Application of (-)-epigallocatechin gallate compound

Abstract

Relates to the field of chemical medicines, in particular to application of (-)-epigallocatechin gallate (EGCG) compounds. In particular to application of EGCG (epigallocatechin gallate) compounds in preparation of inhalation medicines for preventing and / or treating pulmonary fibrosis diseases, an inhalable medicine composition for preventing and / or treating the pulmonary fibrosis diseases and a method for preventing and / or treating the pulmonary fibrosis diseases. The EGCG compound is EGCG or a pharmaceutically acceptable salt, ester, hydrate or solvate thereof. The medicine / medicine composition taking the EGCG compound as the effective component, provided by the embodiment, can be used as an inhalable medicine for preventing and treating the pulmonary fibrosis, the administration dosage can be greatly reduced, a wider treatment window, lower adverse reaction and lower administration frequency are provided, and a new treatment choice is provided for patients with the pulmonary fibrosis.

Description

Application of (-)-epigallocatechin gallate compounds

[0001] priority

[0002] The PCT patent application claims priority of the Chinese patent with application date of November 24, 2022 and application number 202211485124.4. This patent application combines the technical solutions of the above-mentioned patent. Technical Field

[0003] The present disclosure relates to the field of chemical medicine, and in particular to the application of (-)-epigallocatechin gallate compounds. Background Art

[0004] Pulmonary fibrosis (PF) is the terminal clinical manifestation of numerous interstitial lung diseases with diverse etiologies. Characterized by persistent alveolar damage, fibroblast proliferation, and extensive extracellular matrix (ECM) deposition, PF leads to varying degrees of inflammation and fibrosis in the alveoli and interstitium, ultimately causing structural destruction of the lungs and respiratory failure. It is also known as interstitial lung disease (ILD) or diffuse parenchymal lung disease (DPLD). ILD is broadly categorized into four main types: idiopathic interstitial pneumonias (IIPs), PF caused by autoimmune or connective tissue diseases, PF caused by contact or treatment, and sarcoidosis. Idiopathic pulmonary fibrosis (IPF) is the most important and common type of idiopathic interstitial pneumonia (IIPs). Current clinical research on pulmonary fibrosis also focuses on IPF. Clinically, it manifests as progressive dyspnea accompanied by an irritating dry cough. Patients also experience symptoms such as weight loss, fatigue, general malaise, and muscle and joint pain. The median survival time is approximately 2.8 years, and the 5-year survival rate is less than 50%. Most patients die from respiratory failure and secondary lung infections.

[0005] The pathogenesis of pulmonary fibrosis involves multiple factors, including persistent damage to the alveolar epithelium, leading to dysregulation of the intracellular environment of the alveolar epithelium-mesenchymal transition (EMT), activation of signaling pathways such as Transforming Growth Factor-β (TGF-β), Wnt, and Notch, epithelial cell dysfunction and apoptosis, and scar tissue formation, all leading to disruption of the lung's internal environment. Currently, the only drugs approved worldwide for the treatment of pulmonary fibrosis are nintedanib and pirfenidone, both of which are oral preparations. While both can slow the decline in lung function, they cannot reverse disease progression, and both require high oral doses and have severe adverse reactions. Specifically, the dose of nintedanib is 150 mg BID (i.e., 150 mg twice a day), and the most common adverse reaction is gastrointestinal reaction. In clinical studies, the incidence of diarrhea is as high as 61.5%; the dose of pirfenidone is 801 mg TID (i.e., 801 mg three times a day), and the most common adverse reaction is photosensitivity, with an incidence of as high as 51% in clinical studies. These serious adverse reactions are also the most common reason why patients are forced to reduce their dose or even stop taking the drug early.

[0006] Among the numerous cytokines that promote the development of pulmonary fibrosis, TGF-β1 signaling is a key driver of collagen accumulation and fibrotic diseases, and an important regulator of inflammation and epithelial cell proliferation. However, the multifunctional nature of this cytokine has limited the development of TGF-β1 inhibitors as therapeutic agents. Studies have shown that lysyl oxidase-like 2 protein (LOXL2), an extracellular matrix protein, is rarely expressed in healthy adult tissues but is induced in various fibrotic diseases and tumors, secreted by activated fibroblasts, disease-associated smooth muscle cells, endothelial cells, and epithelial cells. Combined inhibition of LOXL2 and TGF-β1 activity by triphenolic hydroxy compounds effectively blocks pathological collagen accumulation in vivo without the toxicity associated with global inhibitors.

[0007] (-)-Epigallocatechin gallate (EGCG) is the most abundant active ingredient in green tea extract (GTE) catechins, accounting for 50-80% of the total catechin content. It is a polyphenolic flavonoid compound with the chemical formula EGCG has multiple biological functions, including preventing cell damage caused by free radicals, antibacterial properties, reducing inflammation, and preventing certain chronic diseases, including heart disease, diabetes, and some cancers. Studies have shown that EGCG can effectively inhibit lysyl oxidase-like protein-2 (LOXL2) and TGFβ receptor 1 and 2 (TGFβR1 / 2) kinases (Ying Wei et al. 2017; Harold A. Chapman et al. 2020). In the currently published studies on pulmonary fibrosis, EGCG is administered orally. Although oral administration of EGCG has certain effects in pulmonary fibrosis model animals and human patients, its intestinal absorption after oral administration is poor, and drug absorption is affected by food. After oral administration, EGCG is hydrolyzed by esterase in saliva, and after entering the blood and liver, it undergoes extensive enzymatic metabolic reactions, such as glucuronidation and sulfation. As a result, the bioavailability of oral EGCG is extremely low, and a very high dose is required to exert its anti-pulmonary fibrosis effect. For example, the dose reported in the literature by Harold A. Chapman, et al. is 600 mg; long-term high-dose medication may also pose a safety risk.

[0008] In addition, there is published evidence from human and animal studies suggesting a link between oral consumption of green tea extract (GTE) and liver damage (Jiang Hu et al., 2018; García-Cortés et al., 2016; Harrison-Dunn, 2016; Teschke et al., 2014). The European Food Safety Authority (EFSA) Scientific Cooperation Programme (ESCO) published a safety assessment of EGCG (green tea extract) in 2018, stating that interventional clinical trial evidence indicates that oral intake of EGCG at doses equal to or greater than 800 mg per day leads to significant increases in serum transaminases, suggesting liver damage and therefore cannot be considered safe. There have also been reports of hepatotoxicity in a product containing 80% green tea extract, equivalent to a daily dose of 375 mg of EGCG (EFSA, 2018). Other clinical studies have shown that oral EGCG can significantly reduce patient weight (I-Ju Chen et al. 2016). In IPF patients, underweight patients have higher mortality rates and shorter survival compared to overweight patients, with median survival rates of approximately 3.6 years and 5.8 years for patients with a BMI <25 and BMI ≥30, respectively (Mazen Alakhras et al. 2007; Nobuyasu Awano et al. 2021). Therefore, the safety and efficacy window for oral EGCG in the treatment of IPF is narrow, with the risk of ineffectiveness at low doses and toxicity at high doses. Due to the complexity of the disease, IPF patients often require dose adjustments based on disease progression, making oral EGCG inadequate for personalized medication. Following oral administration, EGCG undergoes extensive enzymatic metabolism in the body. Although EGCG is not a substrate for the drug-metabolizing CYP450 enzyme system, it can inhibit the activity of multiple CYP enzymes to varying degrees. EGCG can also inhibit drug transporters such as OATP. Therefore, EGCG may have a significant impact on the bioavailability of various drugs. Some researchers have recommended that patients receiving OATP substrate medications, especially those with a narrow therapeutic index, should avoid or at least be cautious about consuming large amounts of GTE or EGCG (Ahmed A. Albassam, 2017). In a study of 26 IPF patients, concomitant oral administration of EGCG reduced nintedanib plasma exposure by approximately 21%, a statistically significant effect that may impair efficacy (GD Marijn Veerman, et al. 2022). Therefore, the aforementioned narrow safe and effective therapeutic window and serious drug-drug interactions limit the clinical application of oral EGCG in the treatment of pulmonary fibrosis.

[0009] Currently, no inhaled drug has been successfully approved for the treatment of IPF worldwide, and there is very little development of related inhaled drugs. Pirfenidone requires very large oral doses to reach effective drug levels in the lungs. The approved dose is 801 mg TID, and the resulting plasma drug levels are very high, resulting in poor patient tolerance. Its nebulized inhalation preparation is being developed, but in the Phase II study, the low dose (50 mg / once a day) was ineffective and the study of this dose was terminated. The high-dose group still under study has a larger inhalation dose and a higher frequency of administration (100 mg / twice a day). And the current clinical performance is not as significantly better than oral administration as expected. The results of animal experiments showed that after pirfenidone was changed to inhalation administration, the half-life of the drug in the lungs was t 1 / 2 In Phase I studies, the pharmacokinetic (PK) performance of humans was basically consistent with the results of animal experiments. A 100 mg nebulized dose of pirfenidone only resulted in a maximum drug concentration of C in the alveolar epithelial lining fluid (ELF). max Compared with the oral dose (801 mg) in the alveolar epithelial lining fluid C max The drug concentration in the lungs was 35 times higher and dropped to a lower lung drug concentration level than oral administration within a short period of time. The systemic exposure was approximately 15 times lower than that reported for oral doses. Pirfenidone still had high plasma drug concentrations (807-1370 ng / mL) after inhalation administration. Therefore, pirfenidone still had a high risk of adverse reactions after switching to inhalation administration. This proves that after inhalation administration of pirfenidone, the drug quickly enters the bloodstream from the lungs, making it difficult to maintain an effective drug concentration level in the lungs for a long time, and it did not achieve the ideal effect that people originally expected.

[0010] It can be seen that not all drugs are suitable for pulmonary delivery. After inhalation administration, most drugs quickly enter the blood due to the inability to maintain effective lung retention, resulting in the inability to maintain high concentrations in the local lungs to exert lasting drug effects. In addition, due to the complex pathological mechanism of pulmonary fibrosis and rapid progression, drug development is very difficult and the failure rate is extremely high. The various growth factors involved in the development of fibrosis, such as TGF-β and platelet-derived growth factor (PDGF), have multiple physiological functions in the body and maintain body homeostasis. The toxic and side effects caused by systemic inhibition of the growth factors are common. Therefore, therapeutic drugs for pulmonary fibrosis should have a wider treatment window and achieve individualized dosing regimens for patients with mild or severe diseases. In particular, therapeutic drugs that can be delivered by inhalation and exert lasting effects in the local lungs are still an urgent need that is not met clinically.

[0011] Therefore, the existing technology still needs to be improved and developed.

[0012] Summary of the Invention

[0013] In view of the above-mentioned deficiencies in the prior art, the purpose of the present disclosure is to provide an application of (-)-epigallocatechin gallate compounds, aiming to solve the problems of the existing clinical application of EGCG in the form of oral administration in the treatment of pulmonary fibrosis, such as extremely low bioavailability, high dosage requirements, narrow safe and effective treatment window, and serious drug interactions with multiple drugs.

[0014] The technical solutions disclosed in this disclosure are as follows:

[0015] In a first aspect, the present disclosure provides a use of a (-)-epigallocatechin gallate compound in the preparation of an inhaled drug for preventing and / or treating pulmonary fibrosis, wherein the (-)-epigallocatechin gallate compound is (-)-epigallocatechin gallate or a pharmaceutically acceptable salt, ester, hydrate or solvate thereof.

