Hydrogen peroxide sensitive covalent cyclodextrin scaffolds, methods of making and uses thereof

Abstract

The present application relates to hydrogen peroxide (H2O2) sensitive covalent cyclodextrin framework and its preparation method and application. Specifically, the covalent cyclodextrin framework is a cyclodextrin-metal organic framework with covalently linked peroxymonosulfate groups and / or covalently modified by phenylboronic acid ester cross-linking agent. The covalent cyclodextrin framework of the present application has hydrogen peroxide sensitivity, can reduce the level of hydrogen peroxide, and shows good antioxidant and anti-inflammatory capacity in vivo and in vitro. It can also play a synergistic therapeutic effect with drug molecules. The covalent cyclodextrin framework of the present application also has suitable biodegradability, good biological safety, and also has a large specific surface area for drug loading, and is an excellent drug carrier.

Description

Technical Field

[0001] This invention relates to the field of medical biomaterials, specifically to a hydrogen peroxide-sensitive covalent cyclodextrin backbone, its preparation method, and its applications. Background Technology

[0002] Pollution, declining air quality, and an aging population are contributing to a rise in respiratory diseases. Chronic obstructive pulmonary disease (COPD) is characterized by persistent respiratory symptoms and incompletely reversible airflow limitation. In recent years, COPD has become a significant global public health issue, with both its prevalence and mortality rates showing an upward trend. The World Health Organization estimates that by 2030, COPD will become the third leading cause of death worldwide. Currently, the number of COPD patients aged 40 and above in my country is approaching 100 million. The pathogenesis of COPD mainly involves chronic inflammatory responses in the airways and lung tissues, oxidative-antioxidant imbalances, protease and antiprotease imbalances, airway remodeling, and genetics. Among these, chronic airway inflammation and oxidative stress play a crucial role in the course of COPD and determine the degree of disease progression. Acute lung injury (ALI) is primarily caused by inflammation, oxidative stress, and impaired lung function, characterized by diffuse alveolar-interstitial edema, refractory hypoxemia, and non-cardiac pulmonary edema. The clinical manifestations of acute respiratory injury (ALI) are progressive hypoxemia and dyspnea. Although there are recognized methods for the prevention or treatment of ALI, it is accompanied by many adverse reactions, with a mortality rate as high as 40%. In the treatment of lung diseases, pulmonary administration is an important route of drug delivery because the lungs have a large surface area and are richly vascularized. Pulmonary administration also avoids the absorption barrier of oral administration. Pulmonary administration for the treatment of respiratory diseases can directly target the lesion site, enhance drug efficacy, reduce toxic side effects, and also reduce the dosage, lowering the economic burden on patients. Dry powder inhalers (DPIs) offer advantages among pulmonary drug delivery formulations, including high inhalation efficiency, ease of use, no propellants or air pollution, and good stability.

[0003] Ulcerative colitis (UC) is an idiopathic, lifelong inflammatory bowel disease of the colonic mucosa with an unpredictable course. Its main clinical manifestations include abdominal pain, uncontrollable diarrhea, bloody and purulent stools, and weight loss, leading to a severe reduction in patients' quality of life and disability. However, the pathogenesis of UC is complex, and there is currently no specific drug for treatment. Conventional treatment involves daily administration of high doses of immunosuppressants or anti-inflammatory drugs, which often results in severe adverse reactions. Therefore, there is an urgent clinical need to develop intelligent delivery systems that can target drugs to the inflamed colonic lesions to enhance drug efficacy and reduce toxicity.

[0004] In summary, there is a need in this field to provide drug carriers that offer excellent biosafety, suitable biodegradability, the ability to target lesions, and the ability to enhance drug efficacy. Summary of the Invention

[0005] The purpose of this invention is to provide a hydrogen peroxide-sensitive covalent cyclodextrin framework (COF) material with excellent biosafety, suitable biodegradability, the ability to target lesions, and the ability to enhance drug efficacy.

[0006] The covalent cyclodextrin backbone of this invention is hydrogen peroxide sensitive, capable of reducing hydrogen peroxide levels, and exhibits excellent antioxidant and anti-inflammatory capabilities both in vivo and in vitro. It can also exert synergistic therapeutic effects with drug molecules. Furthermore, the covalent cyclodextrin backbone of this invention possesses suitable biodegradability, good biosafety, and a large specific surface area suitable for drug loading, making it a high-performance drug carrier.

[0007] In a first aspect, the present invention provides a hydrogen peroxide-sensitive covalent cyclodextrin framework (COF), characterized in that the covalent cyclodextrin framework is a cyclodextrin-metal-organic framework (CD-MOF) having one or more of the following characteristics:

[0008] Having covalently linked peroxyoxalate groups (-OC(=O)-C(=O)-O-); and / or

[0009] Covalent modification via phenylboronic acid ester crosslinking agents.

[0010] In another preferred embodiment, the covalent cyclodextrin backbone is a cyclodextrin-organic metal framework (OC-COF) having covalently linked peroxyoxalate groups.

[0011] In another preferred embodiment, the covalent cyclodextrin backbone is a cyclodextrin-metal-organic backbone (B-COF) covalently modified by a phenylboronic acid ester crosslinking agent.

[0012] In another preferred embodiment, the phenylboronic ester crosslinking agent is BRAP.

[0013] In another preferred embodiment, the peroxyoxalate group is generated by reacting an oxalyl crosslinking agent with a cyclodextrin-metal-organic framework.

[0014] In another preferred embodiment, the molar ratio of CD-MOF to oxalyl crosslinking agent is 1:0.01-40, more preferably 1:0.1-30, more preferably 1:1-20, such as 1:0.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19 or 1:20.

[0015] In another preferred embodiment, the oxalyl crosslinking agent is oxalyl chloride.

[0016] In another preferred embodiment, the molar ratio of CD-MOF to phenylboronic acid ester crosslinking agent is 1:0.01-40, more preferably 1:0.1-30, and even more preferably 1:1-20, such as 1:0.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20.

[0017] In another preferred embodiment, the CD-MOF is a cubic CD-MOF.

[0018] In a second aspect, the present invention provides the use of a covalent cyclodextrin backbone as described in the first aspect of the present invention in the preparation of a pharmaceutical composition.

[0019] In another preferred embodiment, the covalent cyclodextrin skeleton serves as a drug carrier and / or ROS scavenger and / or anti-inflammatory active ingredient in the pharmaceutical composition.

[0020] In another preferred embodiment, the dosage form of the pharmaceutical composition is a liquid formulation or a solid formulation.

[0021] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: tablets, lozenges, suspensions, powders, granules, emulsions, hard or soft capsules, syrups, elixirs, dry powder inhalers, sprays, powder mists or aerosols, solutions, enemas, injections, patches, and dressings.

[0022] In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral dosage form, an inhaled dosage form, or a rectal dosage form.

[0023] In another preferred embodiment, the pharmaceutical composition targets lesion sites, such as sites of inflammation, where H2O2 levels are upregulated.

[0024] In another preferred embodiment, the pharmaceutical composition is used to treat inflammatory diseases, such as pulmonary inflammatory diseases or gastrointestinal inflammatory diseases.

[0025] In a third aspect, the present invention provides a pharmaceutical composition comprising:

[0026] (a) the covalent cyclodextrin backbone as described in claim 1; and

[0027] (b) The first active ingredient loaded on the covalent cyclodextrin backbone.

[0028] In another preferred embodiment, the first active ingredient is a small molecule compound, a protein drug, a nucleic acid, etc.

[0029] In another preferred embodiment, the weight ratio of the first active ingredient to the covalent cyclodextrin backbone in the pharmaceutical composition is 0.001-40 wt%, more preferably 0.01-30 wt%, more preferably 0.1-20 wt%, such as 1 wt%, 2 wt%, 5 wt%, 10 wt%, or 15 wt%.

[0030] In another preferred embodiment, the first active ingredient is a drug for treating lung diseases, such as a bronchodilator, a β2 receptor agonist, an anticholinergic drug, an inhaled corticosteroid, an anti-inflammatory drug, an antibacterial drug, an antiviral drug, or a combination thereof.

[0031] In another preferred embodiment, the first active ingredient is selected from the group consisting of: glucocorticoids such as hydrocortisone, prednisone, prednisolone, methylprednisolone, fluticasone propionate, lodemethasone dipropionate, budesonide, dexamethasone, and beclomethasone; β2 receptor agonists such as salbutamol, terbutaline, salmeterol, formoterol, and indacaterol; cholinergic receptor antagonists such as tiotropium bromide and ipratropium bromide; theophylline drugs such as aminophylline, dihydroxypropyltheophylline, doxophylline, roflumilast, silloster, AWD-12-281, tofimilast, UK-500001, GSK256066, SCH900182, and CHF. 6001, ORG-9935, Zadavirin, benzafentrine, pumafentrine, ensimifentrine, ligustrazine, curcumin, quercetin, resveratrol, tanshinone IIA, paeonol, luteolin, matrine, hesperidin, ginkgolide, andrographolide, steviol glycosides, rutin, tea polyphenols, baicalein, or combinations thereof.

[0032] In another preferred embodiment, the lung disease is selected from the group consisting of: COPD, acute lung injury, and lung infection.

[0033] In another preferred embodiment, the first active ingredient is a drug for treating gastrointestinal diseases (especially inflammatory gastrointestinal diseases, such as inflammatory bowel disease and acute ulcerative colitis), such as glucocorticoids, aminosalicylic acids, active ingredients of traditional Chinese medicine, or combinations thereof.

[0034] In another preferred embodiment, the first active ingredient is selected from the group consisting of prednisolone, dexamethasone, 5-aminosalicylic acid, sulfasalazine, resveratrol, berberine, or combinations thereof.

[0035] In another preferred embodiment, the pharmaceutical composition further includes other pharmaceutically acceptable carriers.

[0036] In another preferred embodiment, the dosage form of the pharmaceutical composition is a liquid formulation or a solid formulation.

[0037] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: tablets, lozenges, suspensions, powders, granules, emulsions, hard or soft capsules, syrups, elixirs, dry powder inhalers, sprays, powder mists or aerosols, solutions, enemas, injections, patches, and dressings.

[0038] In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral dosage form, an inhaled dosage form, or a rectal dosage form.

[0039] In another preferred embodiment, the pharmaceutical composition targets lesion sites, such as sites of inflammation, where H2O2 levels are upregulated.

[0040] In a fourth aspect, the present invention provides a method for preparing a pharmaceutical composition as described in the third aspect, comprising the steps of:

[0041] (i) Provide a solution of the first active ingredient;

[0042] (ii) Add the covalent cyclodextrin backbone and disperse, stir and incubate, filter and dry, thereby loading the first active ingredient onto the covalent cyclodextrin backbone.

[0043] In another preferred embodiment, the incubation temperature is 25-80°C, more preferably 40-60°C.

[0044] In another preferred embodiment, the incubation time is 1-48 hours, more preferably 12-36 hours.

[0045] In a fifth aspect, the present invention provides a method for preparing a hydrogen peroxide-sensitive covalent cyclodextrin backbone as described in the first aspect, comprising the steps of:

[0046] (1) Provides CD-MOF; and

[0047] (2) In an inert solvent and in the presence of an alkaline catalyst, an oxalyl crosslinking agent is reacted with CD-MOF to obtain a covalent cyclodextrin skeleton with peroxyoxalate groups.

[0048] In another preferred embodiment, the method further includes the step of: (3) reacting a phenylboronic acid ester crosslinking agent with the covalent cyclodextrin skeleton modified with peroxyoxalate group in an organic solvent in the presence of a catalyst and a linker, thereby obtaining a covalent cyclodextrin skeleton with peroxyoxalate group and modified by a phenylboronic acid ester crosslinking agent.

[0049] In another preferred embodiment, the molar ratio of the CD-MOF to the oxaloyl crosslinking agent and the covalent cyclodextrin backbone with peroxyoxalate group modification to the phenylboronic acid ester crosslinking agent is independently 1:0.01-40, more preferably 1:0.1-30, more preferably 1:1-20, such as 1:0.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19 or 1:20.

[0050] In another preferred embodiment, the reaction in step (2) has one or more characteristics selected from the group consisting of:

[0051] a) The inert solvent is selected from the group consisting of dichloromethane, tetrahydrofuran, or combinations thereof;

[0052] b) The basic catalyst is selected from the group consisting of pyridine, triethylamine, or combinations thereof;

[0053] c) The reaction temperature is 0-30℃, preferably 20-25℃;

[0054] d) The reaction time is 3-72 h, preferably 24-72 h.

[0055] In another preferred embodiment, the reaction in step (3) has one or more characteristics selected from the group consisting of:

[0056] a) The organic solvent is selected from the group consisting of dichloromethane, tetrahydrofuran, or combinations thereof;

[0057] b) The catalyst is a basic catalyst, preferably selected from the group consisting of pyridine, triethylamine, or combinations thereof;

[0058] c) The linker is selected from the group consisting of diphenyl carbonate (DPC);

[0059] d) The molar ratio of the linker to the phenylboronic acid ester crosslinking agent is 1:0.2-1.2, preferably 1:0.5-0.9;

[0060] e) The reaction temperature is 0-85℃, preferably 60-80℃;

[0061] f) The reaction time is 10-36 h, preferably 12-24 h.

