WO2024109652A1 - (-)-表没食子儿茶素没食子酸酯类化合物的应用 - Google Patents

(-)-表没食子儿茶素没食子酸酯类化合物的应用 Download PDF

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WO2024109652A1
WO2024109652A1 PCT/CN2023/132299 CN2023132299W WO2024109652A1 WO 2024109652 A1 WO2024109652 A1 WO 2024109652A1 CN 2023132299 W CN2023132299 W CN 2023132299W WO 2024109652 A1 WO2024109652 A1 WO 2024109652A1
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time
epigallocatechin gallate
dosage
inhalation
pulmonary fibrosis
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PCT/CN2023/132299
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English (en)
French (fr)
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瞿介明
赵婧雅
徐琳
车琳
李秀娟
权梦雪
梁文青
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长风药业股份有限公司
上海交通大学医学院附属瑞金医院
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Publication of WO2024109652A1 publication Critical patent/WO2024109652A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system

Definitions

  • the present disclosure relates to the field of chemical medicine, and in particular to the application of (-)-epigallocatechin gallate compounds.
  • Pulmonary fibrosis is the terminal clinical manifestation of many interstitial lung diseases with different causes. It is characterized by persistent alveolar damage, fibroblast proliferation and a large amount of extracellular matrix (ECM) deposition, which leads to varying degrees of inflammation and fibrosis in the alveoli and interstitium, and then leads to lung structure destruction and respiratory failure. Therefore, it is also called interstitial lung disease (ILD) or diffuse parenchymal lung disease (DPLD). ILD is mainly divided into four categories: idiopathic interstitial pneumonia (IIPs), interstitial lung fibrosis caused by autoimmune or connective tissue diseases, interstitial lung fibrosis caused by contact or treatment, and sarcoidosis.
  • IIPs idiopathic interstitial pneumonia
  • IIPs interstitial lung fibrosis caused by autoimmune or connective tissue diseases
  • interstitial lung fibrosis caused by contact or treatment
  • sarcoidosis sarcoidosis.
  • Idiopathic pulmonary fibrosis is the most important and common type of idiopathic interstitial pneumonia (IIPs).
  • IPF interstitial pneumonia
  • clinical research on pulmonary fibrosis is mainly focused on IPF, which is clinically manifested as progressive dyspnea.
  • IPF interstitial pneumonia
  • the median survival time is about 2.8 years, and the 5-year survival rate is less than 50%.
  • the pathogenesis of pulmonary fibrosis involves multiple factors, such as persistent damage to the alveolar epithelium leading to dysregulation of the intracellular environment of the alveolar epithelial-mesenchymal transition (EMT), activation of signaling pathways such as transforming growth factor- ⁇ (TGF- ⁇ ), Wnt, Notch, epithelial cell dysfunction and apoptosis, scar tissue formation, etc., which lead to destruction of the intrapulmonary environment.
  • TGF- ⁇ transforming growth factor- ⁇
  • Wnt transforming growth factor- ⁇
  • Notch epithelial cell dysfunction
  • apoptosis scar tissue formation, etc.
  • the only drugs approved worldwide for the treatment of pulmonary fibrosis are nintedanib and pirfenidone, both of which are oral preparations; although both can slow the rate of decline in lung function, they cannot reverse the progression of the disease, and both have large oral doses and serious adverse reactions.
  • the dose of nintedanib is 150 mg BID (i.e. 150 mg, twice a day), and the most common adverse reaction is gastrointestinal reaction.
  • BID i.e. 150 mg, twice a day
  • the most common adverse reaction is gastrointestinal reaction.
  • the incidence of diarrhea is as high as 61.5%
  • the dose of pirfenidone is 801 mg TID (i.e. 801 mg, 3 times a day)
  • the most common adverse reaction is photosensitivity, with an incidence of up to 51% in clinical studies; and these serious adverse reactions are also the most common reasons why patients are forced to reduce the dose or even stop taking the drug early.
  • TGF- ⁇ 1 signaling is a key driver of collagen accumulation and fibrotic diseases, and an important regulator of inflammation and epithelial cell proliferation.
  • TGF- ⁇ 1 inhibitors limits the development of TGF- ⁇ 1 inhibitors as therapeutic agents.
  • Lysyl oxidase-like 2 protein LOXL2
  • LOXL2 Lysyl oxidase-like 2 protein
  • the combined inhibition of LOXL2 and TGF- ⁇ 1 activity by triphenolic hydroxy compounds can effectively block pathological collagen accumulation in vivo without producing the toxicity associated with global inhibitors.
  • EGCG is administered orally.
  • oral administration of EGCG has certain effects in pulmonary fibrosis model animals and human patients, its intestinal absorption is poor after oral administration, and drug absorption is affected by food.
  • EGCG is hydrolyzed by esterase in saliva after oral administration, and undergoes extensive enzymatic metabolic reactions such as glucuronidation and sulfation after entering the blood and liver, resulting in extremely low bioavailability of oral EGCG.
  • a very high dose is required to exert an anti-pulmonary fibrosis effect.
  • the dose reported in the literature by Harold A. Chapman, et al. is 600 mg; long-term high-dose medication may pose a safety hazard.
  • EGCG can undergo extensive enzyme metabolism in the body.
  • EGCG is not a substrate of the drug metabolizing enzyme CYP450 enzyme system, EGCG 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 effect on the bioavailability of a variety of 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 level is very high, resulting in poor patient tolerance.
  • Its nebulized inhalation preparation is being developed, but the Phase II study was terminated due to the ineffectiveness of the low dose (50 mg/once a day).
  • 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.
  • Pirfenidone still had a high plasma drug concentration (807-1370ng/mL) after inhalation administration. Therefore, pirfenidone still had a high risk of adverse reactions after inhalation administration. This proves that after inhalation administration of pirfenidone, the drug quickly enters the blood from the lungs, and it is difficult to maintain an effective drug concentration level in the lungs for a long time, and it did not achieve the ideal effect as people expected at the beginning.
  • therapeutic drugs for pulmonary fibrosis should have a wide treatment window and realize individualized dosing regimens for patients with mild and severe diseases.
  • therapeutic drugs that can be delivered by inhalation and exert lasting effects in the local lungs are still an urgent unmet clinical need.
  • the purpose of the present invention 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 and high dosage.
  • problems such as drug dosage, narrow safe and effective therapeutic window, and serious drug interactions with multiple drugs.
  • the present disclosure provides an application 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.
  • an inhalable pharmaceutical composition for preventing and/or treating pulmonary fibrosis disease 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.
  • 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.
  • the present invention uses EGCG or its pharmaceutically acceptable salts, esters, hydrates or solvates as EGCG compounds to prepare drugs for treating pulmonary fibrosis in the form of inhalation, thereby achieving: (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) broadening 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.
  • Figure 1 is a comparison of the change curves of the drug concentration (Concentration, in ng/mL) in the plasma and lungs 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).
  • 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 oral administration (po, dosage of 60 mg/kg) of EGCG.
  • Figure 3 is a comparison of the curves of the change in drug concentration in rat plasma and lungs over time (Time, in hours) after airway atomization of EGCG solution (dosage of 0.8 mg/kg) and oral administration of EGCG solution (dosage of 60 mg/kg).
