Use of ezh2 as a target in macrophages in preparation of a drug for intervention in pulmonary fibrosis-related interstitial lung disease

Abstract

The application provides an application of EZH2 as a target in macrophages in preparation of a drug for intervention of a pulmonary fibrosis related interstitial lung disease, belongs to the technical field of biological medicine and pulmonary fibrosis intervention, and specifically relates to an application of EZH2 as an epigenetic regulation target in alveolar macrophages in preparation of a drug for preventing and / or reducing pulmonary fibrosis (including but not limited to idiopathic pulmonary fibrosis, connective tissue disease related pulmonary fibrosis, drug-induced pulmonary fibrosis, radiation-induced pulmonary fibrosis, and sarcoidosis related pulmonary fibrosis). The application provides an intervention idea based on a specific cell type, so as to realize prevention, reduction or inhibition of occurrence and development of pulmonary fibrosis, and provide a clear target for subsequent development of a targeted preparation.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and pulmonary fibrosis intervention technology, and relates to the application of EZH2 in macrophages as a target in the preparation of drugs for interstitial lung diseases related to pulmonary fibrosis. Background Technology

[0002] Interstitial lung disease (ILD) is a collective term for approximately 200 different diseases that can lead to inflammation and scarring of the lung tissue. ILD is characterized by progressive dyspnea, cough, hypoxia, impaired lung function, radiographically diffuse bilateral infiltrates, inflammation, fibrosis, limited patient activity, and a decline in quality of life (QOL). Idiopathic pulmonary fibrosis (IPF) is the most aggressive form of ILD, leading to progressive and permanent lung scarring and a poor prognosis. The Global Burden of Disease study indicates that ILD accounted for 0.26% of all-cause mortality in 2017, and ILD-related life loss has increased by 86% over the past 20 years. The estimated 5-year survival rate for ILD patients is 56%, while the 5-year survival rate for IPF patients is only 34%, with a median survival of only 2–4 years.

[0003] Currently, the only drugs available for treating ILD are nintedanib and pirfenidone, and these two drugs can only slow the progression of pulmonary fibrosis, not reverse existing fibrosis. Furthermore, these drugs have side effects (such as gastrointestinal reactions and photosensitive rashes), are expensive, and are not tolerated by some patients.

[0004] Therefore, finding new targets and therapies that can reverse pulmonary fibrosis with better safety and tolerability is of significant clinical importance and urgent need. Increasing research indicates that macrophages play a crucial role in the development and progression of pulmonary fibrosis. Alveolar macrophages, interstitial macrophages, and monocyte-derived alveolar macrophages can secrete pro-fibrotic factors such as TGF-β, IL-13, PDGF, and SPP1, thereby promoting fibroblast activation, myofibroblast differentiation, and collagen deposition.

[0005] EZH2 is a key catalytic subunit of the polycomb repressor complex 2 (PRC2) and participates in epigenetic modifications such as H3K27me3. While some studies suggest that total EZH2 inhibition has anti-fibrotic effects, conflicting results have been obtained in other studies. Current technology remains unclear regarding which cell types of EZH2 are more suitable as drug targets in the context of pulmonary fibrosis, and how to improve cell selectivity while preserving anti-fibrotic effects. Summary of the Invention

[0006] To address the shortcomings of existing research on EZH2 global inhibition strategies, such as insufficient cell selectivity, significant potential impact, and unclear anti-fibrotic mechanisms, this invention provides the application of EZH2 in macrophages as a target in the preparation of drugs for interstitial lung diseases related to pulmonary fibrosis. Specifically, it utilizes EZH2 in alveolar macrophages as an epigenetic regulatory target in the preparation of drugs for the prevention and / or alleviation of pulmonary fibrosis (including but not limited to idiopathic pulmonary fibrosis, connective tissue disease-related pulmonary fibrosis, drug-induced pulmonary fibrosis, radiation-induced pulmonary fibrosis, sarcoidosis-related pulmonary fibrosis, etc.). This invention provides an EZH2 intervention strategy based on specific cell types to prevent, alleviate, or inhibit the occurrence and development of pulmonary fibrosis, and provides a clear target for subsequent targeted formulation development.