[0062] In a fifth aspect, the present invention provides a method for preparing a reactive oxygen species-sensitive covalent cyclodextrin backbone as described in the first aspect of the present invention, comprising the steps of:

[0063] (1) Provides CD-MOF; and

[0064] (2) In an organic solvent, in the presence of a catalyst and a linker, a phenylboronic acid ester crosslinking agent is reacted with CD-MOF to obtain a covalent cyclodextrin skeleton modified with a phenylboronic acid ester crosslinking agent.

[0065] In another preferred embodiment, the method further includes the step of: (3) reacting an oxalyl crosslinking agent with a covalent cyclodextrin skeleton modified by a phenylboronic acid ester crosslinking agent obtained in step (2) in an inert solvent and in the presence of an alkaline catalyst, thereby obtaining a covalent cyclodextrin skeleton having a peroxyoxalate ester group and modified by a phenylboronic acid ester crosslinking agent.

[0066] In another preferred embodiment, the molar ratio of the CD-MOF to the phenylboronic acid ester crosslinking agent and the covalent cyclodextrin backbone modified by the phenylboronic acid ester crosslinking agent to the oxaloyl crosslinking agent is independently 1:0.01-40, more preferably 1:0.1-30, more preferably 1:1-20, such as 1:0.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19 or 1:20.

[0067] In another preferred embodiment, the reaction in step (2) has one or more characteristics selected from the group consisting of:

[0068] a) The organic solvent is selected from the group consisting of dichloromethane, tetrahydrofuran, or combinations thereof;

[0069] b) The catalyst is a basic catalyst, preferably selected from the group consisting of pyridine, triethylamine, or combinations thereof;

[0070] c) The linker is selected from the group consisting of diphenyl carbonate (DPC);

[0071] d) The molar ratio of the linker to the phenylboronic acid ester crosslinking agent is 1:0.2-1.2, preferably 1:0.5-0.9;

[0072] e) The reaction temperature is 0-85℃, preferably 60-80℃;

[0073] f) The reaction time is 10-36 h, preferably 12-24 h.

[0074] In another preferred embodiment, the reaction in step (3) has one or more characteristics selected from the group consisting of:

[0075] a) The inert solvent is selected from the group consisting of dichloromethane, tetrahydrofuran, or combinations thereof;

[0076] b) The basic catalyst is selected from the group consisting of pyridine, triethylamine, or combinations thereof;

[0077] c) The reaction temperature is 0-30℃, preferably 20-25℃;

[0078] d) The reaction time is 3-72 h, preferably 24-72 h.

[0079] In another preferred embodiment, the reactive oxygen species-sensitive covalent cyclodextrin backbone of the present invention is prepared by the method of the fifth or sixth aspect of the present invention.

[0080] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0081] Figure 1 The images are scanning electron microscope (SEM) images of μm-CD-MOF and μm-OC-COF from Example 1.

[0082] Figure 2 Infrared spectra of CD-MOF and OC-COF in Example 1;

[0083] Figure 3 The powder X-ray diffraction patterns of CD-MOF and OC-COF in Example 1 are shown below.

[0084] Figure 4 Thermogravimetric analysis spectra of CD-MOF and OC-COF in Example 1;

[0085] Figure 5 Scanning electron microscope images of nano-CD-MOF and nano-OC-COF in Example 2;

[0086] Figure 6 The cell safety results of OC-COF in Example 4;

[0087] Figure 7 The degradation of OC-COF in aqueous solution for different times is shown in Example 4.

[0088] Figure 8 These are scanning electron microscope (SEM) images of OC-COF hydrolysis at different time points in Example 4;

[0089] Figure 9 This is a graph showing the reduction of hydrogen peroxide content by OC-COF in Example 4;

[0090] Figure 10 This is a graph showing how OC-COF improves cell survival in an oxidative environment, as illustrated in Example 4.

[0091] Figure 11This refers to the in vitro antioxidant effect of OC-COF in Example 4;

[0092] Figure 12 This refers to the in vitro anti-apoptotic effect of OC-COF in Example 4;

[0093] Figure 13 The degradation of RhoB-OC-COF after inhalation into the lungs of rats in Example 4;

[0094] Figure 14 The image shows a scanning electron microscope (SEM) image of LIG@OC-COF in Example 6.

[0095] Figure 15 The powder X-ray diffraction pattern of LIG@OC-COF in Example 6;

[0096] Figure 16 The thermal analysis diagram of LIG@OC-COF in Example 6;

[0097] Figure 17 Thermogravimetric analysis diagram of LIG@OC-COF in Example 6;

[0098] Figure 18 The infrared image of LIG@OC-COF in Example 6;

[0099] Figure 19 This is an in vitro deposition distribution map of LIG@OC-COF in Example 6;

[0100] Figure 20 This demonstrates the in vitro anti-inflammatory effect of LIG@OC-COF in Example 6.

[0101] Figure 21 This describes the absorption of LIG and LIG@OC-COF in alveolar monolayer epithelial cells in Example 6.

[0102] Figure 22 In Example 6, LIG@OC-COF was used to improve lung function in COPD rats.

[0103] Figure 23 Images of lung tissue sections from different groups of COPD rats in Example 6;

[0104] Figure 24 The release of inflammatory factors and antioxidant factors in each group of COPD rats in Example 6;

[0105] Figure 25 The expression of intracellular proteins SOD, Nrf2 and NF-κB in lung tissue of COPD rats in each group in Example 6;

[0106] Figure 26 This is an HE-stained section of lung tissue from rats with acute lung injury in Example 6;

[0107] Figure 27 The levels of inflammatory factors in each group of rats with acute lung injury in Example 6;

[0108] Figure 28 The antioxidant indicators of each group of rats with acute lung injury in Example 6;

[0109] Figure 29 The expression of SOD, NF-κB, and Nrf2 proteins in lung tissues of rats with acute lung injury in Example 6;

[0110] Figure 30 The particle size distribution (a) and scanning electron microscope image (b) of BCOF in Example 7 are shown.

[0111] Figure 31 The images show the 1H NMR spectra of BRAP (a) and BCOF (b) in Example 7, the infrared spectrum (c) of BCOF, and the PXRD spectrum (d) of BCOF.

[0112] Figure 32 The hydrolysis curves (a) of BCOF in H2O2 media at different concentrations (0, 1 and 10 mM) and the pyrolysis curves (b) of BCOF in media at different pH values ​​(1.2, 6.8 and 7.4) are shown in Example 7.

[0113] Figure 33 The results show the cytotoxicity assay of BCOF in Example 7 (a) and the protective effect of BCOF on cells in the environment (b).

[0114] Figure 34 The changes in body weight (a), DAI score (b), and colon length (c) during the UC modeling process in Example 7 are shown.

[0115] Figure 35 Representative in vivo images (a) and quantitative fluorescence analysis (b) of the heart, liver, spleen, lung, kidney, stomach, small intestine and colon tissues of colitis mice after gavage administration of BCOF in Example 7 at different time points; representative in vivo images (c) and quantitative fluorescence analysis (d) of the distribution of BCOF in the colon tissues of healthy and colitis mice 6 hours after gavage administration (n=3);

[0116] Figure 36 The following is a schematic diagram of the dosing regimen for the BCOF efficacy evaluation in Example 7 (a), the change in body weight percentage of mice in each group over 7 days (b), the change in DAI of mice in each group over 7 days (c), representative pictures of mice in each group (d), and quantitative colon length (e) (n=6).

[0117] Figure 37 HE sections of mouse colon tissue from each group in Example 7;

[0118] Figure 38 HE sections of colon, heart, liver, spleen, lung, and kidney tissues from each group of mice in Example 7;

[0119] Figure 39 The release curves of Ber@BCOF in 0mM, 1mM and 10mM H2O2 media (n=3) (a) and the release curves of Ber@BCOF in pH 1.2 and pH 6.8 media (n=3) (b) are shown in Example 9.

[0120] Figure 40 The following are examples of changes in the percentage of body weight (n=6) and the changes in DAI (n=6) of mice over 7 days using PD@BCOF in Example 10 (a) and (b) respectively. Detailed Implementation

[0121] Through long-term and in-depth research, the inventors synthesized a hydrogen peroxide-sensitive covalent cyclodextrin backbone (COF) using a cyclodextrin metal-organic framework (CD-MOF) as a template. This COF exhibits hydrogen peroxide sensitivity and demonstrates excellent antioxidant and anti-inflammatory effects both in vitro and in vivo, and can synergistically enhance drug efficacy. Furthermore, the COF of this invention possesses good biocompatibility, suitable biodegradability, and strong drug-loading capacity, making it highly suitable as a drug carrier for loading active molecules and preparing drug delivery systems targeting specific diseases or routes of administration. Based on these findings, the inventors completed this invention.

[0122] the term

[0123] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0124] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0125] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0126] As used herein, the terms “room temperature” or “normal temperature” refer to a temperature of 4-40°C, preferably 25±5°C.

[0127] Cyclodextrin - Metal-organic framework

[0128] As used in this article, the terms "cyclodextrin-metal-organic framework" and "CD-MOF" are used interchangeably. CD-MOF is a novel, safe, and pharmaceutically usable cubic cyclodextrin-metal-organic framework formed by using cyclodextrin as an organic ligand and metal ions as an inorganic metal center.

[0129] CD-MOF is a green, biodegradable supramolecular material, a porous framework material formed by the coordination of pharmaceutical excipient cyclodextrin and metal ions. CD-MOF is characterized by its porous structure, large specific surface area, and regular morphology. It is also green, edible, and suitable for pharmaceutical use. Furthermore, CD-MOF contains a large number of hydroxyl groups, allowing for functionalization modifications.

[0130] Typically, the cyclodextrin-metal-organic framework is a framework material formed by cyclodextrin and an alkali metal salt; the alkali metal includes, but is not limited to, Li. + K + 、Rb + Cs + Na + Mg 2+ Sn 2+ Ag + Yb + Ba 2+ 、Sr 2+ Ca 2+ La 3+ K is preferred + .

[0131] Typically, the average particle size of the cyclodextrin-metal-organic framework material is 50 nm-50 μm, preferably 100-500 nm (nanometer-scale) or 1-5 μm (micrometer-scale).

[0132] Typically, the preparation of cyclodextrin-metal-organic frameworks (CD-MOFs) (refer to patent CN201610125456.X) involves: mixing a metal salt solution with a cyclodextrin aqueous solution, pre-adding a portion of organic solvent, reacting at a certain temperature for a certain time via solvent vapor diffusion, and then adding a size regulator to obtain the cyclodextrin-based metal-organic framework material; or mixing a metal salt solution with a cyclodextrin aqueous solution, pre-adding a portion of organic solvent, using a solvothermal / microwave / ultrasonic vibration reaction medium to rapidly react the reactants, and then adding a size regulator after a certain reaction time to obtain the cyclodextrin-based metal-organic framework material.

[0133] Typically, the concentration of the metal salt in the metal salt solution is 0.05-0.4M, preferably 0.2M.

[0134] Typically, the concentration of cyclodextrin in the aqueous cyclodextrin solution is 0.013-0.05M, preferably 0.025M.

[0135] Typically, the cyclodextrin is selected from the group consisting of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin, methyl-β-cyclodextrin, carboxymethyl-β-cyclodextrin, or combinations thereof.

[0136] Preferably, the cyclodextrin is γ-cyclodextrin.

[0137] Preferably, the cyclodextrin-metal-organic framework is cubic.

[0138] H2O2-sensitive covalent cyclodextrin backbone

[0139] The covalent cyclodextrin backbone of the present invention is a cyclodextrin-metal-organic framework (CD-MOF) having one or more of the following characteristics: having covalently linked peroxyoxalate groups (-OC(=O)-C(=O)-O-); and / or being covalently modified by a phenylboronic acid ester crosslinking agent.

[0140] As used in this invention, the terms "hydrogen peroxide-sensitive covalent cyclodextrin backbone" and "covalent cyclodextrin backbone" are used interchangeably to refer to the hydrogen peroxide-sensitive covalent cyclodextrin backbone of this invention.

[0141] This invention provides a covalently cyclodextrin backbone (COF) sensitive to H2O2 by covalently modifying CD-MOF. Specifically, CD-MOF is covalently modified with a crosslinking agent to provide peroxyoxalate and / or phenylboronic acid ester groups covalently linked to CD-MOF.

[0142] As used in this invention, the term "oxalate crosslinking agent" refers to a substance capable of reacting with CD-MOF (e.g., with hydroxyl groups) to generate or provide peroxyoxalate groups (-OC(=O)-C(=O)-O-).

[0143] As used in this invention, the term "phenylboronic ester crosslinking agent" refers to a substance capable of reacting with CD-MOF (such as hydroxyl groups) to generate or provide phenylboronic ester groups.

[0144] Oxalic acid crosslinking agents capable of reacting with CD-MOF to generate peroxyoxalate groups are known to those skilled in the art, including but not limited to oxalyl chloride.

[0145] Preferably, the phenylboronic ester crosslinking agent includes, but is not limited to, BRAP.

[0146] The covalent cyclodextrin backbone described in this invention differs from existing covalent cyclodextrin backbones. Covalent cyclodextrin backbones crosslinked with diphenyl carbonate or epichlorohydrin do not exhibit H2O2 responsiveness, while covalent cyclodextrin backbones crosslinked with 3,3'-dimercaptobispropionyl chloride exhibit glutathione responsiveness. There are currently no reports of cubic covalent cyclodextrin backbones with reactive oxygen species responsiveness.

