A compound minocycline hydrochloride-melatonin colon-targeting capsule and a preparation method thereof

Abstract

The application belongs to the technical field of pharmaceutical preparations, and particularly relates to a minocycline hydrochloride-melatonin compound colon-targeting capsule and a preparation method thereof. The capsule comprises pellets and a capsule shell, the pellets comprise a drug core and a colon-targeting sustained-release layer wrapped outside the drug core. The drug core contains active ingredients of minocycline hydrochloride and melatonin; the colon-targeting sustained-release layer contains a specially prepared pH-enzyme colon-targeting response agent and a self-prepared plasticizer. The pH-enzyme response agent is based on hydroxypropyl methylcellulose grafted vanillic acid derivatives, and can be degraded in the colon in response to pH changes and azo reductase; the plasticizer has good compatibility with coating materials, and can effectively reduce drug leakage in the stomach and the front end of the intestine. The capsule prepared by the application can significantly improve the gastrointestinal irritation of minocycline hydrochloride, realize targeted release of the drug in the colon, and utilize the synergistic effect of melatonin to reduce the dosage of antibiotics while showing excellent therapeutic effect on ulcerative colitis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically relating to a minocycline hydrochloride-melatonin compound colon-targeting capsule and its preparation method. Background Technology

[0002] Minocycline hydrochloride is a semi-synthetic tetracycline broad-spectrum antibiotic. Its hydrochloride salt is a yellow crystalline powder with a bitter taste, is hygroscopic, and degrades upon exposure to light. It is readily soluble in alkali metal hydroxide or carbonate solutions, slightly soluble in ethanol, and slightly soluble in water. This product exhibits the highest antibacterial activity among tetracyclines, still possessing bactericidal activity against tetracycline-resistant Staphylococcus aureus, Streptococcus, and Escherichia coli, and exhibits relatively low selective pressure due to resistance mutations.

[0003] Minocycline hydrochloride is rapidly and completely absorbed after oral administration, exhibiting strong tissue penetration and wide distribution throughout the body's organs and fluids. It particularly easily crosses the blood-brain barrier, reaching high concentrations in the cerebrospinal fluid. Long-term clinical use has confirmed that its adverse reactions mainly include gastrointestinal irritation symptoms (such as nausea, vomiting, abdominal pain, and diarrhea) and esophageal ulcers. Among these adverse reactions, gastrointestinal discomfort primarily stems from the drug's direct irritation of the upper gastrointestinal mucosa and small intestinal flora imbalance, while esophageal ulcers are directly related to the local disintegration of the capsule in the esophagus.

[0004] Melatonin can synchronize circadian rhythms, shorten sleep latency, and improve sleep quality in cases of shift work, jet lag, and primary insomnia. It also possesses antioxidant and immunomodulatory effects, but requires low-dose, short-term use; long-term, high-dose suppression of endogenous secretion is detrimental. Patent CN117643569A discloses the synergistic effect of melatonin and minocycline hydrochloride in improving the success rate of periodontitis treatment. However, no further research has been conducted on the synergistic effect of melatonin and minocycline hydrochloride on colitis.

[0005] Therefore, there is an urgent need for a colon-targeted capsule containing minocycline hydrochloride and melatonin, which can achieve targeted release in the colon to overcome the gastric irritation of minocycline hydrochloride and enhance its colon-targeting effect to exert anti-inflammatory efficacy. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide a minocycline hydrochloride-melatonin compound colon-targeting capsule and its preparation method.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A minocycline hydrochloride-melatonin compound colon-targeting capsule includes microspheres and a capsule shell. The microspheres include a drug core and a colon-targeting sustained-release layer wrapped around the drug core, with a mass ratio of 1:(0.1 to 0.15).

[0009] The core comprises the following raw materials in parts by weight: 20-40 parts minocycline hydrochloride, 10-20 parts melatonin, 20-40 parts filler, and 4-11 parts binder I;

[0010] The colon-targeted sustained-release layer comprises the following raw materials in parts by weight: 5-15 parts of binder II, 10-20 parts of pH-enzyme colon-targeted responsive agent, 2-4 parts of plasticizer, 3-5 parts of opacifier, 5-20 parts of sustained-release agent, and 2-10 parts of pore-forming agent.

[0011] Preferably, the pH-enzyme colon-targeting responsive agent is prepared by the following process:

[0012] (1) Vanillic acid and potassium carbonate were added to acetonitrile, and then 4-bromo-2-methoxyaniline was added. The mixture was reacted under heating conditions, and the intermediate 1 was obtained after purification. The structure of intermediate 1 is shown in Formula I:

[0013] ;

[0014] (2) Add 4-aminophenyl acrylate and intermediate 1 to sodium hypochlorite solution, then add hydrochloric acid solution to react, and purify to obtain vanillic acid derivative; the structure of vanillic acid derivative is shown in Formula II:

[0015] ;

[0016] (3) Hydroxypropyl methylcellulose was dissolved in water, heated to 50-60°C under a nitrogen atmosphere, and potassium sulfate was added to initiate the reaction. Then, the vanillic acid derivative was added to continue the reaction. After cooling, the mixture was dialyzed and lyophilized to obtain the pH-enzyme colon-targeting responsive agent.

[0017] Preferably, the molar ratio of vanillic acid, potassium carbonate and 4-bromo-2-methoxyaniline in step (1) is 1:(2.2-2.8):(1.1-1.5); the heating temperature is 60-80°C and the reaction time is 12-18 h.