[0016] In a second aspect, an inhalable pharmaceutical composition for preventing and / or treating pulmonary fibrosis comprises: a (-)-epigallocatechin gallate compound as an active ingredient, and a pharmaceutically acceptable excipient; the (-)-epigallocatechin gallate compound is EGCG or a pharmaceutically acceptable salt, ester, hydrate or solvate thereof.

[0017] In a third aspect, the present disclosure provides a method for preventing and / or treating pulmonary fibrosis, wherein the method comprises: administering the (-)-epigallocatechin gallate compound to a subject by inhalation, wherein the (-)-epigallocatechin gallate compound is (-)-epigallocatechin gallate or a pharmaceutically acceptable salt, ester, hydrate or solvate thereof.

[0018] Beneficial effects: Compared with the existing EGCG used in the form of oral administration for the treatment of pulmonary fibrosis, the present disclosure uses EGCG or its pharmaceutically acceptable salts, esters, hydrates or solvates to prepare EGCG compounds for the treatment of pulmonary fibrosis in the form of inhalation, thereby achieving the following: (1) reducing the dosage and frequency of administration; (2) reducing adverse reactions and side effects; (3) increasing the concentration and residence time of the drug in the lungs; (4) widening the safe and effective treatment window; and (5) having little effect on the bioavailability of other drugs used in combination and no drug-drug interaction with other drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a comparison of the change curves of the plasma and lung drug concentrations (Concentration, in ng / mL) of rats over time (Time, in hour) after airway aerosol administration of EGCG at different doses (inh, the doses were 1.6 mg / kg, 3.2 mg / kg, and 6.4 mg / kg, respectively).

[0020] Figure 2 is a comparison of the change curves of drug concentration (Concentration, in ng / mL) in rat plasma and lungs over time (Time, in hour) after intravenous injection (iv, dosage of 3.2 mg / kg) of EGCG and after oral administration (po, dosage of 60 mg / kg) of EGCG.

[0021] Figure 3 is a comparison of the curves of changes in plasma and lung drug concentrations over time (Time, in hours) in rats after airway atomization of EGCG solution (dosage of 0.8 mg / kg) and oral administration of EGCG solution (dosage of 60 mg / kg).

[0022] Figure 4 is a comparison of the change curves of the lung drug concentration (lung concentration, unit is ng / mL) of rats over time (Time, unit is hour) after airway aerosol administration of low-dose EGCG (dosage doses are 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, and 0.4 mg / kg) and oral administration (dosage dose is 60 mg / kg) of EGCG solution.

[0023] Figure 5 shows the effects of airway atomization and oral administration of EGCG solution on the lung weight index (in %) of mice compared with the pulmonary fibrosis model group (Kruskal-Wallis test, *: p < 0.05, i.e., statistically significant difference; **: p < 0.01, i.e., statistically very significant difference; ***: p < 0.001, i.e., statistically extremely significant difference).

[0024] Figure 6 shows the effects of airway aerosol administration and oral administration of EGCG on the body weight (in g) of mice compared with the mice in the pulmonary fibrosis model group (statistical analysis was performed using two-way ANOVA and Tukey's multiple comparison test).

[0025] Figure 7a shows the effects of airway aerosol administration and oral administration of EGCG on the degree of bleomycin-induced lung fibrosis in mice (Masson staining, 100×);

[0026] FIG7 b shows the effects of airway aerosol administration and oral administration of EGCG on bleomycin-induced pathological structural changes in mouse lung tissue (H&E staining, 100×).

[0027] Figure 8 shows the effects of airway aerosol administration of EGCG solution on alveolar inflammatory cell infiltration, hemorrhage, alveolar expansion, and alveolar fibrous exudate in mice with pulmonary fibrosis compared with oral administration.

[0028] FIG9 shows the effects of airway aerosol administration and oral administration of EGCG on the fibrosis score (Fibrosis Score) of mice compared with the pulmonary fibrosis model group.

[0029] Figure 10 shows the effects of oral gavage and airway aerosol administration of EGCG on the hydroxyproline content (in μg / μL) in the lung tissue of mice compared with the pulmonary fibrosis model group (One-Way ANOVA analysis was used for variance homogeneity analysis, combined with Dunnett's test analysis method for inter-group comparison).

[0030] Figure 11 shows the effects of oral gavage and airway aerosol administration of EGCG, as well as airway aerosol administration of the control drug pirfenidone (AP01), on the hydroxyproline content (in μg / μL) in the lung tissue of rats compared with the rats in the pulmonary fibrosis model group (using Kruskal-Wallis test, *: p < 0.05, i.e., statistically significant difference; **: p < 0.01, i.e., statistically very significant difference; ***: p < 0.001, i.e., statistically extremely significant difference; ****: p < 0.0001, i.e., statistically extremely highly significant difference).

[0031] Figure 12 shows the curve of the change in the average drug concentration (plasma concentration) of EGCG in the blood over time after oral administration of EGCG capsules (150 mg / pill, 4 capsules in total) and nebulized inhalation of EGCG solution (3 mg dose group, 10 mg dose group, 30 mg dose group) to healthy subjects. DETAILED DESCRIPTION

[0032] The present disclosure provides an application of (-)-epigallocatechin gallate compounds. To clarify the objectives, technical solutions, and effects of the present disclosure, the present disclosure is further described below. It should be understood that the specific embodiments described herein are intended only to illustrate the present disclosure and are not intended to limit the present disclosure.

[0033] In a first aspect, the present disclosure provides an embodiment of the use of a (-)-epigallocatechin gallate compound in the preparation of an inhaled drug for treating pulmonary fibrosis, wherein the (-)-epigallocatechin gallate compound is (-)-epigallocatechin gallate or a pharmaceutically acceptable salt, ester, hydrate or solvate thereof.

[0034] In some embodiments, the (-)-epigallocatechin gallate compound is (-)-epigallocatechin gallate or a pharmaceutically acceptable ester thereof; in certain embodiments, the pharmaceutically acceptable ester thereof refers to a fatty acid ester of EGCG, that is, obtained by esterifying at least one hydroxyl group in the EGCG structure with a C1-C30 carboxylic acid.

[0035] In some embodiments, the inhaled drug is formulated into a dosage form for inhalation, wherein the dosage form is selected from a solution, a suspension, an aerosol or a powder inhaler.

[0036] In some embodiments, the inhaled drug is formed by drying the (-)-epigallocatechin gallate compound to form a powder, which is then re-dissolved in a diluent.

[0037] In some embodiments, when the dosage form is a solution, the inhaled drug comprises: the (-)-epigallocatechin gallate compound as an active ingredient, a diluent, and at least one of a pH regulator, an osmotic pressure regulator, and an antioxidant, wherein the (-)-epigallocatechin gallate compound is completely dissolved in the diluent.

[0038] In some embodiments, when the dosage form is a suspension, the inhalation drug comprises: the (-)-epigallocatechin gallate compound as an active ingredient, a diluent, and at least one of a surfactant, a pH adjuster, and a tension adjuster; the (-)-epigallocatechin gallate compound or the (-)-epigallocatechin gallate compound and a carrier suitable for inhalation administration form particles suspended in the diluent.

[0039] In some embodiments, when the dosage form is an aerosol, the inhaled medicine comprises: the (-)-epigallocatechin gallate compound as an active ingredient, a diluent, a propellant, and at least one of a surfactant, a cosolvent, and a pH adjuster.

[0040] In some embodiments, when the dosage form is a powder inhaler, the inhalation drug includes: the (-)-epigallocatechin gallate compound as an active ingredient, a carrier suitable for inhalation administration, and at least one of an excipient and a surfactant.

[0041] In some embodiments, the diluent is one or more of water, ethanol and glycerol; preferably, the diluent is water.

[0042] In some embodiments, the inhaled medication has a pH of 3.0-5.0.

[0043] In some embodiments, the concentration of the (-)-epigallocatechin gallate compound in the inhaled drug is 0.1-25 mg / mL.

[0044] In some embodiments, the inhaled drug comprises the (-)-epigallocatechin gallate compound, or the inhaled drug comprises the (-)-epigallocatechin gallate compound and other anti-pulmonary fibrosis drugs. In some embodiments, the other anti-pulmonary fibrosis drugs are selected from one or more of pirfenidone, nintedanib, glucocorticoids, immunosuppressants, prostacyclin and its analogs, CTGF antibodies, Galectin-3 inhibitors, integrin antagonists, recombinant serum amyloid protein P and its analogs, PDE inhibitors, LPA antagonists, JAK kinase inhibitors, and multiple cytokine receptor TKIs.

[0045] Preferably, in some embodiments, the other anti-pulmonary fibrosis drugs are selected from pirfenidone, nintedanib, BI 1015550 ([1-({(5R)-2-[4-(5-chloropyrimidin-2-yl)piperidin-1-yl]-5-oxido-6,7-dihydrothieno[3,2-d]pyrimidin-4-yl}amino)cyclobutyl]metha nol, CAS No.: 1423719-30-5, ), one or more of treprostinil and its analogs, recombinant serum amyloid P, and LPA antagonists.

[0046] In some embodiments, the inhaled drug can also be used in combination with the other anti-fibrosis drugs mentioned above. The "combination" described in the present invention is a mode of administration, which refers to the administration of at least one dose of (-)-epigallocatechin gallate compound and at least one dose of other compound within a certain time limit, wherein both substances show pharmacological effects. The time limit is a dosing cycle, preferably within 24 hours, more preferably within 12 hours. (-)-epigallocatechin gallate compound and other anti-fibrosis drugs can be administered simultaneously or sequentially. This period includes such treatments, wherein (-)-epigallocatechin gallate compound and other anti-fibrosis drugs are administered by the same route of administration or different routes of administration. The combined administration mode of the present invention is selected from simultaneous administration, independent formulation and co-administration, or independent formulation and sequential administration.

[0047] In some embodiments, the dosage of the (-)-epigallocatechin gallate compound is 0.1-100 mg / time.

[0048] Preferably, in some embodiments, the dosage of the (-)-epigallocatechin gallate compound is 0.1-50 mg / time.

[0049] More preferably, in some embodiments, the dosage of the (-)-epigallocatechin gallate compound is 0.1-30 mg / time.

[0050] Most preferably, in some embodiments, the dosage of the (-)-epigallocatechin gallate compound is 0.1-15 mg / time.

[0051] Exemplarily, the dosage of the (-)-epigallocatechin gallate compound can be selected from 0.1 mg / time, 1 mg / time, 1.5 mg / time, 2 mg / time, 2.5 mg / time, 3 mg / time, 3.5 mg / time, 4 mg / time, 4.5 mg / time, 5 mg / time, 5.5 mg / time, 6 mg / time, 6.5 mg / time, 7 mg / time, 7.5 mg / time, 8 mg / time, 8.5 mg / time, 9 mg / time, 9.5 mg / time, 10 mg / time, 10.5 mg / time, 11 mg / time, 11.5 mg / time, 12 mg / time, 12.5 mg / time, 13 mg / time, 14 mg / time, 15 mg / time, 16 mg / time, 17 mg / time, 18 mg / time, 19 mg / time, 20 mg / time, 21 mg / time, 22 mg / time, 23 mg / time, 24 mg / time, 25 mg / time, 26 mg / time, 27 mg / time, 28 mg / time, 29 mg / time, 30 mg / time, 31 mg / time, 32 mg / time, 33 mg / time, 34 mg / time, 35 mg / time, 36 mg / time, 37 mg / time, 38 mg / time, 39 mg / time, 40 mg / time, 41 mg / time, 42 mg / time, 43 mg / time, 44 mg / time, 45 mg / time, 46 mg / time, 47 mg / time, 48 mg / time, 49 mg / time, 50 mg / time, 51 mg / time, 3mg / time, 13.5mg / time, 14mg / time, 14.5mg / time, 15mg / time, 15.5mg / time, 16mg / time, 16.5mg / time, 17mg / time, 17.5mg / time, 18mg / time, 18.5mg / time, 19mg / time, 19.5mg / time, 20mg / time, 25mg / time, 30mg / time, 35mg / time, 40mg / time, 45mg / time, 50mg / time, 55mg / time, 60mg / time, 65mg / time, 70mg / time, 75mg / time, 80mg / time, 90mg / time, 100mg / time, etc.

