Hirudin nano-drug for relieving chronic asthma and preparation method of hirudin nano-drug

Abstract

The invention belongs to the technical field of nano preparations, and particularly relates to a hirudin nano drug for relieving chronic asthma and a preparation method thereof.The hirudin nano drug is prepared from, by weight, 0.5-1 part of hirudin prodrug, 10-15 parts of stabilizer, 40-80 parts of freeze-drying protective additive and 1.5 parts of histidine; the hirudin prodrug molecule is composed of hirudin, MMP-9 responsive peptide, ROS responsive thioether bond and hydrophobic hexadecanoic acid, can spontaneously assemble to form a nano-sized prodrug under hydrophilic and hydrophobic effects, has stability in systemic circulation and is targeted and enriched at an asthma airway inflammation part; the hirudin with the thrombin activity inhibition effect is responsively released at the focus, so that the effect and the purpose of slowly treating the asthma are achieved.

Description

Technical Field

[0001] This invention belongs to the field of nanoparticle technology, specifically relating to a hirudin nanomedicine for relieving chronic asthma and its preparation method. Background Technology

[0002] Chronic asthma, a complex inflammatory airway disease, involves not only the infiltration and activation of various inflammatory cells but also continuous airway remodeling, leading to reversible airflow limitation and airway hyperresponsiveness. While existing treatments such as corticosteroids and bronchodilators effectively control symptoms, they remain limited in intervening in disease progression, particularly in reversing airway remodeling. In recent years, the exploration of non-classical targets has provided new insights into asthma treatment, with thrombin attracting significant attention due to its central role in inflammation and tissue repair. Thrombin activity is significantly elevated in the airways of asthma patients, activating not only protease-activated receptor-mediated inflammatory signaling pathways but also directly stimulating fibroblast proliferation and airway smooth muscle contraction, making it a key link between inflammation and remodeling. Hirudin, a potent and specific direct inhibitor of thrombin, theoretically can block the pathological process of asthma at multiple stages, inhibiting acute inflammatory responses and potentially delaying or improving airway structural changes, offering potential for etiological treatment of chronic asthma.

[0003] However, directly applying hirudin to the treatment of asthma faces a series of serious challenges. First, its potent systemic anticoagulant activity may lead to a serious risk of bleeding, significantly limiting the safety window for clinical use. Second, as a polypeptide molecule, hirudin is rapidly eliminated from the body with a short half-life, requiring frequent dosing to maintain effective concentrations, which is detrimental to the long-term management of chronic diseases. Furthermore, the lack of targeted systemic delivery means that only a very small amount of the drug accumulates in the lung lesions, limiting efficacy and increasing the risk of systemic side effects. Existing conventional formulations or simple carrier delivery systems cannot simultaneously address these issues, failing to achieve precise drug release and long-term retention at the site of inflammation.

[0004] Therefore, there is an urgent need to transform hirudin into a highly effective and safe anti-asthma drug through structural modification or dosage form improvement, while maintaining its pharmacological activity, and through precise delivery and controlled release, so as to provide new technical means to solve the difficulties in the treatment of chronic asthma. Summary of the Invention

[0005] To address the above issues, this invention provides a hirudin nanomedicine for relieving chronic asthma and its preparation method. Hirudin, an MMP-9 enzyme-responsive peptide, and a hydrophobic tail chain are covalently combined to construct an amphiphilic prodrug molecule. Utilizing its self-assembly properties, core-shell structured nanoparticles are formed, promoting their stability in systemic circulation and targeted accumulation at inflamed airway sites in asthma. The responsive release of active hirudin precisely inhibits abnormally active thrombin in the affected area, providing a novel strategy for the safe and efficient treatment of chronic asthma.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a hirudin nanomedicine for relieving chronic asthma, the hirudin nanomedicine comprising the following raw materials in parts by weight: 0.5-1 parts hirudin prodrug, 10-15 parts stabilizer, 40-80 parts lyophilization protectant and 1.5 parts histidine.