  • Figure 6 shows the effect of airway aerosol administration of EGCG and oral administration 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).
  • FIG7a shows the effects of airway aerosol administration of EGCG and oral administration on the degree of bleomycin-induced lung tissue fibrosis in mice (Masson staining, 100 ⁇ );
  • Figure 7b shows the effects of EGCG administered by airway aerosol and oral administration on bleomycin-induced lung tissue in mice Effects of pathological structural changes (H&E staining, 100 ⁇ ).
  • Figure 8 shows the effects of airway aerosol administration of EGCG solution on alveolar inflammatory cell infiltration, hemorrhage, alveolar expansion, and alveolar fibrous exudation in mice with pulmonary fibrosis compared with oral administration.
  • Figure 9 shows the effect of airway atomization and oral administration of EGCG on the fibrosis score of mice compared with the mice in the pulmonary fibrosis model group.
  • Figure 10 shows the effect of oral gavage and airway atomization of EGCG on the hydroxyproline content (in ⁇ g/ ⁇ L) in the lung tissue of mice compared with the mice in 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).
  • Figure 11 shows the effects of oral administration and airway atomization of EGCG, as well as airway atomization of the control drug pirfenidone (AP01), on the hydroxyproline content (in ⁇ g/ ⁇ L) 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., there is a statistically significant difference; **: p ⁇ 0.01, i.e., there is a very significant statistical difference; ***: p ⁇ 0.001, i.e., there is an extremely significant statistical difference; ****: p ⁇ 0.0001, i.e., there is an extremely highly significant statistical difference).
  • Figure 12 shows the curve of the average drug concentration (plasma concentration) of EGCG in the blood changing with time after oral administration of EGCG capsules (150 mg/capsule, 4 capsules in total) and nebulized inhalation administration of EGCG solution (3 mg dose group, 10 mg dose group, 30 mg dose group) to healthy subjects.
  • the present disclosure provides an application of (-)-epigallocatechin gallate compounds. To make the purpose, technical scheme and effect of the present disclosure clearer and more specific, the present disclosure is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present disclosure and are not used to limit the present disclosure.
  • the present disclosure provides a (-)-epigallocatechin gallate.
  • the invention discloses an application of a compound in preparing an inhalation medicine for treating pulmonary fibrosis, wherein the (-)-epigallocatechin gallate compound is (-)-epigallocatechin gallate or a pharmaceutically acceptable salt, ester, hydrate or solvate thereof.
  • 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.
  • the inhaled drug 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.
  • the inhaled drug is formed by drying the (-)-epigallocatechin gallate compound to form a powder, which is then reconstituted with a diluent.
  • the inhaled drug when the dosage form is a solution, includes: 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, and the (-)-epigallocatechin gallate compound is completely dissolved in the diluent.
  • the inhalation drug when the dosage form is a suspension, includes: 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.
  • the inhaled medicine when the dosage form is an aerosol, includes: 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.
  • the inhalation drug when the dosage form is a powder inhaler, 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.
  • the diluent is one or more of water, ethanol and glycerol; preferably, the diluent is water.
  • the pH of the inhaled medication is 3.0-5.0.
  • the concentration of the (-)-epigallocatechin gallate compound in the inhaled medicament is 0.1-25 mg/mL.
  • the inhaled drug includes the (-)-epigallocatechin gallate compound, or the inhaled drug includes the (-)-epigallocatechin gallate compound and other anti-pulmonary fibrosis drugs.
  • the other anti-pulmonary fibrosis drugs are selected from 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 one or more of multiple cytokine receptor TKIs.
  • the other anti-pulmonary fibrosis drug is 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.
  • the inhaled drug can also be used in combination with 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 compounds 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 treatment, in which (-)-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 described in the present invention is selected from simultaneous administration, independent preparation and co-administration, or independent preparation and sequential administration.
  • the dosage of the (-)-epigallocatechin gallate compound is 0.1-100 mg/time.
  • the dosage of the (-)-epigallocatechin gallate compound is 0.1-50 mg/time.
  • the dosage of the (-)-epigallocatechin gallate compound is 0.1-30 mg/time.
  • the dosage of the (-)-epigallocatechin gallate compound is 0.1-15 mg/time.
  • 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,
  • the oral dosage of EGCG reported in existing literature is 400 mg-600 mg/time.
  • the dosage of the (-)-epigallocatechin gallate compound is 1/1000 to 1/10 of the oral dosage.
  • 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.
  • the pulmonary fibrosis disease is an interstitial lung disease, which includes 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 and one or more of sarcoidosis.
  • the idiopathic interstitial pneumonia includes idiopathic pulmonary fibrosis;
  • the pulmonary interstitial 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 allergic pneumonia;
  • the interstitial fibrosis caused by treatment includes interstitial lung disease caused by chemotherapy, radiotherapy, and some drug treatments.
  • 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.
  • 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.
  • the content of the (-)-epigallocatechin gallate compound in each dose of the inhalable pharmaceutical composition is 0.1-50 mg.
  • the content of the (-)-epigallocatechin gallate compound in each dose of the inhalable pharmaceutical composition is 0.5-30 mg.
  • the content 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, 30 mg.
  • the inhalable pharmaceutical composition is the (-)-epigallocatechin-3-yl
  • the theophylline gallate compound and the pharmaceutically acceptable excipients are prepared into an inhalation preparation using a conventional or special preparation process.
  • the inhalation preparation is an inhalation solution, an inhalation suspension, an aerosol, a powder spray or other inhalation preparations.
  • the inhalation solution is an atomized inhalation solution; the inhalation suspension is an atomized inhalation suspension.
  • Atomized inhalation refers to using 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.
  • the pharmaceutical composition can achieve the purpose of local treatment and systemic treatment by atomized inhalation; for example, it has a strong anti-fibrotic effect and anti-inflammatory effect at the same time.
  • the (-)-epigallocatechin gallate compound is first processed into a powder by a drying process, and the powder is reconstituted with the pharmaceutically acceptable excipients by a diluent before being administered to the subject and then delivered to the lungs of the subject in an aerosolized form.
  • the drying process can be freeze-drying, spray-drying, spray-freeze drying or supercritical fluid technology, etc.
  • 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.
  • the dosage form of the inhalable pharmaceutical composition is an inhalation suspension
  • 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.
  • the inhalation solution or inhalation suspension contains almost no preservative.
  • the concentration of the (-)-epigallocatechin gallate compound 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.
  • the (-)- The concentration of epigallocatechin gallate compounds is exemplarily 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.
  • 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.
  • the propellant is a hydrofluoroalkane compound.
  • the propellant is one or both of 1,1,1,2-tetrafluoroethane (HFA134a for short) and 1,1,1.2,3,3,3-heptafluoropropane (HFA 227 for short).
  • the additive comprises a solvent, and the solvent is 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.
  • the dosage form of the inhalable pharmaceutical composition is a powder inhaler
  • the pharmaceutically acceptable excipients include excipients, carriers and additives.
  • the excipients include one or more of sugars, sugar alcohols, starches, macromolecular polymers, fatty acids or their salts, waxes, calcium sulfate, calcium carbonate, talcum powder, iron oxide and light anhydrous silicic acid.