[0147] Uses, pharmaceutical compositions

[0148] The hydrogen peroxide-sensitive covalent cyclodextrin backbone of the present invention has anti-inflammatory and ROS scavenging activities, and can be used as an active ingredient to prepare anti-inflammatory agents and ROS scavengers, especially for lesion sites (such as inflammatory sites) with high hydrogen peroxide levels.

[0149] Furthermore, the covalent cyclodextrin backbone of the present invention has a strong and excellent drug loading capacity and is targeted to sites where H2O2 levels are upregulated (such as sites of inflammation), and can be used as a drug carrier to achieve drug release with high H2O2 environmental sensitivity.

[0150] The covalent cyclodextrin backbone of the present invention can also be used in combination with other therapeutic agents, which may be used simply in combination with the covalent cyclodextrin backbone of the present invention, or used independently as separate formulations, or loaded into the covalent cyclodextrin backbone of the present invention.

[0151] Furthermore, the inventors unexpectedly discovered that the covalent cyclodextrin backbone of the present invention, after being loaded with drugs, can synergistically exert therapeutic effects (including but not limited to anti-oxidation and anti-inflammation) with other loaded active molecules, thereby enabling better treatment of diseases. Preferably, the disease is one in which H2O2 levels are upregulated.

[0152] As used in this invention, the term "site with increased H2O2 level" refers to a site where the hydrogen peroxide level is 10% or more, 20% or more, 50% or more, or even 100% higher than that of the normal control / site.

[0153] Preferably, the present invention provides a pharmaceutical composition comprising:

[0154] (a) the covalent cyclodextrin backbone as described in claim 1; and

[0155] (b) The first active ingredient loaded in the covalent cyclodextrin backbone.

[0156] The covalent cyclodextrin backbone of the present invention can be used for loading various active ingredients. Those skilled in the art can select the first active ingredient as needed, for example (including but not limited to) small molecule compounds, protein drugs, nucleic acids, etc. Generally, the covalent cyclodextrin backbone of the present invention serves as both a drug delivery carrier and can exert its anti-inflammatory, ROS scavenging activity and / or synergistic therapeutic effect with the first active ingredient.

[0157] In another preferred embodiment, the pharmaceutical composition further includes other pharmaceutically acceptable carriers.

[0158] Preferably, the present invention does not impose particular requirements on the dosage form of the pharmaceutical composition, such as in the form of a liquid or solid dosage form; typically including (but not limited to) tablets, lozenges, suspensions, powders, granules, emulsions, hard or soft capsules, syrups, elixirs, dry powder inhalers, sprays, powder mists or aerosols, solutions, enemas, injections, patches, dressings, etc. Furthermore, the pharmaceutical composition can be administered to the subject via any suitable route, including oral, parenteral, inhalation spray, topical, rectal, nasal, sublingual, vaginal, or via an implantable cartridge, preferably oral, inhalation, or rectal administration.

[0159] The precise amount of active ingredient provided to an individual in a therapeutically effective dose will depend on the route of administration, the type and severity of the disease and / or condition, and individual characteristics such as general health, age, sex, weight, and tolerance to the drug. Those skilled in the art will be able to determine the appropriate dosage based on these and other factors. When administered in combination with other therapeutic agents, the “therapeutically effective amount” of any other therapeutic agent will depend on the type of drug used. Appropriate dosages are known for approved therapeutic agents and can be adjusted by those skilled in the art based on the individual’s condition, the type of condition being treated, and the amount of the compound of the invention used below. Preferably, the compositions should be formulated such that an inhibitory dose of 0.01-100 mg / kg body weight / day can be administered to patients receiving these compositions. In some embodiments, the compositions of the invention provide doses from 0.01 mg to 50 mg. In other embodiments, doses of 0.1 mg-25 mg or 5 mg-40 mg are provided.

[0160] Examples of subjects to which the pharmaceutical compositions or therapeutic agents of the present invention are administered include mammals (e.g., humans, mice, rats, hamsters, rabbits, cats, dogs, cattle, sheep, monkeys, etc.).

[0161] Preparation method

[0162] The hydrogen peroxide-sensitive covalent cyclodextrin backbone of the present invention can be prepared by synthetic methods commonly used in the art, as long as covalently linked peroxyoxalate groups and / or phenylboronic acid ester groups can be formed on the cyclodextrin metal-organic backbone.

[0163] Specifically, the method for preparing the covalent cyclodextrin backbone of the present invention may include the following steps:

[0164] (a) In an inert solvent and in the presence of a basic catalyst, an oxalyl crosslinking agent is reacted with a CD-MOF (or a covalent cyclodextrin backbone modified with a phenylboronic acid ester crosslinking agent obtained in step (b)) to obtain a peroxyoxalate-modified covalent cyclodextrin backbone; and / or

[0165] (b) In an organic solvent, in the presence of a catalyst and a linker, BRAP is reacted with CD-MOF (or the peroxyoxalate-modified covalent cyclodextrin backbone obtained in step (a)) to obtain a phenylboronic acid ester-modified covalent cyclodextrin backbone.

[0166] In another preferred embodiment, the reaction in step (a) has one or more characteristics selected from the group consisting of:

[0167] a) The inert solvent is selected from the group consisting of dichloromethane, tetrahydrofuran, or combinations thereof;

[0168] b) The basic catalyst is selected from the group consisting of pyridine, triethylamine, or combinations thereof;

[0169] c) The reaction temperature is 0-30℃, preferably 20-25℃;

[0170] d) The reaction time is 3-72 h, preferably 24-72 h.

[0171] In another preferred embodiment, the reaction in step (b) has one or more characteristics selected from the group consisting of:

[0172] a) The organic solvent is selected from the group consisting of dichloromethane, tetrahydrofuran, or combinations thereof;

[0173] b) The catalyst is a basic catalyst, preferably selected from the group consisting of pyridine, triethylamine, or combinations thereof;

[0174] c) The linker is selected from the group consisting of diphenyl carbonate (DPC);

[0175] d) The molar ratio of the connector to BRAP is 1:0.2-1.2, preferably 1:0.5-0.9;

[0176] e) The reaction temperature is 0-85℃, preferably 60-80℃;

[0177] f) The reaction time is 10-36 h, preferably 12-24 h.

[0178] The present invention has the following main advantages:

[0179] The cyclodextrin backbone material prepared in this invention exhibits high hydrogen peroxide responsiveness. As a drug carrier, it reduces the concentration of reactive oxygen species at inflammatory sites by reacting with hydrogen peroxide, thus alleviating the progression of inflammatory diseases and synergistically exerting antioxidant and anti-inflammatory therapeutic effects with drug molecules. It remains stable in a simulated gastrointestinal pH environment, effectively improving cell survival in oxidative stress conditions. The preparation process is simple, controllable, and biocompatible, exhibiting good biodegradability under physiological conditions and not causing cumulative toxicity or inflammatory reactions after administration.

[0180] The dry powder inhaler prepared using OC-COF as an inhalable carrier, as described in this invention, exhibits superior lung deposition rate. After inhalation, it reaches the alveoli, helping to reduce the dosage, promoting drug absorption in the lungs, and significantly improving the therapeutic effect of pulmonary administration. For example, the LIG@OC-COF dry powder inhaler, used to treat COPD and acute lung injury, reduces inflammatory cell infiltration in the lungs, decreases the release of inflammatory factors, lowers oxidative factors, increases antioxidant factors, and improves lung function after administration.

[0181] The BCOF carrier material involved in this invention loads anti-inflammatory bowel disease drugs, thereby enhancing drug efficacy and reducing toxicity, and greatly improving the treatment effect of ulcerative colitis.

[0182] Compared with the prior art, the carrier described in this invention has the ability to load drugs in multiple cavities, high hydrogen peroxide responsiveness, and regular particle morphology.

[0183] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0184] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0185] Example 1

[0186] Weigh 14.584 g of γ-CD and 5.049 g of KOH at a molar ratio of 1:8, add 450 mL of ultrapure water (18.2 MΩ·cm), sonicate to dissolve, and filter through a 0.8 μm water membrane to obtain γ-CD-KOH mother liquor. Measure 100 mL of the mother liquor and 60 mL of methanol at a ratio of 5:3 (v / v) into a sealed container, shake vigorously to mix thoroughly, and place in a 60℃ water bath. After the solution becomes clear, heat for 20 min. Add 640 mg of PEG 20000 to every 100 mL of the mother liquor, heat at 60℃ to dissolve, shake to mix thoroughly, and then let stand in a cold water bath for 2–3 h. Centrifuge (4000 rpm, 5 min), wash the lower precipitate twice with 160 mL of anhydrous ethanol and anhydrous methanol, and vacuum dry (80℃, 2 h) to obtain micron-sized γ-CD-MOF (μm-CD-MOF).

[0187] Using CD-MOF as a template and oxalyl chloride as a crosslinking agent, a reactive oxygen species-sensitive covalent cyclodextrin backbone OC-COF was synthesized. 422.4 mg of the synthesized CD-MOF was weighed into a round-bottom flask. In 5 mL of dichloromethane and 338 μL of triethylamine as a catalyst, oxalyl chloride and CD-MOF were reacted at 25 °C at a molar ratio of 1:12 for 24 h. After the reaction was complete, the precipitate was obtained by centrifugation at 4000 rpm for 5 min. The precipitate was washed once each with 75% ethanol, 50% ethanol, and water, centrifuged, pre-frozen at -80 °C for 4 h, and freeze-dried for 24 h to obtain the peroxalate bonded covalent cyclodextrin backbone (OC-COF).

[0188] Scanning electron microscopy results ( Figure 1 The results show that CD-MOF has a regular cubic shape and uniform particle size (2-3 μm); the OC-COF obtained after crosslinking still maintains the original regular shape and particle size.

[0189] FT-IR results ( Figure 2 (Displayed at 1750cm) -1 The characteristic peak of oxaloyl diketone appeared at the location, while no peak appeared in CD-MOF, confirming that cross-linked OC-COF has been successfully prepared using oxaloyl chloride as a cross-linking agent.

[0190] PXRD results ( Figure 3 The results show that the cross-linked OC-COF has lost the crystallinity of the original CD-MOF and is an amorphous polymer.

[0191] Thermogravimetric analysis results ( Figure 4The results show that CD-MOF loses about 10% of its weight between 0 and 200°C, mainly due to the loss of free water molecules, as CD-MOF is highly hygroscopic. In contrast, OC-COF is relatively stable between 0 and 200°C, indicating that OC-COF contains virtually no moisture. When the temperature exceeds 200°C, both OC-COF and CD-MOF begin to decompose, suggesting that OC-COF exhibits good thermal stability during routine storage and use, making it a relatively stable drug delivery carrier.

[0192] Example 2

[0193] Weigh 14.584 g of γ-CD and 5.049 g of KOH at a molar ratio of 1:8, add 450 mL of ultrapure water (18.2 MΩ·cm), sonicate to dissolve, and filter through a 0.8 μm water membrane to obtain γ-CD-KOH mother liquor. Measure 100 mL of the mother liquor and 60 mL of methanol at a ratio of 5:3 (v / v) into a sealed container, shake vigorously to mix thoroughly, and place in a 60 °C water bath. After the solution becomes clear, heat for 20 min. Then, add 80 mL of a methanol solution (8 mg / mL) containing the size regulator PEG 20000 to every 100 mL of the mother liquor, continue heating for 1 h, let stand in a cold water bath for 2 h, centrifuge (4500 rpm, 5 min), discard the supernatant, wash the lower precipitate twice each with 160 mL of anhydrous ethanol and anhydrous methanol, and vacuum dry (80 °C, 2 h) to obtain nano-sized γ-CD-MOF (nano-CD-MOF).

[0194] Using CD-MOF as a template and oxalyl chloride as a crosslinking agent, a reactive oxygen species-sensitive covalent cyclodextrin backbone OC-COF was synthesized. 422.4 mg of the synthesized CD-MOF was weighed into a round-bottom flask. In 5 mL of dichloromethane and 338 μL of triethylamine as a catalyst, oxalyl chloride and CD-MOF were reacted at 25 °C at a molar ratio of 1:12 for 24 h. After the reaction was complete, the precipitate was obtained by centrifugation at 4000 rpm for 5 min. The precipitate was washed once each with 75% ethanol, 50% ethanol, and water, centrifuged, pre-frozen at -80 °C for 4 h, and freeze-dried for 24 h to obtain the peroxalate bonded covalent cyclodextrin backbone (OC-COF).

[0195] Nanoscale microscopic results are shown Figure 2 The particle sizes of nano-CD-MOF and nano-OC-COF are approximately 500 nm. The OC-COF, with its regular cubic morphology, allows for adjustable particle size to meet different drug delivery requirements. Compared to spherical particles, the cubic shape effectively evades phagocytosis and clearance by macrophages.

[0196] Example 3

[0197] CD-MOF was prepared according to Example 1. Using CD-MOF as a template and oxalyl chloride as a crosslinking agent, a reactive oxygen species-sensitive covalent cyclodextrin backbone OC-COF was synthesized. 422.4 mg of the synthesized CD-MOF was weighed into a round-bottom flask. In a certain volume of organic solvent, in the presence of a catalyst, oxalyl chloride and CD-MOF were reacted at 25°C at a specific molar ratio for 24 h. After the reaction was complete, the precipitate was obtained by centrifugation at 4000 rpm for 5 min. The precipitate was washed once each with 75% ethanol, 50% ethanol, and water, centrifuged, pre-frozen at -80°C for 4 h, and freeze-dried for 24 h to obtain the peroxyoxalate bonded covalent cyclodextrin backbone (OC-COF). Specific preparation parameters are shown in Table 1.