[0052] The oral dosage of EGCG reported in existing literature is 400mg-600mg / time. In some embodiments, the dosage of the (-)-epigallocatechin gallate compound is 1 / 1000 to 1 / 10 of the oral dosage.

[0053] Preferably, in some embodiments, the dosage of the (-)-epigallocatechin gallate compound is 1 / 600 to 1 / 10 of the oral dosage. More preferably, in some embodiments, the dosage of the (-)-epigallocatechin gallate compound is 1 / 200 to 1 / 10 of the oral dosage.

[0054] In some embodiments, the pulmonary fibrosis disease is an interstitial lung disease, which includes one or more of idiopathic interstitial pneumonia, pulmonary fibrosis caused by autoimmune or connective tissue diseases, pulmonary fibrosis associated with contact or occupational exposure, pulmonary fibrosis caused by treatment, and sarcoidosis.

[0055] In some embodiments, the idiopathic interstitial pneumonia includes idiopathic pulmonary fibrosis; the interstitial pulmonary fibrosis caused by autoimmune or connective tissue diseases includes lupus, scleroderma, polymyositis or dermatomyositis, and interstitial lung disease associated with rheumatoid arthritis; the interstitial fibrosis associated with contact or occupational exposure includes asbestosis, silicosis, and hypersensitivity pneumonitis; the interstitial fibrosis caused by treatment includes interstitial lung disease caused by chemotherapy, radiotherapy, and some drug treatments.

[0056] In a second aspect, the embodiments of the present disclosure provide an inhalable pharmaceutical composition for preventing and / or treating pulmonary fibrosis, which comprises: a (-)-epigallocatechin gallate compound as an active ingredient, and a pharmaceutically acceptable excipient; the (-)-epigallocatechin gallate compound is EGCG or a pharmaceutically acceptable salt, ester, hydrate or solvate thereof.

[0057] In some embodiments, the content of the (-)-epigallocatechin gallate compound in the inhalable pharmaceutical composition may be 0.1-100 mg, 0.1-80 mg, 0.1-70 mg, 0.1-60 mg, 0.1-50 mg, 0.1-30 mg, 0.1-15 mg, etc.

[0058] In some embodiments, the content of the (-)-epigallocatechin gallate compound in each dose of the inhalable pharmaceutical composition is 0.1-50 mg.

[0059] In some embodiments, the content of the (-)-epigallocatechin gallate compound in each dose of the inhalable pharmaceutical composition is 0.5-30 mg.

[0060] In some embodiments, the amount of the (-)-epigallocatechin gallate compound in each dose of the inhalable pharmaceutical composition is, for example, 0.5 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, 10.5 mg, 11 mg, 11.5 mg, 12 mg, 12.5 mg, 13 mg, 13.5 mg, 14 mg, 14.5 mg, 15 mg, 15.5 mg, 16 mg, 16.5 mg, 17 mg, 17.5 mg, 18 mg, 18.5 mg, 19 mg, 19.5 mg, 20 mg, 25 mg, or 30 mg.

[0061] In one embodiment, the inhalable pharmaceutical composition is prepared into an inhalation preparation by combining the (-)-epigallocatechin gallate compound and the pharmaceutically acceptable excipients using a conventional or special preparation process. The inhalation preparation is an inhalation solution, inhalation suspension, aerosol, powder inhalation or other inhalation preparation.

[0062] Furthermore, in some embodiments, the inhalation solution is a nebulized inhalation solution; the inhalation suspension is a nebulized inhalation suspension. Nebulized inhalation refers to the use of an atomizing device to disperse the pharmaceutical composition into tiny droplets, suspending them in a gas and inhaling them into the respiratory tract and lungs. Nebulized inhalation of the pharmaceutical composition can achieve both local and systemic therapeutic effects; for example, it can simultaneously exhibit strong anti-fibrotic and anti-inflammatory effects.

[0063] In some embodiments, the (-)-epigallocatechin gallate compound is first processed into a powder through a drying process, and before administration to a subject, the powder is reconstituted with the pharmaceutically acceptable excipient using a diluent and then delivered to the subject's lungs in an aerosolized form. The drying process can be freeze-drying, spray-drying, spray-freeze drying, or supercritical fluid technology, etc.

[0064] In some embodiments, the inhalable pharmaceutical composition is in the form of an inhalation solution, and the pharmaceutically acceptable excipient is selected from one or more of a surfactant, a pH regulator, an antioxidant, a preservative, an osmotic pressure regulator, a metal ion complexing agent, water, and an additive. In other embodiments, the inhalable pharmaceutical composition is in the form of an inhalation suspension, and the pharmaceutically acceptable excipient is selected from one or more of a surfactant, a pH regulator, an antioxidant, a preservative, an osmotic pressure regulator, a metal ion complexing agent, water, and an additive. Furthermore, in some embodiments, the inhalation solution or inhalation suspension contains virtually no preservatives.

[0065] In some embodiments, the concentration of the (-)-epigallocatechin gallate compound in the inhalation solution and the inhalation suspension is 0.1-35 mg / mL, 0.1-30 mg / mL, 0.1-25 mg / mL, 0.5-25 mg / mL, 0.5-15 mg / mL, 0.5-10 mg / mL, 0.5-5 mg / mL, etc.

[0066] In some embodiments, the concentration of the (-)-epigallocatechin gallate compound in the inhalation solution and the inhalation suspension is, for example, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, and 25 mg / mL.

[0067] In some embodiments, the dosage form of the inhalable pharmaceutical composition is an aerosol, and the pharmaceutically acceptable excipient is selected from one or more of a self-solvent, a surfactant, a propellant, and an additive.

[0068] Furthermore, in some embodiments, the propellant is a hydrofluoroalkane compound. Preferably, the propellant is one or both of 1,1,1,2-tetrafluoroethane (HFA134a) and 1,1,1.2,3,3,3-heptafluoropropane (HFA 227). Furthermore, in some embodiments, the additive comprises a solvent selected from one or more of glycerol, propylene glycol, polyethylene glycol, ethanol, or oleic acid; preferably, the solvent is one or both of ethanol and propylene glycol.

[0069] In some embodiments, the inhalable pharmaceutical composition is in the form of a powder inhaler, and the pharmaceutically acceptable excipients include excipients, carriers, and additives. Furthermore, in some embodiments, the excipients include one or more of sugars, sugar alcohols, starches, macromolecular polymers, fatty acids or their salts, waxes, calcium sulfate, calcium carbonate, talc, iron oxide, and light anhydrous silicic acid. The sugars include one or more of lactose, glucose, white sugar, trehalose, and sucrose; the sugar alcohols include one or more of erythritol, mannitol, and sorbitol; the macromolecular polymers include one or more of crystalline cellulose, methylcellulose, hydroxypropyl cellulose, carboxymethylcellulose calcium, hydroxypropyl methylcellulose, sodium carboxymethyl ether cellulose, pullulan, dextrin, gum arabic, agar, gelatin, tragacanth gum, sodium alginate, polyvinyl pyrrolidone, and polyvinyl alcohol; and the fatty acids may be one or more of palmitic acid, stearic acid, and oleic acid.

[0070] Preferably, in some embodiments, the excipient is selected from one or more of saccharides, sugar alcohols, macromolecular polymers, and calcium carbonate. The saccharide is lactose or sucrose; the sugar alcohol is erythritol, sorbitol, or mannitol; and the macromolecular polymer is carboxymethylcellulose calcium, pullulan, polyvinyl pyrrolidone, or methylcellulose. Most preferably, the excipient is lactose or erythritol.

[0071] In some embodiments, the carrier includes lactose, glucose, fructose, sucrose, maltose, dextran, erythritol, sorbitol, mannitol, calcium sulfate, calcium carbonate, talc or iron oxide, etc.

[0072] In some embodiments, the epigallocatechin gallate compound may be in a crystalline or amorphous state.

[0073] In some embodiments, when preventing and / or treating pulmonary fibrosis, the inhalable pharmaceutical composition is delivered to the respiratory tract and lungs of the subject through aerosol droplets or micronized particles emitted by an inhalation drug delivery device. Furthermore, in some embodiments, the average particle size of the aerosol droplets is 0.5-10 μm; illustratively, the average particle size of the aerosol droplets may be 1-8 μm, 1-5 μm, 1-3 μm, 2-3 μm, etc. Further in some embodiments, the average particle size of the micronized particles is less than or equal to 20 μm; preferably less than or equal to 10 μm, more preferably 1-9 μm, and most preferably 3-8 μm; micronized particles within this particle size range can reach the respiratory tract (such as bronchi) and lungs of the subject.

[0074] In a third aspect, an embodiment of the present disclosure provides a method for preventing and / or treating pulmonary fibrosis, wherein the method comprises: administering the (-)-epigallocatechin gallate compound to a subject by inhalation, wherein the (-)-epigallocatechin gallate compound is (-)-epigallocatechin gallate or a pharmaceutically acceptable salt, ester, hydrate or solvate thereof.

[0075] Specifically, in some embodiments, the method may include delivering an effective amount of the (-)-epigallocatechin gallate compound required for treatment to the lungs of a mammal by inhalation. Pulmonary fibrosis diseases may include: idiopathic interstitial pneumonia, pulmonary interstitial fibrosis caused by autoimmune or connective tissue diseases, pulmonary interstitial fibrosis caused by contact or treatment, and interstitial lung diseases such as sarcoidosis. Among them, the idiopathic interstitial pneumonia includes idiopathic pulmonary fibrosis. The interstitial fibrosis caused by autoimmune or connective tissue diseases includes lupus, scleroderma, polymyositis or dermatomyositis, and interstitial lung diseases related to rheumatoid arthritis. The interstitial fibrosis related to contact or occupational exposure includes asbestosis, silicosis, and hypersensitivity pneumonitis. The treatment-related interstitial fibrosis includes interstitial lung diseases caused by chemotherapy, radiotherapy, and some drug treatments.

[0076] In some embodiments, the dosage of the (-)-epigallocatechin gallate compound is 0.01-2.0 mg / kg. Specifically, the dosage of the active ingredient EGCG in the test animal in the treatment method can be 0.01-2.0 mg / kg, which is converted to a proposed clinical dosage of 0.1-100 mg for human patients. The corresponding dosage is converted with reference to the methods reported in clinical practice or literature (E Boger, et al. 2016; Ramon Hendrickx, et al. 2018; Therese Ericsson, et al. 2017). Taking into account the patient's weight and other physiological and pathological conditions, in some embodiments, the dosage for humans can be 0.1-80 mg, 0.1-70 mg, 0.1-60 mg, 0.1-50 mg, 0.1-30 mg, 0.1-15 mg, etc. In some embodiments, the dosage for human administration can be, for example, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, 10.5 mg, 11 mg, 11.5 mg, 12 mg, 12.5 mg, 13 mg, 13.5 mg, 14 mg, 14.5 mg, 15 mg, 15.5 mg, 16 mg, 16.5 mg, 17 mg, 17.5 mg, 18 mg, 18.5 mg, 19 mg, 19.5 mg, 20 mg, 25 mg, 30 mg, etc.