[0007] Furthermore, the stabilizer is selected from any one of poloxamer 188, soybean lecithin, human serum albumin, and polyethylene glycol 4000.

[0008] Furthermore, the freeze-drying protectant is selected from one or more of sucrose, trehalose, mannitol, and hydroxyethyl starch.

[0009] Furthermore, the hirudin prodrug comprises the following raw materials: hirudin, specific peptide, 2-aminoethanethiol and 16-bromohexadecanoic acid, wherein the mass ratio of hirudin, specific peptide, 2-aminoethanethiol and 16-bromohexadecanoic acid is 10:1.5:0.2:0.5.

[0010] Furthermore, the amino acid sequence of the specific peptide is Fmoc-FFAGLDD, where Fmoc is an amino protecting group.

[0011] Furthermore, the preparation method of the hirudin prodrug is as follows: S1: After dissolving 2-aminoethanethiol, triphenylmethyl chloride and DIPEA (N,N-diisopropylethylamine) are added to protect the thiol group of 2-aminoethanethiol. The reaction is carried out at room temperature for 30 min to form thiol (triphenylmethyl) and the modified solution is obtained. S2: After dissolving hirudin and specific peptide together, they are linked by dehydration condensation reaction of the amino group at the N-terminus of hirudin and the carboxyl group at the C-terminus of the specific peptide to form a specific peptide-hirudin conjugate, and the conjugate solution is obtained. The conjugate solution is mixed with the modification solution to carry out an amidation reaction, which promotes the dehydration condensation of the carboxyl group at the C-terminus of hirudin and the amino group in the triphenylmethyl-protected 2-aminoethanethiol to form a specific peptide-hirudin-thiol (triphenylmethyl) block conjugate. After purification, the reaction solution is obtained. S3: Prepare a cleavage solution by taking TFA (trifluoroacetic acid), Tis (triisopropylsilane) and water and add it to the reaction solution. React at room temperature for 1 h to cleave the triphenylmethyl protecting group, expose the thiol group, and form a specific peptide-hirudin-thiol. Then add pre-cooled ice-cold ether to it, centrifuge and collect the precipitate to obtain the intermediate. S4: After dissolving the intermediate and 16-bromohexadecanoic acid together, the mixture was reacted at 40°C under alkaline conditions for 6 h. The exposed thiol was in the form of anions, which attacked the carbon atom at the end of 16-bromohexadecanoic acid. The bromide ion left, and nucleophilic substitution occurred, forming a thioether bond, to obtain a specific peptide-hirudin-thioether bond-hexadecanoic acid complex. The Fmoc protecting group of the N-terminal amino group of the specific peptide was then removed by cleavage, purified, and lyophilized to obtain the hirudin prodrug.

[0012] This invention also provides a method for preparing hirudin nanomedicine to relieve chronic asthma, the specific steps of which are as follows: Step 1: Dissolve the hirudin prodrug in an organic solvent to obtain an organic phase. Use a microsyringe to slowly inject the organic phase into an aqueous solvent. The hydrophobic part of the hirudin prodrug molecule spontaneously aggregates inward to form a hydrophobic core, while the hydrophilic part, namely the specific peptide and hirudin, forms a hydrophilic shell with water, self-assembling into nanoparticles with a shell-core structure. After injection, stir at room temperature to promote the evaporation of the organic solvent and obtain the prodrug solution. Step 2: Dissolve the stabilizer, lyophilization protectant and histidine, adjust the pH to 6.5 to obtain an auxiliary solution, and add the prodrug solution to the auxiliary solution while stirring. Mix well to obtain the prescription solution. Step 3: Filter, sterilize, and freeze-dry the prescription solution to obtain hirudin nanomedicine.