  • the sugars include one or more of lactose, glucose, white sugar, trehalose, sucrose, etc.; the sugar alcohols include one or more of erythritol, mannitol, sorbitol, etc.; the macromolecular polymers include crystalline cellulose, methylcellulose, hydroxypropyl cellulose, carboxymethyl cellulose calcium, hydroxypropyl methylcellulose, sodium carboxymethyl ether cellulose, amylopectin, dextrin, gum arabic, agar, gelatin, tragacanth gum, sodium alginate, polyvinyl pyrrolidone, polyvinyl alcohol, etc.; the fatty acid can be one or more of palmitic acid, stearic acid, oleic acid, etc.
  • the excipient is selected from one or more of sugars, sugar alcohols, macromolecular polymers and calcium carbonate.
  • the sugar is lactose or sucrose; the sugar alcohol is erythrose.
  • the macromolecular polymer is calcium carboxymethylcellulose, pullulan, polyvinyl pyrrolidone or methylcellulose. The best excipient is lactose or erythritol.
  • the carrier includes lactose, glucose, fructose, sucrose, maltose, dextran, erythritol, sorbitol, mannitol, calcium sulfate, calcium carbonate, talc or iron oxide, etc.
  • the epigallocatechin gallate compound may be in a crystalline or amorphous state.
  • the inhalable pharmaceutical composition when preventing and/or treating pulmonary fibrosis, is delivered to the respiratory tract and lungs of the subject through aerosol droplets or micronized particles emitted by an inhalation drug delivery device.
  • the average particle size of the aerosol droplets is 0.5-10 ⁇ m; illustratively, the average particle size of the aerosol droplets may be 1-8 ⁇ m, 1-5 ⁇ m, 1-3 ⁇ m, 2-3 ⁇ m, etc.
  • the average particle size of the micronized particles is less than or equal to 20 ⁇ m; preferably less than or equal to 10 ⁇ m, more preferably 1-9 ⁇ m, and most preferably 3-8 ⁇ m; micronized particles within this particle size range can reach the respiratory tract (such as bronchi) and lungs of the subject.
  • 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.
  • 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 fibrosis caused by autoimmune or connective tissue diseases, pulmonary fibrosis caused by contact or treatment, and interstitial lung diseases such as sarcoidosis.
  • the idiopathic interstitial pneumonia includes idiopathic pulmonary fibrosis.
  • Interstitial fibrosis caused by autoimmune or connective tissue diseases includes lupus, scleroderma, polymyositis or dermatomyositis, and interstitial lung disease associated with rheumatoid arthritis.
  • Interstitial fibrosis associated with contact or occupational exposure includes asbestosis, silicosis, and hypersensitivity pneumonitis.
  • Interstitial fibrosis associated with treatment includes interstitial lung disease caused by chemotherapy, radiotherapy, and some drug treatments.
  • the dosage of the (-)-epigallocatechin gallate compound is 0.01-2.0 mg/kg.
  • the dosage of the active ingredient EGCG in the treatment method can be 0.01-2.0 mg/kg for the test animal, which is converted to the proposed clinical dosage of human patients of 0.1-100 mg, and the corresponding dosage is converted with reference to the method reported in the clinic or literature (E Boger, et al. 2016; Ramon Hendrickx, et al. 2018; Therese Ericsson, et al. 2017).
  • 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.
  • the dosage for human administration can be illustratively 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.
  • the treatment method includes delivering (-)-epigallocatechin gallate compounds to the patient's lungs at a dosage of 1/1000 to 1/10 of the oral dosage.
  • it can be 1/600 to 1/10 of the oral dosage.
  • it can be 1/200 to 1/10 of the oral dosage, and can be 1/50 to 1/10 of the oral dosage.
  • the dosage of the (-)-epigallocatechin gallate compound may 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.
  • the dosage of the (-)-epigallocatechin gallate compound is 0.5-15 mg/time.
  • 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, 25m
  • the treatment method includes delivering 0.1-100 mg of (-)-epigallocatechin gallate compounds to the patient's lungs; illustratively, 0.1-80 mg of (-)-epigallocatechin gallate compounds, 0.1-70 mg of (-)-epigallocatechin gallate compounds, 0.1-60 mg of (-)-epigallocatechin gallate compounds, 0.1-50 mg of (-)-epigallocatechin gallate compounds, or 0.1-50 mg of (-)-epigallocatechin gallate compounds, 0.1-30 mg of (-)-epigallocatechin gallate compounds, etc.
  • 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.
  • the (-)-epigallocatechin-2-yl is administered to a subject by inhalation.
  • the invention discloses a method for delivering (-)-epigallocatechin gallate compounds, comprising: first delivering (-)-epigallocatechin gallate compounds to the lungs of a subject at a lower dose as a starting dose, and then delivering (-)-epigallocatechin gallate compounds to the lungs of the subject in a dose increasing manner according to the patient's condition.
  • 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.
  • the active agent is selected from pirfenidone, nintedanib, glucocorticoids, immunosuppressants, prostacyclin and its analogs, CTGF antibodies, Galectin-3 inhibitors, integrin antagonists, recombinant serum amyloid 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.
  • the prostacyclin and its analogs can be selected from Treprostinil and Iloprost;
  • the CTGF antibody can be selected from Pamrevlumab;
  • the Galectin-3 inhibitor can be selected from GB0139;
  • the integrin antagonist is an integrin ( ⁇ v ⁇ 1, ⁇ v ⁇ 6) antagonist, and the integrin antagonist can be selected from PLN-74809;
  • the recombinant Pentraxin-2 can be selected from PRM-151;
  • the PDE inhibitor can be selected from BI 1015550;
  • the LPA antagonist can be selected from BMS-986278.
  • 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.
  • 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 150 mg. The dosage is less than or equal to 100 mg.
  • the daily dosage administered to a subject can 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, 14 mg, 13.5 mg, 13 mg, 12.5 mg, 12 mg, 11.5 mg, 11 mg, 10.5 mg, 10 mg, 9.5 mg, 9 mg, 8.5 mg, 8 mg, 7.5 mg, 7 mg, 6.5 mg, 6 mg, 5.5 mg, 5 mg, 4.5 mg, 4 mg, 3.5 mg, 3 mg, 2.5 mg, 1 mg, 0.5 mg, 0.2 mg, 0.1 mg, etc.
  • the inhalation administration refers to delivering the drug to the subject using an inhalation administration device loaded with the drug.
  • the inhalation administration device is an atomizer, a pressurized metered dose inhaler, a dry powder inhaler, or a soft mist inhaler.
  • the dosage dosage described in the present disclosure can be the dosage output from the inhalation device (i.e., the device delivered dosage); it can also be the dosage delivered to the patient's lungs (i.e., the lung dosage) converted according to the delivery efficiency of the inhalation drug delivery device generally recognized in the art of about 25%-75%.
  • 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.
  • the drug/drug composition provided by the embodiments of the present disclosure with (-)-epigallocatechin gallate compounds as the active ingredient can be administered to a subject by inhalation (e.g., delivered), and after delivery, the (-)-epigallocatechin gallate compounds can be effectively deposited in the lungs of the subject, so that the drug concentration in the lungs of the subject is higher and the systemic blood drug concentration is lower, thereby avoiding potential side effects such as hepatotoxicity.
  • a larger span can be created between efficacy and toxicity, so that the drug has a wide therapeutic window when administered by inhalation.