[0018] Preferably, in step (2), the ratio of intermediate 1,4-aminophenyl acrylate, sodium hypochlorite solution, and hydrochloric acid solution is 1 mmol: (1-1.5) mmol: (3.7-4.5) mL: (0.4-0.8) mL; the concentration of sodium hypochlorite solution is 10 wt%, and the concentration of hydrochloric acid solution is 2.5 mol / L; the reaction time is 3-5 h.

[0019] Preferably, the mass ratio of hydroxypropyl methylcellulose, potassium sulfate and vanillic acid derivative in step (3) is 1:(0.012-0.016):(3.2-4); the initiation time is 12-15 min; the reaction time is 0.5-1 h; and the molecular weight cutoff for dialysis is 10 kDa.

[0020] Preferably, the plasticizer is prepared by the following process:

[0021] Phthalic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, and 4-hydroxy-2-pyrrolidone were added to acetonitrile and reacted under heating conditions. After purification, the plasticizer was obtained. The structure of the plasticizer is shown in Formula III.

[0022] .

[0023] Preferably, the molar ratio of phthalic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine and 4-hydroxy-2-pyrrolidone is 1:(2.5-2.8):(5-5.6):(2-2.5); the heating temperature is 40-60°C and the reaction time is 0.5-1.5 h.

[0024] Preferably, the filler is selected from lactose and microcrystalline cellulose; both adhesive I and adhesive II are povidone K30; the light-blocking agent is titanium dioxide; the slow-release agent is selected from polyacrylate and ethyl cellulose; and the pore-forming agent is selected from polysorbate and polyethylene glycol 6000.

[0025] A method for preparing the above-mentioned minocycline hydrochloride-melatonin compound colon-targeting capsule includes the following steps:

[0026] (a) Preparation of the core: According to the weight proportions of each raw material in the core, minocycline hydrochloride, melatonin, filler and part of binder I are mixed to obtain a mixed powder; the remaining binder I is dissolved in water to form an adhesive liquid and added to the mixed powder, and the core is obtained after stirring, extrusion and drying.

[0027] (b) Preparation of microcapsules: According to the weight proportions of each raw material in the colon-targeted sustained-release layer, binder II, pH-enzyme colon-targeted responsive agent, plasticizer, opacifier, sustained-release agent and pore-forming agent are added to an ethanol-water solution to obtain a mixture; the mixture is coated onto the surface of the drug core obtained in step (a) by a fluidized bed bottom spraying process to form a colon-targeted sustained-release layer, and microcapsules are obtained after drying;

[0028] (c) Capsule preparation: The microparticles are filled into capsule shells to obtain the minocycline hydrochloride-melatonin compound colon-targeting capsules.

[0029] Preferably, each minocycline hydrochloride-melatonin compound colon-targeting capsule contains 100–120 mg of minocycline hydrochloride.

[0030] The present invention has the following advantages over the prior art:

[0031] 1. This invention prepares a compound colon-targeting preparation by combining minocycline hydrochloride and melatonin. By adding a pH-enzyme colon-targeting responsive agent and a self-made plasticizer, the gastrointestinal irritation of minocycline hydrochloride is improved, achieving targeted release of the drug in the colon, reducing the dosage of antibiotics while reducing the risk of drug resistance, and utilizing the anti-inflammatory, antioxidant and circadian rhythm regulating effects of melatonin to enhance the therapeutic effect on colitis.

[0032] 2. The pH-enzyme colon-targeting responsive agent of this invention is a graft copolymer obtained by free radical grafting of carboxylated vanillic acid derivative side chains onto hydroxypropyl methylcellulose (HPMC) as the main chain. This material exhibits excellent film-forming properties and shows significant swelling differences in different pH environments of the gastrointestinal tract: in the acidic environment of the stomach, the carboxyl groups of the side chains are protonated, reducing the hydrophilicity of the material and limiting the swelling degree, effectively inhibiting drug release; when reaching the terminal ileum and ascending colon, the carboxyl groups are deprotonated, generating electrostatic repulsion, and the copolymer rapidly swells to form continuous diffusion channels, achieving drug release. Furthermore, the diazo groups introduced by azotization of the side chains can be specifically cleaved by azoreductase secreted by anaerobic bacteria in the colon, achieving dual pH-enzyme triggering and enhancing colon-targeting. In addition, the ester groups of the grafted side chains have structures similar to the ester groups in plasticizers, and can reduce drug leakage in the acidic phase through dipole-dipole interactions; the anisole group, with its hydrophobic aromatic ether structure, synergistically inhibits gastric acid swelling with the protonated carboxyl groups, and its rigid aromatic ring enhances the mechanical properties of the membrane and is compatible with ester plasticizers, reducing the risk of acid leakage.