[0007] The technical solution of the present invention is as follows: This invention, by inhibiting EZH2 in macrophages, preferably by inhibiting the expression and / or activity of EZH2 in macrophages, can alleviate bleomycin-induced pulmonary inflammatory infiltration, radiographic consolidation, and collagen deposition, thereby achieving intervention in pulmonary fibrosis. Simultaneously, this invention also found that inhibiting neutrophils and fibroblasts has no effect on pulmonary fibrosis lesions and may even worsen them.

[0008] The objective of this invention is achieved through the following technical solution: Application of EZH2 in macrophages as a target in the preparation of drugs to intervene in pulmonary fibrosis-related interstitial lung disease.

[0009] Furthermore, EZH2 can be used as an intervention agent, specifically small molecule inhibitors, siRNA, shRNA, antisense oligonucleotides, CRISPR / Cas or CRISPRi systems, protein degraders, or other bioactive substances that can downregulate EZH2 expression or function.

[0010] Furthermore, the target is used as a reagent to inhibit EZH2 expression and / or activity in macrophages, for the preparation of drugs to prevent, alleviate or inhibit pulmonary fibrosis.

[0011] Furthermore, the drug is a pharmaceutical composition or delivery system, and EZH2 contains a delivery carrier with macrophage targeting, preferably a liposome, lipid nanoparticle, polymer nanoparticle, exosome, or viral carrier; the carrier may carry surface modification groups that facilitate local delivery to the lungs or uptake by macrophages; the content of EZH2 in the drug is typically 0.01 wt%-95.0 wt%.

[0012] Furthermore, the preparation of the drug for intervening in pulmonary fibrosis-related interstitial lung disease is a drug for treating idiopathic pulmonary fibrosis, connective tissue disease-related pulmonary fibrosis, drug-related pulmonary fibrosis, radiation-related pulmonary fibrosis, or other pulmonary fibrosis-related interstitial lung diseases.

[0013] Furthermore, the drug can be administered via inhalation, nebulization, intratracheal administration, intravenous administration, or other methods suitable for pulmonary enrichment.

[0014] Generally, pharmaceuticals are used clinically only after being formulated into a pharmaceutical composition. The pharmaceutical composition described in this invention can be prepared according to methods known in the art, and can be formulated into any dosage form suitable for human or animal use by combining the pharmaceutical composition of this invention with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants.

[0015] Dosage forms can be liquid, solid, or semi-solid. Liquid dosage forms can include solutions (including true solutions and colloidal solutions), emulsions (including o / w, w / o, and double emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments, etc.; solid dosage forms can include tablets (including regular tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, microcapsules, pellets, suppositories, films, patches, aerosols, and sprays, etc.; semi-solid dosage forms can include ointments, gels, and pastes, etc.

[0016] The drug of this invention can be formulated into conventional formulations, sustained-release formulations, controlled-release formulations, targeted formulations, and various microparticle delivery systems. To formulate the drug of this invention into tablets, a wide range of excipients known in the art can be used, including diluents, binders, wetting agents, disintegrants, lubricants, and flow aids. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, isopropanol, etc.; binders can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, croscarmellose, croscarmellose sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; lubricants and flow aids can be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0017] Tablets can also be further processed into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets.

[0018] To formulate the drug delivery unit into capsules, the active ingredient of the present invention can be mixed with a diluent and a disintegrant, and the mixture can be placed directly into hard or soft capsules. Alternatively, the active ingredient of the present invention can be first formed into granules or microspheres with a diluent, binder, and disintegrant, and then placed into hard or soft capsules. The diluents, binders, wetting agents, disintegrants, and disintegrants used to prepare tablets of the present invention can also be used to prepare capsules of the present invention.

[0019] To prepare the drug of this invention into an injectable formulation, water, ethanol, isopropanol, propylene glycol, or mixtures thereof can be used as solvents, and appropriate amounts of commonly used solubilizers, co-solvents, pH adjusters, and osmotic pressure regulators can be added. Solubilizers or co-solvents can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; pH adjusters can be phosphates, acetates, hydrochloric acid, sodium hydroxide, etc.; osmotic pressure regulators can be sodium chloride, mannitol, glucose, phosphates, acetates, etc. If preparing a lyophilized powder for injection, mannitol, glucose, etc., can also be added as a support agent.