[0077] The oral dosage of EGCG reported in existing literature is 400mg-600mg / time. In some embodiments, the treatment method includes delivering a dose of (-)-epigallocatechin gallate compound to the patient's lungs, which may be 1 / 1000 to 1 / 10 of the oral dosage. Preferably, it may be 1 / 600 to 1 / 10 of the oral dosage. Exemplarily, it may be 1 / 200 to 1 / 10 of the oral dosage, or 1 / 50 to 1 / 10 of the oral dosage.

[0078] In some embodiments, the dosage of the (-)-epigallocatechin gallate compound can be 0.1-80 mg / time, 0.1-70 mg / time, 0.1-50 mg / time, 0.5-30 mg / time, 0.5-15 mg / time, etc.

[0079] Most preferably, the dosage of the (-)-epigallocatechin gallate compound is 0.5-15 mg / time.

[0080] For example, the dosage of the (-)-epigallocatechin gallate compound is 0.1 mg / time, 1 mg / time, 1.5 mg / time, 2 mg / time, 2.5 mg / time, 3 mg / time, 3.5 mg / time, 4 mg / time, 4.5 mg / time, 5 mg / time, 5.5 mg / time, 6 mg / time, 6.5 mg / time, 7 mg / time, 7.5 mg / time, 8 mg / time, 8.5 mg / time, 9 mg / time, 9.5 mg / time, 10 mg / time, 10.5 mg / time, 11 mg / time, 11.5 mg / time, 12 mg / time, 12.5 mg / time, 13 mg / time, 13.5mg / time, 14mg / time, 14.5mg / time, 15mg / time, 15.5mg / time, 16mg / time, 16.5mg / time, 17mg / time, 17.5mg / time, 18mg / time, 18.5mg / time, 19mg / time, 19.5mg / time, 20mg / time, 25mg / time, 30mg / time, 35mg / time, 40mg / time, 45mg / time, 50mg / time, 55mg / time, 60mg / time, 65mg / time, 70mg / time, 75mg / time, 80mg / time, 90mg / time, 100mg / time, etc.

[0081] Further, in some embodiments, the treatment method comprises delivering 0.1-100 mg of (-)-epigallocatechin gallate compound to the patient's lungs; illustratively, 0.1-80 mg of (-)-epigallocatechin gallate compound, 0.1-70 mg of (-)-epigallocatechin gallate compound, 0.1-60 mg of (-)-epigallocatechin gallate compound, 0.1-50 mg of (-)-epigallocatechin gallate compound, or 0.1-50 mg of (-)-epigallocatechin gallate compound, 0.1-30 mg of (-)-epigallocatechin gallate compound, etc. Exemplary, the pulmonary delivery dose is 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, 10.5 mg, 11 mg, 11.5 mg, 12 mg, 12.5 mg, 13 mg, 13.5 mg, 14 mg, 14.5 mg, 15 mg, 15.5 mg, 16 mg, 16.5 mg, 17 mg, 17.5 mg, 18 mg, 18.5 mg, 19 mg, 19.5 mg, 20 mg, 25 mg, 30 mg, etc.

[0082] In some embodiments, administering the (-)-epigallocatechin gallate compound to the subject by inhalation comprises: first delivering the (-)-epigallocatechin gallate compound to the lungs of the subject at a lower dose as an initial dose, and then delivering the (-)-epigallocatechin gallate compound to the lungs of the subject in an increasing dose manner according to the patient's condition.

[0083] In some embodiments, the method further comprises administering an active agent to the subject by inhalation, the active agent being administered simultaneously, separately, or sequentially with the (-)-epigallocatechin gallate compound.

[0084] In some embodiments, the active agent is selected from pirfenidone, nintedanib, glucocorticoids, immunosuppressants, prostacyclin and its analogs, CTGF antibodies, Galectin-3 inhibitors, integrin antagonists, recombinant serum amyloid protein P (referred to as recombinant pentraxin-2, such as PRM-151) and its analogs, PDE inhibitors, LPA antagonists, JAK kinase inhibitors, and one or more of various cytokine receptor TKIs. Furthermore, in some embodiments, the prostacyclin and its analogs may be selected from Treprostinil and Iloprost; the CTGF antibody may be selected from Pamrevlumab; the Galectin-3 inhibitor may be selected from GB0139; the integrin antagonist is an integrin (αvβ1, αvβ6) antagonist, and the integrin antagonist may be selected from PLN-74809; the recombinant Pentraxin-2 may be selected from PRM-151; the PDE inhibitor may be selected from BI 1015550; and the LPA antagonist may be selected from BMS-986278.

[0085] In some preferred embodiments, the active agent is selected from one or more of pirfenidone, nintedanib, BI1015550, treprostinil and its analogs, recombinant serum amyloid protein P, and LPA antagonists.

[0086] In some embodiments, the frequency of administration is 1 time / 2 days, 1 time / day or 2 times / day. That is, the (-)-epigallocatechin gallate compound is administered to the subject once every 2 days, once a day or twice a day. Further, in some embodiments, the dosage administered to the subject per day is less than or equal to 150 mg; preferably, the dosage administered to the subject per day is less than or equal to 100 mg. Exemplarily, the dosage administered to the subject per day may be 150 mg, 140 mg, 120 mg, 100 mg, 80 mg, 70 mg, 60 mg, 50 mg, 40 mg, 35 mg, 30 mg, 25 mg, 20 mg, 19.5 mg, 19 mg, 18.5 mg, 18 mg, 17.5 mg, 17 mg, 16.5 mg, 16 mg, 15.5 mg, 15 mg, 14.5 mg g, 14mg, 13.5mg, 13mg, 12.5mg, 12mg, 11.5mg, 11mg, 10.5mg, 10mg, 9.5mg, 9mg, 8.5mg, 8mg, 7. 5mg, 7mg, 6.5mg, 6mg, 5.5mg, 5mg, 4.5mg, 4mg, 3.5mg, 3mg, 2.5mg, 1mg, 0.5mg, 0.2mg, 0.1mg, etc.

[0087] In some embodiments, the inhalation administration refers to delivering the drug to the subject using an inhalation administration device loaded with the drug.

[0088] In some embodiments, the inhalation drug delivery device is an atomizer, a pressurized metered dose inhaler, a dry powder inhaler, or a soft mist inhaler.

[0089] It should be understood that the dosage dose described in the present disclosure can be the dose output from the inhalation device (i.e., the device-delivered dose); it can also be the dose delivered to the patient's lungs (i.e., the lung dose) calculated based on the delivery efficiency of the inhalation drug delivery device generally known in the art of approximately 25%-75%.

[0090] When the subject is a human, most preferably, the dosage of the (-)-epigallocatechin gallate compound is 1-15 mg / time; when administered by inhalation, the dosage of the (-)-epigallocatechin gallate compound administered to the subject is 3-50 mg / time.

[0091] The drugs / drug compositions provided by the embodiments of the present disclosure, which contain (-)-epigallocatechin gallate compounds as active ingredients, can be administered to a subject (e.g., delivered) by inhalation. After delivery, the (-)-epigallocatechin gallate compounds can be effectively deposited in the subject's lungs, resulting in higher drug concentrations in the subject's lungs and lower systemic blood drug concentrations, thereby avoiding potential side effects such as hepatotoxicity. This can create a wider gap between efficacy and toxicity, resulting in a wide therapeutic window for the drug when administered by inhalation.

[0092] Moreover, the (-)-epigallocatechin gallate compounds provided by the inhaled administration of the drug / drug composition of this embodiment at a higher lung concentration can quickly and more effectively regulate the activity of key proteins related to pulmonary fibrosis and inflammation, such as α-smooth muscle actin (α-SMA), SNAI1, type I collagen (collagen I), Fibronectin, phosphorylated SMAD3 (pSMAD3), and phosphorylated SMAD2 (pSMAD2) in lung tissue, resulting in better drug efficacy; while some proteins in lung tissue cannot be effectively inhibited after oral administration of EGCG. After inhaled administration of the drug / drug composition of this embodiment, the (-)-epigallocatechin gallate compounds can be maintained in the lungs of the subject at a higher drug concentration for a longer time, providing feasibility for reducing the frequency of administration and increasing the compliance of the subject. In addition, by inhaled administration of the drug / drug composition of this embodiment, the problem of drug-drug interactions between orally administered EGCG and other IPF therapeutic drugs is avoided.

[0093] In general, the drugs / drug compositions provided in the embodiments of the present disclosure, which contain (-)-epigallocatechin gallate compounds as active ingredients, are used as inhalable drugs for the prevention and treatment of pulmonary fibrosis. They can greatly reduce the dosage, provide a wider treatment window, lower adverse reactions, and lower dosing frequency, and will provide new treatment options for patients with pulmonary fibrosis, with great social and economic benefits.

[0094] The technical solution of the present disclosure is described in detail below through specific embodiments.

[0095] Example 1 Pharmacokinetic study of EGCG in rat plasma after airway atomization, intravenous injection, and oral administration

[0096] 1.1 Preparation

[0097] Material: EGCG, content >98wt%.

[0098] Preparation of airway aerosol drug delivery solution: Accurately weigh the EGCG compound and add solvent 1 to prepare a drug solution, a transparent and clear liquid; wherein the components of solvent 1 are purified water, citric acid and sodium citrate.

[0099] Preparation of solution for intravenous injection and oral administration: Accurately weigh the EGCG compound and add physiological saline to prepare a solution.

[0100] Apparatus: Nebulizer for liquid pulmonary drug delivery.

[0101] Animals: Clean-grade male SD rats were provided by Qinglongshan Animal Breeding Farm, Jiangning District, Nanjing, with a body weight range of 160-200 g.

[0102] 1.2 Administration method and dosage

[0103] Intratracheal atomization: Rats in each airway atomization group were anesthetized by isoflurane inhalation and fixed in a mouse holder. An anesthesia laryngoscope was used to press the base of the animal's tongue to expose the glottis. A lung micro-liquid atomizer needle (blunt) containing a certain amount of EGCG solution was gently inserted into the trachea to atomize the test sample into the lungs. The needle was then quickly withdrawn, the mouse was removed from the holder, and the head was rotated left and right to ensure that the drug solution was distributed as evenly as possible in each lung lobe. The airway atomization dose was 1.6 mg / kg, 3.2 mg / kg, and 6.4 mg / kg, with 5 mice in each dose group.

[0104] Oral gavage: Determine the dosage for each animal based on their body weight. Use a syringe of appropriate specifications and a gavage hose to draw out the appropriate dosage for each animal. Administer the drug via oral gavage at a dose of 60 mg / kg for a total of 5 animals.

[0105] Intravenous injection: Administered via tail vein of rats at a dose of 3.2 mg / kg, for a total of 5 rats.

[0106] 1.3 Sample collection and processing methods

[0107] Blood was collected from the retinal venous plexus in each experimental group before administration (0 min) and 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 6 h after administration (8 blood collection points in total), with approximately 50-100 μL of whole blood collected at each blood collection point.

[0108] Blood was collected at fixed time points, placed in sodium heparin anticoagulant tubes, centrifuged, and plasma was collected in centrifuge tubes and frozen at -70°C for testing.

[0109] 1.4 Analytical conditions and calculation methods of pharmacokinetic parameters

[0110] Mass spectrometry conditions: UPLC-MS / MS. Chromatographic conditions: HPLC. Pharmacokinetic parameters for each animal were calculated using WinNonlin pharmacokinetic software and the statistical moment method.

