine group (gavage with colchicine), Comparative Example 1 (gavage with a mixture of colchicine and ferulic acid), and Example 1 (gavage with a mixture of colchicine and ferulic acid from Example 1). The blank group and the model group were administered an equal volume of physiological saline by gavage.

[0073] At 48 h post-drug administration, rats were anesthetized by intraperitoneal injection of pentobarbital to ensure no spontaneous movement. Blood was then collected from the abdominal aorta of each group, with at least 8 mL collected from each rat. The blood samples were centrifuged at 3000 rpm for 15 minutes at 4°C, and the serum was separated, aliquoted, and flash-frozen. The rat ELISA assay was performed according to the instructions. The levels of various inflammatory factors (TNF-α, IL-1β, and IL-6) in the samples were calculated using a standard curve.

[0074] The results are as follows Figures 9-11 As shown, where, Figure 9 The levels of IL-1β in the serum of rats in each group after drug administration. Figure 10 The levels of IL-6 in the serum of rats in each group after drug administration. Figure 11 The levels of TNF-α in the serum of rats in each group after drug administration were shown. It was found that, compared with the blank group, the concentrations of IL-1β, IL-6, and TNF-α in the serum of rats in the model group were significantly increased, indicating successful modeling and inducing a strong inflammatory response. Compared with the model group, the colchicine group, Comparative Example 1 group, and Example 1 group all downregulated the levels of the three inflammatory factors to varying degrees. Among them, the anti-inflammatory effect of Comparative Example 1 group was superior to that of the colchicine group. Most importantly, the anti-inflammatory effect of Example 1 group was the most significant, indicating that the colchicine and ferulic acid co-amorphous compound provided by this invention has excellent therapeutic effects on gout in rats.

[0075] Performance Test 8 The effect of the colchicine and ferulic acid co-amorphous compound prepared in Example 1 on the generation of reactive oxygen species in cardiomyocytes was investigated.

[0076] Experimental method: When the density of cardiomyocytes in the culture flask reached 80%, the cells were seeded into dishes and diluted to 4 × 10⁴ cells with 10% FBS DMEM. 4 Cells / mL. Take a sterile 96-well plate and seed each well with 100 μL of cell suspension (4 × 10⁻⁶ cells / mL). 3 (Number of samples per well). The wells were divided into a blank group, a colchicine group, Example 1 group, and a comparative example group. The wells were incubated at 37°C and 5% CO2 for 24 hours. After serum withdrawal and 24 hours of incubation, the wells were treated with the added drug. The original culture medium was aspirated from the wells. The blank group was treated with 1% FBS medium (100 μL / well), while the treated wells were treated with 1% FBS medium and the corresponding drug (30 nmol of colchicine, 100 μL / well). The wells were incubated at 37°C and 5% CO2 for 24 hours.

[0077] Dilute DCFH-DA 1:1000 with serum-free culture medium to a final concentration of 10 μmol / L. Remove the cell culture medium and add an appropriate volume of diluted DCFH-DA. Add 70 μL to each well and incubate at 37°C for 40 min. Without discarding the staining solution, add 10 μL of Hoechst 33342 staining solution diluted 1:100 to each well and stain the nuclei at 37°C in the dark for 10 min. Wash the cells three times with serum-free cell culture medium to thoroughly remove any DCFH-DA that has not yet entered the cells. Add 100 μL of PBS to each well and observe the stained cells under a fluorescence microscope, taking pictures to capture images.

[0078] Reactive oxygen species (ROS) staining using a fluorescent probe (DCFH-DA) can directly evaluate the intensity of oxidative damage to cardiomyocytes caused by compounds, providing direct evidence of oxidative stress for evaluating drug cardiotoxicity. Results are as follows: Figure 12 As shown, Figure 12 The staining results of reactive oxygen species (ROS) generated in different groups after stimulation of cardiomyocytes by different drugs are shown. Among them, Hoechst 33342: blue fluorescent staining of cell nuclei, used to locate cells; DCFH-DA: green fluorescent probe of reactive oxygen species (ROS); Merge: the above two images are superimposed. The deeper the green fluorescence, the more reactive oxygen species are generated, indicating that the level of oxidative stress in cardiomyocytes is higher (the more severe the degree of oxidative damage).