[0013] The beneficial effects achieved by this invention are as follows: The hirudin nanomedicine for relieving chronic asthma prepared in this invention binds one end of hirudin to a matrix metalloproteinase MMP-9 responsive peptide, and the other end is linked to a hydrophobic tail chain of hexadecanoic acid via a thioether bond, forming an amphiphilic prodrug molecule. This molecule can spontaneously assemble into core-shell structured nanoparticles in the aqueous phase, which not only solves the problems of poor stability and short half-life of hirudin itself in vivo circulation, but also achieves passive targeting and accumulation at the site of inflammation due to the nanosize effect. After intravenous administration, its nanosize characteristics enable more effective accumulation in the inflamed areas of the lungs. In the unique microenvironment of the asthmatic airways, overexpressed matrix metalloproteinase-9 first recognizes and cleaves specific peptides on the surface of the nanomedicine, completing initial activation. After cleavage, the nano-assembly morphology of the drug changes, exposing internal thioether bonds and hydrophobic chains. Subsequently, high concentrations of reactive oxygen species (ROS) at the lesion site further cleave the thioether bonds connecting hirudin to the hydrophobic tail chain. This dual-response mechanism ensures the precise and controllable release of active hirudin at the target site. When it does not reach the target lesion site, the nanomedicine cannot undergo specific enzymatic cleavage and remains in a pharmacologically inactive "prodrug" state, persisting in the body for a long time through blood circulation, providing a slow therapeutic effect on asthma. In the pathological process of asthma, thrombin is not only a key enzyme in the coagulation pathway but also a powerful inflammatory mediator and pro-fibrotic factor. The released active hirudin can effectively block thrombin-induced inflammatory cell infiltration, reduce airway hyperresponsiveness, and inhibit abnormal contraction and proliferation of airway smooth muscle. This responsive, nanoscale prodrug design offers a novel treatment strategy for alleviating chronic asthma that is both highly effective and safe. Attached Figure Description

[0014] Figure 1 This is a schematic diagram illustrating the mechanism of action of the hirudin nanomedicine for relieving chronic asthma according to the present invention. Figure 2 The results of transmission electron microscopy examination of the hirudin nanomedicine prepared in Example 4; Figure 3 The results of transmission electron microscopy examination of the hirudin nanomedicine prepared in Example 4 after treatment with MMP-9 enzyme. Detailed Implementation

[0015] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0017] The mechanism of action of the hirudin nanomedicine for relieving chronic asthma is illustrated in the following embodiments. Figure 1 Unless otherwise specified, all methods are conventional. Unless otherwise specified, all materials used in the following examples are new materials purchased from the market. Specifically, the hirudin used was in lyophilized powder form with a purity ≥98%; the specific peptide amino acid sequence used was Fmoc-FFAGLDD, synthesized using solid-phase synthesis, where Fmoc is the amino protecting group of F (phenylalanine), which was not removed during synthesis; the purity of 2-aminoethanethiol and 16-bromohexadecanoic acid used was ≥95%; poloxamer 188 used was pharmaceutical injection grade; the phosphatidylcholine content in the soybean lecithin used was ≥90%; the hydroxyethyl starch used was a low molecular weight specification; the histidine used was pharmaceutical injection L-histidine; and all water used was injection grade water.