[0033] 3. The present invention further includes excipients such as plasticizers, light-blocking agents, and pore-forming agents in the colon-targeted sustained-release layer. The plasticizer is formed by esterification of phthalic acid and 4-hydroxy-2-pyrrolidone. Its five-membered lactam structure is similar to the pyrrolidone unit of povidone, which is used as a binder. It has good compatibility and can significantly improve the flexibility and extensibility of the coating, avoid cracking when the tablet core absorbs water and swells, and reduce drug leakage in the acid phase. At the same time, it does not affect the swelling jump of the copolymer at the pH of the colon, ensuring the targeted release of the colon-targeted sustained-release layer. Attached Figure Description

[0034] Figure 1 FT-IR chromatograms of hydroxypropyl methylcellulose and pH-enzyme colon-targeting responsive agent in Preparation Example 1 of this invention;

[0035] Figure 2 The expression of IL-4 in the serum and colon tissue of rats in each group in Experiment Example 2 of this invention;

[0036] Figure 3 The expression of TNF-α in the serum and colon tissue of rats in each group in Experiment Example 2 of this invention;

[0037] Figure 4 The expression of MPO in the serum and colon tissue of rats in each group in Experiment Example 2 of this invention is shown. Detailed Implementation

[0038] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels. In the following embodiments, the polyacrylate resin used is Eutec RS 100; the polysorbate used is Tween 80; and both adhesive I and adhesive II are povidone K30.

[0039] Preparation Example 1

[0040] Preparation Example 1 provides a pH-enzyme colon-targeting responsive agent, prepared by the following process:

[0041]

[0042] (1) Vanillic acid (1.68 g, 10 mmol) and potassium carbonate (3.60 g, 26 mmol) were added to 100 mL of acetonitrile, stirred evenly, and then 4-bromo-2-methoxyaniline (2.62 g, 13 mmol) was slowly added. The mixture was heated to 70 °C and reacted for 15 h. The resulting mixture was extracted with ethyl acetate and distilled water. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate: dichloromethane = 1:1, v / v) to obtain intermediate 1 (2.41 g, yield 83.38%). 1 H NMR (C 15 H 15 NO5, 400 MHz, DMSO): δ 12.62 (s, 1H), 7.75-7.69 (m,2H), 7.45 (d, 1H), 6.78 (d, 1H), 6.49-6.45 (m, 2H), 4.92 (s, 2H), 3.88 (s,3H), 3.82 (s, 3H); HRMS(ESI+): [M+H] + The calculation yields 290.10, and the value is found to be 290.10.

[0043] (2) Intermediate 1 (2.89 g, 10 mmol), 4-aminophenyl acrylate (1.96 g, 12 mmol), 2.5 M hydrochloric acid solution (6 mL) and 10 wt% sodium hypochlorite solution (41 mL, 55 mmol) were added to a reaction flask and reacted at room temperature for 4 h. Ethyl acetate was added to the reaction mixture for quenching. The aqueous layer was extracted with ethyl acetate. The organic layers were combined, dried with anhydrous sodium sulfate and concentrated. The mixture was purified by rapid column chromatography (n-heptane: ethyl acetate = 80:20, v / v) to obtain vanillic acid derivative (3.53 g, yield 78.80%). 1 H NMR (C 24 H 20 N2O7, 400 MHz, DMSO): δ 12.62 (s, 1H), 7.88 (d, 3H), 7.75-7.69 (m, 2H), 7.45 (q, 3H), 6.84 (dd, 2H), 6.26 (dd, 1H), 6.12 (dd, 1H), 5.76 (dd, 1H), 4.05 (s, 3H), 3.82 (s, 3H); HRMS(ESI+): [M+H] + The result is 449.13, and the value is 449.10.

[0044] (3) Weigh 2.5 g of hydroxypropyl methylcellulose and dissolve it in 50 g of water. Heat the reaction solution to 55 °C under a nitrogen atmosphere and add 0.03 g of potassium sulfate (KPS) to initiate the reaction for 14 min. Then add 9 g of vanillic acid derivative to the reaction solution. After the addition is complete, continue the reaction at 55 °C for 1 h. After cooling to room temperature, use a dialysis bag (10 kDa) to collect the sample. The lyophilized portion is the pH-enzyme colon-targeting response agent.

[0045] FT-IR spectra of hydroxypropyl methylcellulose and pH-enzyme colon-targeting responsive agents are shown below. Figure 1 As shown, from Figure 1 It can be seen that the pH-enzyme colon-targeting responsive agent is effective at 1430 cm⁻¹. -1 The characteristic peak at 1250 cm⁻¹ corresponds to N=N stretching vibration. -1 The characteristic peak at 1100 cm⁻¹ corresponds to the asymmetric stretching of Ar-OC. -1 The characteristic peak at 3200 cm⁻¹ corresponds to the symmetric stretching of Ar-OC. -1 The characteristic peak at 1680 cm⁻¹ corresponds to the stretching vibration of the OH group in the carboxyl group. -1 The characteristic peak corresponds to the stretching vibration of the carbon-oxygen double bond in the carboxyl group; the above results confirm the successful preparation of the pH-enzyme colon-targeting responsive agent.

[0046] Preparation Example 2

[0047] Preparation Example 2 provides a pH-enzyme colon-targeting responsive agent, prepared by the following process:

[0048] (1) Vanillic acid (1.68 g, 10 mmol) and potassium carbonate (3.04 g, 22 mmol) were added to 100 mL of acetonitrile, stirred until homogeneous, and then 4-bromo-2-methoxyaniline (2.22 g, 11 mmol) was slowly added. The mixture was heated to 60 °C and reacted for 18 h. The resulting mixture was extracted with ethyl acetate and distilled water. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate: dichloromethane = 1:1, v / v) to obtain intermediate 1 (2.10 g, yield 72.66%). 1 The H NMR and HRMS results are the same as those in Preparation Example 1.