[0079] It can be observed that the blank group without drug stimulation generated very little reactive oxygen species (ROS), while the colchicine group damaged cardiomyocytes and generated significantly more ROS. The mixture of colchicine and ferulic acid in Comparative Example 1 contained ferulic acid, which significantly reduced ROS generation. Furthermore, ROS generation was further reduced in Example 1, indicating that the colchicine and ferulic acid co-amorphous compound provided by this invention can effectively alleviate colchicine-induced ROS damage to cardiomyocytes.

[0080] Performance Test 9 The effect of the colchicine and ferulic acid co-amorphous compound prepared in Example 1 on the mitochondrial membrane potential of cardiomyocytes was investigated.

[0081] Experimental method: When the density of cardiomyocytes in the culture flask reached 80%, the cells were seeded into dishes and diluted to 4 × 10⁴ cells with 10% FBS DMEM. 4 Cells / mL. Take a sterile 96-well plate and seed each well with 100 μL of cell suspension (4 × 10⁻⁶ cells / mL). 3 The wells were divided into a blank group, a colchicine group, Example 1 group, and a comparative example group. The wells were incubated at 37°C and 5% CO2 for 24 hours. After serum withdrawal and 24 hours of incubation, the wells were treated with the added drug. The original culture medium was aspirated from the wells. The blank group was treated with 1% FBS medium (100 μL / well), while the drug-treated groups were treated with 1% FBS medium and the corresponding drug (30 nmol of colchicine, 100 μL / well). The wells were incubated at 37°C and 5% CO2 for 24 hours.

[0082] Discard the old culture medium and prepare working solution at a ratio of 1:5000. Add 100 μL of Mitotracker dye working solution to each well, gently agitate to completely cover the cells, then add 10 μL of Hoechst 33342 dye solution diluted 1:100 to each well and incubate at 37°C for 15 min in the dark. Aspirate the dye working solution, wash 2-3 times with serum-free culture medium for 5 min each time, observe using a fluorescence microscope, and take pictures.

[0083] MitoTracker staining, by specifically labeling changes in mitochondrial morphology and membrane potential, can directly reflect the degree of mitochondrial membrane potential depolarization under drug intervention, and has important applications in compound toxicity evaluation. Results are as follows: Figure 13 As shown, Figure 13 The effects of different drugs on the mitochondrial membrane potential of cardiomyocytes are shown. Hoechst 33342: blue fluorescent staining of the cell nucleus for cell localization; MitoTracker: red fluorescent probe for mitochondrial membrane potential; Merge: the overlay of the two images above. The intensity of red fluorescence is positively correlated with mitochondrial membrane potential—the lighter the red, the lower the membrane potential, indicating that the cardiomyocytes are in an early apoptotic state.

[0084] It was observed that the mitochondrial membrane potential was higher in the blank group, while it significantly decreased in the colchicine group after the addition of colchicine, indicating that some cardiomyocytes may have undergone early apoptosis. In contrast, the mixture of colchicine and ferulic acid in Comparative Example 1 showed a certain protective effect on cardiomyocytes, and the mitochondrial membrane potential in Example 1 was higher than that in Comparative Example 1, indicating that the colchicine and ferulic acid amorphous compound provided by this invention has a certain protective effect against cardiomyocyte apoptosis.

[0085] In summary, the inherent dissolution rate of the colchicine and ferulic acid co-amorphous compound provided by this invention is reduced to about 15% of that of colchicine. By controlling the dissolution rate, this invention can not only effectively avoid the phenomenon of excessive blood drug concentration, but also achieve slow drug release, thereby significantly reducing toxic side effects and improving drug safety.