[0111] 1.5 Experimental results and data analysis

[0112] The pharmacokinetic parameters of EGCG in rat plasma after airway aerosol administration (inh, doses of 1.6 mg / kg, 3.2 mg / kg, and 6.4 mg / kg, respectively), intravenous administration (iv, dose of 3.2 mg / kg), and oral gavage (po, dose of 60 mg / kg) are shown in Table 1. A comparison of the time-dependent changes in drug concentrations in rat plasma and lungs following airway aerosol administration of EGCG at different doses (inh, doses of 1.6 mg / kg, 3.2 mg / kg, and 6.4 mg / kg, respectively) is shown in Figure 1. A comparison of the time-dependent changes in drug concentrations in rat plasma and lungs following intravenous administration (iv, dose of 3.2 mg / kg) and oral gavage (po, dose of 60 mg / kg) of EGCG is shown in Figure 2. The experimental results shown in Table 1 and Figures 1 and 2 show that the bioavailability of EGCG is significantly improved after inhalation (such as airway atomization); when the same plasma exposure level is reached, the dose ratio of oral administration by gavage and inhalation administration is 37.5 times different (po 60 mg / kg: inh 1.6 mg / kg), indicating that inhalation administration greatly reduces the dosage of EGCG.

[0113] Table 1 Pharmacokinetic parameters of EGCG in rat plasma after airway aerosol administration, intravenous injection, and oral administration

[0114] Example 2 Study on the changes in drug concentrations in rat plasma and lungs after airway atomization and oral administration of EGCG

[0115] As shown in Example 1, after inhalation (such as airway atomization) of 1.6 mg / kg EGCG and oral administration of 60 mg / kg EGCG, the AUC of the drug in rat plasma was 0-t Based on this, this example studies the changes in plasma and lung drug concentrations over time in rats after a single airway aerosol administration of a lower dose (0.8 mg / kg) compared to oral administration of EGCG (60 mg / kg). Both the inhalation and oral administration groups were studied using 5 rats.

[0116] 2.1 Blood Collection Method: Blood was collected via femoral artery bleeding at 30 minutes, 2 hours, 4 hours, and 6 hours after administration (a total of four blood collection points). After blood collection, the lungs were lavaged twice with 0.9 wt% normal saline injected through the trachea. The lungs were then removed, dried with filter paper, and stored in a -20°C refrigerator for testing.

[0117] 2.2 Plasma sample processing method: Take 1 mL of plasma sample and mix it with 3 mL of ethyl acetate solution, vortex and oscillate, centrifuge, evaporate the supernatant, then reconstitute it with reconstitution solution, vortex and oscillate, and take 100 μL of sample to measure the blood drug concentration.

[0118] 2.3 Lung tissue sample processing method: Take lung tissue samples, weigh them, add 3 times normal saline (w:v) to homogenize, take 1 mL of homogenate sample and mix it with 3 mL of ethyl acetate solution, vortex and oscillate, centrifuge, evaporate the supernatant, reconstitute it with reconstitution solution, vortex and oscillate, and take 100 μL of sample to measure the drug concentration in the lungs.

[0119] 2.4 Experimental results and data analysis

[0120] After inhalation administration (such as airway atomization) of EGCG solution (dosage of 0.8 mg / kg) and oral administration of EGCG solution (dosage of 60 mg / kg), the C max and AUC 0-t See Table 2 below; after airway atomization of EGCG solution (dosage of 0.8 mg / kg) and oral administration of EGCG solution (dosage of 60 mg / kg), the curves of drug concentration changes over time in rat plasma and lungs are compared as shown in Figure 3. The experimental results shown in Table 2 and Figure 3 show that after inhalation administration of 0.8 mg / kg and oral administration of 60 mg / kg EGCG solution, the drug concentration level in plasma and the curve of change over time are similar; after inhalation administration, the drug concentration in the lungs is as high as 100 times that in the plasma, indicating that the drug concentration in the lungs after inhalation administration is very high compared with the systemic circulation. Inhalation administration can further broaden the safe and effective window of EGCG in anti-pulmonary fibrosis.

[0121] Table 2 C in plasma and lung after inhalation administration of 0.8 mg / kg and oral administration of 60 mg / kg EGCG solution max and AUC 0-t

[0122] Example 3 Pharmacokinetic study of EGCG in rat plasma and lungs after low-dose airway aerosol administration and oral gavage

[0123] As described in Example 1, rats were given different doses of EGCG solution by gavage or airway aerosol administration to study the pharmacokinetic parameters of the drug in the rat lungs. The airway aerosol administration doses were 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, and 0.4 mg / kg, with 6-8 rats in each dose group. The oral administration dose was 60 mg / kg, with a total of 6 rats.

[0124] The blood collection method and sample processing method were as described in Example 2. The blood collection time points in this experiment were 5 minutes, 2 hours, 6 hours, 8 hours, 10 hours, and 16 hours after administration, and approximately 3.5 mL of whole blood was collected at each blood collection point.

[0125] 3.1 Experimental results and data analysis

[0126] The curves of drug concentration changes over time in rat lungs after airway aerosol administration of low-dose EGCG (dosages were 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, and 0.4 mg / kg) and oral administration (dosage was 60 mg / kg) of EGCG solution are shown in Figure 4. As shown in Figure 4: (1) When the inhalation dose was above 0.2 mg / kg, the highest drug concentration C max The lung drug concentration was above 1 μg / mL (e.g., 1-20 μg / mL); while the lung concentration and the average plasma drug concentration measured after oral administration were less than 100 ng / mL; this indicates that the lung drug concentration is very high after inhalation administration of EGCG solution. (2) Even at a very low inhalation dose (0.05 mg / kg), the lung drug concentration can reach a drug concentration equivalent to that after oral administration (60 mg / kg), and the inhalation dose is only 1 / 1200 of the oral dose; this indicates that the delivery of EGCG by inhalation greatly broadens the effective therapeutic dose window of the drug. (3) After airway atomization of 0.1, 0.2, and 0.4 mg / kg EGCG solution, the lung drug concentration can be maintained above the corresponding oral peak concentration (32 ng / mL) within 16 hours (greater than 50 ng / mL); this indicates that when EGCG solution is inhaled, a higher drug concentration level can be maintained in the lung for a longer period of time.

[0127] The average C in lung tissue after airway aerosol administration and oral administration of EGCG solution max and AUC 0-t See Table 3 below. The average C max and AUC 0-t The data further showed that after inhalation, the EGCG solution has a good retention effect in the lungs, preventing the drug from being rapidly absorbed into the blood circulation after administration, and greatly increasing the local drug concentration in the lung tissue. From this, it can be inferred that the anti-IPF activity of EGCG is likely driven by its high exposure in the lung tissue; thus, compared with oral administration, the safe and effective window of EGCG for inhalation administration is significantly expanded. At the same time, inhalation delivery of EGCG provides the possibility of extending the dosing interval and achieving more flexible treatment plans.

[0128] Table 3 Average C in lung tissue after airway aerosol administration and oral administration of EGCG solution max and AUC 0-t

[0129] Example 4 Effect of inhaled EGCG on bleomycin-induced pulmonary fibrosis in mice

[0130] 4.1 Experimental Animals

[0131] Fifty-six C57BL / 6J mice, SPF grade, male, 7-10 weeks old, weighing 20-35 g, were purchased from Beijing Biotechnology Co., Ltd.

[0132] 4.2 Main Reagents

[0133] Bleomycin hydrochloride for injection (Bleomycin, BLM, 15 mg / vial, batch number 20067411): Hanhui Pharmaceutical Co., Ltd.

[0134] Solvent 1: Components include purified water, citric acid, sodium citrate, transparent and clear liquid.

[0135] Solvent 2: Sodium chloride injection (batch number K21080307), purchased from Hunan Kelun Pharmaceutical Co., Ltd.

[0136] Hydroxyproline (HYP) kit: purchased from Merck Sigma-Aldrich.

[0137] 4.3 Construction of animal grouping model

[0138] The mice were randomly divided into a sham operation group (normal control group), a pulmonary fibrosis model group, an oral gavage group, and airway aerosol administration groups 1-4, with 8 mice in each group. On day 1 (abbreviated as D), the normal control group mice were given vehicle 2 (sodium chloride injection, 1 mL / kg) via intratracheal aerosol, while the mice in the other groups were given bleomycin (2 mg / kg, 1 mL / kg) via intratracheal aerosol, once in the morning and once in the afternoon, to establish the model.

[0139] 4.4 Dosage and Frequency

[0140] The oral dose for mice was 100 mg / kg (the equivalent oral dose for mice was calculated based on the FDA guidelines for body surface area conversion, based on the oral dose of 60 mg / kg in rats). Four doses, 0.2 mg / kg, 0.4 mg / kg, 0.8 mg / kg, and 1.6 mg / kg, were administered via airway aerosol. The normal control group and the pulmonary fibrosis model group received intra-airway aerosolization of the vehicle once daily, starting on day 10 after model establishment.

[0141] 4.5 Specimen Collection

[0142] On day 22, mice in each group were anesthetized with intraperitoneal injection of chloral hydrate (50 mg / mL, 0.1 mL / 10 g) and sacrificed by exsanguination from the abdominal aorta. All animals underwent autopsy, lung tissue was preserved, and whole lung weight was measured (including animals found dead or euthanized at the point of death). The left lung was excised and stored below -60°C for hydroxyproline content determination. The remaining right lung tissue and bronchioles were fixed in 10% neutral buffered formalin solution and subjected to routine histological processing: paraffin embedding, sectioning, slide preparation, and HE and Masson staining.

[0143] 4.6 Detection indicators

[0144] (1) Lung coefficient

[0145] Lung coefficient = lung weight (mg) / body weight (g). In the model group, the massive infiltration of inflammatory cells in the early stage, the massive proliferation of fibroblasts in the late stage, and the excessive deposition of collagen can all lead to an increase in the lung coefficient. Therefore, the lung coefficient can indirectly reflect the degree of inflammation and fibrosis in mice with pulmonary fibrosis.

[0146] (2) Mouse weight

[0147] Animals were weighed upon arrival, before grouping, on the day of modeling, after the first dose, and every three days thereafter. Animals were also weighed before planned euthanasia and when found dead or dying. Changes in mouse body weight can indirectly reflect the effects of drug administration on mice. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test.

[0148] (3) Pathological examination

[0149] Light microscopy was used to grade lung lesions, including inflammatory cell infiltration and fibrosis, on hematoxylin and eosin (HE)-stained sections. Masson staining was used to assess the degree of lung fibrosis. Fibrosis, inflammatory cell infiltration, alveolar hemorrhage, dilatation, epithelial hyperplasia, and fibrous exudates were diagnosed and classified using standardized terminology using a 4-grade system (mild, mild, moderate, and severe).

[0150] (4) Determination of collagen content in lung tissue

[0151] That is, hydroxyproline content determination, hydroxyproline in lung tissue is detected according to the operating instructions of the kit.

[0152] (5) Statistical analysis

[0153] The results can be determined by any statistical test method known in the art, such as the Student's t test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test. SPSS (13.0) statistical software package was used for test analysis. The results are expressed as mean ± standard error. Overall differences were evaluated. One-way ANOVA analysis was used to analyze the homogeneity of variance between group means, and Dunnett's test was used for inter-group comparisons. When the normality test of the parameter failed, Kruskal-Wallis was used for inter-group comparisons. Two-way ANOVA and Tukey's test were used for multiple comparisons between groups. Differences considered to be statistically significant are indicated with asterisks (*P < 0.05; **P < 0.01; ***P < 0.001).