[0049] (2) Intermediate 1 (2.89 g, 10 mmol), 4-aminophenyl acrylate (1.63 g, 10 mmol), 2.5 M hydrochloric acid solution (4 mL), and 10 wt% sodium hypochlorite solution (37 mL, 50 mmol) were added to a reaction flask and reacted at room temperature for 3 h. Ethyl acetate was added to the reaction mixture for quenching. The aqueous layer was extracted with ethyl acetate, and the organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and purified by rapid column chromatography (n-heptane:ethyl acetate = 80:20, v / v) to obtain the vanillic acid derivative. 1 The H NMR and HRMS results are the same as those in Preparation Example 1.

[0050] (3) Weigh 2.5 g of hydroxypropyl methylcellulose and dissolve it in 50 g of water. Heat the reaction solution to 50 °C under a nitrogen atmosphere and add 0.03 g of potassium sulfate (KPS) to initiate the reaction for 15 min. Then add 8 g of vanillic acid derivative to the reaction solution. After the addition is complete, continue the reaction at 50 °C for 1 h. After cooling to room temperature, use a dialysis bag (10 kDa) to collect the sample. The lyophilized sample is the pH-enzyme colon-targeting response agent.

[0051] Preparation Example 3

[0052] Preparation Example 3 provides a pH-enzyme colon-targeting responsive agent, prepared by the following process:

[0053] (1) Vanillic acid (1.68 g, 10 mmol) and potassium carbonate (3.88 g, 28 mmol) were added to 100 mL of acetonitrile, stirred until homogeneous, and then 4-bromo-2-methoxyaniline (3.03 g, 15 mmol) was slowly added. The mixture was heated to 80 °C and reacted for 12 h. The resulting mixture was extracted with ethyl acetate and distilled water. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / dichloromethane = 1 / 1, v / v) to obtain intermediate 1 (2.48 g, yield 85.80%). 1 The H NMR and HRMS results are the same as those in Preparation Example 1.

[0054] (2) Intermediate 1 (2.89 g, 10 mmol), 4-aminophenyl acrylate (2.45 g, 15 mmol), 2.5M hydrochloric acid solution (8 mL), and 10 wt% sodium hypochlorite solution (45 mL, 60 mmol) were added to a reaction flask and reacted at room temperature for 5 h. Ethyl acetate was added to the reaction mixture for quenching. The aqueous layer was extracted with ethyl acetate, and the organic layers were combined, dried over anhydrous sodium sulfate, concentrated, and purified by rapid column chromatography (n-heptane / ethyl acetate = 80 / 20, v / v) to obtain vanillic acid derivative (3.59 g, yield 80.14%). 1 The H NMR and HRMS results are the same as those in Preparation Example 1.

[0055] (3) Weigh 2.5 g of hydroxypropyl methylcellulose and dissolve it in 50 g of water. Heat the reaction solution to 60 °C under a nitrogen atmosphere and add 0.04 g of potassium sulfate (KPS) to initiate the reaction for 12 min. Then add 10 g of vanillic acid derivative to the reaction solution. After the addition is complete, continue the reaction at 60 °C for 0.5 h. After cooling to room temperature, use a dialysis bag (10 kDa) to collect the sample. The lyophilized sample is the pH-enzyme colon-targeting response agent.

[0056] Preparation Example 4

[0057] Preparation Example 4 provides a plasticizer prepared by the following process:

[0058]

[0059] Phthalic acid (1.66 g, 10 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 5 g, 26 mmol), 4-dimethylaminopyridine (DMAP, 6.48 g, 53 mmol), and 4-hydroxy-2-pyrrolidone (2.32 g, 23 mmol) were added to 20 mL of acetonitrile, stirred until homogeneous, and heated to 50°C for 1 h. Acetonitrile was removed under reduced pressure, and the residue was dissolved in ethyl acetate. The ethyl acetate phase was washed successively with 1M hydrochloric acid, saturated sodium bicarbonate, and saturated brine. After drying with anhydrous magnesium sulfate and filtering, the concentrate was purified by column chromatography (n-heptane / ethyl acetate = 5 / 1, v / v) to obtain the plasticizer (2.75 g, yield: 82.82%). 1 H NMR (C 16 H 16 N2O6, 400 MHz, DMSO): δ 8.07-8.04 (m, 2H), 7.73-7.71 (m, 2H), 5.30-5.27 (m, 4H), 3.96 (q, 2H), 3.71 (q, 2H), 2.76 (q, 2H),2.54 (q, 2H); HRMS(ESI+): [M+H] + The calculation yields 333.10, and the value is found to be 333.10.

[0060] Preparation Example 5

[0061] Preparation Example 5 provides a plasticizer prepared by the following process:

[0062] Phthalic acid (1.66 g, 10 mmol), EDCI (4.79 g, 25 mmol), DMAP (6.15 g, 50 mmol), and 4-hydroxy-2-pyrrolidone (2.02 g, 20 mmol) were added to 20 mL of acetonitrile, stirred until homogeneous, and heated to 40°C for 1.5 h. Acetonitrile was removed under reduced pressure, and the residue was dissolved in ethyl acetate. The ethyl acetate phase was washed successively with 1M hydrochloric acid, saturated sodium bicarbonate, and saturated brine. After drying with anhydrous magnesium sulfate and filtering, the concentrate was purified by column chromatography (n-heptane / ethyl acetate = 5 / 1, v / v) to obtain the plasticizer (2.48 g, yield 74.69%).