  • the higher lung concentration provided by inhalation administration of the drug/pharmaceutical composition of this embodiment (-)-epigallocatechin gallate compounds can quickly and more effectively regulate the key protein activities related to pulmonary fibrosis and inflammation, such as ⁇ -smooth muscle actin ( ⁇ -SMA), SNAI1, type I collagen (collagen I), Fibronectin, phosphorylated SMAD3 (pSMAD3), phosphorylated SMAD2 (pSMAD2) in lung tissue, and produce better drug effects; while some proteins in lung tissue cannot be effectively inhibited after oral administration of EGCG.
  • ⁇ -SMA smooth muscle actin
  • SNAI1 type I collagen
  • Fibronectin phosphorylated SMAD3
  • pSMAD2 phosphorylated SMAD2
  • (-)-epigallocatechin gallate compounds can be maintained in the lungs of the subject at a higher drug concentration for a longer time, which provides feasibility for reducing the frequency of administration and increasing the compliance of the subject.
  • inhalation administration of the drug/drug composition of this embodiment the problem of drug-drug interaction between orally administered EGCG and other IPF therapeutic drugs is also avoided.
  • the drugs/drug compositions with (-)-epigallocatechin gallate compounds as active ingredients are used as inhalable drugs for the prevention and treatment of pulmonary fibrosis, which can greatly reduce the dosage, provide a wider treatment window, lower adverse reactions, and lower dosage frequency, and will provide new treatment options for patients with pulmonary fibrosis, with great social and economic benefits.
  • Example 1 Pharmacokinetic study of EGCG in rat plasma after airway atomization, intravenous injection, and oral administration
  • Preparation of airway aerosol drug delivery solution accurately weigh the EGCG compound, 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.
  • Preparation of solution for intravenous injection and oral administration Accurately weigh the EGCG compound and add physiological saline to prepare a solution.
  • Nebulizer for liquid pulmonary administration Nebulizer for liquid pulmonary administration.
  • Airway atomization The mice in each airway atomization group were fixed on the mouse holder after isoflurane inhalation anesthesia.
  • the anesthesia laryngoscope pressed the root of the animal's tongue to expose the glottis, and the lung micro-liquid atomizer needle (blunt) with a certain amount of EGCG solution was gently inserted into the trachea to atomize the test sample into the lungs, and then the needle was quickly pulled out, the mouse was removed from the holder, and the head was turned left and right to make the drug solution as evenly distributed as possible in each lung lobe.
  • the airway atomization dosage was 1.6mg/kg, 3.2mg/kg, and 6.4mg/kg, with 5 mice in each dosage group.
  • Oral gavage The dosage for each animal is determined according to the animal's weight. A syringe of appropriate specifications and a gavage hose are used to extract the dosage for each animal, and the drug is administered orally. The dosage is 60 mg/kg, for a total of 5 animals.
  • Intravenous injection The drug was injected into the tail vein of rats. The dose was 3.2 mg/kg, and a total of 5 rats were used.
  • 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 ⁇ L of whole blood at each blood collection point.
  • 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.
  • Mass spectrometry conditions UPLC-MS/MS was used for determination. Chromatographic conditions: HPLC was used for determination. WinNonlin pharmacokinetic professional software was used to calculate the corresponding pharmacokinetic parameters of each animal using the statistical moment method.
  • Airway aerosol administration (inh, the dosage is 1.6mg/kg, 3.2mg/kg, 6.4mg/kg), intravenous injection (iv, the dosage is 3.2mg/kg), oral gavage (po, the dosage is 60
  • EGCG was administered by airway atomization at different doses (inh, the doses were 1.6 mg/kg, 3.2 mg/kg, and 6.4 mg/kg)
  • the pharmacokinetic parameters in rat plasma are shown in Table 1.
  • Example 1 after inhalation (such as airway atomization) administration of 1.6 mg/kg EGCG and oral administration of 60 mg/kg EGCG, the AUC 0-t produced by the drug in rat plasma is similar.
  • this example studies the changes in drug concentration in rat plasma and lungs over time compared with single airway atomization administration of a lower dose (0.8 mg/kg) and oral administration of EGCG (60 mg/kg). Five rats were used for the study in both the inhalation administration group and the oral administration group.
  • Blood collection method Blood was collected by femoral artery bleeding at 30 min, 2 h, 4 h, and 6 h after administration (4 blood collection points in total). After blood collection, 0.9 wt% saline was injected through the trachea to lavage the lungs twice, and then the The whole lung was taken, dried with filter paper, and placed in a -20°C refrigerator for testing.
  • 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 ⁇ L of sample to measure the blood drug concentration.
  • Lung tissue sample processing method Take lung tissue samples, weigh them, add 3 times normal saline (w:v) to homogenize, take 1 mL of the 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 ⁇ L for injection to measure the drug concentration in the lung.
  • rats were given different doses of EGCG solution by gavage or airway atomization to study the pharmacokinetic parameters of the drug in the lungs of rats.
  • the airway atomization dose was 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 gavage dose was 60 mg/kg, with a total of 6 rats.
  • the blood collection method and sample processing method are as described in Example 2.
  • the blood collection time points in this experiment are 5 minutes, 2 hours, 6 hours, 8 hours, 10 hours, and 16 hours after administration, and about 3.5 mL of whole blood is collected at each blood collection point.
  • the drug concentration in the lungs 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.
  • the lung drug concentration can be maintained above the corresponding peak concentration (32 ng/mL) after oral administration within 16 hours (greater than 50 ng/mL); this indicates that after inhalation of EGCG solution, a higher drug concentration level can be maintained in the lung for a longer period of time.
  • the average C max and AUC 0-t in lung tissue after airway atomization and oral administration of EGCG solution are shown in Table 3.
  • the average C max and AUC 0-t data in Table 3 further show that after inhalation of EGCG solution, EGCG solution has a good retention effect in the lungs, avoiding the rapid absorption of the drug into the blood circulation after administration, and greatly improving the local drug concentration in the lung tissue. It can be inferred that EGCG has anti- IPF activity is likely driven by its high exposure in lung tissue; thus, compared with oral administration, the safe and effective window of EGCG by inhalation is significantly expanded. At the same time, inhalation delivery of EGCG provides the possibility of extending the dosing interval and achieving a more flexible treatment plan.
  • mice A total of 56 C57BL/6J mice, SPF grade, male, 7-10 weeks old, weighing 20-35 g, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.
  • Bleomycin hydrochloride for injection (Bleomycin, BLM, 15 mg/vial, batch number 20067411): Hanhui Pharmaceutical Co., Ltd.
  • Solvent 1 The components are purified water, citric acid, sodium citrate, transparent liquid.
  • Solvent 2 Sodium chloride injection (batch number K21080307), purchased from Hunan Kelun Pharmaceutical Co., Ltd.
  • Hydroxyproline (HYP) kit purchased from Merck Sigma-Aldrich.
  • mice were randomly divided into sham operation group (normal control group), pulmonary fibrosis model group, oral administration group, and airway aerosol administration group 1-4, with 8 mice in each group.
  • D On day (abbreviated as D), the mice in the normal control group were given solvent 2 (sodium chloride injection, 1 mL/kg) by airway aerosolization, and the mice in the other groups were given solvent 2 (sodium chloride injection, 1 mL/kg) by airway aerosolization.