[0154] 4.7 Experimental Results and Data Analysis

[0155] 4.7.1 Effect of inhaled EGCG on the lung coefficient in mice with bleomycin-induced pulmonary fibrosis

[0156] Compared with the pulmonary fibrosis model group, the effects of airway aerosol administration of EGCG solution and oral administration of EGCG solution on the lung weight index of mice (using Kruskal-Wallis test, *: p < 0.05, i.e., statistically significant difference; **: p < 0.01, i.e., extremely statistically significant difference; ***: p < 0.001, i.e., extremely statistically significant difference) are shown in Figure 5. After modeling, the lung index of mice in the model group was significantly higher than that of the normal control group (**: P < 0.01). As shown in Figure 5, compared with the lung index of mice in the model group, the lung index of mice in each airway aerosol administration group and oral administration group was significantly reduced, especially the lung index of diseased mice in the airway aerosol administration group with a dose of 0.2 mg / kg and 0.4 mg / kg (**: P < 0.01; ***: P < 0.001), which was significantly reduced. However, the oral administration group did not form a significant difference with the model group.

[0157] 4.7.2 Effect of inhaled EGCG on body weight in mice with bleomycin-induced pulmonary fibrosis

[0158] Compared with the mice in the pulmonary fibrosis model group, the effects of airway aerosol administration of EGCG and oral administration on the body weight of mice (statistically analyzed by using two-way ANOVA and Tukey's multiple comparison test) are shown in Figure 6. As shown in Figure 6: (1) After bleomycin induction, the body weight of mice in each group decreased significantly. (2) When the experimental drug was administered orally or inhaled starting on D10, the body weight of mice showed an upward trend, but the body weight of the oral administration group increased slowly, while the body weight of mice in the 0.2 mg / kg and 0.4 mg / kg airway aerosol administration groups could basically return to the baseline level on D22, and formed a significant difference with the model group (*P < 0.05; **P < 0.01; ***P < 0.001). This shows that compared with oral administration (such as oral administration), inhalation administration of an appropriate amount of EGCG has less effect on the body weight of mice with pulmonary fibrosis, that is, inhalation administration of an appropriate amount of EGCG can reduce the side effects on mice with pulmonary fibrosis.

[0159] 4.7.3 Effects of inhaled EGCG solution on pathological changes in lung tissue of mice with bleomycin-induced pulmonary fibrosis

[0160] On D22, the lung tissue sections of the healthy control group (control) mice, the pulmonary fibrosis model (model) group mice, the oral gavage 100 mg / kg (i.e., po 100) EGCG group mice, and the inhalation administration groups of each dose (inh.0.2, inh.0.4, inh.0.8, inh.1.6) mice were subjected to Masson's staining and H&E staining, and the results are shown in Figures 7a and 7b, respectively.

[0161] As shown in Figures 7a and 7b, the lung tissue structure of the healthy control / blank group (control) mice was clear, the alveolar septa were uniform, there was no water edema, no obvious myofibroblasts, no inflammation or pulmonary fibrosis, and no obvious exudation in the alveolar cavity; the alveolar structure of the pulmonary fibrosis model group (model) mice was severely damaged, with severe atrophy and collapse, thickened alveolar septa, hyaline membrane formation, and obvious alveolar congestion. A large number of inflammatory cells infiltrated around the small airways, the vascular endothelium was damaged, fibroblasts proliferated abnormally, collagen fibers were significantly deposited in the pulmonary interstitium, and the lung tissue became solid.

[0162] Furthermore, Masson's staining results, shown in Figure 7a, indicate that compared with the model group, EGCG administered via airway aerosol significantly reduced collagen deposition, with a clear dose-response relationship observed across all inhaled doses. In particular, inhaled doses above 0.4 mg / kg significantly reduced the degree of pulmonary fibrosis in mice, with doses of 0.8 mg / kg and 1.6 mg / kg achieving a level close to that of healthy control mice. Oral administration of EGCG slightly reduced collagen deposition in the lungs of mice, but this did not significantly differ from the model group, and the effect was significantly weaker than that of airway aerosol administration. H&E staining results, shown in Figure 7b, indicate that airway aerosol administration of EGCG significantly reduced alveolar interstitial proliferation and restored alveolar morphology, with the most pronounced effects observed at doses of 0.4 mg / kg and 0.8 mg / kg. Oral administration of EGCG slightly improved histological changes in the lungs, but this was significantly weaker than that of airway aerosol administration and did not significantly differ from the model group.

[0163] 4.7.4 Effects of inhaled EGCG solution on alveolar inflammation and pulmonary fibrosis in mice with bleomycin-induced pulmonary fibrosis

[0164] Compared with oral administration, the effects of airway aerosol administration of EGCG on alveolar inflammatory cell infiltration, hemorrhage, alveolar expansion, and alveolar fibrous exudation in mice with pulmonary fibrosis are shown in Figure 8. The comparison of the alveolar inflammation scores (Score) of each group shown in Figure 8 shows that: (1) Compared with the model group, inhalation of EGCG solution significantly inhibited the further development of alveolar inflammation in the lung tissue, but there was no significant difference between the oral administration group and the pulmonary fibrosis model group, and there was even a tendency to aggravate alveolar hemorrhage and alveolar epithelial hyperplasia, indicating that oral administration of EGCG solution had a poor inhibitory effect on alveolar inflammation. (2) In terms of inhibiting alveolar expansion, all inhalation administration groups showed significant differences from the pulmonary fibrosis model group (the score of inh 1.6 mg / kg was *: p < 0.05, the scores of inh 0.2 mg / kg and inh 0.8 mg / kg were both **: p < 0.01, and the score of inh 0.4 mg / kg was ****: p < 0.0001), among which the inhalation administration group with a dose of 0.4 mg / kg had the best effect (score ****: p < 0.0001). (3) In terms of inhibiting alveolar hemorrhage, the inhalation administration groups with doses of 0.2 mg / kg and 0.4 mg / kg of EGCG solution were significantly better than the oral administration group. (4) In terms of inhibiting alveolar fibrous exudation, the inhalation administration group with a dose of 1.6 mg / kg of EGCG solution showed significant differences from the pulmonary fibrosis model group. (5) In terms of inhibiting inflammatory cell infiltration, oral administration (such as gavage) of EGCG solution had no anti-inflammatory effect, while the inhalation administration groups of all doses reduced the infiltration of inflammatory cells, especially the inhalation administration group of EGCG solution at a dose of 0.4 mg / kg had the best anti-inflammatory effect (score*: p < 0.05).

[0165] Comparison of mice in the pulmonary fibrosis model group, the effects of airway aerosol administration of EGCG and oral administration on the fibrosis score of mice are shown in Figure 9. The comparison of the pulmonary fibrosis degree scores shown in Figure 9 shows that: The oral administration group and the inhalation administration groups of each dose can significantly reduce the pulmonary fibrosis score of mice. When the inhalation dose is above 0.4 mg / kg, the pulmonary fibrosis can be significantly reduced (score is *p < 0.05), and the inhalation administration groups of 0.2 mg / kg, 0.4 mg / kg, and 0.8 mg / kg showed a good dose-effect relationship. However, the oral administration group did not form a significant difference with the pulmonary fibrosis model group.

[0166] These results indicate that even at a relatively low inhaled dose (0.2 mg / kg), EGCG solution can produce anti-inflammatory effects in the lungs. The anti-fibrotic effect gradually increases with increasing inhaled doses, and the anti-fibrotic effect is particularly pronounced at a dose of 1.6 mg / kg. This suggests that the effective dose range for inhaled EGCG is very wide.

[0167] 4.7.5 Effect of inhaled EGCG solution on hydroxyproline content in lung tissue of mice with pulmonary fibrosis

[0168] Table 4 shows the data on the inhibition of bleomycin-induced hydroxyproline content by airway aerosol administration of EGCG and oral administration of EGCG. Figure 10 shows the effects of oral administration of EGCG and airway aerosol administration on hydroxyproline content in lung tissue of mice compared with the pulmonary fibrosis model group (one-way ANOVA analysis for homogeneity of variance, combined with Dunnett's test for inter-group comparison). Table 4 shows that on day 22, hydroxyproline content in lung tissue of mice with pulmonary fibrosis was significantly increased (***: p < 0.001). Airway aerosol administration of 0.2 mg / kg, 0.4 mg / kg, and 0.8 mg / kg EGCG solution dose-dependently reduced hydroxyproline content in lung tissue. Table 4 and Figure 10 show that, particularly at the inhalation dose of 0.8 mg / kg, inhalation of EGCG solution significantly reduced hydroxyproline content in lung tissue compared with oral administration (***: p < 0.001). The drug exposure in plasma after the inhalation (such as airway atomization) dose of 0.8 mg / kg described in Example 2 is equivalent to the oral (such as gavage) dose of 60 mg / kg EGCG solution in rats (equivalent to an oral dose of 100 mg / kg in mice). One-Way ANOVA analysis method was used for variance homogeneity analysis, and Dunnett's test analysis method was combined for inter-group comparison. Unless otherwise specified, the statistical analysis method used was the Student's T test. *: p < 0.05, i.e., there is a statistically significant difference; **: p < 0.01, i.e., there is a very significant difference in statistics; ***: p < 0.001, i.e., there is an extremely significant difference in statistics.

[0169] Table 4 Inhibition of hydroxyproline content in the lungs of mice induced by bleomycin by airway aerosol administration of EGCG and oral administration of EGCG

[0170] Example 5 Effect of inhaled EGCG on bleomycin-induced pulmonary fibrosis in rats

[0171] 5.1 Research Objectives

[0172] Compared with oral gavage administration of EGCG and intratracheal aerosol administration of pirfenidone solution, the effects of different doses of intratracheal aerosol administration of EGCG solution on the collagen content in the lung tissue of rats with bleomycin-induced pulmonary fibrosis model were detected, and pathological tissue sections were observed to evaluate the therapeutic effect of inhaled EGCG on rat pulmonary fibrosis.

[0173] 5.2 Main Reagents

[0174] The solvent 1 of the EGCG atomized solution, the modeling agent, and the solvent 2 of the modeling agent are as described in Example 4. The positive control drug is pirfenidone (purity 99%, pharmaceutical grade). The solvent 3 of the positive control drug is composed of NaCl, citric acid, etc., and is a transparent and clear liquid.

[0175] 5.3 Model construction and grouped drug administration

[0176] A total of 88 SPF-grade SD rats aged 6 to 9 weeks and weighing approximately 200 to 300 g were randomly divided into a sham operation group (normal control group) (8 rats), a pulmonary fibrosis model control group (10 rats), an oral administration of EGCG group 1 (10 rats), an oral administration of EGCG group 2 (10 rats), airway aerosol administration of EGCG solution groups 1-4 (10 rats in each group, a total of 40 rats), and an airway aerosol administration of pirfenidone solution group (10 rats).

[0177] On day 1, the normal control group mice were given vehicle 2 (sodium chloride injection, 1 mL / kg) via intratracheal aerosolization, while the mice in the other groups were given bleomycin (2.5 mg / kg, 1 mL / kg) via intratracheal aerosolization, once in the morning and afternoon, to establish the model. The animals in each group were weighed on day 8. From D10 to D28, the normal control group and the pulmonary fibrosis model group were given solvent 1 by airway aerosolization, and the rats in the other groups were given the test sample EGCG or the control sample pirfenidone once a day. The dosage and administration method of each group are as follows: the dosage of EGCG in EGCG groups 1 and 2 was 30 mg / kg and 60 mg / kg, respectively, by oral gavage; the dosage of EGCG in groups 1-4 was 0.05 mg / kg, 0.4 mg / kg, 0.8 mg / kg, and 1.6 mg / kg, respectively; the dosage of the control drug pirfenidone solution group in airway aerosolization was 0.9 mg / kg. The dosage design of this group was based on the published literature of MW Surber, et al (ATS POSTER, 2014).