[0018] In the following examples and comparative examples, the hirudin prodrug comprises the following raw materials in parts by weight: 10 parts hirudin, 1.5 parts specific peptide, 0.2 parts 2-aminoethanethiol, and 0.5 parts 16-bromohexadecanoic acid. The specific preparation method is as follows: S1: Dissolve 0.2 parts of 2-aminoethanethiol in 5 parts of DMF (N,N-dimethylpropionamide), then add 0.5 parts of triphenylmethyl chloride and 0.8 parts of DIPEA to protect the thiol group of 2-aminoethanethiol. React at room temperature for 30 min to form triphenylmethyl-2-aminoethanethiol, and obtain the modified solution. S2: Dissolve 10 parts of hirudin and 1.5 parts of specific peptide in 100 parts of DMF-water mixed solvent (1:1 volume ratio). Add 1.5 parts of HBTU (tetramethylurea hexafluorophosphate) and 3 parts of DIPEA for activation. The peptide is linked by dehydration condensation reaction of the amino group at the N-terminus of hirudin and the carboxyl group at the C-terminus of the specific peptide. After reacting for 2 hours, a specific peptide-hirudin conjugate is formed, and the conjugate solution is obtained. Mix the conjugate solution with the modification solution for amidation reaction to promote the dehydration condensation of the carboxyl group at the C-terminus of hirudin and the amino group in the triphenylmethyl-protected 2-aminoethanethiol to form a specific peptide-hirudin-thiol (triphenylmethyl) block conjugate. After purification, the reaction solution is obtained. S3: Prepare a cleavage solution by mixing TFA, Tis and water (volume ratio 95:2.5:2.5) and add it to the reaction solution. React at room temperature for 1 h. Acidic TFA will cleave the triphenylmethyl protecting group, exposing the thiol group, forming a specific peptide-hirudin-thiol. Then add 10 times the volume of ice-cold diethyl ether pre-cooled at 4°C to precipitate a white solid. Centrifuge to collect the precipitate and obtain the intermediate. S4: The intermediate and 0.5 parts of 16-bromohexadecanoic acid were dissolved together in 100 parts of DMF and reacted at 40℃ under alkaline conditions for 6 h. The exposed thiol was in the form of anion and attacked the carbon atom at the end of 16-bromohexadecanoic acid. The bromide ion left and nucleophilic substitution occurred, forming a thioether bond, to obtain a specific peptide-hirudin-thioether bond-hexadecanoic acid complex. Then, 20% (v / v) piperidine DMF solution was added to it and shaken for 15 min to cleave and remove the Fmoc protecting group of the N-terminal amino group of the specific peptide. The mixture was purified by dialyzing in a 10 kDa dialysis bag and lyophilized to obtain the hirudin prodrug.

[0019] Example 1: This example provides a hirudin nanomedicine for relieving chronic asthma. The hirudin nanomedicine comprises the following raw materials in parts by weight: 0.5 parts of hirudin prodrug, 10 parts of poloxamer 188, 40 parts of sucrose, and 1.5 parts of histidine.

[0020] This embodiment also provides a method for preparing hirudin nanomedicine to relieve chronic asthma, the specific steps of which are as follows: Step 1: Dissolve 0.5 parts of hirudin prodrug in 5 parts of DMF to obtain an organic phase. Use a microsyringe to slowly inject the organic phase into 50 parts of water for injection. The hydrophobic part of the hirudin prodrug molecule spontaneously aggregates inward to form a hydrophobic core, while the hydrophilic part, namely the specific peptide and hirudin, forms a hydrophilic shell with water, self-assembling into nanoparticles with a shell-core structure. After injection, stir at 100 rpm at room temperature for 6 h to promote the evaporation of organic solvent and obtain the prodrug solution. Step 2: Dissolve 10 parts of poloxamer 188, 40 parts of sucrose and 1.5 parts of histidine in 500 parts of water for injection, adjust the pH to 6.5 to obtain an auxiliary solution, add the prodrug solution to the auxiliary solution while stirring, mix well to obtain the prescription solution; Step 3: Filter, sterilize, and freeze-dry the prescription solution to obtain hirudin nanomedicine.

[0021] Example 2: This example provides a hirudin nanomedicine for relieving chronic asthma. The hirudin nanomedicine comprises the following raw materials in parts by weight: 0.8 parts hirudin prodrug, 12 parts soybean lecithin, 60 parts trehalose, and 1.5 parts histidine.

[0022] This embodiment also provides a method for preparing hirudin nanomedicine to relieve chronic asthma, the specific steps of which are as follows: Step 1: Dissolve 0.8 parts of hirudin prodrug in 5 parts of DMF to obtain an organic phase. Use a microsyringe to slowly inject the organic phase into 50 parts of water for injection. After injection, stir at 100 rpm at room temperature for 6 h to obtain the prodrug solution. Step 2: Dissolve 12 parts of soybean lecithin, 60 parts of trehalose and 1.5 parts of histidine in 500 parts of water for injection, adjust the pH to 6.5 to obtain an auxiliary solution, and add the prodrug solution to the auxiliary solution while stirring, mix well to obtain the prescription solution; Step 3: Filter, sterilize, and freeze-dry the prescription solution to obtain hirudin nanomedicine.