  • the mice were given bleomycin (2 mg/kg, 1 mL/kg) once in the morning and once in the afternoon to construct the model.
  • mice The oral dose for mice was 100 mg/kg (the oral dose of 60 mg/kg for rats was converted into the equivalent oral dose for mice by body surface area according to FDA guidelines), and the airway atomization was divided into four doses of 0.2 mg/kg, 0.4 mg/kg, 0.8 mg/kg, and 1.6 mg/kg.
  • the normal control group and the pulmonary fibrosis model group were atomized with the solvent 1, 1 time/day in the airway, and the drug was started on D10 after modeling.
  • mice in each group were anesthetized by intraperitoneal injection of chloral hydrate (50 mg/mL, 0.1 mL/10 g) on D22, and then bled from the abdominal aorta. All animals were dissected, lung tissues were preserved, and the weight of the whole lung was weighed (including animals found dead or euthanized at the end of life). The left lung was cut and stored in a refrigerator below -60°C for the detection of hydroxyproline content. 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, HE staining and Masson staining.
  • Lung coefficient lung weight (mg) / body weight (g).
  • the massive infiltration of inflammatory cells in the early stage of the model group, 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.
  • mice were weighed once after receipt, before grouping, on the day of modeling, on the first administration, and every 3 days thereafter; before planned euthanasia, the animals were also weighed when they were 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 and Tukey's multiple comparison test.
  • the hydroxyproline content is determined by detecting hydroxyproline in lung tissue according to the operating instructions of the kit.
  • mice in the pulmonary fibrosis model group Compared with the mice in the pulmonary fibrosis model group, the effects of airway atomization and intragastric 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. statistically extremely significant difference; ***: p ⁇ 0.001, i.e. statistically extremely significant difference) are shown in Figure 5. After modeling, the lung coefficients of mice in the model group were significantly higher than those in the normal control group (**: P ⁇ 0.01).
  • the lung coefficients of mice in the airway atomization and intragastric administration groups were significantly reduced, especially the lung coefficients of diseased mice in the airway atomization groups with a dosage of 0.2 mg/kg and 0.4 mg/kg (**: P ⁇ 0.01; ***: P ⁇ 0.001) were significantly reduced; while the intragastric administration group did not form a significant difference with the model group.
  • the lung tissue sections of the healthy control group (control) mice, the pulmonary fibrosis model (model) group, the group dosed with 100 mg/kg (p.o. 100) EGCG by oral gavage, and the groups dosed with various inhalation doses (inh.0.2, inh.0.4, inh.0.8, inh.1.6) were subjected to Masson’s staining and H&E staining, and the results are shown in Figures 7a and 7b, respectively.
  • the lung tissue structure of the mice in the healthy control/blank group was clear, the alveolar septa were uniform, there was no water edema, no obvious myofibroblasts, no inflammation and pulmonary fibrosis, and no obvious exudation in the alveolar cavity; the alveolar structure of the mice in the pulmonary fibrosis model group (model) 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.
  • the inhalation EGCG solution group with a dose of 1.6 mg/kg showed significant differences from the pulmonary fibrosis model group.
  • oral administration such as gavage
  • the inhalation administration groups of all doses reduced the infiltration of inflammatory cells, especially the inhalation administration group of 0.4 mg/kg EGCG solution had the best anti-inflammatory effect (score*: p ⁇ 0.05).
  • mice in the pulmonary fibrosis model group the effects of airway atomization and intragastric administration of EGCG on the fibrosis scores of mice are shown in Figure 9. From the comparison of the pulmonary fibrosis degree scores shown in Figure 9, it can be seen that:
  • the oral administration group and the inhalation administration groups of various doses can significantly reduce the lung fibrosis score of mice. When the inhalation dose is above 0.4 mg/kg, the lung 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 lung fibrosis model group.
  • EGCG solution can produce anti-inflammatory effects in the lungs at a relatively low dose (0.2 mg/kg); the anti-fibrosis effect gradually increases with the increase of the inhaled dose; the anti-fibrosis effect of the inhaled EGCG solution with a dose of 1.6 mg/kg is particularly significant. It can be seen that the effective dose range of inhaled EGCG is very wide.
  • the inhalation (such as airway atomization) dosage of 0.8 mg/kg described in Example 2 is equivalent to the exposure of the drug in plasma after oral administration (such as gavage) of 60 mg/kg EGCG solution to rats (equivalent to an oral dosage 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 the Student's T test. *: p ⁇ 0.05, that is, there is a statistically significant difference; **: p ⁇ 0.01, that is, there is a very significant difference in statistics; ***: p ⁇ 0.001, that is, there is an extremely significant difference in statistics.
  • 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.
  • a total of 88 SPF SD rats aged 6 to 9 weeks and weighing about 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 intragastric administration of EGCG group 1 (10 rats), an intragastric 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).
  • mice On D1, the normal control group mice were given solvent 2 (sodium chloride injection, 1 mL/kg) by airway aerosolization, and the mice in the other groups were given bleomycin (2.5 mg/kg, 1 mL/kg) by airway aerosolization, once in the morning and afternoon, to build the model. The animals in each group were weighed on D8. 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 The patients were given EGCG or pirfenidone as a control, once a day.
  • solvent 2 sodium chloride injection, 1 mL/kg
  • bleomycin 2.5 mg/kg, 1 mL/kg
  • the dosage and administration method of each group were as follows: the dosage of EGCG group 1 and group 2 for oral administration was 30 mg/kg and 60 mg/kg, respectively; the dosage of EGCG airway atomization 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 airway atomization control drug pirfenidone solution group was 0.9 mg/kg.
  • the dosage design of this group was based on the published literature of MWSurber, et al (ATS POSTER, 2014).
  • 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 then cool, then adjust the pH of the solution to 3-4 with citric acid and sodium citrate, then pre-filter the EGCG solution and perform aseptic filtration, and aseptically fill it to obtain an EGCG atomized solution.
  • the difference between it and the prescription described in 6.1 is that it also contains an appropriate amount of EDTA, and the preparation method is the same as described in 6.2.
  • Example 7 Single dose escalation test of EGCG aerosol inhalation solution in healthy subjects
  • 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 to compare it with nebulized solution placebo and oral EGCG capsules.
  • EGCG nebulized inhalation solution ie, the EGCG nebulized solution containing EDTA as described in Example 6
  • an initial concentration of 10 mg/mL which was diluted according to different doses before administration.
  • Oral control group EGCG capsules, each weighing 150 mg, in which the EGCG content is about 94% of the total weight.
  • EGCG nebulization inhalation administration There were three groups for EGCG nebulization inhalation administration: (1) 3 mg dose group, before administration, 1 mL of the starting drug 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 drug 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.
  • Blood samples were collected from subjects through an indwelling intravenous needle at 13 blood collection points, including 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 nebulization 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.
  • AE Adverse event monitoring
  • physical examination physical examination
  • vital signs temperature, blood pressure, pulse, respiration
  • blood oxygen saturation cardiac function
  • cardiac function lung function.
  • Female subjects with fertility should practice contraception for at least one month before screening and promise to take contraceptive measures throughout the study period and within 3 months after the end of the study; male subjects promise to take contraceptive measures throughout the study period and within 3 months after the end of the study and guarantee not to donate sperm.
  • Forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) ⁇ 80% of predicted values, FEV1/FVC ⁇ 0.7, and normal chest radiograph.
  • ALT Alanine aminotransferase
  • AST aspartate aminotransferase
  • GTT gamma-glutamyltransferase
  • UPN upper limit of normal
  • bilirubin total bilirubin exceeding the upper limit of normal
  • Alcoholism is defined as >21 units of alcohol per week (one unit equals 284 mL of beer or 25 mL of 40% liquor or 125 mL of wine).
  • pulmonary administration has a faster absorption rate.
  • the drug quickly enters the systemic circulation through the highly permeable pulmonary capillaries and alveolar surfaces and quickly reaches a very high peak value.
  • the peak time Tmax of the drug concentration absorbed into the blood after inhalation of EGCG is relatively late, and the Cmax level is relatively low.
  • the drug concentration entering the blood circulation system after inhalation is much lower than the drug level in plasma after oral administration.
  • the inhalation dose is 1/56 of the oral dose, while the plasma Cmax after inhalation is about 1/227 of the Cmax after oral administration.
  • the drug retention amount and retention time in the lungs can be significantly increased, and the drug concentration in plasma can be greatly reduced.
  • aerosol inhalation of EGCG can greatly reduce the risk of systemic adverse reactions while improving the therapeutic effect of lung drugs.
  • the application of (-)-epigallocatechin gallate compounds provided by the present disclosure directly administers EGCG to 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 dosages, it can produce significant anti-inflammatory and anti-pulmonary fibrosis effects, significantly expanding the safe treatment window of EGCG for IPF; after inhalation administration of EGCG, 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.

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Abstract

涉及化学医药领域，一种(-)-表没食子儿茶素没食子酸酯(EGCG)类化合物的应用，具体涉及一种EGCG类化合物在制备预防和/或治疗肺纤维化疾病的吸入药物中的应用、一种预防和/或治疗肺纤维化疾病的可吸入药物组合物、以及一种预防和/或治疗肺纤维化疾病的方法，其中所述应用中，EGCG类化合物为EGCG或其药学上可接受的盐、酯、水合物或溶剂化物。实施例提供的以EGCG类化合物为有效成分的药物/药物组合物作为可吸入药物用于预防和治疗肺纤维化中，可极大降低给药剂量，提供更宽的治疗窗口、更低的不良反应、更低的给药频次，将为肺纤维化患者提供新的治疗选择。

Description

(-)-表没食子儿茶素没食子酸酯类化合物的应用
优先权
所述PCT专利申请要求申请日为2022年11月24日，申请号为202211485124.4的中国专利优先权，本专利申请结合了上述专利的技术方案。
技术领域
本公开涉及化学医药领域，尤其涉及(-)-表没食子儿茶素没食子酸酯类化合物的应用。
背景技术
肺纤维化(Pulmonary Fibrosis，PF)是许多病因各异的肺间质疾病的终末期临床表现，是以肺泡持续性损伤、成纤维细胞增殖及大量细胞外基质(ExtraCellular Matrix，ECM)沉积为特征，从而导致肺泡和肺间质出现不同程度的炎症和纤维化，进而导致肺结构破坏和呼吸衰竭的一种疾病，所以也称间质性肺疾病(Interstitial Lung Disease，ILD)或弥漫性实质性肺疾病(Diffuse Parenchymal Lung Disease，DPLD)。ILD主要分为四大类：特发性间质性肺炎(Idiopathic Interstitial Pneumonia，IIPs)，自身免疫或结缔组织疾病引起的肺间质纤维化，接触或治疗引起的肺间质纤维化，以及结节病。特发性肺纤维化(Idiopathic Pulmonary Fibrosis，IPF)是特发性间质性肺炎(IIPs)中最为重要且最常见的一种，目前对肺纤维化的临床研究也主要集中在IPF方面，临床上表现为进行性呼吸困难 伴刺激性干咳，且患者会出现体重下降、易疲劳、全身不适、肌肉关节疼痛等症状，中位生存期约为2.8年，5年生存率不足50％，患者多死于呼吸衰竭和继发肺部感染。
肺纤维化的发病机制涉及多种因素如持续性损伤肺泡上皮致肺泡上皮-间充质转化(Epithelial-Mesenchymal Transition，EMT)的细胞内环境失调，转化生长因子-β(Transforming Growth Factor-β，TGF-β)、Wnt、Notch等信号通路激活、上皮细胞功能障碍和细胞凋亡、疤痕组织形成等导致的肺内环境破坏。目前世界范围内获批的用于治疗肺纤维化疾病的药物只有尼达尼布(Nintedanib)和吡非尼酮(Pirfenidone)，均为口服制剂；虽然两者可以延缓肺功能下降速度，但不能逆转病情进展，且两者的口服剂量大和不良反应严重。具体的，尼达尼布剂量为150mg BID(即：150毫克，每日2次)，最常见的不良反应为胃肠道反应，在临床研究中，腹泻的发生率高达61.5％；吡非尼酮剂量为801mg TID(即：801毫克，每日3次)，最常见的不良反应为光过敏症，在临床研究中的发生率高达51％；而这些严重的不良反应也是造成患者被迫降低剂量甚至提前停药的最常见原因。
