Selenium-based anti-postmenopausal osteoporosis medicine as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medical preparations, and particularly discloses a selenium-based anti-postmenopausal osteoporosis medicine and a preparation method and application thereof.The selenium-based anti-postmenopausal osteoporosis medicine comprises a main active component, a secondary active component and an auxiliary active component, the main active component is a nano-selenium-phytoestrogen compound and is formed by covalent coupling of selenomethionine and soy isoflavone, the particle size of the main active component is 50-200 nm, and the particle size of the auxiliary active component is 100-200 nm; the mass of the selenium element accounts for 5-30% of the mass of the compound; the auxiliary active component is a collagen peptide-calcium chelate and is prepared from marine collagen peptide and calcium citrate through chelation reaction, and the mass ratio of the calcium element to the collagen peptide is (1: 3)-(1: 5); the targeted delivery carrier is folic acid modified chitosan nanoparticles and is used for loading the main active component and the auxiliary active component; the auxiliary materials comprise a disintegrating agent, a lubricating agent and a pH-sensitive enteric coating material; through the synergistic effect of the main active ingredient nano-selenium-phytoestrogen compound and the auxiliary active ingredient collagen peptide-calcium chelate, bone formation can be promoted and bone resorption can be inhibited at the same time, and dual-function treatment is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical preparations, and particularly relates to a selenium-based anti-postmenopausal osteoporosis drug, its preparation method and application. Background Art

[0002] Postmenopausal osteoporosis (PMO) is a bone metabolism imbalance disease caused by a sudden drop in estrogen levels, characterized by bone mass reduction and bone microstructure damage, which significantly increases the risk of fractures. Currently, the main clinical treatment methods include: calcium agents and vitamin D, estrogen replacement therapy (HRT), single-target drugs (such as RANKL inhibitors), etc.

[0003] However, among the above methods, calcium agents and vitamin D, as basic treatment drugs, although they help to enhance bone strength, their treatment effects are relatively limited and it is difficult to meet the comprehensive needs of patients for disease improvement; bisphosphonate drugs, such as alendronate sodium, although they can effectively inhibit bone resorption, their long-term use may be accompanied by side effects such as gastrointestinal discomfort and bone pain; estrogen replacement therapy (HRT), although it can supplement the estrogen level in the body and thus relieve bone mass loss, long-term use may increase the risk of hormone-related tumors such as breast cancer and endometrial cancer, which severely limits its clinical application; in addition, single-target drugs, such as RANKL inhibitors, although they have a clear anti-osteoporosis effect, their high treatment costs and problems such as immune reactions that may occur during the treatment process also limit their wide application;

[0004] The limitations of the above treatment regimens indicate that single-mechanism drugs or traditional dosage forms are difficult to meet the comprehensive requirements of PMO treatment for high efficiency, safety and targeting, and there are the following core contradictions that have not been resolved: the lack of synergistic effect of dual regulation of bone metabolism "formation - resorption"; the coexistence of low bioavailability and off-target toxicity of active ingredients; the absorption bottleneck of basic calcium agents and the difficulty in balancing long-term drug safety. Summary of the Invention

[0005] The purpose of the present invention is to provide a selenium-based anti-postmenopausal osteoporosis drug, its preparation method and application to solve the problems raised in the above background art.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] A selenium-based anti-postmenopausal osteoporosis drug, comprising the following components:

[0008] The main active ingredient is a nano-selenium - phytoestrogen complex, formed by covalent coupling of selenomethionine and soy isoflavone, with a particle size of 50 - 200 nm and the mass percentage of selenium element accounting for 5% - 30% of the complex;

[0009] The auxiliary active ingredient is a collagen peptide-calcium chelate, which is prepared by the chelation reaction of marine collagen peptide and calcium citrate. The mass ratio of calcium element to collagen peptide is 1:3 - 1:5;

[0010] The targeted delivery carrier is folic acid-modified chitosan nanoparticles, which load the main active ingredient and the auxiliary active ingredient;

[0011] The excipients include a disintegrant, a lubricant and a pH-sensitive enteric coating material.

[0012] Preferably, the preparation method of the nano-selenium-phytoestrogen complex includes:

[0013] a) Dissolve selenomethionine and soy isoflavones in an alkaline ethanol solution at a molar ratio of 1:1 - 1:3;

[0014] b) Add the cross-linking agent EDC / NHS and react for 6 - 12 hours under nitrogen protection to form a covalent conjugate;

[0015] c) Control the particle size of the complex to 50 - 200 nm by the anti-solvent crystallization technique using supercritical CO 2 anti-solvent crystallization technology.