[0198] Table 1 Preparation parameters of OC-COF

[0199] Molar ratio (CD-MOF:OC) Organic solvents and volume Catalyst and dosage 1:8 5 mL of dichloromethane 225 μL of triethylamine 1:12 5 mL of dichloromethane Triethylamine 338 μL 1:16 5 mL of dichloromethane 450 μL of triethylamine 1:12 5mL tetrahydrofuran Triethylamine 338 μL 1:12 5 mL of dichloromethane pyridine 290 μL 1:12 5mL tetrahydrofuran pyridine 290 μL

[0200] Example 4

[0201] (1) Biosafety of OC-COF (prepared in Example 1)

[0202] Lung cells in logarithmic growth phase, including A549-II alveolar epithelial cells, WI26-VA4-I alveolar epithelial cells, MHS-alveolar macrophages, and Calu-3 bronchial epithelial cells, were seeded at a specific density in 96-well plates (200 μL per well). After 12 h of incubation, the complete culture medium was discarded, and 180 μL of serum-free culture medium was added, followed by 20 μL of OC-COF diluted with PBS at different concentrations, resulting in final OC-COF concentrations of 1000, 400, 160, 64, 25.6, 10.24, 4.096, and 1.6384 μg / mL in the wells. A blank control group containing only culture medium and a control group containing both cells and culture medium were also included. After culturing for another 24 h, 15 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for a further period. The culture was terminated, and the absorbance (A) at 450 nm was measured using a microplate reader to calculate the cell viability (n=6 for each group). The seeding plate density and incubation time after adding CCK8 solution were adjusted according to the cell type.

[0203]

[0204] The results are as follows Figure 6 The results showed that cross-linked OC-COF had no significant inhibitory effect on cell proliferation or cytotoxicity on A549, WI26-VA4, MHS and Calu-3 cells. Even at the highest concentration of 1000 μg / mL, the cells still had a survival rate of more than 95%, which proved that OC-COF has good biocompatibility and biosafety as a drug carrier for lung administration.

[0205] (2) Evaluation of biodegradability

[0206] A 20 mg / mL OC-COF solution was prepared and incubated on a shaker at 37°C and 100 rpm for different times. The condition of the solution at different time points was recorded by photographing to evaluate the degradation of OC-COF in in vitro physiological media. Hydrolysis samples at different time points were also observed by scanning electron microscopy.

[0207] Images of OC-COF incubated in PBS medium for different times ( Figure 7 It can be seen that OC-COF slowly hydrolyzes in in vitro physiological media. A significant color change was observed in the suspension after 12 hours of incubation in PBS, and it became essentially clear after 24 hours, demonstrating that OC-COF is a biodegradable carrier material. Figure 8 SEM imaging of hydrolyzed samples at different time points revealed that OC-COF maintained its intact morphology in aqueous solution before 4 hours. At 8 hours, the edges of the cubes became less distinct, and the entire structure became rounded, indicating significant hydrolysis. At 12 hours, the small hydrolyzed particles aggregated into larger particles. One possible reason is that the peroxate bonds hydrolyze in an aqueous environment, thus enabling the biodegradability of OC-COF.

[0208] (3) H2O2 removal experiment of OC-COF

[0209] Concentration-dependent effect of OC-COF on H2O2 aqueous solution: 30% H2O2 solution was diluted with PBS to 100 μM to prepare OC-COF solutions of different masses with concentrations of 1, 2, 3, 4, 5, 6, and 7 mg / mL. PBS aqueous solution containing 100 μM H2O2 without OC-COF was used as the control group, and pure PBS solution was used as the blank group. The solutions were incubated on a shaker (37℃, 100 rpm) for 1, 2, 4, 8, 12, and 24 h, and samples were taken (n=3). The samples were centrifuged at 12000 rpm, and the supernatant was collected. The consumption of H2O2 by different concentrations of OC-COF at different time points was detected using a hydrogen peroxide kit.

[0210] from Figure 9It was found that OC-COF of the same concentration degraded in PBS aqueous solution containing 100 μM H2O2 at different time intervals. After 2 hours of incubation, the H2O2 concentration began to decrease; after 4 hours, the H2O2 concentration decreased significantly, and gradually decreased with increasing reaction time; after 24 hours, the H2O2 concentration approached 0, demonstrating that the interaction between OC-COF and H2O2 is time-dependent, with OC-COF gradually reducing the H2O2 concentration with increasing reaction time. Furthermore, with increasing OC-COF concentration, the amount of H2O2 consumed by OC-COF also gradually increased, showing a clear concentration-dependent relationship with H2O2 content. This fully verifies that OC-COF has a hydrogen peroxide responsiveness and can reduce the concentration of H2O2 in the solution.

[0211] (4) Cell protection effect of OC-COF in hydrogen peroxide environment

[0212] H2O2-damaged MHS cells were used as a cell model of oxidative stress injury. MHS cells are mouse alveolar macrophages, semi-suspended cells with typical macrophage characteristics. MHS cells in the logarithmic growth phase were injected with a certain concentration (3 × 10⁻⁶) of H2O2. 5 Cells were seeded in 96-well plates with 200 μL of 1640 complete medium per well and cultured at 37°C and 5% CO2 for 12 h. The complete medium was then removed, and 160 μL of 1640 medium was added, followed by 20 μL of OC-COF diluted with PBS at different concentrations, resulting in final OC-COF concentrations of 400, 200, 100, 50, 25, 12.5, 6.25, and 0 μg / mL. Then, 20 μL of H2O2 diluted with PBS was added, bringing the final H2O2 concentration in each well to 100 μM, with a final volume of 200 μL per well. Cells were then cultured at 37°C and 5% CO2 for another 24 h. Cells were then removed, and 15 μL of CCK8 reagent was added, followed by incubation for a further time. The OD value was measured at a detection wavelength of 450 nm. The protective effect of different concentrations of OC-COF on cells under corresponding H2O2 conditions was investigated.

[0213] like Figure 10 In a 100 μM H2O2 environment, the survival rate of cells in the positive control group (i.e., OC-COF at 0 μg / mL) was only 45%. When the concentration of OC-COF was increased to 200 μg / mL, the cell survival rate was comparable to that of the normal control group. However, at the lowest concentration of 6.25 μg / mL OC-COF, the cell survival rate was significantly higher than that of the positive control group, indicating that OC-COF can reduce the concentration of H2O2 in the cellular environment and protect cells from oxidative stimulation by ROS.

[0214] (5) In vitro antioxidant effect

[0215] MHS cells in logarithmic growth phase were injected at a rate of 3 × 10⁻⁶. 5 2 mL of OC-COF was inoculated into each well of a 6-well plate at a concentration of 100 μM H2O2. The plate was incubated for 12 hours. The original culture medium was then removed, and 1640 medium containing 100 μM H2O2 was added. Simultaneously, different concentrations of OC-COF diluted with PBS were added to achieve final concentrations of 1000, 500, and 250 μg / mL. The plates without OC-COF served as positive controls. A control without 100 μM H2O2 was also included. H2O2 and OC-COF were used as negative controls. After incubation for 24 h, the original culture medium was removed, and 1 mL of 10 mM dichlorofluorescein diacetate (DCFH-DA) prepared with phenol red-free 1640 medium was added to each well. The cells were incubated at 37 °C with 5% CO2 for 30 min, with gentle shaking every 10 min to ensure that DCFH-DA could fully interact with the cells. After incubation, the culture medium was removed, and the cells were washed 6 times with phenol red-free 1640 medium with gentle shaking to remove any unbound DCFH-DA molecules that had not entered the cells. The final wash was performed at 1000 rpm for 5 min, and the lower precipitate was collected by centrifugation. The cells were resuspended in 0.5 mL of PBS, and intracellular fluorescence was detected by BD Calibur flow cytometry within 30 min.

[0216] Quantitative analysis was performed by detecting the fluorescence intensity of reactive oxygen species in MHS cells using flow cytometry, such as... Figure 11 When 100 μM H2O2 was added to the cells, the fluorescence shift was significant compared to the negative control group. When OC-COF was added, the fluorescence shift weakened. Quantitative analysis revealed that 250 μg / mL OC-COF reduced the intracellular fluorescence intensity by 75%, 500 μg / mL OC-COF reduced it by 88%, and 1000 μg / mL OC-COF reduced it by 92%, indicating that OC-COF can significantly reduce the intracellular reactive oxygen species level.

[0217] (6) In vitro anti-apoptotic effect of OC-COF

[0218] MHS cells in logarithmic growth phase were injected at a rate of 3 × 10⁻⁶. 52 mL of the culture medium was seeded into each well of a 6-well plate and incubated for 12 h. The original culture medium was then removed, and 1800 μL of 1640 medium was added, followed by 100 μL of H2O2 diluted with PBS to bring the final H2O2 concentration in the wells to 100 μM. Simultaneously, 100 μL of OC-COF diluted with PBS at different concentrations was added to bring the final OC-COF concentrations in the wells to 1000, 500, and 250 μg / mL. Cells without OC-COF served as a positive control. Cells without 100 μM H2O2 and OC-COF served as a negative control. Cells with 10% DMSO served as a positive control for apoptosis induction. Cells stained without apoptosis induction, Annvexin V, and PI were also established. After incubation for 24 h, the cell suspension was pipetted, and cells were collected by centrifugation at 1000 rpm for 3 min. 1 mL of the culture medium was then used to seed the cells. Wash cells twice with PBS pre-cooled to 4°C. Add 100 μL of 1×Binding buffer to the cell pellet after the last wash, then add 5 μL of Annexin V-FITC, mix gently, and stain at room temperature in the dark for 30 min. Add 10 μL of PI, mix gently, and stain at room temperature in the dark for 10 min. Add 400 μL of 1×Binding buffer to each tube, mix well, and then use flow cytometry to detect the cells within 1 hour.

[0219] Flow cytometry analysis results showed that, Figure 12 When cells are in an H2O2 environment, the stimulation of reactive oxygen species (ROS) significantly enhances apoptosis. In a 100 μM H2O2 environment for 24 hours, the apoptosis rate of alveolar macrophages (MHS) was 33.3%, while the apoptosis rate of normal cells was 4.82%, indicating that ROS stimulation induces apoptosis. When OC-COF was added, 250 μg / mL OC-COF reduced the apoptosis rate by 6%, and 1000 μg / mL OC-COF reduced the apoptosis rate by 26%, indicating that OC-COF consumes H2O2, thus weakening the extracellular ROS stimulation and reducing the apoptosis rate.

[0220] (7) Lung deposition and degradation of OC-COF

[0221] Rhodamine B (RhoB) was covalently linked to the OC-COF molecule to obtain RhoB-OC-COF. RhoB-OC-COF was administered to rats via lung inhalation at a dose of 40 mg / kg. Lung tissue was dissected at 0 min before administration, and at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after administration. The fluorescence in the lungs was observed using a small animal in vivo imaging system, and the deposition and degradation of OC-COF in the lungs were evaluated based on the fluorescence intensity.

[0222] like Figure 13 As shown, there was no fluorescence in the lungs 0 min before administration, and the fluorescence intensity in the lungs was strongest 5 min after administration, indicating that OC-COF can be successfully deposited in the lungs. As the administration time increased, the fluorescence intensity in the lungs weakened, indicating that OC-COF was gradually hydrolyzed and metabolized in the lungs. After 24 h, the fluorescence intensity in the lungs was very weak, indicating that OC-COF has good biodegradability.

[0223] Example 5

[0224] (1) Preparation of tetramethylpyrazine-loaded OC-COF dry powder inhaler: Weigh 4g of tetramethylpyrazine, add 5mL of anhydrous ethanol, heat to 50℃ to dissolve, then add 1g of OC-COF (prepared in Example 1), stir at 50℃ and 400rpm for 24h, filter with a Buchner funnel, wash with an appropriate amount of anhydrous ethanol, and vacuum dry at 50℃ for 12h to obtain tetramethylpyrazine-loaded OC-COF powder, denoted as LIG@OC-COF. High performance liquid chromatography (HPLC) was used with the following conditions: water:methanol = 40%:60%, 0.6mL / min, 35℃, detection wavelength 280nm, and injection volume 10μL. The drug loading of LIG@OC-COF was determined to be 24.68±0.65%.

[0225] (2) Preparation of Roflumilast-loaded OC-COF dry powder inhaler: Weigh 150 mg of roflumilast, add 5 mL of anhydrous ethanol, heat to 40 °C to dissolve, then add 500 mg of OC-COF (prepared in Example 4), stir at 50 °C and 400 rpm for 24 h, filter with a Buchner funnel, wash with an appropriate amount of anhydrous ethanol, and vacuum dry at 40 °C for 12 h to obtain roflumilast-loaded OC-COF powder, denoted as ROF@OC-COF. High performance liquid chromatography (HPLC) was used with the following conditions: 5 mM NaH2PO4 / H2O:ACN = 20%:80%, 1 mL / min, 25 °C, detection wavelength 214 nm, and injection volume 10 μL. The drug loading of ROF@OC-COF was determined to be 5.66 ± 0.05%.