[0086] This invention uses ferulic acid as a drug ligand, which has multiple pharmacological activities such as anti-oxidation, antithrombosis, lipid-lowering, myocardial oxygen consumption reduction, antibacterial, antiviral, and anticancer effects. It also has low toxicity and high safety, and has a uric acid-lowering effect. The amorphous compound of colchicine and ferulic acid, through uniform dispersion at the molecular level, has a better inhibitory effect on key pro-inflammatory factors IL-1β, IL-6, and TNF-α than colchicine alone or a mixture of colchicine and ferulic acid. The amorphous compound exerts a synergistic effect, significantly improving the therapeutic effect on gout, and can also reduce the cardiocytotoxicity of colchicine.

[0087] Furthermore, the reduction in the glass transition temperature (Tg) of the colchicine and ferulic acid co-amorphous compound of the present invention is beneficial to enhancing the transdermal permeability of the drug. The co-amorphous compound can maintain the drug in a supersaturated state on the skin surface, and its permeation-enhancing effect far exceeds the expected gain brought about by the improvement of solubility alone, showing good application prospects in transdermal formulations.

[0088] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0089] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An amorphous compound of colchicine and ferulic acid, characterized in that, The molar ratio of colchicine to ferulic acid in the amorphous compound of colchicine and ferulic acid is 1-3:

1.

2. The colchicine and ferulic acid co-amorphous compound according to claim 1, characterized in that, The molar ratio of colchicine to ferulic acid in the amorphous compound of colchicine and ferulic acid is 1:

1.

3. The colchicine and ferulic acid co-amorphous compound according to claim 1 or 2, characterized in that, The equilibrium solubility of the colchicine and ferulic acid co-amorphous compound in water at 37°C, calculated as colchicine, is 17.9 mg / mL. And / or, the inherent dissolution rate of the colchicine and ferulic acid co-amorphous compound in water at 37°C, calculated as colchicine, is 0.8 mg / (min·cm). 2 ).

4. The method for preparing the amorphous compound of colchicine and ferulic acid as described in any one of claims 1-3, characterized in that, The preparation method includes: The prescribed amounts of colchicine and ferulic acid were soaked in the first solvent, ground until a gel-like consistency was obtained, and the solvent was allowed to evaporate completely to obtain the amorphous product of colchicine and ferulic acid. Alternatively, the preparation method includes: The prescribed amounts of colchicine and ferulic acid were dissolved in a second solvent and dried to obtain the colchicine and ferulic acid co-amorphous compound.

5. The preparation method according to claim 4, characterized in that, The ratio of the total mass of colchicine and ferulic acid to the volume of the first solvent is 59.36 mg:(1-2)mL; Alternatively, the ratio of the total mass of colchicine and ferulic acid to the volume of the second solvent is 59.36 mg:(1-3) mL; And / or, the first solvent and the second solvent each independently include any one or more of ethanol, methanol, acetone, and acetonitrile.

6. The preparation method according to claim 4 or 5, characterized in that, The drying process is vacuum drying, the temperature of which is 40-50℃, and the drying time is 3-5 hours.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the colchicine and ferulic acid amorphous compound according to any one of claims 1-3 or the colchicine and ferulic acid amorphous compound prepared by the preparation method according to any one of claims 4-6, and pharmaceutical excipients.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical excipients include any one or more of the following: diluents, disintegrants, binders, lubricants, isotonic agents, and buffers.

9. The pharmaceutical composition according to claim 7 or 8, characterized in that, The dosage forms of the pharmaceutical composition include creams, patches, gels, capsules, injections, granules, and tablets.

10. The use of the colchicine and ferulic acid amorphous compound as described in any one of claims 1-3, or the colchicine and ferulic acid amorphous compound prepared by the preparation method described in any one of claims 4-6, or the pharmaceutical composition described in any one of claims 7-9, in the preparation of medicaments for treating gout, gouty arthritis, familial Mediterranean fever, skin diseases, cardiovascular diseases, and antifibrosis.

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