[0016] Preferably, the preparation method of the collagen peptide-calcium chelate includes:

[0017] a) Mix marine collagen peptide (molecular weight ≤ 3000 Da) and calcium citrate at a mass ratio of 3:1 - 5:1;

[0018] b) Under the conditions of pH 7.5 - 8.5 and temperature 40 - 50 °C, carry out ultrasonic oscillation reaction for 2 - 4 hours to form a chelate;

[0019] c) Separate and purify by an ultrafiltration membrane to retain the product with a molecular weight cut-off ≥ 10 kDa.

[0020] Preferably, the folic acid-modified chitosan nanoparticles are prepared by the following steps:

[0021] a) Dissolve chitosan (degree of deacetylation ≥ 90%) in an acetic acid solution, add folic acid-polyethylene glycol-NHS active ester, and react to form a folic acid-modified chitosan derivative;

[0022] b) Adopt the ion gel method to cross-link the derivative solution with sodium tripolyphosphate to form nanoparticles with a particle size of 80 - 150 nm;

[0023] c) Load the main active ingredient and the auxiliary active ingredient by electrostatic adsorption, and the drug loading amount ≥ 15%.

[0024] Preferably, the pH-sensitive enteric coating material is hydroxypropyl methylcellulose phthalate (HPMCP), and the coating weight gain is 3%-8% of the total weight of the composition, so that the active ingredient is released in an intestinal pH≥5.5 environment.

[0025] A preparation method of the selenium-based anti-postmenopausal osteoporosis drug as described in any one of the above, comprising the following steps:

[0026] a) Mix the nano-selenium-phytoestrogen complex and the collagen peptide-calcium chelate in a mass ratio of 1:2-1:4, disperse them in a folic acid-modified chitosan nanoparticle suspension, and complete the loading by ultrasonic treatment for 30-60 minutes;

[0027] b) Mix the loaded nanoparticles with microcrystalline cellulose and cross-linked carboxymethyl cellulose sodium, and apply HPMCP enteric coating by fluidized bed coating technology;

[0028] c) Control the inlet air temperature at 40-50°C, the material temperature ≤35°C, and the spraying rate of the coating solution at 5-10 mL / min during the coating process.

[0029] Preferably, in step a), the ultrasonic treatment frequency is 20-40 kHz, the power density is 50-100 W / L, and the solid content of the nanoparticle suspension is 5%-10%.

[0030] An application of the selenium-based anti-postmenopausal osteoporosis drug as described in any one of the above in the preparation of a dual-functional drug that simultaneously promotes bone formation and inhibits bone resorption.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] (1) Through the synergistic effect of the main active ingredient nano-selenium-phytoestrogen complex and the auxiliary active ingredient collagen peptide-calcium chelate, it can simultaneously promote bone formation and inhibit bone resorption, achieving dual-functional treatment. The nano-selenium-soybean isoflavone complex activates the Wnt / β-catenin signaling pathway and promotes osteoblast differentiation, thereby enhancing bone formation; while the collagen peptide-calcium chelate inhibits the RANKL / OPG ratio and reduces osteoclast activity, effectively inhibiting bone resorption. This dual regulatory mechanism makes the drug have higher efficacy in the treatment of postmenopausal osteoporosis.

[0033] (2) Using folic acid-modified chitosan nanoparticles as a targeted delivery carrier can target the folate receptor highly expressed in bone tissue, thereby increasing the local drug concentration, enhancing the bioavailability of the drug and reducing off-target toxicity. In addition, this carrier also has good stability and biocompatibility, which can ensure the effective delivery and function of the drug in the body. At the same time, the application of the pH-sensitive enteric coating material hydroxypropyl methylcellulose phthalate (HPMCP) enables the drug to be released in an intestinal pH ≥ 5.5 environment, further improving the targeting and bioavailability of the drug. The combination of these characteristics makes the drug provided by the present invention have higher safety and effectiveness in the treatment of postmenopausal osteoporosis. Description of the Drawings

[0034] Figure 1 It is a process flow chart of the present invention. Detailed Embodiments

[0035] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0036] Example 1:

[0037] Please refer to Figure 1 As shown, a selenium-based drug for treating postmenopausal osteoporosis comprises the following components:

[0038] The main active ingredient is a nano-selenium-phytoestrogen complex, which is formed by covalent coupling of selenomethionine and soy isoflavones, with a particle size of 50 - 200 nm and the mass percentage of selenium element accounting for 5% - 30% of the complex;

[0039] The auxiliary active ingredient is a collagen peptide-calcium chelate, which is prepared by chelation reaction of marine collagen peptide and calcium citrate, and the mass ratio of calcium element to collagen peptide is 1:3 - 1:5;

[0040] The targeted delivery carrier is folic acid-modified chitosan nanoparticles, which load the main active ingredient and the auxiliary active ingredient;

[0041] The excipients include a disintegrant, a lubricant and a pH-sensitive enteric coating material.

[0042] In an embodiment of the present invention, the preparation method of the nano-selenium-phytoestrogen complex includes:

[0043] a) Dissolve selenomethionine and soy isoflavones in an alkaline ethanol solution at a molar ratio of 1:1 - 1:3;

[0044] b) Add crosslinking agents EDC / NHS and react for 6 - 12 hours under nitrogen protection to form a covalent conjugate;

[0045] c) Use supercritical CO2 anti-solvent crystallization technology to control the particle size of the complex to 50 - 200 nm.

[0046] In one embodiment of the present invention, the preparation method of the collagen peptide-calcium chelate includes:

[0047] a) Mix marine collagen peptide (molecular weight ≤ 3000 Da) and calcium citrate in a mass ratio of 3:1 - 5:1;

[0048] b) Under the conditions of pH 7.5 - 8.5 and temperature 40 - 50 °C, carry out ultrasonic oscillation reaction for 2 - 4 hours to form a chelate;

[0049] c) Separate and purify through an ultrafiltration membrane to retain the product with a molecular weight cut-off ≥ 10 kDa.

[0050] In one embodiment of the present invention, the folic acid-modified chitosan nanoparticles are prepared by the following steps:

[0051] a) Dissolve chitosan (degree of deacetylation ≥ 90%) in an acetic acid solution, add folic acid-polyethylene glycol-NHS active ester, and react to form a folic acid-modified chitosan derivative;

[0052] b) Adopt the ion gel method to crosslink the derivative solution with sodium tripolyphosphate to form nanoparticles with a particle size of 80 - 150 nm;

[0053] c) Load the main active ingredient and the auxiliary active ingredient through electrostatic adsorption, and the drug loading amount ≥ 15%.

[0054] In one embodiment of the present invention, the pH-sensitive enteric coating material is hydroxypropyl methylcellulose phthalate (HPMCP), and the coating weight gain is 3% - 8% of the total weight of the composition, so that the active ingredient is released in an intestinal environment with pH ≥ 5.5.

[0055] A preparation method of a selenium-based anti-postmenopausal osteoporosis drug as described in any one of the above, comprising the following steps:

[0056] a) Mix the nano-selenium-phytoestrogen complex and the collagen peptide-calcium chelate in a mass ratio of 1:2 - 1:4, disperse them in the folic acid-modified chitosan nanoparticle suspension, and complete the loading by ultrasonic treatment for 30 - 60 minutes;

[0057] b) Mix the loaded nanoparticles with microcrystalline cellulose and croscarmellose sodium, and apply the HPMCP enteric coating by fluidized bed coating technology;

[0058] c) During the film coating process, control the inlet air temperature at 40 - 50 °C, the material temperature ≤ 35 °C, and the spraying rate of the film coating solution at 5 - 10 mL / min.

[0059] In one embodiment of the present invention, in step a), the ultrasonic treatment frequency is 20 - 40 kHz, the power density is 50 - 100 W / L, and the solid content of the nanoparticle suspension is 5% - 10%.

[0060] Use of a selenium-based anti-postmenopausal osteoporosis drug as described in any one of the above in the preparation of a dual-functional drug that simultaneously promotes bone formation and inhibits bone resorption, and the curative effect is achieved through the following mechanism:

[0061] a) The nano-selenium - soy isoflavone complex activates the Wnt / β-catenin signaling pathway to promote osteoblast differentiation;

[0062] b) The collagen peptide - calcium chelate inhibits the RANKL / OPG ratio and reduces osteoclast activity;

[0063] c) The folic acid-modified nanoparticles target the folate receptor highly expressed in bone tissue to increase the local drug concentration.