[0023] Example 3: This example provides a hirudin nanomedicine for relieving chronic asthma. The hirudin nanomedicine comprises the following raw materials in parts by weight: 1 part hirudin prodrug, 15 parts human serum albumin, 80 parts mannitol and 1.5 parts histidine.

[0024] This embodiment also provides a method for preparing hirudin nanomedicine to relieve chronic asthma, the specific steps of which are as follows: Step 1: Dissolve 1 part of hirudin prodrug in 5 parts of DMF to obtain an organic phase. Use a microsyringe to slowly inject the organic phase into 50 parts of water for injection. After injection, stir at 100 rpm at room temperature for 6 hours to obtain the prodrug solution. Step 2: Dissolve 15 parts of human serum albumin, 80 parts of mannitol and 1.5 parts of histidine in 500 parts of water for injection, adjust the pH to 6.5 to obtain an auxiliary solution, add the prodrug solution to the auxiliary solution while stirring, mix well to obtain the prescription solution; Step 3: Filter, sterilize, and freeze-dry the prescription solution to obtain hirudin nanomedicine.

[0025] Example 4: This example provides a hirudin nanomedicine for relieving chronic asthma. The hirudin nanomedicine comprises the following raw materials in parts by weight: 0.6 parts of hirudin prodrug, 15 parts of polyethylene glycol 4000, 80 parts of hydroxyethyl starch, and 1.5 parts of histidine.

[0026] This embodiment also provides a method for preparing hirudin nanomedicine to relieve chronic asthma, the specific steps of which are as follows: Step 1: Dissolve 0.6 parts of hirudin prodrug in 5 parts of DMF to obtain an organic phase. Use a microsyringe to slowly inject the organic phase into 50 parts of water for injection. After injection, stir at 100 rpm at room temperature for 6 hours to obtain the prodrug solution. Step 2: Dissolve 15 parts of polyethylene glycol 4000, 80 parts of hydroxyethyl starch and 1.5 parts of histidine in 500 parts of water for injection, adjust the pH to 6.5 to obtain an auxiliary solution, add the prodrug solution to the auxiliary solution while stirring, mix well to obtain the prescription solution; Step 3: Filter, sterilize, and freeze-dry the prescription solution to obtain hirudin nanomedicine.

[0027] Example 5: This example provides a hirudin nanomedicine for relieving chronic asthma. The hirudin nanomedicine comprises the following raw materials in parts by weight: 1 part hirudin prodrug, 12 parts poloxamer 188, 30 parts sucrose, 30 parts mannitol, and 1.5 parts histidine.

[0028] This embodiment also provides a method for preparing hirudin nanomedicine to relieve chronic asthma, the specific steps of which are as follows: Step 1: Dissolve 1 part of hirudin prodrug in 5 parts of DMF to obtain an organic phase. Use a microsyringe to slowly inject the organic phase into 50 parts of water for injection. After injection, stir at 100 rpm at room temperature for 6 hours to obtain the prodrug solution. Step 2: Dissolve 12 parts of loxamer 188, 30 parts of sucrose, 30 parts of mannitol and 1.5 parts of histidine in 500 parts of water for injection, adjust the pH to 6.5 to obtain an auxiliary solution, add the prodrug solution to the auxiliary solution while stirring, mix well to obtain the prescription solution; Step 3: Filter, sterilize, and freeze-dry the prescription solution to obtain hirudin nanomedicine.

[0029] Morphological examination: 1 mg of the hirudin nanoparticle prepared in Example 4 was weighed and dissolved in 1 mL of water for injection to obtain a nano solution. 10 μL of the solution was added dropwise to a copper mesh, and after precipitation for 1 min, the surface liquid was absorbed with filter paper. The copper mesh was then dried under an electric heating lamp. When the surface liquid was about to disappear, the heating was stopped. 5 μL of a 2% phosphotungstic acid solution was added, and after precipitation for 1 min, the surface liquid was absorbed with filter paper and dried in a cool place. TEM (transmission electron microscopy) was performed at 100 kV, and the results are as follows. Figure 2 As shown.