在促进肺纤维化形成的众多细胞因子中，TGF-β1信号传导是胶原蛋白积聚和纤维化疾病的关键驱动因素，也是炎症和上皮细胞增殖的重要调控因子，但也因这种多功能细胞因子的性质而限制了TGF-β1抑制剂作为治疗剂的发展。研究表明，赖氨酰氧化酶样蛋白-2(Lysyl oxidase-like 2 protein，LOXL2)是一种细胞外基质蛋白，在健康成人组织中很少表达，但在多种纤维化疾病和肿瘤中可被诱导，由活化的成纤维细胞、疾病相关的平滑肌细胞、内皮细胞和上皮细胞等分泌。三酚羟基类化合物对LOXL2和TGF-β1活性的联合抑制可有效阻断体内病理性胶原积聚，而不产生与全局抑制剂相关的毒性。
(-)-表没食子儿茶素没食子酸酯((-)-Epigallocatechin gallate， EGCG)是绿茶提取物(Green Tea Extract，GTE)儿茶素中含量最多的一种活性成分，占儿茶素总含量的50-80％，是一种多酚类黄酮化合物，其化学结构式为EGCG具有多种生物学功能，可防止自由基引起的细胞损伤，抗菌，还可以减少炎症并预防某些慢性病，包括心脏病、糖尿病和一些癌症等。已有研究表明EGCG可有效抑制赖氨酰氧化酶样蛋白-2(LOXL2)和TGFβ受体1和2(TGFβR1/2)激酶。(Ying Wei，et al.2017；Harold A.Chapman,et al.2020)。目前公开的EGCG在肺纤维化相关的研究中，EGCG是以口服形式给药，虽EGCG口服给药在肺纤维化模型动物和人类患者中具有一定的效果，但其口服后肠道吸收差、且药物吸收受食物影响，EGCG口服后在唾液中即被酯酶水解，进入血液和肝脏后又发生广泛的酶代谢反应，如葡萄糖醛酸化、硫酸化等，导致口服EGCG的生物利用度极低，需要很高的剂量才有可能发挥抗肺纤维化作用，例如Harold A.Chapman,et al文献报道的剂量是600mg；长期大剂量服药又可能存在安全性隐患。
另外，有已发表的人类或动物研究的证据表明，口服绿茶提取物(GTE)与肝损伤之间存在联系(Jiang Hu,et al.,2018；García-Cortés et al.,2016；Harrison-Dunn,2016；Teschke et al.,2014)。欧洲食品安全局(EFSA)科学合作项目(ESCO)于2018年发布的关于绿茶提取物EGCG的安全评估中指出，介入性临床试验证据表明，每天口服摄入等于或高于800mg剂量的EGCG，会导致受试者血清转氨酶的显著增加，这表明具有肝损伤，是不能被视为安全的。也有报道称，一种含有80％绿茶提取物日剂量相当于 375mg EGCG的产品，也出现了肝毒性(EFSA,2018)。另有临床研究表明口服EGCG可显著减轻患者体重(I-Ju Chen,et al.2016)，对于IPF患者，相较于高体重患者，低体重的患者死亡率更高生存期更短，BMI＜25和BMI≧30的患者，中位生存期分别约为3.6年和5.8年(Mazen Alakhras，et al.2007；Nobuyasu Awano,et al.2021)。因此，口服EGCG治疗IPF的安全有效窗口较窄，存在低剂量无效高剂量出现毒性的风险，而由于疾病的复杂性，IPF患者常常需要根据病情的进展调整用药剂量，口服EGCG无法满足患者个体化用药的需求。EGCG口服后在体内可经过广泛的酶代谢。虽然EGCG不是药物代谢酶CYP450酶系的底物，但EGCG可在不同程度上抑制多种CYP酶活性。EGCG还可抑制OATP等药物转运蛋白。因此EGCG对多种药物的生物利用度都可能存在显著影响，还有研究者建议正在接受OATP底物的药物治疗的患者，特别是那些治疗指数较窄的患者，应避免或至少谨慎地摄入大量的GTE或EGCG(Ahmed A.Albassam,2017)。在一项26例IPF患者的研究中，患者同时口服EGCG可使尼达尼布血浆暴露量减少约21％，这是一种具有统计学意义的作用，可能会损伤疗效(G.D.Marijn Veerman,et al.2022)。可见，因存在上述安全有效的治疗窗口窄、严重的药物间相互作用等缺陷，限制了口服EGCG在治疗肺纤维化疾病方面的临床应用。
目前世界范围内尚无治疗IPF的吸入药物成功获批，相关吸入药物的开发也极少。吡非尼酮是需要口服非常大的剂量才能在肺部达到有效的药物水平，获批的剂量为801mg TID，而由此产生的血浆药物水平很高，导致患者耐受性差；其雾化吸入制剂正在被开发，但在Phase II研究中因低剂量(50mg/一天一次)无效而被终止了该剂量的研究，仍在研究中的高剂量组的吸入剂量较大且给药频次较高(100mg/一天两次)。且目前的临床表现并未如预期那样显著地优于口服给药。动物实验结果表明，吡非尼酮改为吸入方式施用后，药物在肺部的半衰期t1/2仅约为10min。在Phase I研究 中，人体药代动力学(Pharmacokinetics，PK)表现与动物实验结果基本一致，100mg雾化剂量的吡非尼酮仅使得肺泡上皮衬液(epithelial lining fluid，ELF)中的药物最大浓度Cmax比口服剂量(801mg)在肺泡上皮衬液中的Cmax高35倍，并在短时间内降到比口服更低的肺部药物浓度水平，全身暴露量比口服剂量报告的低约15倍，吡非尼酮吸入给药后仍然有较高的血浆药物浓度(807～1370ng/mL)，因此改为吸入给药后吡非尼酮仍然存在较高的不良反应风险。由此证明，吡非尼酮吸入给药后，药物从肺部快速进入血液，很难在肺部长时间内维持在有效的药物浓度水平，并未像人们当初所预期那样达到理想的效果。
可见并非所有药物都适合肺部递送，绝大多数药物在吸入给药后由于无法保持有效的肺内滞留而快速进入血液，导致无法在肺局部维持较高浓度而发挥持久的药效。另外，由于肺纤维化疾病的病理机制复杂，进展快，药物开发难度很大失败率极高；纤维化发展过程中涉及的多种生长因子，如TGF-β、血小板衍生生长因子(Platelet derived growth factor，PDGF)在机体内具有多种生理功能，维持机体稳态，通过系统性抑制所述生长因子造成的毒副作用屡见不鲜。因此肺纤维化疾病的治疗药物应具有较宽的治疗窗口、实现轻重症患者个体化给药方案，尤其是可通过吸入方式递送并在肺局部发挥持久效用的治疗药物，仍是目前临床上未被满足的迫切需求。
因此，现有技术还有待于改进和发展。
发明内容
鉴于上述现有技术的不足，本公开的目的在于提供一种(-)-表没食子儿茶素没食子酸酯类化合物的应用，旨在解决现有的EGCG以口服给药形式在治疗肺纤维化疾病方面的临床应用存在生物利用度极低、需要很高的给 药剂量、安全有效的治疗窗口窄、与多种药物间存在严重的药物间相互作用等问题。
本公开的技术方案如下：
第一方面，本公开提供一种(-)-表没食子儿茶素没食子酸酯类化合物在制备预防和/或治疗肺纤维化疾病的吸入药物中的应用，其中，所述(-)-表没食子儿茶素没食子酸酯类化合物为(-)-表没食子儿茶素没食子酸酯或其药学上可接受的盐、酯、水合物或溶剂化物。
第二方面，一种预防和/或治疗肺纤维化疾病的可吸入药物组合物，其中，包括：(-)-表没食子儿茶素没食子酸酯类化合物作为有效成分，以及药学上可接受的辅料；所述(-)-表没食子儿茶素没食子酸酯类化合物为EGCG或其在药学上可接受的盐、酯、水合物或溶剂化物。