[0064] In one embodiment of the present invention, the administration dose of the drug is oral 1 - 2 times a day, each time providing 50 - 100 μg of selenium element and 200 - 400 mg of calcium element, and the bone density increase rate is ≥ 30% higher than that of a single selenium preparation (measured by dual-energy X-ray absorptiometry).

[0065] Example two: Preparation of nano-selenium - soy isoflavone complex

[0066] Dissolve selenomethionine (1 mmol) and soy isoflavone (2 mmol) in an ethanol - water mixed solution (volume ratio 3:1) with pH 8.5, add the cross-linking agent EDC (1.2 mmol) and NHS (0.6 mmol), stir and react for 10 hours under nitrogen protection at 25 °C. The reaction solution is subjected to supercritical CO 2 anti-solvent crystallization (pressure 15 MPa, temperature 35 °C, CO 2 flow rate 20 L / min) treatment to obtain a nano-complex with a particle size of 80 - 150 nm;

[0067] Characterization: The coupling efficiency detected by HPLC is ≥ 95%, the average particle size measured by dynamic light scattering (DLS) is 120 nm, and XPS confirms that the selenium element exists stably in the form of Se - C bonds.

[0068] Example three: Preparation and properties of collagen peptide - calcium chelate

[0069] Dissolve marine collagen peptide with a molecular weight of 2000 Da (3 g) and calcium citrate (1 g) in deionized water, adjust the pH to 8.0, and perform ultrasonic treatment (frequency 40 kHz, power 100 W) at 45 °C for 3 hours. The reaction solution is purified by a 10 kDa ultrafiltration membrane and freeze-dried to obtain a white powder;

[0070] Calcium ion chelation rate: The calcium retention rate determined by atomic absorption spectroscopy is 92.5% (the calcium retention rate without chelation in the control group is 43.8%).

[0071] In vitro simulated digestion: The calcium release is ≤10% in gastric juice (pH 2.0) for 2 hours and ≥85% in intestinal juice (pH 6.8) for 4 hours.

[0072] Example 3: Preparation and drug loading of folic acid-modified chitosan nanoparticles

[0073] Dissolve chitosan with a deacetylation degree of 95% (2% w / v) in 1% acetic acid solution, add folic acid-polyethylene glycol-NHS (molar ratio 1:1.5), and react at 40 °C for 6 hours. After dialysis, it is freeze-dried to obtain folic acid-modified chitosan;

[0074] Mix the above derivative with sodium tripolyphosphate (TPP) at a mass ratio of 4:1 and form nanoparticles by ionic crosslinking (particle size 100 ± 20 nm);

[0075] Disperse the nanocomposite in Example 1 (50 mg) and the chelate in Example 2 (150 mg) in the nanoparticle suspension, perform ultrasonic treatment (30 kHz, 60 W / L) for 40 minutes, and centrifuge to collect the drug-loaded nanoparticles;

[0076] Drug loading determination: The drug loading is 18.3% and the encapsulation efficiency is 89.5% measured by HPLC.

[0077] Example 4: Preparation and release characteristics of enteric-coated tablets

[0078] Mix the drug-loaded nanoparticles (200 mg) with microcrystalline cellulose (150 mg) and croscarmellose sodium (50 mg), and use a fluidized bed coater (inlet air temperature 45 °C, material temperature 32 °C) to coat with an HPMCP ethanol solution (8% w / v), and the coating weight gain is 5%;

[0079] Release test (USP II method, rotation speed 50 rpm):

[0080] Gastric juice stage (0.1M HCl, 2 hours): The cumulative release of selenium and calcium is ≤8%;

[0081] Intestinal juice stage (pH 6.8 phosphate buffer, 12 hours): The release of selenium is 92.5% and the release of calcium is 88.7%.

[0082] Example 5: Verification of Bifunctional Efficacy Using an Animal Model

[0083] An OVX (ovariectomized) osteoporosis rat model (n = 30) was selected and randomly divided into:

[0084] Control group (normal saline);

[0085] Positive drug group (alendronate sodium, 1 mg / kg);

[0086] Experimental group (tablets of Example 4, selenium 50 μg / kg + calcium 300 mg / kg).

[0087] After continuous administration for 12 weeks, the following were detected:

[0088] Bone mineral density (BMD): The lumbar BMD of the experimental group increased by 38.2% compared with the control group (the positive drug group increased by 25.7%).