[0226] (3) Preparation of quercetin-loaded OC-COF dry powder inhaler: Weigh 100 mg of quercetin, add 5 mL of anhydrous ethanol, heat to 60 °C to dissolve, then add 500 mg of OC-COF (prepared in Example 4), stir at 60 °C and 400 rpm for 24 h, filter with a Buchner funnel, wash with an appropriate amount of anhydrous ethanol, and vacuum dry at 60 °C for 12 h to obtain quercetin-loaded OC-COF powder, denoted as QUE@OC-COF. High performance liquid chromatography (HPLC) was used with the following conditions: methanol:0.4% phosphoric acid aqueous solution = 60%:40%, 0.6 mL / min, 35 °C, detection wavelength of 370 nm, and injection volume of 20 μL. The drug loading of QUE@OC-COF was determined to be 8.94 ± 0.07%.

[0227] (4) Preparation of curcumin-loaded OC-COF dry powder inhaler: Weigh 50 mg of curcumin, add 5 mL of anhydrous ethanol, heat to 40 °C to dissolve, then add 382 mg of OC-COF (prepared in Example 4), stir at 40 °C and 400 rpm for 24 h, filter with a Buchner funnel, wash with an appropriate amount of anhydrous ethanol, and vacuum dry at 40 °C for 12 h to obtain curcumin-loaded OC-COF powder, denoted as CUR@OC-COF. The drug loading of CUR@OC-COF was measured to be 6.57 ± 0.08% at 428 nm using a UV-Vis spectrophotometer.

[0228] Example 6

[0229] (1) Characterization of LIG@OC-COF

[0230] The surface morphology of sample LIG@OC-COF (Example 5) was observed using a field emission electron microscope (FET) and energy dispersive spectroscopy (EDS) system. Figure 14 SEM results showed that the drug-loaded powder particles were still uniform cubic particles with a particle size of 2-3 μm. The drug loading process did not change the morphology and size of the original OC-COF particles. The particle size of the drug-loaded powder was within 1-5 μm, which met the particle size requirements of dry powder inhalers.

[0231] The PXRD patterns of physical mixtures of LIG, OC-COF, LIG@OC-COF, and LIG and OC-COF were determined using X-ray powder diffraction. Figure 15 It can be seen that tetramethylpyrazine has diffraction peaks at 10.01°, 16.56°, 17.22°, 20.20°, 21.15°, 22.35°, 24.80°, 27.40°, 27.88°, and 29.05°, indicating that the drug is in a crystalline state. OC-COF, on the other hand, has almost no diffraction peaks, indicating that it is in an amorphous state. The drug-loaded powder LIG@OC-COF also has no diffraction peaks, while the physical mixture of tetramethylpyrazine and OC-COF has the same diffraction peaks as the pure drug, proving that LIG@OC-COF is not a simple physical mixture, and that drug molecules may have entered the cavity of the OC-COF after loading.

[0232] Thermal analysis of physical mixtures of LIG, OC-COF, LIG@OC-COF, and LIG and OC-COF was performed using differential scanning calorimetry. Figure 16The DSC curves showed characteristic endothermic peaks at 86.30℃ and 118.93℃ for tetramethylpyrazine, indicating that the tetramethylpyrazine raw material exists in a certain crystalline form. OC-COF showed almost no endothermic peaks, further indicating its amorphous state. In the LIG / OC-COF physical mixture, OC-COF affected the endothermic peak of LIG, causing its original endothermic peak at 118.93℃ to shift slightly to the right. The drug-loaded powdered LIG@OC-COF showed almost no endothermic peaks, further confirming the interaction between LIG and OC-COF. This suggests that LIG may be encapsulated within the OC-COF cavity, rather than being a simple physical mixture.

[0233] Thermogravimetric analysis (TGA) was performed on physical mixtures of LIG, OC-COF, LIG@OC-COF, and LIG and OC-COF using a thermogravimetric analyzer. Figure 17 The tetramethylpyrazine active pharmaceutical ingredient (TMP) loses approximately 100% of its weight in the 100–150°C range because it has a low melting point of 77–80°C and is prone to sublimation, making it unstable at high temperatures. OC-COF, on the other hand, is relatively stable in the 0–200°C range, but begins to slowly decompose above 200°C. The drug-loaded powder LIG@OC-COF loses approximately 25% of its weight in the 100–150°C range, representing the loss of TMP due to heating. Weight loss above 150°C is due to the decomposition of OC-COF. The weight loss curve of the physical mixture LIG / OC-COF is basically consistent with that of the TMP, further confirming that the drug-loaded powder differs from a simple physical mixture. There is an interaction between OC-COF and TMP.

[0234] like Figure 18 In the infrared spectrum of the active pharmaceutical ingredient ligustrazine, the range of 3000–2850 cm⁻¹ is... -1 The strong absorption in the range is due to the stretching vibration absorption of methyl and methylene (CH2) groups, 2850–2700 cm⁻¹. -1 The moderate-intensity absorption within the range is the absorption band of the methylene (N-CH2) group attached to the nitrogen atom of the piperazine ring, around 1500 cm⁻¹. -1 The absorption bands within this range are C=N. The infrared spectrum of OC-COF shows the 3650–3200 cm⁻¹ range. -1 The absorption within the range is due to the stretching vibration of the hydroxyl group (OH); the characteristic absorption peak at 1750 nm is that of C=O. In the infrared spectrum of the drug-loaded powder LIG@OC-COF, the characteristic absorption peak of tetramethylpyrazine basically disappeared or weakened, while in the infrared spectrum of the physical mixture LIG / OC-COF, the characteristic absorption peak of tetramethylpyrazine still existed. This proves that there is an interaction between the drug molecules and the carrier material after drug loading, and the drug is loaded into the cavity of OC-COF, which is different from simple physical mixtures.

[0235] (2) Aerodynamic evaluation of LIG@OC-COF

[0236] The emptying rate was determined according to the method specified in the 2020 edition of the Chinese Pharmacopoeia. Empty shells of HPMC No. 3 capsules were accurately weighed and recorded as W1. Each capsule was then filled with 10 mg of LIG@OC-COF, and the mass of the capsules after filling with powder was accurately weighed and recorded as W2. Each capsule was placed in an inhalation device for an NGI experiment at a flow rate of 65 L / min. The total mass of the empty capsules and residual drug was measured and recorded as W3. The emptying rate was calculated using the following formula:

[0237]

[0238] In the formulation design, the LIG@OC-COF dry powder inhaler is available in a strength of 10 mg / capsule. Emptying rate testing results show that the emptying rate of LIG@OC-COF is 97.73 ± 0.66%, which meets the emptying rate requirement for dry powder inhalers (greater than 90%) stipulated in the 2015 edition of the Chinese Pharmacopoeia.

[0239] Accurately weigh 10 mg of LIG@OC-COF and fill it into HPMC capsules (size 3), 10 capsules per group, for a total of 3 groups. Before starting the experiment, add 15 mL of methanol-water (50:50, v / v) to the pre-separator, connect all parts of the NGI as required, connect the NGI to the flow system, and adjust the flow control valve to achieve a stable flow rate of Q through the system. out (This experiment used a flow rate of 64.8 mL / min). At this flow rate, the pressure ratio (P3 / P2) before and after the control valve should be less than 0.5 to ensure the instrument is suitable for evaluating the aerodynamics of inhaled powder aerosols under these conditions. Turn off the vacuum pump, remove the flow system, connect the inhalation device to the NGI, turn on the vacuum pump, and simultaneously set the evacuation time on the TPK2000-R to 3.7 s. Place one capsule filled with the sample into the inhalation device, press the buttons on both sides of the device with your fingers to puncture the capsule, and connect the inhalation device horizontally to the end of the artificial larynx of the NGI via the adapter. Press the start button to begin the inhalation test. After 10 s, remove the inhalation device and repeat the test with 10 capsules. Turn off the vacuum pump. After the test, wash the inhalation device, adapter, artificial larynx, pre-separator, and 8 collection trays sequentially with 50% methanol aqueous solution. Transfer the washings to volumetric flasks, add an appropriate amount of 1M NaOH solution to hydrolyze the carrier OC-COF and fully release the drug, then add an equal volume of 1M HCl, and finally bring the volume to a final volume. Centrifuge the sample at 12000 rpm, collect the supernatant, and analyze it according to the determination conditions under the "Chromatographic Conditions" section. Record the peak area. Separately, prepare a standard curve using the ligustrazine reference standard stock solution and perform the same determination. Plot the in vitro deposition distribution map of the drug administered to the lungs. Figure 19The results indicate that LIG@OC-COF has excellent in vitro lung deposition effect, and the fine particle fraction was calculated. The fine particle percentage of LIG@OC-COF is 71.02±1.80%, which meets the requirements for lung deposition rate of dry powder inhalers used to treat lung diseases.

[0240] (3) In vitro anti-inflammatory effects of LIG@OC-COF

[0241] MHS cells in logarithmic growth phase were seeded at a specific cell density in 96-well plates with complete culture medium and incubated for 12 hours to allow for full cell adhesion. The supernatant was then aspirated, and 160 μL of 1640 culture medium was added, followed by 20 μL of LPS solution diluted with PBS at a specific concentration. Simultaneously, 20 μL of different concentrations of LIG, OC-COF, and LIG@OC-COF solutions prepared with PBS were added. A positive control group was established with LPS and culture medium, while a negative control group was established with culture medium only. The total volume in all wells was 200 μL. The cells were incubated at 37°C and 5% CO2 for 12 hours. 100 μL of the cell culture supernatant was then collected and analyzed according to the ELISA kit instructions. Finally, the absorbance at 450 nm was measured using a microplate reader, and the release of pro-inflammatory factors in each group was calculated (n=3 for each group).

[0242] like Figure 20 It is known that LPS stimulation can significantly increase the release of pro-inflammatory factors TNF-α, IL-6, and IL-1β from alveolar macrophages, while the release of inflammatory factors is reduced when LIG is administered. The carrier OC-COF alone can also significantly reduce the release of inflammatory factors. The drug-loaded powder has the best effect and the most obvious in vitro anti-inflammatory effect.

[0243] from Figure 20 The values ​​of TNF-α and IL-1β in the LIG@OC-COF group (410 μg / mL) were significantly lower than those of LIG (200 μg / mL) and OC-COF (620 μg / mL) used alone, suggesting that LIG and OC-COF produced a synergistic therapeutic effect in the LIG@OC-COF group. The OC-COF of the present invention has a good effect on improving the efficacy of drugs.

[0244] (4) Evaluation of LIG@OC-COF absorption in a lung cell model

[0245] Inhaled drug particles, after being cleared by cilia and prevented from being phagocytosed by macrophages, pass through the alveoli into the pulmonary interstitium. They can then penetrate the interstitium via epithelial cells and are subsequently transported into the bloodstream. Calu-3 bronchial epithelial cells are commonly used to evaluate the characteristics of drug absorption in the lungs. This section first uses the CCK8 assay to screen for safe dosage concentrations for transport experiments. Then, a lung cell transport model is established and validated. Finally, HPLC is used to determine the concentrations of LIG and LIG@OC-COF in the Calu-3 cell model at different time points, calculating transmembrane transport and apparent permeability coefficients to elucidate the drug absorption mechanism and investigate the effect of the carrier OC-COF on LIG transport and absorption.

[0246] Based on the experimental results of CCK-8 cells, the low and high concentrations of LIG in the transport experiment were set at 50 and 200 μg / mL, respectively. After obtaining a dense monolayer cell membrane, the culture medium in the upper and lower chambers was discarded. The cell surface was washed with HBSS preheated to 37°C. Then, 0.5 mL of sample solution was added to the AP side and 1.5 mL of HBSS was added to the BL side of the cell model that met the transport conditions. Incubation was continued. At 30, 60, 90, and 120 min, 200 μL of solution was aspirated from the BL side, and the same volume of HBSS was added simultaneously. The LIG content in the receiving cell at different time points was determined by HPLC, with each concentration replicated in triplicate. The apparent permeability coefficient (Papp) was calculated, and a curve was plotted with the sampling time as the x-axis and the corresponding Papp value at the y-axis.

[0247]

[0248] V: Volume of the receiving cell (mL); Area: Surface area of ​​the cell monolayer (cm²) 2 ); time: total transfer time (s); [drug] acceaptor: drug concentration in the receiving pool; [drug] initial donor: drug concentration added.

[0249] Apparent permeability coefficient (Papp) is a parameter that measures the ability of a drug to be absorbed across a membrane. A higher Papp value indicates that the drug is more easily absorbed by the body. For example... Figure 21 Based on LIG concentrations, the Papp values ​​of high-concentration (200 μg / mL) and low-concentration (50 μg / mL) LIG and LIG@OC-COF were both greater than 1 × 10⁻⁶ in both the monolayer alveolar epithelial cell model and the monolayer bronchial epithelial cell model. -6 The cm / s indicates that ligustrazine is completely absorbed in the lungs, and OC-COF does not affect the complete absorption of ligustrazine by lung cells.

[0250] (5) Evaluation of the efficacy of LIG@OC-COF in a rat COPD model

[0251] Establishment of a COPD rat model: Based on the principle that "exertion depletes qi" (from *Suwen*), rats were forced to swim for 30 minutes daily in a constant-temperature water bath at (43±1)℃ to induce fatigue and deplete lung qi; then they were placed in a 1m... 3 Inside the fumigation chamber, rats were exposed to cigarettes for 1 hour daily, using 20 filter-free Hongmei brand cigarettes each time. Finally, the rats were placed in a hypoxic environment at normal pressure (5 hours daily), with nitrogen gas introduced and the oxygen concentration adjusted to (10.0±0.5)% using an automatic oxygen analyzer. A CO2 sensor maintained the CO2 level in the chamber at 0.03%. A temperature sensor and its control circuit maintained the chamber temperature at a constant 22℃-24℃. Vapor was absorbed by color-changing silica gel (which can be reused after drying). The device had a small hole connecting to the outside to ensure normal pressure within the chamber. The chamber was made of transparent plexiglass, allowing observation of the rats' activity, eating, and drinking. Modeling was performed 6 days a week, with a break on the 7th day, for a total of 4 weeks.