[0030] Enzyme responsiveness assessment: Take the nano-solution prepared in the morphology assessment experiment, add 0.05 mg of MMP-9 enzyme to it, dissolve it, and then drop 10 μL onto a copper grid. Observe the TEM morphology changes. The results are shown in the figure. Figure 3 .

[0031] Figure 2 The results showed that the hirudin nanomedicine prepared in Example 4 could self-assemble into a nanoparticle-like structure under physiological conditions, with a spherical morphology, a diameter of about 150 nm, and a relatively uniform shape.

[0032] Figure 3 The results showed that when MMP-9 enzyme was added, the spherical morphology of hirudin nanomedicine in the sample disappeared. That is, after MMP-9 specifically cleaved the specific peptide on the outside of the nanomedicine, the original nanostructure was destroyed, the self-assembly behavior changed, and the hydrophobic long chains in the original nanoparticle core were exposed. This enabled the nanomedicine to respond to the high ROS environment again, further release hirudin, improve the therapeutic effect, and reduce systemic toxicity.

[0033] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0034] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A hirudin nano-drug for alleviating chronic asthma, characterized in that, The hirudin nanomedicine comprises the following raw materials by weight: 0.5-1 part of hirudin prodrug, 10-15 parts of stabilizer, 40-80 parts of freeze-drying protective agent and 1.5 parts of histidine; The hirudin prodrug comprises the following raw materials: hirudin, specific peptide, 2-aminoethanethiol and 16-bromohexadecanoic acid, and the preparation method of the hirudin prodrug is as follows: S1: dissolving 2-aminoethanethiol and then protecting the thiol group to obtain a modified liquid; S2: dissolving hirudin and specific peptide together and then performing dehydration condensation reaction to obtain a combination liquid, mixing the combination liquid with the modified liquid to obtain a reaction liquid; S3: preparing a cleavage liquid by using TFA, Tis and water, adding the cleavage liquid into the reaction liquid for cleavage, adding diethyl ether into the reaction liquid, centrifuging to collect the precipitate to obtain an intermediate; S4: mixing the intermediate with 16-bromohexadecanoic acid, purifying and freeze-drying to obtain the hirudin prodrug.

2. The hirudin nano-drug for alleviating chronic asthma according to claim 1, wherein the hirudin nano-drug is administered in an amount of 0.1-0.5 mg / kg. The amino acid sequence of the specific peptide is Fmoc-FFAGLDD.

3. The hirudin nanomedicine for relieving chronic asthma according to claim 1, characterized in that, The mass ratio of the hirudin, specific peptide, 2-aminoethanethiol and 16-bromohexadecanoic acid is 10:1.5:0.2:0.

5.

4. The hirudin nano-drug for alleviating chronic asthma according to claim 1, wherein the hirudin nano-drug is administered in an amount of 0.1-0.5 mg / kg. The stabilizer is selected from any one of poloxamer 188, soybean lecithin, human serum albumin and polyethylene glycol 4000; The freeze-drying protective agent is selected from one or more of sucrose, trehalose, mannitol and hydroxyethyl starch.

5. A method for preparing the hirudin nano-drug for alleviating chronic asthma according to any one of claims 1-4, characterized by, The specific preparation method is as follows: Step 1: dissolving the hirudin prodrug in an organic solvent to obtain an organic phase, injecting the organic phase into an aqueous solvent, stirring to obtain a prodrug solution; Step 2: dissolving the stabilizer, freeze-drying protective agent and histidine, adjusting pH, and mixing with the prodrug solution to obtain a prescription liquid; Step 3: filtering, sterilizing and freeze-drying the prescription liquid to obtain the hirudin nanomedicine.

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