[0063] Preparation Example 6

[0064] Preparation Example 6 provides a plasticizer prepared by the following process:

[0065] Phthalic acid (1.66 g, 10 mmol), EDCI (5.36 g, 28 mmol), DMAP (6.84 g, 56 mmol), and 4-hydroxy-2-pyrrolidone (2.53 g, 25 mmol) were added to 25 mL of acetonitrile, stirred until homogeneous, and heated to 60°C for 0.5 h. Acetonitrile was removed under reduced pressure, and the residue was dissolved in ethyl acetate. The ethyl acetate phase was washed successively with 1 M hydrochloric acid, saturated sodium bicarbonate, and saturated brine. After drying with anhydrous magnesium sulfate and filtering, the concentrate was purified by column chromatography (n-heptane / ethyl acetate = 5 / 1, v / v) to obtain the plasticizer (2.82 g, yield 84.93%).

[0066] Example 1

[0067] Example 1 provides a minocycline hydrochloride-melatonin compound colon-targeting capsule, comprising micro-pellets and a capsule shell, wherein:

[0068] The microcapsules consist of a drug core and a colon-targeted sustained-release layer encapsulating the drug core, with a mass ratio of 1:0.12.

[0069] The core contains the following raw materials in parts by weight: 30 parts minocycline hydrochloride, 15 parts melatonin, 30 parts lactose, and 7 parts povidone K30 (binder I).

[0070] The colon-targeted sustained-release layer comprises the following raw materials in parts by weight: 10 parts of povidone K30 (adhesive II), 15 parts of the pH-enzyme colon-targeted responsive agent of Preparation Example 1, 3 parts of plasticizer of Preparation Example 4, 4 parts of titanium dioxide, 12 parts of polyacrylic acid resin, and 6 parts of Tween 80.

[0071] Example 1 also provides a method for preparing the above-mentioned minocycline hydrochloride-melatonin compound colon-targeting capsule, comprising the following steps:

[0072] (a) Preparation of the core: According to the weight proportions of each raw material in the core, minocycline hydrochloride, melatonin, lactose and part of binder I (5 parts) are sieved through a 100-mesh sieve and mixed evenly to obtain a mixed powder; the remaining binder I (2 parts) is dissolved in water to form an adhesive liquid and added to the mixed powder. The mixture is stirred for 3-5 minutes at a speed of 100 rpm and a shearing speed of 1200 rpm to obtain a soft material; the soft material is placed in a rounding machine (extrusion frequency 25 Hz, shearing frequency 15 Hz / min) to obtain wet pellets, and the wet pellets are dried at 60-65℃ until the moisture content is <2.5-3% to obtain the core;

[0073] (b) Preparation of microcapsules: According to the weight proportions of each raw material in the colon-targeted sustained-release layer above, binder II, pH-enzyme colon-targeted responsive agent, plasticizer, titanium dioxide, polyacrylate resin, and polysorbate are dissolved in a mixed solution of 160 parts of pure water and 500 parts of ethanol to obtain a mixture; place the drug powder obtained in step (a) in a fluidized bed (inlet air temperature 45℃, preheated for 10 min), spray the mixture at a spray pressure of 0.1 MPa, a flow rate of 2 ml / min, and a weight gain of 12%, and continue drying for 20 min after spraying, and screen to obtain 16-24 mesh capsules to obtain microcapsules;

[0074] (c) The above microparticles are loaded into No. 0 hard capsule shells (the capsule shell material is hydroxypropyl methylcellulose) to obtain minocycline hydrochloride-melatonin compound colon-targeting capsules, each capsule containing 100 mg of minocycline hydrochloride.

[0075] Example 2

[0076] Example 2 provides a minocycline hydrochloride-melatonin compound colon-targeting capsule, comprising micro-pellets and a capsule shell, wherein:

[0077] The microcapsule comprises a drug core and a colon-targeted sustained-release layer encapsulating the drug core, with a mass ratio of 1:0.1.

[0078] The core contains the following raw materials in parts by weight: 20 parts minocycline hydrochloride, 10 parts melatonin, 20 parts microcrystalline cellulose, and 4 parts povidone K30 (binder I).

[0079] The colon-targeted sustained-release layer comprises the following raw materials in parts by weight: 5 parts of povidone K30 (adhesive II), 10 parts of the pH-enzyme colon-targeted responsive agent of Preparation Example 2, 2 parts of plasticizer of Preparation Example 5, 3 parts of titanium dioxide, 5 parts of ethyl cellulose, and 2 parts of polyethylene glycol 6000.

[0080] Example 2 also provides a method for preparing the above-mentioned minocycline hydrochloride-melatonin compound colon-targeting capsule, comprising the following steps:

[0081] (a) Preparation of the core: According to the weight proportions of each raw material in the core, minocycline hydrochloride, melatonin, microcrystalline cellulose and part of binder I (3 parts) are sieved through a 100-mesh sieve and mixed evenly to obtain a mixed powder; the remaining binder I (1 part) is dissolved in water to form an adhesive liquid and added to the mixed powder. The mixture is stirred for 3-5 min at a rotation speed of 100 rpm and a shearing speed of 1200 rpm to obtain a soft material; the soft material is placed in a rounding machine (extrusion frequency 20 Hz, shearing frequency 20 Hz / min) to obtain wet pellets; the wet pellets are dried at 60-65℃ until the moisture content is <2.5-3% to obtain the core.