[0178] 5.4 Specimen Collection

[0179] On day 29, all animals were euthanized and dissected, and tissues were preserved. During autopsy, the lungs, trachea, and bronchi were observed for abnormalities. Left lung tissue was obtained and stored below -60°C for analysis of hydroxyproline content. Right lung tissue was prepared for pathological sectioning and stained with hematoxylin and eosin and Masson staining for observation of lung pathomorphology and assessment of the degree of pulmonary fibrosis.

[0180] 5.5 Effect of inhaled EGCG solution on hydroxyproline content in lung tissue of rats with pulmonary fibrosis

[0181] The data on the inhibition of bleomycin-induced hydroxyproline content in the lungs of rats by oral gavage of EGCG, airway aerosol administration of EGCG solution, and airway aerosol administration of pirfenidone solution are shown in Table 5 and Figure 11 below:

[0182] Each group was compared with the model group, and the Kruskal-Wallis test was used for statistical analysis. *: p < 0.05, indicating a statistically significant difference; **: p < 0.01, indicating a statistically very significant difference; ***: p < 0.001, indicating a statistically extremely significant difference; ****: p < 0.0001, indicating a statistically very highly significant difference.

[0183] On day 28, compared with the normal control group, the hydroxyproline content in the lung tissue of rats in the pulmonary fibrosis model group was significantly increased (****: p < 0.0001). Although the hydroxyproline content in the lungs of rats decreased after oral administration of EGCG and airway aerosol administration of the control drug pirfenidone solution, it did not reach a statistically significant difference compared with the model control group. However, after inhalation of EGCG, the hydroxyproline content in the lungs of rats was significantly reduced (statistically significant compared with the model control group, ****: p < 0.0001 for the 0.4 mg / kg inhalation EGCG group, and ***: p < 0.001 for the 0.8 mg / kg and 1.6 mg / kg inhalation EGCG groups). Even at a very low dose of 0.05 mg / kg, the hydroxyproline content in the lung tissue of rats with pulmonary fibrosis was significantly reduced (*: p < 0.05). These results indicate that EGCG administered by inhalation can significantly reduce the degree of pulmonary fibrosis in model animals, providing a wide effective therapeutic dose window.

[0184] Table 5 Inhibition of bleomycin-induced pulmonary hydroxyproline content in rats after oral gavage of EGCG, airway aerosol administration of EGCG solution, and airway aerosol administration of pirfenidone solution

[0185] Example 6 Preparation of EGCG atomized solution

[0186] 6.1 Prescription

[0187] 6.2 Preparation method: Mix the EGCG raw material and excipients in a liquid preparation tank, add water for injection to 1000 ml, stir to dissolve and cool, then adjust the pH of the solution to 3-4 with citric acid and sodium citrate, then pre-filter the EGCG solution and sterile filter it, and then aseptically fill it to obtain an EGCG atomized solution.

[0188] In another example of preparing an EGCG nebulized solution, the difference from the formulation described in 6.1 is that it also contains an appropriate amount of EDTA, and the preparation method is the same as that described in 6.2.

[0189] Example 7 Single Dose Escalation Study of EGCG Nebulized Inhalation Solution in Healthy Subjects

[0190] A randomized, double-blind, placebo-controlled clinical study was conducted in 22 healthy subjects to evaluate the safety, tolerability, and pharmacokinetic properties of EGCG nebulized solution after a single inhalation in healthy subjects, and compared with nebulized solution placebo and oral EGCG capsules.

[0191] Trial drug:

[0192] Experimental group: EGCG nebulized inhalation solution (ie, the EGCG nebulized solution containing EDTA as described in Example 6), with an initial concentration of 10 mg / mL, which was diluted according to different doses before administration.

[0193] Placebo group: blank preparation

[0194] Oral control group: EGCG capsules, each weighing 150 mg, of which the EGCG content was approximately 94% of the total weight.

[0195] Grouping and drug administration:

[0196] As shown in Table 6 below, 4 subjects were randomly assigned to the oral control group and given 600 mg EGCG capsules (4 capsules in total, containing approximately 564 mg of EGCG) in an open-blind manner.

[0197] There were three groups for EGCG nebulization inhalation: (1) 3 mg dose group: before administration, 1 mL of the starting solution was diluted with diluent to 1 mg / mL, and 3 mL was added to the nebulizer for administration. (2) 10 mg dose group: before administration, 1 mL of the starting solution was diluted with diluent to 3.33 mg / mL, and 3 mL was added to the nebulizer for administration. (3) 30 mg dose group: without dilution, 3 mL was directly added to the nebulizer for administration. Six subjects were randomly assigned to each nebulization inhalation dose group, starting from the low dose. Two subjects in each group were set as sentinels (1:1 randomization, 1 EGCG nebulization inhalation group, 1 placebo group). They were observed for ≥24 hours. The researchers conducted safety assessments on the subjects, and after the researchers' permission, the remaining four subjects were given medication (3:1 randomization, 3 EGCG nebulization inhalation groups, 1 placebo group).

[0198] Table 6 Grouping information of oral control group and EGCG aerosol inhalation group

[0199] Blood sample collection:

[0200] Blood samples were collected from subjects at 13 blood collection points through an indwelling intravenous needle at 0h (within 1h before administration) and 15min, 30min, 1h, 1.5h, 2h, 3h, 4h, 6h, 8h, 10h, 12h, and 24h after administration (starting from the start of nebulized inhalation or the completion of oral administration of EGCG capsules). 4mL of blood was collected each time, and plasma was extracted for PK testing, backup, and metabolite identification.

[0201] Safety Assessment:

[0202] Adverse event (AE) monitoring; physical examination; vital signs (temperature, blood pressure, pulse, respiration) and blood oxygen saturation; cardiac function; pulmonary function.

[0203] Screening of subjects: inclusion criteria

[0204] The research subjects should meet the following criteria:

[0205] Healthy adult subjects aged 18-59 years (inclusive), male or female.

[0206] · Understand the research procedures and methods, voluntarily participate in this trial, and sign the written informed consent.

[0207] Body mass index (BMI = weight / height squared (kg / m 2 )): 18≤BMI<28; male weight ≥50.0kg and <90.0kg, female weight ≥45.0kg and <90.0kg.

[0208] No clinically significant abnormalities confirmed by medical history, physical examination, laboratory tests, vital signs or electrocardiogram, and medically healthy as determined by the investigator.

[0209] Agree to use effective contraceptive measures throughout the study. Female subjects of childbearing potential should practice contraception for at least one month prior to screening and commit to using contraception throughout the study and for three months after the study's conclusion. Male subjects commit to using contraception throughout the study and for three months after the study's conclusion and guarantee not to donate sperm.

[0210] Forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) ≥ 80% of predicted values, FEV1 / FVC ≥ 0.7, and a normal chest radiograph.

[0211] Agree not to consume any coffee, tea, or beverages or foods containing coffee or tea from 48 hours before to 48 hours after administration.

[0212] Ability to use the inhalation device correctly and effectively during the screening period and the study.

[0213] No medication was used in the 7 days before the dose and no other medication was used in the 24 hours after the dose.

[0214] Screening of subjects: exclusion criteria

[0215] Subjects who meet any of the following criteria will be excluded from this study:

[0216] ·A history of significant medical conditions that the investigator believes may adversely affect participation.

[0217] Female subjects who are pregnant or breastfeeding.

[0218] Female subjects of childbearing potential who have not used contraception for at least 30 days before dosing; male subjects who are unwilling to use contraceptive measures (or cannot guarantee not to donate sperm) and female subjects of childbearing potential during the trial and within 90 days after dosing.

[0219] Allergic to the study drug or any of its ingredients.

[0220] Tested positive for COVID-19, HIV, Hepatitis B, and Hepatitis C.

[0221] Patients whose alanine aminotransferase (ALT), aspartate aminotransferase (AST), or gamma-glutamyltransferase (GGT) levels exceed the upper limit of normal (ULN) at screening / baseline visit, or whose total bilirubin levels exceed the upper limit of normal (ULN).

[0222] Smoking or using e-cigarettes within 6 months before the first dose.

[0223] History of drug abuse or alcoholism within three months prior to the screening period, with alcoholism defined as >21 units of alcohol per week (one unit equals 284 mL of beer, 25 mL of 40% liquor, or 125 mL of wine).

[0224] · Blood / plasma donation ≥ 400 mL within 3 months prior to administration.

[0225] Test results:

[0226] All subjects showed no significant abnormalities in their physical examinations, vital signs, cardiac function, or pulmonary function. Only a few subjects in the placebo and nebulized administration groups experienced localized symptoms in the oropharynx and respiratory tract, including itchy throat, dry mouth, bitter taste in the mouth, and cough. These symptoms were mild and disappeared after drug discontinuation: 1 of 4 subjects in the 10mg dose group and 1 of 2 subjects in the placebo group; 2 of 4 subjects in the 30mg dose group; and no subjects in the 3mg dose group experienced these symptoms. The researchers concluded that EGCG nebulized inhalation was generally well tolerated and had a high safety factor.

[0227] The time-dependent curves of the average plasma EGCG concentrations after administration are shown in Figure 12. The experimental results in Figure 12 indicate that after inhalation administration, there is a good correlation between plasma drug exposure and the administered dose. The multiple difference in plasma drug exposure after inhalation and oral administration is much higher than the multiple difference between the oral and inhalation doses.

[0228] Generally speaking, compared with oral administration, pulmonary administration has a faster absorption rate. The drug quickly enters the systemic circulation through the highly permeable pulmonary capillaries and alveolar surface and quickly reaches a very high peak. However, surprisingly, the time it takes for the drug concentration absorbed into the blood after inhalation of EGCG to reach its peak is T max Later, and C max When the lung exposure is equal to or better than that of oral administration, that is, when the dose produces equal or better therapeutic effects, the drug concentration entering the blood circulation system after inhalation is much lower than the drug level in plasma after oral administration. For example, in the 10 mg dose group, the inhalation dose is 1 / 56 of the oral dose, and the plasma C max After oral administration, C max This also indicates that EGCG aerosol inhalation can significantly increase the drug retention and retention time in the lungs, greatly reducing the drug concentration in plasma. The above results suggest that aerosol inhalation of EGCG can improve the therapeutic effect of pulmonary drugs while greatly reducing the risk of systemic adverse reactions.

[0229] In summary, the application of (-)-epigallocatechin gallate compounds provided by the present disclosure directly administers EGCG to the lung tissue by inhalation, which increases the exposure of the drug in the lung tissue, greatly enhances the ratio of the drug concentration in the lung tissue to the plasma concentration, improves the efficacy and reduces the potential hepatotoxicity; even at very low inhalation doses, it can produce significant anti-inflammatory and anti-pulmonary fibrosis effects, significantly expanding the safe treatment window of EGCG for IPF; when EGCG is inhaled, the drug can be maintained in the lungs at a high effective concentration for a long time, and the concentration is higher than the drug concentration level that can be achieved in the lung tissue after oral administration, which provides feasibility for reducing the frequency of administration, increasing patient compliance, and realizing individualized dosing regimens. In addition, by inhalation administration, the problem of drug-drug interaction between oral EGCG and other IPF therapeutic drugs can also be avoided.

[0230] It should be understood that the application of the present disclosure is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description, and all such improvements and changes should fall within the scope of protection of the claims attached to the present disclosure.