[0252] COPD Animal Grouping and Administration: Successfully modeled COPD rats were divided into a model group, a tetramethylpyrazine (TMP) gavage group, an OC-COF blank carrier DPI group, and a LIG@OC-COF DPI group. TTP was administered at a dose of 8.4 mg / kg. Based on the TMP loading of 24.68% in LIG@OC-COF, the dose for the LIG@OC-COF DPI group was 34.04 mg / kg. The dose for the OC-COF blank carrier DPI group was the same as the OC-COF dose in the LIG@OC-COF DPI group, therefore the OC-COF blank carrier dose was 25.64 mg / kg. The TTP gavage group received a dose of 42 mg / kg, five times the DPI dose. After modeling, the DPI group received daily intratracheal administration, the TTP control group received gavage, and the COPD model group and healthy groups received daily gavage with saline, for one week.

[0253] Pulmonary function testing and lung tissue morphology observation: After one week of drug administration, rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (30 mg / kg). The trachea in the neck was cut open, and an endotracheal tube with a three-way valve was inserted into the midline of the trachea. The rats were placed in a closed plethysmometer equipped with a pulmonary function testing instrument, and pulmonary function indicators such as forced expiratory volume in 0.3 seconds (FEV0.3), forced vital capacity (FVC), and (FEV0.3 / FVC)% were measured and recorded. Left upper lung tissue from each group of rats was collected, fixed in formalin, routinely dehydrated, cleared, paraffin-embedded, and embedded. Sections were prepared at 5 μm, stained with hematoxylin and eosin (HE), and the morphological changes of the lung tissue in each group of rats were observed under a light microscope. Figure 22Compared with the normal group, the lung function parameters of FEV0.3, FVC, and FEV0.3 / FVC in the model group rats were significantly decreased, proving that the COPD model was successfully established. Compared with the model group, the lung function parameters of the LIG@OC-COF DPI group were improved (P<0.001), and the lung function parameters of the ligustrazine gavage group were also improved, but the OC-COF DPI group showed no significant improvement. Figure 23 In the normal group, the alveolar structure of rats was normal, with no obvious compensatory emphysema formation and no significant thickening of the pulmonary artery walls. In the model group, the pulmonary vessels of rats were significantly dilated, with thickened vessel walls and a large number of lymphocytes and a small number of neutrophils infiltrating the surrounding area; the alveolar septa were widened, and a small number of inflammatory cells infiltrated the pulmonary interstitium; a large number of alveoli were dilated, and even alveolar walls ruptured and fused, resulting in compensatory emphysema. Compared with the model group, the LIG@OC-COF DPI group showed significantly improved pulmonary vascular smooth muscle cell proliferation and alveolar dilation, a small number of inflammatory cells infiltrated, and no obvious emphysema.

[0254] ELISA was used to detect the levels of IL-8, TNF-α, MDA, and SOD, while Western blotting was used to detect the expression of NF-κB and Nrf2. Blood was collected from the abdominal aorta of each group of rats, and ELISA was used to detect the levels of IL-8, TNF-α, MDA, and SOD in rat serum. The procedures were strictly performed according to the instructions of each kit. Lung tissue was collected from each group of rats, and lung tissue proteins were extracted and quantified using the BCA method. After sample loading, electrophoresis, membrane transfer, and blocking, the membrane was incubated with primary antibody overnight at 4°C, followed by incubation with secondary antibody at room temperature for 2 hours. After washing, chemiluminescent reagent was added, and X-ray film was exposed and developed to record the images. The results were analyzed using a gel imaging analysis system, and the relative expression levels of NF-κB and Nrf2 were calculated.

[0255] like Figure 24 Compared with the normal group, the serum TNF-α and IL-8 levels in the model group rats were significantly increased. Compared with the model group, the IL-8 and TNF-α levels in the LIG@OC-COF DPI group were significantly decreased (P<0.001), and the levels in the ligustrazine gavage group and the OC-COF group were also significantly decreased, but the decrease was most significant in the LIG@OC-COF group. Compared with the normal group, the SOD level in the model group rats was significantly decreased and the MDA level was significantly increased. Compared with the model group, the SOD level in each treatment group was significantly increased and the MDA level was significantly decreased, but the treatment effect of the LIG@OC-COF group was the best. Although there was no significant difference in IL-8 and MDA levels between the LIG group and the LIG@OC-COF group, the synergistic effect of OC-COF, due to the administration of LIG@OC-COF via dry powder inhalation, reduced the drug dosage of LIG by 5 times. Western blot detection of protein expression levels, such as... Figure 25Compared with the normal group, the model group showed increased NF-κB expression and decreased SOD and Nrf2 expression. Compared with the model group, all treatment groups showed decreased NF-κB expression and increased SOD and Nrf2 expression, but LIG@OC-COF showed the most significant effect. In summary, LIG@OC-COF DPI treatment in COPD rats significantly reduced the levels of inflammatory factors and enhanced antioxidant capacity, which is related to the NF-κB / Nrf2 signaling pathway.

[0256] (6) Efficacy evaluation of LIG@OC-COF in a rat model of acute lung injury

[0257] Establishment of an acute lung injury model in rats: Rats were lightly anesthetized with isoflurane and fixed in a supine position on a control board at a 45° angle. The rat's larynx was irradiated using a laryngoscope, and the tongue was pulled out of the mouth from the corner of the mouth using forceps. The rat's mouth was opened using a rat laryngoscope, and a micro-needle containing a solution was quickly inserted into the trachea the moment the glottis opened. The syringe was immediately pushed to deliver lipopolysaccharide solution to the rat's lungs at a dose of 8 mg / kg. The micro-needle was then removed, and the animal was placed in a vertical position and gently rotated for 30 seconds to ensure even distribution of the spray throughout the lungs, thus inducing acute lung injury in rats. An equal volume of physiological saline solution was sprayed into the normal control group using the same method.

[0258] Animal grouping and administration: Rats with successfully induced acute lung injury were randomly divided into a model group, a tetramethylpyrazine (TMP) gavage group, a dexamethasone (DEX) gavage group, a LIG@OC-COF DPI group, and an OC-COF DPI group. TTMP was administered at a dose of 8.4 mg / kg. Based on the TMP loading of 24.68% in LIG@OC-COF, the dose in the LIG@OC-COF DPI group was 34.04 mg / kg. The dose in the OC-COF blank carrier DPI group was consistent with the OC-COF dose in the LIG@OC-COF DPI group, therefore the OC-COF blank carrier dose was 25.64 mg / kg. The dose in the TTMP gavage group was 42 mg / kg, five times that of the DPI group. Dexamethasone, a commonly used clinical drug for treating acute lung injury, was administered as a positive control at a dose of 3 mg / kg. Administration was performed 2 hours after inducing the acute lung injury model in rats. The DPI group was administered the medication via airway, while the tetramethylpyrazine control group was administered it via gavage. In both the acute lung injury model group and the control group, air was injected into the lungs using the same method, and the medication was administered once. Six hours later, blood was collected from the abdominal aorta of the rats, and the rats were euthanized. The thoracic cavity was then percutaneously dissected layer by layer, and the lungs were carefully dissected. Blood samples were centrifuged at 4000 rpm for 10 min, and the supernatant was collected. Serum was stored at -20°C for the determination of inflammatory factors in ELISA experiments. Lung tissue samples were used for HE staining experiments, and Western blot was used to detect protein expression levels.

[0259] HE staining of rat lung sections: as shown Figure 26 In normal rats, no obvious abnormalities were observed in lung tissue structure. In the acute lung injury model group, the lung tissue showed extensive inflammatory cell infiltration, significant alveolar wall thickening, diffuse interstitial and alveolar edema, and even congestion and consolidation. Lung tissue pathology in the LIG@OC-COF DPI group showed significant improvement in inflammatory cell infiltration, congestion, and edema of the alveoli and interstitium. In contrast, the ligustrazine gavage group and the OC-COF DPI group showed significant improvement in interstitial congestion, slight alveolar wall thickening, and slight improvement in inflammatory cell infiltration.

[0260] Measurement of inflammatory factors in rat serum: Inflammatory factors in serum were detected using an ELISA kit, and the results are as follows. Figure 27 Compared with the normal control group, the levels of inflammatory factors IL-6, IL-1β, IL-8, and TNF-α in the model group were significantly increased after LPS induction, indicating that the release of pro-inflammatory cytokines was induced, and the rat acute lung injury model was successfully established. Compared with the model group, the levels of inflammatory factors IL-6, IL-1β, IL-8, and TNF-α in the drug-treated groups were significantly reduced. Among them, the LIG@OC-COF DPI group had the best efficacy (p < 0.001), indicating that LIG@OC-COF pulmonary inhalation can effectively reduce the inflammatory response in rats with acute lung injury. Ligustrazine and OC-COF alone can also reduce the level of inflammatory factors, and the synergistic effect of the two is the best. After LIG is loaded into OC-COF, the dose of LIG is reduced by 5 times by dry powder inhalation, and there is also a significant difference compared with the positive control group DEX.

[0261] Rat serum antioxidant markers: such as Figure 28 Compared with the control group, the plasma MDA content in the model group rats was significantly increased and the SOD content was significantly decreased, indicating that ALI can cause an imbalance in the oxidative-antioxidant balance, thereby inducing oxidative stress damage. Compared with the model group, the MDA content in each treatment group decreased and the SOD content increased. The LIG@OC-COF DPI group showed the most significant effect, indicating that LIG@OC-COF has a certain alleviating effect on oxidative stress damage in ALI rats. Intracellular antioxidant protein SOD was detected, such as... Figure 29 The model group showed decreased SOD protein expression, which increased after LIG@OC-COF treatment.

[0262] Studies on the effects of LIG@OC-COF on the rat ALI signaling pathway: such as Figure 29Compared with the control group, the expression of Nrf2 in the lung tissue of rats in the model group was decreased, while the expression of NF-κB protein was increased. Compared with the model group, the expression of NF-κB protein was significantly decreased and the expression of Nrf2 was significantly increased in all treatment groups, with LIG@OC-COF showing the most significant effect. Therefore, OC-COF has certain anti-inflammatory and good antioxidant effects. Tetramethylpyrazine also has a good anti-inflammatory effect. When OC-COF and tetramethylpyrazine are used in combination and administered directly to the lungs via dry powder inhalation, the dosage of tetramethylpyrazine is reduced by 5 times, and a good synergistic anti-inflammatory and antioxidant effect is achieved. The reduction of inflammation and oxidative stress levels in ALI rats by LIG@OC-COF may be related to the regulation of the NF-κB / Nrf2 signaling pathway. Compared with the normal control group, the expression of NF-κB protein in the model group was increased, and the difference was statistically significant. Compared with the model group, NF-κB protein expression was significantly reduced in the tetramethylpyrazine gavage group, the LIG@OC-COF DPI group, and the dexamethasone group. NF-κB protein expression was also reduced in the OC-COF DPI group, but the difference was not statistically significant. This suggests that OC-COF has a certain anti-inflammatory effect, and tetramethylpyrazine has a good anti-inflammatory effect. The combination of OC-COF and tetramethylpyrazine can exert a good synergistic anti-inflammatory effect, indicating that LIG@OC-COF's reduction of inflammatory cytokine concentration in lung tissue is related to the inhibition of NF-κB expression.

[0263] Example 7

[0264] (1) Preparation of a covalent cyclodextrin backbone (BCOF) with reactive oxygen species sensitivity

[0265] Preparation of CD-MOF: The molar ratio of γ-CD to KOH was 1:6. Appropriate amounts of γ-CD (6.48 g) and KOH (2.24 g) were weighed and dissolved in 200 mL of pure water, sonicated until completely dissolved, and then filtered through a 0.8 μm aqueous filter membrane to obtain the mother liquor. 100 mL of the mother liquor was added to 1.28 mg of PEG 20000. The solution became turbid. The mixture was thoroughly shaken and heated in a water bath (60 °C) until the precipitate completely dissolved again. After the system became clear, it was allowed to stand at room temperature to allow the crystals to fully separate and precipitate. The precipitate was centrifuged, the supernatant was discarded, and the precipitate was washed twice each with twice the volume of ethanol and methanol to remove impurities, yielding CD-MOF.

[0266] Preparation of BRAP: The reaction substrates were 4-hydroxymethylphenylboronic acid and 1,1,1-tris(hydroxymethyl)ethane. A certain amount of the reactants (2.28 g of 4-hydroxymethylphenylboronic acid) was weighed in a 1:1 molar ratio and dispersed in 30 mL of anhydrous tetrahydrofuran. Under nitrogen protection, the mixture was stirred at 500 rpm at room temperature for 24 h. Then, 600 mg of anhydrous sodium sulfate was added, and stirring was continued overnight. After the reaction was completed, the mixture was filtered through a 0.45 μm organic filter membrane, and the solvent was removed by rotary evaporation at 100 rpm and 50 °C. The mixture was then dried under nitrogen and vacuum dried at 30 °C for 2 h to obtain a white BRAP powder.