[0020] In addition, colorants, preservatives, flavorings, tasters or other additives may be added to pharmaceutical preparations if necessary.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. The application of EZH2 in macrophages as a target in the preparation of drugs for interstitial lung diseases related to pulmonary fibrosis, as described in this invention. Animal experimental results of this invention show that, in the context of EZH2 macrophage-specific deficiency, BLM-induced pulmonary consolidation, inflammatory infiltration and collagen deposition are significantly reduced, indicating that EZH2 is an effective target for intervening in pulmonary fibrosis.

[0022] 2. The application of EZH2 in macrophages as a target in the preparation of drugs for interstitial lung disease related to pulmonary fibrosis, as described in this invention, emphasizes a specific cell type intervention route compared with overall EZH2 inhibition, providing a clearer development direction for improving targeting and reducing the adverse effects of non-target cells.

[0023] 3. This invention can be implemented through gene-level intervention or converted into small molecule, nucleic acid drugs and targeted delivery formulations, and has good drug development translatability.

[0024] 4. This invention can form a multi-level application layout of "target use - drug composition - targeted delivery - indication refinement". Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the specific embodiments of this disclosure or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0026] Figure 1 These are representative micro-CT images of the lungs of macrophage-specific EZH2 knockout mice and control mice on day 21 after BLM induction in the experimental examples of this invention; Figure 2 This is a pathological image of mouse lung tissue from an experimental example of this invention; Figure 3 This is a graph verifying the EZH2 knockout efficiency in the experimental examples of this invention; Figure 4 This is a pathological image illustrating the effect of conditional EZH2 knockout in centriole cells on pulmonary fibrosis in an experimental example of this invention. Figure 5 This is a pathological image showing the effect of conditional EZH2 knockout in fibroblasts on pulmonary fibrosis in an experimental example of this invention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1: Application of EZH2 in macrophages as a target in the preparation of drugs to intervene in pulmonary fibrosis-related interstitial lung disease: The inhibition of EZH2 in macrophages can be achieved by using siRNA, shRNA, antisense oligonucleotides, or CRISPRi systems targeting EZH2 and delivering them via lipid nanoparticles, viral vectors, or exosomes.

[0029] Example 2: Application of EZH2 in macrophages as a target in the preparation of drugs to intervene in pulmonary fibrosis-related interstitial lung disease: By employing a delivery system with macrophage recognition ligands, preferential uptake of alveolar macrophages or mononuclear-derived alveolar macrophages is achieved, thereby improving cell type selectivity.

[0030] Example 3: Application of EZH2 in macrophages as a target in the preparation of drugs to intervene in pulmonary fibrosis-related interstitial lung disease: The traditional small molecule inhibitor approach can be replaced by protein degrading agents, inhibitory peptides, aptamers, or other bioactive molecules that can reduce EZH2 function.

[0031] Example 4: Application of EZH2 in macrophages as a target in the preparation of drugs to intervene in pulmonary fibrosis-related interstitial lung disease: Combining macrophage EZH2 intervention reagents with existing antifibrotic drugs can achieve better overall anti-inflammatory and antifibrotic effects.

[0032] Example 5: Application of EZH2 in macrophages as a target in the preparation of drugs to intervene in pulmonary fibrosis-related interstitial lung disease: According to existing process requirements, it is prepared into an atomizing agent.

[0033] Example 6: Application of EZH2 in macrophages as a target in the preparation of drugs to intervene in pulmonary fibrosis-related interstitial lung disease: It is prepared into an injectable form according to existing process requirements.

[0034] Experimental example: Myeloid-specific Ezh2 deficiency reduces BLM-induced pulmonary fibrosis.