[0082] (b) Preparation of microcapsules: According to the weight proportions of each raw material in the colon-targeted sustained-release layer above, binder II, pH-enzyme colon-targeted responsive agent, plasticizer, titanium dioxide, ethyl cellulose, and polyethylene glycol 6000 are dissolved in a mixed solution of 160 parts pure water and 500 parts ethanol to obtain a mixture; the drug powder obtained in step (a) is placed in a fluidized bed (inlet air temperature 45℃, preheated for 10 min), and the mixture is sprayed at a spray pressure of 0.1 MPa, a flow rate of 2 ml / min, and a weight gain of 10%; after spraying, continue drying for 20 min, and screen to obtain 16-24 mesh capsules to obtain microcapsules;

[0083] (c) The above microparticles are loaded into No. 0 hard capsule shells (the capsule shell material is hydroxypropyl methylcellulose) to obtain minocycline hydrochloride-melatonin compound colon-targeting capsules, each capsule containing 120 mg of minocycline hydrochloride.

[0084] Example 3

[0085] Example 3 provides a minocycline hydrochloride-melatonin compound colon-targeting capsule, comprising micro-pellets and a capsule shell, wherein:

[0086] The microcapsules consist of a drug core and a colon-targeted sustained-release layer encapsulating the drug core, with a mass ratio of 1:0.15.

[0087] The core contains the following raw materials in parts by weight: 40 parts minocycline hydrochloride, 20 parts melatonin, 40 parts lactose, and 11 parts povidone K30 (binder I).

[0088] The colon-targeted sustained-release layer comprises the following raw materials in parts by weight: 15 parts of povidone K30 (adhesive II), 20 parts of the pH-enzyme colon-targeted responsive agent of Preparation Example 3, 4 parts of plasticizer of Preparation Example 6, 5 parts of titanium dioxide, 20 parts of polyacrylic acid resin, and 10 parts of polysorbate.

[0089] Example 3 also provides a method for preparing the above-mentioned minocycline hydrochloride-melatonin compound colon-targeting capsule, comprising the following steps:

[0090] (a) Preparation of the core: According to the weight proportions of each raw material in the core, minocycline hydrochloride, melatonin, lactose and part of binder I (8 parts) are sieved through a 100-mesh sieve and mixed evenly to obtain a mixed powder; the remaining binder I (3 parts) is dissolved in water to form a binder solution and added to the mixed powder. The mixture is stirred for 3-5 minutes at a rotation speed of 100 rpm and a shearing speed of 1200 rpm to obtain a soft material; the soft material is placed in a rounding machine (extrusion frequency 30 Hz, shearing frequency 20 Hz / min) to obtain wet pellets, and the wet pellets are dried at 60-65℃ until the moisture content is <2.5-3% to obtain the core;

[0091] (b) Preparation of microcapsules: According to the weight proportions of each raw material in the colon-targeted sustained-release layer above, binder II, pH-enzyme colon-targeted responsive agent, plasticizer, titanium dioxide, polyacrylate resin, and polysorbate are dissolved in a mixed solution of 160 parts of pure water and 500 parts of ethanol to obtain a mixture; place the drug powder obtained in step (a) in a fluidized bed (inlet air temperature 45℃, preheated for 10 min), spray the mixture at a spray pressure of 0.1 MPa, a flow rate of 2 ml / min, and a weight gain of 15%, and continue drying for 20 min after spraying, and screen to obtain 16-24 mesh capsules to obtain microcapsules;

[0092] (c) The above microparticles are loaded into No. 0 hard capsule shells (the capsule shell material is hydroxypropyl methylcellulose) to obtain minocycline hydrochloride-melatonin compound colon-targeting capsules, each capsule containing 110 mg of minocycline hydrochloride.

[0093] Comparative Example 1

[0094] Comparative Example 1 is basically the same as Example 1, except that the plasticizer is replaced with dimethyl phthalate.

[0095] Comparative Example 2

[0096] Comparative Example 2 is essentially the same as Example 1, except that the pH-enzyme colon-targeting responsive agent is replaced with hydroxypropyl methylcellulose.

[0097] Experimental Example 1

[0098] According to the second method of dissolution and release determination method - paddle method in General Chapter 0931 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, the drug release characteristics of the capsules obtained in Examples 1-3 and Comparative Examples 1-2 were evaluated.

[0099] ① Weigh 50 mg of capsules from Examples 1-3 and Comparative Examples 1-2 into dissolution vessels. The volume of the dissolution medium is 750 mL, the rotation speed is 100 r / min, and the temperature is (37±0.5)℃. First, release the capsules in simulated gastric fluid at pH=1.2 for 2 h, then in simulated small intestinal fluid at pH=6.8 for 4 h. The volume of the dissolution medium in both cases meets the leakage conditions. Take 1 mL samples at 2 h and 6 h, and immediately add 1 mL of the same volume and temperature medium. Filter the collected samples through a 0.45 μm filter membrane and determine the contents of minocycline hydrochloride and melatonin using LC-MS / MS. Calculate the cumulative release rate. The results are shown in Table 1.

[0100] ② Weigh 50 mg of capsules from Examples 1-3 and Comparative Examples 1-2 into dissolution vessels. The volume of the dissolution medium is 750 mL, the rotation speed is 100 r / min, and the temperature is (37±0.5)℃. Dissolution is carried out for 10 h in phosphate buffer (pH=7.4) containing 4% rat cecal contents, with the dissolution medium volume meeting the leakage conditions. Samples of 1 mL were taken at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h, and immediately followed by the addition of 1 mL of the same temperature and volume of medium. The collected samples were filtered through a 0.45 μm filter membrane, and the contents of minocycline hydrochloride and melatonin were determined by LC-MS / MS. The cumulative release rate was calculated, and the results are shown in Table 2.