[0267] Preparation of BCOF: A certain amount of the reaction substrate (361 mg of CD-MOF) was weighed according to the molar ratio of CD-MOF:BRAP:DPC = 1:6:7 and dispersed in 5 mL of anhydrous N,N-dimethylformamide. 225 μL of triethylamine was added, and the mixture was stirred at 80 °C and 500 rpm for 24 h. After the reaction was complete, the system was allowed to stand at room temperature, and an appropriate amount of 95% ethanol was added to terminate the reaction. The mixture was stirred thoroughly, centrifuged at 4000 rpm for 5 min, and the supernatant was discarded. The precipitate was washed three times each with anhydrous ethanol and pure water, while simultaneously being ultrasonically dispersed. After washing, the precipitate was dispersed in pure water and pre-frozen at -80 °C for more than 4 h. Finally, solid powder BCOF was obtained by freeze-drying (-50 °C, 10 mTorr, 12 h). The hydrolysis rate of 5 mg / mL BCOF was still less than 5% after being placed at room temperature in pure water for 24 hours, but it could be completely hydrolyzed by sonication for 10 minutes in 100 mM H2O2 medium, which preliminarily indicates that BCOF forms and is sensitive to H2O2.

[0268] (2) Characterization of BCOF

[0269] The morphology of the samples was characterized using scanning electron microscopy (SEM). The particle size distribution and surface charge of the samples were characterized using dynamic light scattering with a Malvern viscometer-particle size analyzer. The results are as follows: Figure 30 As shown: DLS measurements revealed that BCOF particles were at the nanoscale (269.0 ± 19.2 nm), with a negative surface charge (-27.3 ± 9.92 mV) and a dispersion coefficient of 0.110 in aqueous solution. SEM analysis showed that the BCOF particles were uniform in size, approximately 100 nm, and nearly cubic in shape.

[0270] The synthesis of BRAP and BCOF was verified using a Bruker AVANCE NEO 600 1H NMR spectrometer. 1 The H-NMR spectrum indicates that ( Figure 31(a, b) The NMR spectra of the hydrolysis products of BCOF in hydrogen peroxide contain both the characteristic peaks of the BRAP cleavage product HBA (δ = 6.8–7.2 ppm; δ = 4.5 ppm) and the characteristic peak of cyclodextrin protons at δ = 5.0 ppm.

[0271] The sample was measured at wavenumbers of 4000-400 cm⁻¹ using a Thermo Nicolet IS 5 infrared spectrometer. -1 The infrared absorption spectrum is within the range. FTIR results show that ( Figure 31 .c), BCOF at 1750cm -1 The peak at 1263 cm⁻¹ represents the -C=O group. -1 and 1029cm -1 The peak at 1680-1580 cm⁻¹ represents the -CO- in the ester bond, and the peak at 1680-1580 cm⁻¹ represents the -CO- in the ester bond. -1 The presence of characteristic peaks of the benzene ring in the BRAP molecule, combined with the results of the 1H NMR spectrum, further confirms the successful synthesis of BCOF.

[0272] The crystal structure of BCOF was characterized using a Bruker D8 Advance powder X-ray diffractometer. The PXRD pattern showed that ( Figure 31 .d) The crystal peaks of CD-MOF are obvious, reflecting its specific crystal form, while the synthesized BCOF exhibits an amorphous state. This may be because the coordinating K was removed during the crosslinking process. + This disrupts the crystallinity of CD-MOF.

[0273] (3) Evaluation of hydrogen peroxide sensitivity and in vitro stability of BCOF

[0274] The hydrolysis rate of BCOF in different concentrations of H2O2 media was quantitatively analyzed by high performance liquid chromatography (HPLC). 5 mg of BCOF powder was accurately weighed and dispersed in 5 mL of PBS aqueous solution with different pH values ​​(pH = 1.2, 6.8, and 7.4) and different concentrations of H2O2 (0, 1, and 10 mM H2O2 at pH = 7.4). The mixture was incubated in a constant temperature shaking incubator at 37℃ and 100 rpm. 100 μL samples were taken at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h, and the same volume of the corresponding medium was simultaneously replenished. The samples were centrifuged at 12000 rpm for 5 min, and 50 μL of the supernatant was injected into HPLC to determine the HBA content. The cumulative release percentage was calculated based on the HBA standard curve. A hydrolysis percentage curve was plotted with time t (h) on the x-axis and cumulative release amount on the y-axis. Each experiment was performed in triplicate. The results showed that (…). Figure 32a) After 24 hours, the hydrolysis rates of BCOF in 0 mM, 1 mM, and 10 mM H2O2 media were 0.53%, 35.83%, and 89.45%, respectively, further demonstrating that BCOF possesses good concentration- and time-dependent H2O2-sensitive cleavage capabilities. Furthermore, Figure 32 The results showed that the hydrolysis rate of BCOF was less than 5% after 24 h in media with pH = 1.2, 6.8 and 7.4, indicating that BCOF has good stability in the three media and can be applied to oral drug delivery systems.

[0275] (4) Evaluation of BCOF cytotoxicity

[0276] RAW264.7 cells in logarithmic growth phase were selected for the experiment. RAW264.7 cells in logarithmic growth phase were seeded at a certain density in 96-well plates, with a final volume of 200 μL per well, and cultured for 24 h. The culture medium was removed, and 200 μL of a series of BCOF samples at concentrations of 400, 200, 100, and 50 μg / mL were added. A blank group (containing only culture medium) and a control group (cells and culture medium) were also set up. After culturing for another 24 h, 15 μL of CCK-8 solution was added to each well, and incubation was continued at 37°C for 1.5 h. Culture was terminated, and the absorbance (A) value at 450 nm was measured using a microplate reader. Cell viability was calculated using the following formula (n=6). The cytotoxicity evaluation of Caco-2 cells was performed using the same procedure as for RAW264.7 cells. The results showed that ( Figure 33 a) BCOF showed that the survival rate of both cell types was above 90% within the range of 0–400 μg / mL, indicating that BCOF has good biocompatibility within this range.

[0277]

[0278] RAW264.7 cells in logarithmic growth phase were selected for the experiment. These cells were seeded at a specific density in 96-well plates, with a final volume of 200 μL per well, and cultured for 24 h. The culture medium was then removed, and 100 μL of H2O2 diluted with DMEM was added, bringing the final H2O2 concentration in the wells to 1 mM. Next, 100 μL of BCOF and HBA sequence concentrations were added, with concentrations set at 0.33, 0.16, 0.08, and 0 μM. A blank group (containing only culture medium) and a control group (containing both cells and culture medium) were also included. After culturing for another 24 h, 15 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 1.5 h. The culture was then terminated, and the absorbance (A) at 450 nm was measured using a microplate reader. Cell viability was calculated using the formula (n=6).

[0279] The results show that ( Figure 33b): After co-incubating RAW264.7 cells with 1 mM H2O2 for 24 h, the cell viability was as low as 79.45%. The addition of 0.08, 0.16, and 0.33 μM BCOF significantly improved cell viability, increasing it to 91.39%, 96.97%, and 99.66% respectively (P<0.5). However, the addition of 0.16 and 0.33 μM HBA only increased cell viability to 82.54% and 85.52%, respectively, which was not significantly different from the negative control group (co-incubation with only H2O2). This result indicates that in an H2O2 environment, BCOF has a stronger cell-protective effect than the same dose of HBA, significantly improving cell viability. The reason may be that BCOF can be sensitively cleaved to generate HBA under the stimulation of H2O2. That is, the scavenging of H2O2 and the product HBA play a synergistic role in producing strong anti-inflammatory and antioxidant effects, which can significantly improve cell survival rate. This lays the foundation for verifying the anti-inflammatory effect of BCOF in the in vivo treatment of colitis.

[0280] (5) Biological tissue distribution of BCOF and evaluation of colon tissue targeting

[0281] An acute ulcerative colitis model was established using 6-8 week old male SPF-grade Balb / c mice. The mice were acclimatized for one week, with fasting but free access to water for 12 hours before the experiment. Then, the model mice were fed a 3.5% (w / v) DSS aqueous solution instead of regular drinking water for 7 days. During the modeling process, the mice's diurnal rhythm was maintained, the room temperature and humidity were kept constant, bedding was changed regularly, and they were fed normally. Fresh DSS drinking water was provided every two days to prevent spoilage. Starting from day 0, the same experimenter weighed and recorded the mice's weight and observed their condition and fecal characteristics daily at the same time. The Disease Activity Index (DAI) was scored based on the mice's weight, fecal blood loss, and fecal morphology. DAI is an important evaluation standard for characterizing the severity of colitis. The final score is the sum of all factors; a higher DAI score indicates more severe colitis symptoms. After DSS treatment, mice with the same reduction in weight and DAI were selected as the experimental model mice.

[0282]

[0283] For seven consecutive days, mice in the model group were given free access to a 3.5% DSS aqueous solution, and their body weight, fecal symptoms, and mental state were carefully observed and recorded daily. The results showed that ( Figure 34(ac): During the modeling process, the weight of healthy mice remained almost unchanged for 7 days, their mental state was active, and their fecal index remained almost unchanged. However, the weight of mice in the model group gradually decreased from day 4, and the weight of mice in the model group decreased sharply from day 5 to day 7. This indicates that the disease of UC deteriorated rapidly in the middle and late stages of its development. On day 7, the weight of mice in the model group decreased significantly to 74.93%, their fur became sparse and dull, their mental state became lethargic, and they developed dark red bloody stools and mucus-like loose stools. Their DAI score increased significantly to 10, and there were blood-crusted feces at the anus. Dissecting the healthy group and the model group mice and measuring their colons, the results showed that the average length of the colon in the healthy group was 9.36 cm, while the average length of the colon in the UC model group mice was only 5.46 cm, which was significantly shortened to 58.3%. All of the above indicators show that the model was successfully established.

[0284] Evaluation of BCOF distribution in biological tissues: The in vivo tissue distribution of BCOF in mice with acute ulcerative colitis was investigated using a small animal in vivo fluorescence imaging system (IVIS). After successful establishment of the acute ulcerative colitis (UC) mouse model, mice were fasted for 12 hours but allowed free access to water. They were weighed and administered an appropriate amount of Cy5-labeled BCOF by gavage. The mice were sacrificed at 2, 6, 12, and 24 hours after administration, and tissues from the heart, liver, spleen, lung, kidney, stomach, small intestine, and colon were removed. The enrichment content of nanoparticles in each organ tissue was analyzed using the mouse imaging system IVIS (n=3).

[0285] The results show ( Figure 35 BCOF was mainly distributed in the gastrointestinal tract of mice with acute ulcerative colitis, while it was rarely distributed in the heart, liver, spleen, lungs, and kidneys. At 24 hours, BCOF was almost absent in the heart, liver, spleen, lungs, and kidneys, indicating that BCOF did not accumulate in the major organs. This also indirectly confirms that BCOF has good biocompatibility with the heart, liver, spleen, lungs, and kidneys. In addition, the content of BCOF in the colon tissue reached its highest level at 6 hours, accounting for about 70.2% of the total content.

[0286] Evaluation of BCOF targeting in colonic tissue: After successful establishment of an acute ulcerative colitis mouse model, mice were randomly divided into groups (n=3), fasted for 12 hours but allowed free access to water, weighed, and administered the same dose of Cy5-labeled BCOF to the healthy group and UC group by gavage. Six hours after administration, mice were sacrificed, and colonic tissue was harvested. Imaging analysis of particle enrichment in colonic tissue was performed using an IVIS mouse imaging system to evaluate the potential of different BCOF carriers for the treatment of ulcerative colitis. (Parameter settings: excitation wavelength / emission wavelength = 646 / 660nm; pixels: 8, F / Stop: 1). Results showed ( Figure 35At 6 hours, the distribution of BCOF in the colonic tissue of colitis mice was about 5 times that of the colonic tissue of healthy mice, indicating that BCOF has a certain targeting ability to colonic inflammation sites.

[0287] Pharmacodynamic evaluation of BCOF: UC (colonial dysplasia) was induced in mice using a 3% DSS aqueous solution with free access to water. Simultaneously, mice were administered an appropriate dose of BCOF via gavage. The efficacy was initially evaluated by measuring mouse weight loss, disease index, colon morphology, colon pathological sections, and inflammatory factor expression levels. Experimental groups and administration: Control group: Healthy mice were administered physiological saline via gavage; Model group: UC model mice were administered physiological saline via gavage; BCOF group: UC model mice were administered BCOF via gavage at a dose of 150 mg / kg. Administration regimen: Model group mice had free access to 3% DSS aqueous solution from day 0, while control group mice had free access to distilled water. All mice were administered the drug according to their experimental groups from day 0, once daily for 7 consecutive days. From day 0, mouse weight and water intake were recorded daily, and fecal observation, including fecal morphology and blood in stool, was recorded. The disease index (DAI) score was also recorded. Colon length: After euthanizing the mice, the portion from the cecal-colonic junction to the anus was cut off. The colon was removed and placed on a clean white cardboard for observation of its morphology and length measurement. Colon histopathological analysis: Approximately 0.5 cm of colonic tissue was taken from 1 cm from the mouse anus, rinsed with 4°C ice-cold saline, blotted dry with filter paper, and fixed with 4% paraformaldehyde tissue fixative for subsequent HE staining. Pathological sections were prepared and observed under a microscope. The remaining colonic segment was placed at -80°C for the detection of inflammatory markers such as MPO and TNF-α.