[0089] Biomechanics: The maximum load of the femur in the experimental group increased by 42.3%, significantly higher than that of the positive drug group (29.1%).

[0090] Histology: TRAP staining showed that the number of osteoclasts in the experimental group decreased by 57%, which was better than that of the positive drug group (decreased by 39%).

[0091] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A selenium-based anti-postmenopausal osteoporosis drug, characterized in that: Contains the following ingredients: The main active ingredient is a nano-selenium-phytoestrogen complex, which is formed by covalent coupling of selenomethionine and soy isoflavones. Its particle size is 50-200nm, and the mass proportion of selenium is 5%-30% of the complex. The auxiliary active ingredient is collagen peptide-calcium chelate, which is prepared by chelating marine collagen peptide and calcium citrate, and the mass ratio of calcium element to collagen peptide is 1:3-1:5; The targeted delivery carrier is folic acid-modified chitosan nanoparticles loaded with the main active ingredient and the auxiliary active ingredient; Excipients include disintegrants, lubricants and pH-sensitive enteric coating materials.

2. A selenium-based anti-postmenopausal osteoporosis drug according to claim 1, characterized in that: The preparation method of the nano-selenium-phytoestrogen complex comprises: a) dissolving selenomethionine and soybean isoflavones in an alkaline ethanol solution at a molar ratio of 1:1-1:3; b) adding cross-linking agent EDC / NHS, reacting for 6-12 hours under nitrogen protection to generate a covalent conjugate; c) Supercritical CO2 anti-solvent crystallization technology is used to control the particle size of the composite to 50-200nm.

3. A selenium-based anti-postmenopausal osteoporosis drug according to claim 1, characterized in that: The preparation method of the collagen peptide-calcium chelate comprises: a) mixing marine collagen peptide (molecular weight ≤ 3000Da) and calcium citrate at a mass ratio of 3:1-5:1; b) reacting under ultrasonic oscillation at pH 7.5-8.5 and temperature 40-50° C. for 2-4 hours to form a chelate; c) separating and purifying by ultrafiltration membrane, and retaining products with a molecular weight of ≥10 kDa.

4. A selenium-based anti-postmenopausal osteoporosis drug according to claim 1, characterized in that: The folic acid-modified chitosan nanoparticles are prepared by the following steps: a) dissolving chitosan (deacetylation degree ≥ 90%) in acetic acid solution, adding folic acid-polyethylene glycol-NHS active ester to react and generate folic acid-modified chitosan derivatives; b) cross-linking the derivative solution with sodium tripolyphosphate using an ion gel method to form nanoparticles with a particle size of 80-150 nm; c) The main active ingredient and the auxiliary active ingredient are loaded by electrostatic adsorption, and the loading amount is ≥15%.

5. A selenium-based anti-postmenopausal osteoporosis drug according to claim 1, characterized in that: The pH-sensitive enteric coating material is hydroxypropyl methylcellulose phthalate (HPMCP), and the coating weight gain is 3%-8% of the total weight of the composition, so that the active ingredient is released under an intestinal pH environment of ≥5.

5.

6. A method for preparing the selenium-based anti-postmenopausal osteoporosis drug according to any one of claims 1 to 5, characterized in that: The following steps are involved: a) mixing the nano-selenium-phytoestrogen complex with the collagen peptide-calcium chelate in a mass ratio of 1:2-1:4, dispersing the mixture in a suspension of folic acid-modified chitosan nanoparticles, and subjecting the mixture to ultrasonic treatment for 30-60 minutes to complete the loading; b) mixing the loaded nanoparticles with microcrystalline cellulose and croscarmellose sodium, and applying HPMCP enteric coating using fluidized bed coating technology; c) During the coating process, the inlet air temperature is controlled at 40-50°C, the material temperature is ≤35°C, and the coating liquid spray rate is 5-10mL / min.

7. The method for preparing a selenium-based anti-postmenopausal osteoporosis drug according to claim 6, characterized in that: In step a), the ultrasonic treatment frequency is 20-40 kHz, the power density is 50-100 W / L, and the solid content of the nanoparticle suspension is 5%-10%.

8. Use of the selenium-based anti-postmenopausal osteoporosis drug according to any one of claims 1 to 5 in the preparation of a dual-function drug that simultaneously promotes bone formation and inhibits bone resorption.

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