Claims

1. Use of a (-)-epigallocatechin gallate compound in the preparation of an inhalation drug for preventing and / or treating pulmonary fibrosis, wherein: The (-)-epigallocatechin gallate compound is (-)-epigallocatechin gallate or a pharmaceutically acceptable salt, ester, hydrate or solvate thereof.

2. The use according to claim 1, wherein The inhalation medicine is formulated into a dosage form for inhalation, and the dosage form is selected from a solution, a suspension, an aerosol or a powder inhaler.

3. The use according to claim 1, wherein The inhalation medicine is formed by drying the (-)-epigallocatechin gallate compound to form powder, which is then redissolved in a diluent.

4. The use according to claim 2, wherein: When the dosage form is a solution, the inhalation drug comprises: the (-)-epigallocatechin gallate compound as an active ingredient, a diluent, and at least one of a pH regulator, an osmotic pressure regulator, and an antioxidant, wherein the (-)-epigallocatechin gallate compound is completely dissolved in the diluent; When the dosage form is a suspension, the inhalation drug comprises: the (-)-epigallocatechin gallate compound as an active ingredient, a diluent, and at least one of a surfactant, a pH regulator, and a tension regulator; the (-)-epigallocatechin gallate compound or the (-)-epigallocatechin gallate compound and a carrier suitable for inhalation administration form particles suspended in the diluent; When the dosage form is an aerosol, the inhaled medicine comprises: the (-)-epigallocatechin gallate compound as an active ingredient, a diluent, a propellant, and at least one of a surfactant, a cosolvent, and a pH adjuster; When the dosage form is a powder inhaler, the inhalation medicine comprises: the (-)-epigallocatechin gallate compound as an active ingredient, a carrier suitable for inhalation administration, and an adjuvant. At least one of a shaping agent and a surfactant.

5. The use according to claim 3 or 4, wherein Satisfy at least one of the following conditions (1)-(3): (1) The diluent is one or more of water, ethanol and glycerol; (2) The pH value of the inhaled drug is 3.0-5.

0. (3) The concentration of the (-)-epigallocatechin gallate compound in the inhaled drug is 0.1-25 mg / mL.

6. The use according to claim 1, wherein The inhaled medicine includes the (-)-epigallocatechin gallate compound, or the inhaled medicine includes the (-)-epigallocatechin gallate compound and other anti-pulmonary fibrosis drugs.

7. The use according to claim 6, wherein: The other anti-pulmonary fibrosis drugs are selected from one or more of pirfenidone, nintedanib, glucocorticoids, immunosuppressants, prostacyclin and its analogs, CTGF antibodies, Galectin-3 inhibitors, integrin antagonists, recombinant serum amyloid protein P and its analogs, PDE inhibitors, LPA antagonists, JAK kinase inhibitors, and multiple cytokine receptor TKIs.

8. The use according to claim 7, wherein: The other anti-pulmonary fibrosis drugs are selected from one or more of pirfenidone, nintedanib, BI 1015550, treprostinil and its analogs, recombinant serum amyloid protein P, and LPA antagonists.

9. The use according to claim 1, wherein: The dosage of the (-)-epigallocatechin gallate compound is 0.1-100 mg / time.

10. The use according to claim 9, wherein: The dosage of the (-)-epigallocatechin gallate compound is 0.1-50 mg / time.

11. The use according to claim 10, wherein: The dosage of the (-)-epigallocatechin gallate compound is 0.1-30 mg / time.

12. The use according to claim 11, wherein: The dosage of the (-)-epigallocatechin gallate compound is 0.1-15 mg per time.

13. The use according to claim 9, wherein: The dosage of the (-)-epigallocatechin gallate compound is selected from 0.1 mg / time, 1 mg / time, 1.5 mg / time, 2 mg / time, 2.5 mg / time, 3 mg / time, 3.5 mg / time, 4 mg / time, 4.5 mg / time, 5 mg / time, 5.5 mg / time, 6 mg / time, 6.5 mg / time, 7 mg / time, 7.5 mg / time, 8 mg / time, 8.5 mg / time, 9 mg / time, 9.5 mg / time, 10 mg / time, 10.5 mg / time, 11 mg / time, 11.5 mg / time, 12 mg / time, 12.5 mg / time, 13 mg / time, g / time, 13.5mg / time, 14mg / time, 14.5mg / time, 15mg / time, 15.5mg / time, 16mg / time, 16.5mg / time, 17mg / time, 17.5mg / time, 18mg / time, 18.5mg / time, 19mg / time, 19.5mg / time, 20mg / time, 25mg / time, 30mg / time, 35mg / time, 40mg / time, 45mg / time, 50mg / time, 55mg / time, 60mg / time, 65mg / time, 70mg / time, 75mg / time, 80mg / time, 90mg / time or 100mg / time.

14. The use according to claim 1, wherein: The dosage of the (-)-epigallocatechin gallate compound is 1 / 1000 to 1 / 10 of the oral dosage.

15. The use according to claim 14, wherein: The dosage of the (-)-epigallocatechin gallate compound is 1 / 600 to 1 / 10 of the oral dosage.

16. The use according to any one of claims 1 to 15, wherein: The pulmonary fibrosis disease is an interstitial lung disease, which includes one or more of idiopathic interstitial pneumonia, pulmonary interstitial fibrosis caused by autoimmune or connective tissue diseases, pulmonary interstitial fibrosis associated with contact or occupational exposure, pulmonary interstitial fibrosis caused by treatment, and sarcoidosis.

17. The use according to claim 16, wherein: The idiopathic interstitial pneumonia includes idiopathic pulmonary fibrosis; The pulmonary interstitial fibrosis caused by the autoimmune or connective tissue disease includes lupus, scleroderma, polymyositis or dermatomyositis, and interstitial lung disease associated with rheumatoid arthritis; The contact or occupational exposure-related interstitial fibrosis includes asbestosis, silicosis, and hypersensitivity pneumonitis; The treatment-induced interstitial fibrosis includes interstitial lung disease induced by chemotherapy, radiotherapy and some drug treatments.

18. An inhalable pharmaceutical composition for preventing and / or treating pulmonary fibrosis, wherein: include: (-)-epigallocatechin gallate compound is used as an active ingredient, and a pharmaceutically acceptable excipient; the (-)-epigallocatechin gallate compound is EGCG or a pharmaceutically acceptable salt, ester, hydrate or solvate thereof.

19. The pharmaceutical composition according to claim 18, wherein The content of the (-)-epigallocatechin gallate compound in each dose of the inhalable pharmaceutical composition is 0.1-50 mg.

20. The pharmaceutical composition according to claim 19, wherein The content of the (-)-epigallocatechin gallate compound in each dose of the inhalable pharmaceutical composition is 0.5-30 mg.

21. The pharmaceutical composition according to any one of claims 18 to 20, wherein The dosage form of the inhalable pharmaceutical composition is an inhalation solution, an inhalation suspension, an aerosol or a powder inhalation.

22. The pharmaceutical composition according to claim 21, wherein When the dosage form of the inhalable pharmaceutical composition is an inhalation solution, the pharmaceutically acceptable excipient is selected from one or more of a surfactant, a pH regulator, an antioxidant, a preservative, an osmotic pressure regulator, a metal ion complexing agent, water and an additive; When the dosage form of the inhalable pharmaceutical composition is an inhalation suspension, the pharmaceutically acceptable excipients are selected from surfactants, pH regulators, antioxidants, preservatives, osmotic pressure regulators, One or more of a metal ion complexing agent, water, and additives; When the dosage form of the inhalable pharmaceutical composition is an aerosol, the pharmaceutically acceptable excipient is selected from one or more of a self-solvent, a surfactant, a propellant and an additive; When the dosage form of the inhalable pharmaceutical composition is a powder inhaler, the pharmaceutically acceptable excipients include excipients, carriers and additives.

23. A method for preventing and / or treating pulmonary fibrosis, wherein: The method comprises: administering the (-)-epigallocatechin gallate compound to a subject by inhalation administration, wherein the (-)-epigallocatechin gallate compound is (-)-epigallocatechin gallate or a pharmaceutically acceptable salt, ester, hydrate or solvate thereof.

24. The method of claim 23, wherein: The dosage of the (-)-epigallocatechin gallate compound is 0.01-2.0 mg / kg.

25. The method of claim 23, wherein: The dosage of the (-)-epigallocatechin gallate compound is 0.1-80 mg / time.

26. The method of claim 25, wherein: The dosage of the (-)-epigallocatechin gallate compound is 0.1-50 mg / time.

27. The method of claim 26, wherein: The dosage of the (-)-epigallocatechin gallate compound is 0.1-30 mg / time.

28. The method of claim 27, wherein: The dosage of the (-)-epigallocatechin gallate compound is 0.1-15 mg per time.

29. The method of claim 25, wherein: The dosage of the (-)-epigallocatechin gallate compound is selected from 0.1 mg / time, 1 mg / time, 1.5 mg / time, 2 mg / time, 2.5 mg / time, 3 mg / time, 3.5 mg / time, 4 mg / time, 4.5 mg / time, 5 mg / time, 5.5 mg / time, 6 mg / time, 6.5 mg / time, 7 mg / time, 7.5 mg / time, 8 mg / time, 8.5 mg / time, 9 mg / time, 9.5 mg / time, 10 mg / time, 10.5 mg / time, 11 mg / time, 11.5 mg / time. times, 12 mg / time, 12.5 mg / time, 13 mg / time, 13.5 mg / time, 14 mg / time, 14.5 mg / time, 15 mg / time, 15.5 mg / time, 16 mg / time, 16.5 mg / time, 17 mg / time, 17.5 mg / time, 18 mg / time, 18.5 mg / time, 19 mg / time, 19.5 mg / time, 20 mg / time, 25 mg / time, 30 mg / time, 35 mg / time, 40 mg / time, 45 mg / time, 50 mg / time, 55 mg / time, 60 mg / time, 65 mg / time, 70 mg / time, 75 mg / time, 80 mg / time, 90 mg / time or 100 mg / time.

30. The method of claim 23, wherein: The dosage of the (-)-epigallocatechin gallate compound is 1 / 1000 to 1 / 10 of the oral dosage.

31. The method of claim 30, wherein: The dosage of the (-)-epigallocatechin gallate compound is 1 / 600 to 1 / 10 of the oral dosage.

32. The method of claim 23, wherein: Also includes: Other anti-pulmonary fibrosis drugs are administered to the subject by inhalation administration, and the other anti-pulmonary fibrosis drugs are administered simultaneously, independently or sequentially with the (-)-epigallocatechin gallate compound.

33. The method of claim 32, wherein: The other anti-pulmonary fibrosis drugs are selected from one or more of pirfenidone, nintedanib, glucocorticoids, immunosuppressants, prostacyclin and its analogs, CTGF antibodies, Galectin-3 inhibitors, integrin antagonists, recombinant serum amyloid protein P and its analogs, PDE inhibitors, LPA antagonists, JAK kinase inhibitors, and multiple cytokine receptor TKIs.

34. The method of claim 33, wherein: The other anti-pulmonary fibrosis drugs are selected from one or more of pirfenidone, nintedanib, BI 1015550, treprostinil and its analogs, recombinant serum amyloid protein P, and LPA antagonists.

35. The method of any one of claims 23 to 34, wherein: The frequency of administration is 1 time / 2 days, 1 time / day or 2 times / day.

36. The method of any one of claims 23 to 34, wherein: The inhalation administration refers to delivering the drug to the subject using an inhalation administration device loaded with the drug.

37. The method of claim 36, wherein: The inhalation drug delivery device is an atomizer, a pressure metered dose inhaler, a dry powder inhaler or a soft mist inhaler.

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