[0288] The results show that ( Figure 36 Compared to healthy mice, mice in the UC model group showed a significant decrease in average body weight to 79.01% (P<0.001), a significant increase in average DAI score to 9.17 (P<0.001), and a significant shortening of average colon length to 5.46 cm (P<0.001) on day 7. However, compared to the UC model group, after 7 days of continuous gavage administration of BCOF particles, the UC condition in the mice improved. On day 7, the average body weight of mice in the BCOF group decreased to 86.70% (P<0.05), the average DAI score increased to 5.60 (P<0.05), and the average colon length was 6.30 cm (P<0.05). HE section analysis of colon tissue showed that ( Figure 37The colons of healthy mice exhibited normal colonic mucosal structure with abundant goblet cells and no inflammatory cell infiltration. The colons of UC model mice showed extensive inflammatory cell infiltration and a reduction in goblet cells. Compared to the UC model mice, the colons of BCOF-treated mice showed mild inflammatory cell infiltration and an increase in goblet cells. BCOF effectively reduced the lesion area in the colonic tissue of UC mice by approximately 7.5%, indicating an improvement in colonic inflammation, consistent with changes in body weight, DAI score, and colon length. Furthermore, HE section results from various organs of the mice showed… Figure 38 Compared with healthy mice, the BCOF group mice did not show obvious inflammation and necrosis in any of their organs, indicating that BCOF has good biocompatibility.

[0289] Example 8

[0290] CD-MOF and BRAP were prepared according to Example 7.

[0291] A certain amount of reaction substrate (361 mg of CD-MOF) was weighed according to a specific molar ratio of CD-MOF:BRAP:DPC and dispersed in 5 mL of anhydrous N,N-dimethylformamide. 225 μL of catalyst was added, and the mixture was stirred at a specific temperature and 500 rpm for a specific reaction time (Table 2). After the reaction was complete, the system was allowed to stand at room temperature, and an appropriate amount of 95% ethanol was added to terminate the reaction. The mixture was stirred thoroughly, centrifuged at 4000 rpm for 5 min, and the supernatant was removed. The precipitate was washed three times each with anhydrous ethanol and pure water, while simultaneously being ultrasonically dispersed. After washing, the precipitate was dispersed in pure water and pre-frozen at -80℃ for more than 4 h. Finally, solid powder BCOF was obtained by freeze-drying (-50℃, 10 mTorr, 12 h). The hydrolysis rate of 5 mg / mL BCOF was still less than 5% after being placed at room temperature in pure water for 24 hours, but it could be completely hydrolyzed by sonication for 10 minutes in 100 mM H2O2 medium, which preliminarily indicates that BCOF forms and is sensitive to H2O2.

[0292] Table 2 Preparation parameters of BCOF

[0293] CD-MOF: BRAP: DPC catalyst reaction temperature reaction time 1:4:7 Triethylamine 60℃ 24h 1:8:7 Triethylamine 60℃ 24h 1:12:7 Triethylamine 60℃ 24h 1:6:7 Triethylamine 80℃ 24h 1:6:7 Pyridine 60℃ 24h 1:6:7 Triethylamine 60℃ 12h

[0294] Example 9

[0295] (1) Preparation of BCOF-supported berberine composition

[0296] 0.02 g of berberine (Ber) was weighed, dissolved in pure water, and diluted to 6 mL to prepare a saturated Ber solution. 200 mg of BCOF was weighed and dispersed in 6 mL of the saturated Ber solution. The solution was stirred at 37 °C and 500 rpm for 6 h. After stopping stirring, the solution was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the precipitate was washed three times with 10 mL of pure water. The precipitate was then dispersed in pure water and pre-frozen at -80 °C for more than 4 h. Finally, the solid powder Ber@BCOF was obtained by freeze-drying (-50 °C, 10 mTorr, 12 h). The drug loading was determined to be 8.03% by high-performance liquid chromatography.

[0297] (2) Drug release behavior of the drug delivery system in media with different concentrations of H2O2 and different pH values

[0298] Using PBS buffer solution at pH 7.4 as the drug release medium, 5 mg of Ber@BCOF was accurately weighed and dispersed in 5 mL of PBS aqueous solution with different pH values ​​(pH 1.2, 6.8, and 7.4) and different concentrations of H2O2 (0, 1, and 10 mM H2O2 at pH 7.4). The experiment was conducted in a constant temperature shaking incubator at 37℃ and 100 rpm. 100 μL samples were taken at 15 min, 0.5 h, 1 h, 2 h, 4 h, and 8 h, and the same volume of the corresponding medium was simultaneously replenished. The samples were centrifuged at 12000 rpm for 5 min, and 80 μL of the supernatant was injected into high-performance liquid chromatography (HPLC) to determine the Ber content. The cumulative release percentage was calculated based on the Ber standard curve. A drug release curve was plotted with time t (h) on the x-axis and cumulative release amount on the y-axis. Each group of experiments was performed in triplicate.

[0299] The results show that ( Figure 39 a) With increasing H2O2 concentration and time, the release rate of Ber@BCOF gradually increased. At 8 hours, the release rates of Ber@BCOF in 0mM, 1mM, and 10mM H2O2 media were 24.98%, 62.26%, and 74.70%, respectively, demonstrating that Ber@BCOF exhibits good concentration- and time-dependent H2O2-sensitive drug release capabilities. This lays the foundation for responsive drug release in high-concentration H2O2 environments at inflammatory sites. Furthermore, Figure 40The results showed that at 8 h, the release rates of Ber@BCOF in media at pH 1.2 and pH 6.8 were 42.50% and 17.95%, respectively. Since the solubility of Ber in acidic media is higher than in neutral media, it is easier for Ber in the Ber@BCOF drug delivery system to detach from its interaction with the carrier and be released into the acidic medium at pH 1.2. Nevertheless, the release rate of Ber@BCOF in the pH 1.2 medium was still much lower than that in the 1 mM H2O2 and 10 mM H2O2 media, indicating that Ber@BCOF can be applied to oral drug delivery systems and exhibits sensitive drug release in the inflammatory environment of high concentration H2O2.

[0300] Example 10

[0301] (1) Preparation of BCOF-supported prednisolone composition

[0302] 1.5 g of prednisolone (PD) was weighed, dissolved in pure water, and diluted to 50 mL to prepare a saturated PD solution. 700 mg of BCOF was weighed and dispersed in 25 mL of the saturated PD solution. The solution was stirred at 37 °C and 500 rpm for 12 h. After stopping stirring, the solution was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the precipitate was washed three times with 10 mL of pure water. The precipitate was then dispersed in pure water and pre-frozen at -80 °C for more than 4 h. Finally, the solid powder PD@BCOF was obtained by freeze-drying (-50 °C, 10 mTorr, 12 h). The drug loading was determined to be 12.93% by high-performance liquid chromatography.

[0303] (2) Pharmacodynamic evaluation of PD@BCOF

[0304] UC (ulcerative colitis) was induced in mice using a 3.5% DSS aqueous solution with free access to drinking water. PD@BCOF was administered by gavage on day 4 of modeling. The efficacy was initially evaluated by measuring mouse weight loss, disease index, and colon length. Experimental groups and administration: Normal group: healthy mice were administered physiological saline by gavage; UC group: UC model mice were administered physiological saline by gavage; PD group: UC model mice were administered free drug by gavage at a dose of 1 mg / kg; PD@BCOF group: UC model mice were administered PD@BCOF (PD@BCOF powder dispersed in a 0.2 mL suspension with physiological saline) by gavage at a dose of 1 mg / kg. Administration regimen: Model mice had free access to 3.5% DSS aqueous solution from day 0 to day 6, while control mice had free access to distilled water. All mice were administered the drug according to their experimental groups starting from day 4, once daily by gavage. Starting from day 0, the weight and water intake of the mice were recorded for 7 consecutive days. The feces were observed, and the morphology and presence of blood in the stool were recorded. The disease index (DAI) score was also recorded. Colon length: After the mice were sacrificed on day 7, the portion from the cecum-colon junction to the anus was cut off, the mouse colon was removed, placed on a clean white cardboard, its morphology was observed, and its length was measured with a ruler.

[0305] The therapeutic effect of PD@BCOF on colitis was preliminarily evaluated using the percentage change in body weight and the DAI score. The results showed that ( Figure 40 Compared to healthy mice, mice in the UC model group showed a significant decrease in average body weight to 79.06% and a significant increase in average DAI score to 9.33 on day 7, indicating that all mice in the model group exhibited obvious colitis symptoms. However, compared to the UC model group, after gavage administration of PD and PD@BCOF particles starting from day 4, mice in the PD group showed a decrease in average body weight to 83.90% and an increase in average DAI score to 6.80 on day 7, which was not significantly different from the UC group, indicating that 1 mg / kg of free PD did not have a therapeutic effect on colitis symptoms. However, mice in the PD@BCOF group showed only a decrease in average body weight to 88.49% (P<0.05) and an increase in average DAI score to 5.50 (P<0.01) on day 7, demonstrating a significant therapeutic effect compared to the UC model group. The above results indicate that when the PD dosage is only 1 mg / kg, PD@BCOF shows twice the therapeutic effect on UC compared to free PD. This is because the BCOF carrier has good targeting of the inflamed colonic site and can enrich PD at the inflamed colonic site, thus making low-dose PD show good efficacy against UC. In other words, the BCOF carrier can reduce the dosage and toxic side effects of PD, achieving the effect of synergistic effect and reduced toxicity.

[0306] Example 11

[0307] Preparation of BCOF-loaded resveratrol composition: 6.5 g of resveratrol (RES) was weighed, dissolved in anhydrous ethanol, and diluted to 100 mL to prepare a saturated RES solution. 300 mg of BCOF was weighed and dispersed in 8 mL of saturated PD solution, and stirred at 37 °C and 500 rpm for 12 h. After stopping stirring, the mixture was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the precipitate was washed three times with 10 mL of pure water. The precipitate was dispersed in pure water and pre-frozen at -80 °C for more than 4 h. Finally, solid powder RES@BCOF was obtained by freeze-drying (-50 °C, 10 mTorr, 12 h). The drug loading was determined to be 10.30% by high performance liquid chromatography.

[0308] discuss

[0309] This invention utilizes a cyclodextrin metal-organic framework (CD-MOF) as a template, reacting oxalyl chloride with the hydroxyl groups in the CD-MOF to synthesize a covalently linked cyclodextrin framework (OC-COF) consisting of peroxyoxalate groups. BCOF is obtained by reacting BRAP with the hydroxyl groups in the CD-MOF. By controlling the particle size of the CD-MOF, OC-COF and BCOF with different particle sizes can be synthesized to meet various drug delivery requirements.

[0310] Given the continuous release of high-concentration, high-level ROS at inflammatory sites, this invention designs a hydrogen peroxide-sensitive covalent cyclodextrin backbone material as a drug carrier. This material not only significantly depletes the ROS concentration at the lesion site through reaction with hydrogen peroxide but also efficiently and targetedly delivers drugs to the inflamed site, exerting a synergistic anti-inflammatory and antioxidant effect. The peroxate bond in OC-COF and the benzene-boron bond in BCOF can react with ROS. Upon reaching the inflamed site, OC-COF / BCOF reduces ROS levels, thereby exerting an anti-inflammatory effect, alleviating the progression of inflammatory diseases, and synergistically enhancing the therapeutic effect with drugs. In other words, using the COF of this invention as a drug carrier can reduce drug dosage, improve therapeutic efficacy, and reduce drug side effects.

[0311] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (a) A hydrogen peroxide-sensitive covalent cyclodextrin framework, which is a cyclodextrin-metal-organic framework (CD-MOF) with covalently linked peroxyoxalate groups -OC(=O)-C(=O)-O-; and the hydrogen peroxide-sensitive covalent cyclodextrin framework has a regular cubic morphology; and (b) A first active ingredient loaded on the covalent cyclodextrin backbone, wherein the active ingredient is tetramethylpyrazine.

2. The pharmaceutical composition according to claim 1, characterized in that, In the pharmaceutical composition, the weight ratio of the first active ingredient to the hydrogen peroxide-sensitive covalent cyclodextrin backbone is 1 wt%-40 wt%.

3. The pharmaceutical composition according to claim 1, characterized in that, The peroxyoxalate group is generated by reacting an oxalyl crosslinking agent with a cyclodextrin-metal-organic framework.

4. The pharmaceutical composition according to claim 3, characterized in that, The oxalyl crosslinking agent is oxalyl chloride. .

5. The pharmaceutical composition according to claim 4, characterized in that, The molar ratio of the cyclodextrin-metal-organic framework to the oxaloyl crosslinking agent is 1:(1-20).

6. Use of the pharmaceutical composition of claim 1 in the preparation of a medicament for anti-inflammatory purposes.

7. Use of a hydrogen peroxide-sensitive covalent cyclodextrin backbone in the preparation of pharmaceutical compositions targeting colon tissue; The covalent cyclodextrin backbone is a cyclodextrin-metal-organic framework (CD-MOF) covalently modified by a phenylboronic acid ester crosslinking agent; and The phenylboronic acid ester crosslinking agent is BRAP. ; The cyclodextrin-metal-organic framework mentioned above is a cubic cyclodextrin-metal-organic framework (CD-MOF).

8. The use as described in claim 7, characterized in that, The pharmaceutical composition comprises: (a) The hydrogen peroxide-sensitive covalent cyclodextrin backbone as described in claim 7; and (b) The first active ingredient loaded on the covalent cyclodextrin backbone; The first active ingredient is selected from the following group: prednisolone, dexamethasone, 5-aminosalicylic acid, sulfasalazine, resveratrol, berberine, or a combination thereof.

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