[0035] (1) Experimental animals and genotypes: EZH2 flox / flox EZH2 was obtained by crossing with Lyz2-Cre transgenic mice. flox / flox Lyz2-Cre + Mice (denoted as cKO) and EZH2 flox / flox Lyz2-Cre - Lignal control mice; The experimental animals were C57BL / 6J background mice, 8-10 weeks old, male mice, 12 cKO mice and 12 littermate control mice; (2) Group design: It is preferred to set up four groups, namely control + saline group, cKO + saline group, control + BLM group and cKO + BLM group, to distinguish the genotype effect and the modeling effect; the mice were randomly divided into groups, with 6 mice in each group. (3) Establishment of pulmonary fibrosis model: Mice were anesthetized with 1% sodium pentobarbital (0.8g / Kg) and then injected with BLM (MCE, 2mg / kg) via tracheal spray (tracheal spray needle, Shanghai Yuyan Instruments) in a volume of 50 μL; the blank control group was injected with an equal volume of physiological saline. (4) Tissue sampling and testing: Micro-CT scans were performed on day 21 after BLM administration to assess the extent of lung lesions; mice were then sacrificed, and the left lung was fixed in 4% paraformaldehyde and embedded in paraffin for sectioning, while the right lung was used for RNA or protein extraction; fibrosis-related indicators such as Col1a1, Acta2, Fn1, and Tgfb1 were detected simultaneously. (5) Pathological evaluation: The thickness of paraffin sections is preferably 4 μm. HE staining is performed to observe inflammatory infiltration and alveolar structural changes, and Masson staining is performed to observe collagen deposition. It is preferred to further use Ashcroft score, collagen area percentage or image analysis software for quantitative evaluation, and use one-way ANOVA to compare the differences among multiple groups. (6) Experimental results: Compared with the control + BLM group, the cKO + BLM group showed a reduction in the extent of pulmonary consolidation and ground-glass opacities on micro-CT; HE staining showed a reduction in alveolar septal widening, inflammatory cell infiltration, and alveolar structural damage; Masson staining showed a significant reduction in blue collagen deposition; and the Aschroft score of the cKO group (2±0.82) was significantly lower than that of the control group (4±0.89) (P=0.0001); inhibition of EZH2 in neutrophils and fibroblasts aggravated pulmonary fibrosis or showed no change; these results indicate that reducing the level of EZH2 in macrophages can significantly alleviate the BLM-induced pulmonary fibrosis phenotype. Figure 1 These are representative micro-CT images of the lungs of macrophage-specific EZH2 knockout mice and control mice on day 21 after BLM induction. It can be seen that after BLM modeling, the knockout group (EZH2...) flox / flox Lyz2-CRE + Compared to the control group (EZH2) flox / flox Lyz2-CRE - The amount of pulmonary consolidation and ground-glass opacities was significantly reduced; Figure 2 This is a pathological image of mouse lung tissue. HE staining shows the knockout group (EZH2) after BLM modeling. flox / flox Lyz2-CRE + Compared to the control group (EZH2) flox / flox Lyz2-CRE - Inflammatory infiltration and alveolar structural destruction were reduced; Masson staining showed that the knockout group (EZH2) after BLM modeling was reduced. flox / flox Lyz2-CRE + Compared to the control group (EZH2) flox / flox Lyz2-CRE - Blue collagen deposition was significantly reduced; Figure 3This is a validation plot for EZH2 knockout efficiency. Western blot results show that the knockout group (EZH2) flox / flox Lyz2-CRE + Compared to the control group (EZH2) flox / flox Lyz2-CRE - EZH2 protein expression was significantly reduced; Figure 4 This is a pathological image showing the effect of conditional EZH2 knockout on pulmonary fibrosis in centrioles. HE staining shows the knockout group (EZH2) after BLM modeling. flox / flox Ly6g-CRE + Compared to the control group (EZH2) flox / flox Ly6g-CRE - Inflammatory infiltration and alveolar structural damage were not significantly reduced; Masson staining showed that the knockout group (EZH2) after BLM modeling was not significantly reduced. flox / flox Ly6g-CRE + Compared to the control group (EZH2) flox / flox Ly6g-CRE - No significant reduction was observed in blue collagen deposition; Figure 5 Pathological image showing the effect of conditional EZH2 knockout on pulmonary fibrosis in fibroblasts. HE staining shows the knockout group (EZH2) after BLM modeling. flox / flox Col1a2-CRE + Compared to the control group (EZH2) flox / flox Col1a2-CRE - Inflammatory infiltration and alveolar structural destruction were significantly increased; Masson staining showed that the knockout group (EZH2) after BLM modeling was significantly reduced. flox / flox Col1a2-CRE + Compared to the control group (EZH2) flox / flox Col1a2-CRE - Blue collagen deposition increased significantly.