[0101] Table 1

[0102]

[0103] Table 2

[0104]

[0105] As shown in Table 1, the capsules obtained in Examples 1 to 3 basically did not release in the artificial gastric fluid at pH 1.2, and only released a small amount in the artificial small intestinal fluid at pH 6.8. The cumulative release within 6 hours was less than 10%, while the cumulative release of Comparative Examples 1 and 2 was greater than 10% within 6 hours.

[0106] As shown in Table 2, the capsules obtained in Examples 1-3 exhibited rapid drug release rates in media containing rat cecal contents, with near-complete release within 12 hours. Combined with the results in Table 1, this demonstrates that the minocycline hydrochloride-melatonin compound capsules prepared in this invention exhibit significant colon-targeting activity, and their in vitro release behavior meets the requirements for colon-targeting. In contrast, Comparative Example 2, which replaced the pH-enzyme colon-targeting responsive agent with non-enzyme-responsive hydroxypropyl methylcellulose, showed a cumulative release rate of only about 22% within 12 hours, indicating that the drug cannot be effectively released in the colonic environment without pH-enzyme-responsive materials.

[0107] The pH-enzyme colon-targeting responsive agent of this invention is a graft copolymer obtained by free radical grafting of carboxylated vanillic acid derivative side chains onto hydroxypropyl methylcellulose (HPMC) as the main chain. This material exhibits excellent film-forming properties and shows significant swelling differences in different pH environments of the gastrointestinal tract: in the acidic environment of the stomach (pH=1.2), the carboxyl groups of the side chains are protonated, reducing the hydrophilicity of the material and limiting the swelling degree, effectively inhibiting drug release (see Table 1, Example 1 and Comparative Example 2); when it reaches the terminal ileum to the ascending colon (pH=7.4), the carboxyl groups are deprotonated, generating electrostatic repulsion, and the copolymer rapidly swells to form continuous diffusion channels, achieving drug release (see Table 2). Furthermore, the diazo groups introduced by azotization of the side chains can be specifically cleaved by azoreductase secreted by anaerobic bacteria in the colon, thereby achieving dual pH-enzyme triggering and enhancing colon-targeting.

[0108] The plasticizer used in the colon-targeted sustained-release layer of the present invention is formed by esterification of phthalic acid and 4-hydroxy-2-pyrrolidone. Its five-membered lactam structure is similar to the pyrrolidone unit of povidone as a binder, and has good compatibility. It can significantly improve the flexibility and extensibility of the coating, avoid cracking when the tablet core absorbs water and expands, and reduce drug leakage in the acid stage (see Table 1 Example 1 and Comparative Example 1).

[0109] Experimental Example 2

[0110] 1. Laboratory animals and grouping

[0111] Eighty healthy male SPF-grade SD rats (weighing 170±10 g) were randomly divided into eight groups of ten each: blank group, model group, positive control group, Examples 1-3 groups, and Comparative Examples 1-2 groups. The rats were acclimatized for three days before the experiment.

[0112] 2. Modeling

[0113] A 5% sodium dextran solution was prepared using distilled water as a modeling agent. Except for the control group, which drank distilled water, all other groups drank the 5% sodium dextran solution for 7 consecutive days to establish a rat model of ulcerative colitis.

[0114] 3. Administration method

[0115] Starting from day 8, the administration was initiated, and the modeling agent, sodium dextran solution, was replaced with distilled water for the rats' daily drinking water. The blank control group and the model group were administered physiological saline by gavage; the positive control group was administered sulfasalazine suspension (0.1 g / mL, prepared by adding sulfasalazine to 4% hydroxypropyl methylcellulose solution) by gavage at a dose of 100 mg / kg; groups 1-3 of Examples and groups 1-2 of Comparative Examples were administered the corresponding capsules by gavage at a dose of 100 mg crude drug / kg. The administration was repeated once daily for 6 consecutive days.

[0116] 4. Detection Indicators

[0117] After administration on day 13, rats were fed normally for 24 hours. On day 14, 1 mL of blood was collected from the retroocular venous plexus, and the rats were sacrificed. After being left at room temperature for 2 hours, the rats were centrifuged at 3000 r / min for 10 min to collect serum. The expression levels of IL-4, TNF-α, and MPO in the serum were detected according to the kit instructions. A certain mass of colon tissue was taken and prepared into a 10% tissue homogenate with physiological saline. This homogenate was then diluted to 5% with physiological saline, and the expression levels of IL-4, TNF-α, and MPO in the tissue homogenate were measured according to the kit instructions. The results of IL-4 expression levels in serum and tissue homogenate are shown in the table below. Figure 2 The expression levels of TNF-α in serum and tissue homogenates are shown in the following figures. Figure 3 The expression levels of MPO in serum and tissue homogenates are shown in the following results. Figure 4 .

[0118] 5. Experimental Results

[0119] like Figure 2 , 3 As shown in Figures 4 and 5, compared to the blank group, the levels of TNF-α and MPO in the serum and colon tissue of rats in the model group were significantly increased, while the level of IL-4 was significantly decreased. Compared to the model group, the levels of TNF-α and MPO in the serum and colon tissue of rats in the positive control group and Examples 1-3 were all decreased, while the level of IL-4 was all increased. However, the effects of Comparative Examples 1-2 were significantly less than those in Example 1. These results indicate that the capsule formulation prepared in this invention can target the lesion site in the colon and effectively inhibit the inflammatory response.

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A minocycline hydrochloride-melatonin compound colon-targeting capsule, comprising micro-pellets and a capsule shell, characterized in that: The microcapsule comprises a drug core and a colon-targeted sustained-release layer wrapped around the drug core, with a mass ratio of 1:(0.1 to 0.15). The core comprises the following raw materials in parts by weight: 20-40 parts minocycline hydrochloride, 10-20 parts melatonin, 20-40 parts filler, and 4-11 parts binder I; The colon-targeted sustained-release layer comprises the following raw materials in parts by weight: 5-15 parts of binder II, 10-20 parts of pH-enzyme colon-targeted responsive agent, 2-4 parts of plasticizer, 3-5 parts of opacifier, 5-20 parts of sustained-release agent, and 2-10 parts of pore-forming agent; The pH-enzyme colon-targeting responsive agent is prepared by the following process: (1) Vanillic acid and potassium carbonate were added to acetonitrile, and then 4-bromo-2-methoxyaniline was added. The mixture was reacted under heating conditions, and the intermediate 1 was obtained after purification. The structure of intermediate 1 is shown in Formula I: ； (2) Add 4-aminophenyl acrylate and intermediate 1 to sodium hypochlorite solution, then add hydrochloric acid solution to react, and purify to obtain vanillic acid derivative; the structure of vanillic acid derivative is shown in Formula II: ； (3) Hydroxypropyl methylcellulose was dissolved in water, heated to 50-60°C under a nitrogen atmosphere, and potassium sulfate was added to initiate the reaction. Then, the vanillic acid derivative was added to continue the reaction. After cooling, the mixture was dialyzed and lyophilized to obtain the pH-enzyme colon-targeting responsive agent. The structure of the plasticizer is shown in Formula III: 。 2. The minocycline hydrochloride-melatonin compound colon-targeting capsule according to claim 1, characterized in that, In step (1), the molar ratio of vanillic acid, potassium carbonate and 4-bromo-2-methoxyaniline is 1:(2.2-2.8):(1.1-1.5); the heating temperature is 60-80℃ and the reaction time is 12-18 h.

3. The minocycline hydrochloride-melatonin compound colon-targeting capsule according to claim 1, characterized in that, In step (2), the ratio of intermediate 1,4-aminophenyl acrylate, sodium hypochlorite solution, and hydrochloric acid solution is 1 mmol: (1-1.5) mmol: (3.7-4.5) mL: (0.4-0.8) mL; the concentration of sodium hypochlorite solution is 10 wt%, and the concentration of hydrochloric acid solution is 2.5 mol / L; the reaction time is 3-5 h.

4. The minocycline hydrochloride-melatonin compound colon-targeting capsule according to claim 1, characterized in that, In step (3), the mass ratio of hydroxypropyl methylcellulose, potassium sulfate and vanillic acid derivative is 1:(0.012-0.016):(3.2-4); the initiation time is 12-15 min; the reaction time is 0.5-1 h; and the molecular weight cutoff for dialysis is 10 kDa.

5. The minocycline hydrochloride-melatonin compound colon-targeting capsule according to claim 1, characterized in that, The plasticizer is prepared by the following process: Phthalic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine and 4-hydroxy-2-pyrrolidone were added to acetonitrile, reacted under heating conditions, and the plasticizer was obtained after purification.

6. The minocycline hydrochloride-melatonin compound colon-targeting capsule according to claim 5, characterized in that, The molar ratio of phthalic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine, and 4-hydroxy-2-pyrrolidone is 1:(2.5-2.8):(5-5.6):(2-2.5); the heating temperature is 40-60°C, and the reaction time is 0.5-1.5 h.

7. The minocycline hydrochloride-melatonin compound colon-targeting capsule according to claim 1, characterized in that, The filler is selected from lactose and microcrystalline cellulose; both adhesive I and adhesive II are povidone K30; the light-blocking agent is titanium dioxide; the slow-release agent is selected from polyacrylate and ethyl cellulose; and the pore-forming agent is selected from polysorbate and polyethylene glycol 6000.

8. A method for preparing the minocycline hydrochloride-melatonin compound colon-targeting capsule according to any one of claims 1 to 7, characterized in that, Includes the following steps: (a) Preparation of the core: According to the weight proportions of each raw material in the core, minocycline hydrochloride, melatonin, filler and part of binder I are mixed to obtain mixed powder; The remaining adhesive I was dissolved in water to form an adhesive solution, which was then added to the mixed powder. After stirring, extrusion, and drying, a core was obtained. (b) Preparation of microcapsules: According to the weight proportions of each raw material in the colon-targeted sustained-release layer, binder II, pH-enzyme colon-targeted responsive agent, plasticizer, opacifier, sustained-release agent and pore-forming agent are added to an ethanol-water solution to obtain a mixture; the mixture is coated onto the surface of the drug core obtained in step (a) by a fluidized bed bottom spraying process to form a colon-targeted sustained-release layer, and microcapsules are obtained after drying; (c) Capsule preparation: The microparticles are filled into capsule shells to obtain the minocycline hydrochloride-melatonin compound colon-targeting capsules.

9. The preparation method of the minocycline hydrochloride-melatonin compound colon-targeting capsule according to claim 8, characterized in that, Each minocycline hydrochloride-melatonin compound colon-targeting capsule contains 100-120 mg of minocycline hydrochloride.

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