[0036] (7) Explanation: Given that the Lyz2-Cre system is used in this embodiment, the influence of neutrophils cannot be ruled out; however, the inhibition of EZH2 expression by neutrophils has no effect on improving pulmonary fibrosis.

[0037] Results and Discussion: 1. The application of EZH2 in macrophages as a target in the preparation of drugs to intervene in pulmonary fibrosis-related interstitial lung diseases, as described in this invention, does not advocate in general terms that "overall inhibition of EZH2 is beneficial." Instead, it focuses the research on EZH2 in macrophages, thereby making the technical solution more targeted and more conducive to the subsequent development of targeted delivery drugs.

[0038] 2. This invention focuses on the application of reagents that inhibit EZH2 expression and / or activity in macrophages in the preparation of drugs for the prevention and / or relief of pulmonary fibrosis.

[0039] 3. The drug of the present invention is a pharmaceutical composition comprising an EZH2 intervention reagent and a macrophage-targeting delivery carrier, the composition being used to prevent and / or alleviate pulmonary fibrosis; wherein the disease is idiopathic pulmonary fibrosis, connective tissue disease-associated pulmonary fibrosis, drug-associated pulmonary fibrosis, radiation-associated pulmonary fibrosis, or other pulmonary fibrosis-associated interstitial lung diseases.

[0040] 4. Any of the above-described uses or compositions of the present invention, wherein the drug is administered by inhalation, nebulization, intratracheal administration or other methods of local lung enrichment.

[0041] 5. Any of the above-described uses or compositions of the present invention, wherein the EZH2 intervention reagent is used in combination with existing antifibrotic drugs, anti-inflammatory drugs or immunomodulatory drugs.

[0042] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. Application of EZH2 in macrophages as a target in the preparation of drugs to intervene in pulmonary fibrosis-related interstitial lung disease.

2. The application of EZH2 in macrophages as a target according to claim 1 in the preparation of drugs for intervening in pulmonary fibrosis-related interstitial lung diseases, characterized in that, EZH2 can be used as an intervention agent, including small molecule inhibitors, siRNA, shRNA, antisense oligonucleotides, CRISPR / Cas or CRISPRi systems, protein degraders, or other bioactive substances that can downregulate EZH2 expression or function.

3. The application of EZH2 in macrophages as a target according to claim 1 in the preparation of drugs for interstitial lung diseases related to pulmonary fibrosis, characterized in that, The target is intended to be used as a reagent to inhibit EZH2 expression and / or activity in macrophages, for the preparation of drugs to prevent, alleviate or inhibit pulmonary fibrosis.

4. The application of EZH2 in macrophages as a target according to claim 1 in the preparation of drugs for intervening in pulmonary fibrosis-related interstitial lung diseases, characterized in that, The drug is a pharmaceutical composition or delivery system. EZH2 contains a macrophage-targeting delivery carrier, including liposomes, lipid nanoparticles, polymer nanoparticles, exosomes, or viral vectors. The carrier carries surface-modifying groups that facilitate local delivery to the lungs or uptake by macrophages. The content of EZH2 in the drug is 0.01 wt%-95.0 wt%.

5. The application of EZH2 in macrophages as a target according to claim 1 in the preparation of drugs for intervening in pulmonary fibrosis-related interstitial lung diseases, characterized in that, The preparation of drugs for interstitial lung diseases related to pulmonary fibrosis is a preparation of drugs for treating idiopathic pulmonary fibrosis, connective tissue disease-related pulmonary fibrosis, drug-related pulmonary fibrosis, radiation-related pulmonary fibrosis, or other pulmonary fibrosis-related interstitial lung diseases.

6. The application of EZH2 in macrophages as a target according to claim 1 in the preparation of drugs for intervening in pulmonary fibrosis-related interstitial lung diseases, characterized in that, Drugs can be administered via inhalation, nebulization, intratracheal administration, intravenous administration, or other methods suitable for pulmonary enrichment.

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