第三方面，本公开提供一种预防和/或治疗肺纤维化疾病的方法，其中，所述方法包括：通过吸入给药向受试者施用所述(-)-表没食子儿茶素没食子酸酯类化合物，所述(-)-表没食子儿茶素没食子酸酯类化合物为(-)-表没食子儿茶素没食子酸酯或其药学上可接受的盐、酯、水合物或溶剂化物。
有益效果：相对于现有的EGCG以口服给药形式用于治疗肺纤维化，本公开通过将EGCG或其药学上可接受的盐、酯、水合物或溶剂化物的EGCG类化合物用于制备治疗肺纤维化的以吸入形式给药的药物，从而实现：(1)降低给药剂量和给药频次；(2)减少不良反应和副作用；(3)提高药物在肺部的浓度和停留时间；(4)拓宽安全有效的治疗窗口；(5)对组合使用的其他药物的生物利用度影响小且与其他药物之间不发生药物-药物的相互作用。
附图说明
图1为以不同给药剂量气道雾化给药(inh,给药剂量分别为1.6mg/kg、3.2mg/kg、6.4mg/kg)EGCG后，大鼠血浆和肺部药物浓度(Concentration，单位为ng/mL)随时间(Time，单位为hour)的变化曲线对比图。
图2为静脉注射给药(iv,给药剂量为3.2mg/kg)EGCG后与灌胃给药(po,给药剂量为60mg/kg)EGCG后，大鼠血浆和肺部药物浓度(Concentration，单位为ng/mL)随时间(Time，单位为hour)的变化曲线对比图。
图3为气道雾化给药EGCG溶液(给药剂量为0.8mg/kg)及灌胃给药EGCG溶液(给药剂量为60mg/kg)后，大鼠血浆和肺部药物浓度随时间(Time，单位为hour)的变化曲线对比图。
图4为气道雾化给药低剂量EGCG(给药剂量分别为0.05mg/kg、0.1mg/kg、0.2mg/kg、0.4mg/kg)及灌胃给药(给药剂量为60mg/kg)EGCG溶液后，大鼠肺部药物浓度(lung concentration，单位为ng/mL)随时间(Time，单位为hour)的变化曲线对比图。
图5为与肺纤维化模型组小鼠对比，气道雾化给药EGCG溶液及灌胃给药EGCG溶液后对小鼠的肺重指数(单位为％)的影响(采用Kruskal-Wallis test检验，*:p＜0.05,即统计学上具有显著性差异；**：p＜0.01，即统计学上具有极显著性差异；***：p＜0.001，即统计学上具有极其显著的差异)。
图6为与肺纤维化模型组小鼠对比，气道雾化给药EGCG及灌胃给药后对小鼠的体重(单位为g)的影响(通过使用双因素ANOVA和Tukey多重比较检验进行统计分析)。
图7a为气道雾化给药EGCG和灌胃给药对博来霉素诱导的小鼠肺组织纤维化程度的影响(Masson染色，100×)；
图7b为气道雾化给药EGCG和灌胃给药对博来霉素诱导的小鼠肺组织 病理结构改变的影响(H&E染色，100×)。
图8为与灌胃给药相比，气道雾化给药EGCG溶液对肺纤维化小鼠肺泡炎细胞浸润、出血、肺泡扩张、肺泡纤维样渗出的影响。
图9为与肺纤维化模型组小鼠对比，气道雾化给药EGCG及灌胃给药后对小鼠纤维化评分(Fibrosis Score)的影响。
图10为与肺纤维化模型组小鼠对比，灌胃和气道雾化给药EGCG对小鼠肺组织中羟脯氨酸含量(单位为μg/μL)的影响(采用One-Way ANOVA分析法进行方差齐性分析，并结合Dunnett's test分析方法进行组间比较)。
图11为与肺纤维化模型组大鼠对比，灌胃和气道雾化给药EGCG、以及气道雾化给予对照药吡非尼酮(AP01)，对大鼠肺组织中羟脯氨酸含量(单位为μg/μL)的影响(采用Kruskal-Wallis test检验，*:p＜0.05,即统计学上具有显著性差异；**：p＜0.01，即统计学上具有极显著性差异；***：p＜0.001，即统计学上具有极其显著的差异；****：p＜0.0001，即统计学上具有极其高度显著的差异)。
图12为向健康受试者口服给药EGCG胶囊(150mg/粒，共4粒)和雾化吸入给药EGCG溶液(3mg剂量组，10mg剂量组，30mg剂量组)后，血液中EGCG的平均药物浓度(plasma concentration)随时间的变化曲线。
具体实施方式
本公开提供一种(-)-表没食子儿茶素没食子酸酯类化合物的应用，为使本公开的目的、技术方案及效果更加清楚、明确，以下对本公开进一步详细说明。应当理解的是，此处所描述的具体实施例仅用以解释本公开，并不用于限定本公开。
第一方面，本公开实施例提供一种(-)-表没食子儿茶素没食子酸酯类 化合物在制备治疗肺纤维化疾病的吸入药物中的应用，其中，所述(-)-表没食子儿茶素没食子酸酯类化合物为(-)-表没食子儿茶素没食子酸酯或其药学上可接受的盐、酯、水合物或溶剂化物。
在一些实施例中，所述(-)-表没食子儿茶素没食子酸酯类化合物为(-)-表没食子儿茶素没食子酸酯或其药学上可接受的酯；在某些实施例中，所述其药学上可接受的酯是指EGCG的脂肪酸酯，也即，由EGCG结构中至少一个羟基与C1-C30的羧酸酯化得到。
在一些实施例中，所述吸入药物被配制为可供吸入的剂型，所述剂型选自溶液、混悬液、气雾剂或粉雾剂。
在一些实施例中，所述吸入药物是由所述(-)-表没食子儿茶素没食子酸酯类化合物经干燥工艺加工形成的粉末、再经稀释剂复溶形成。
在一些实施例中，所述剂型为溶液时，所述吸入药物包括：作为活性成分的所述(-)-表没食子儿茶素没食子酸酯类化合物，稀释剂，以及pH调节剂、渗透压调节剂和抗氧化剂中的至少一种，所述(-)-表没食子儿茶素没食子酸酯类化合物完全溶解于所述稀释剂中。
在一些实施例中，所述剂型为混悬剂时，所述吸入药物包括：作为活性成分的所述(-)-表没食子儿茶素没食子酸酯类化合物，稀释剂，以及表面活性剂、pH调节剂、张力调节剂中的至少一种，所述(-)-表没食子儿茶素没食子酸酯类化合物或所述所述(-)-表没食子儿茶素没食子酸酯类化合物与可适用于吸入给药的载体形成颗粒悬浮于稀释剂中。
在一些实施例中，所述剂型为气雾剂时，所述吸入药物包括：作为活性成分的所述(-)-表没食子儿茶素没食子酸酯类化合物，稀释剂，抛射剂，以及表面活性剂、助溶剂和pH调节剂中的至少一种。
在一些实施例中，所述剂型为粉雾剂时，所述吸入药物包括：作为活性成分的所述(-)-表没食子儿茶素没食子酸酯类化合物，可适用于吸入给药的载体，以及赋形剂和表面活性剂中的至少一种。
在一些实施例中，所述稀释剂为水、乙醇和甘油中的一种或多种；优选的，所述稀释剂为水。
在一些实施例中，所述吸入药物的pH值为3.0-5.0。
在一些实施例中，所述吸入药物中、所述(-)-表没食子儿茶素没食子酸酯类化合物的浓度为0.1-25mg/mL。
在一些实施例中，所述吸入药物包括所述(-)-表没食子儿茶素没食子酸酯类化合物，或所述吸入药物包括所述(-)-表没食子儿茶素没食子酸酯类化合物和其他抗肺纤维化药物。在一些实施例中，所述其他抗肺纤维化药物选自吡非尼酮、尼达尼布、糖皮质激素、免疫抑制剂、前列环素及其类似物、CTGF抗体、Galectin-3抑制剂、整合素拮抗剂、重组血清淀粉样蛋白P及其类似物、PDE抑制剂、LPA拮抗剂、JAK激酶抑制剂、多种细胞因子受体TKI中的一种或多种。
优选的，在一些实施例中，所述其他抗肺纤维化药物选自吡非尼酮、尼达尼布、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，)、曲前列环素及其类似物、重组血清淀粉样蛋白P、LPA拮抗剂中的一种或多种。
在一些实施例中，所述吸入药物还可与上述的其他抗纤维化药物联用。本发明中所述的“联用”是一种给药方式，是指在一定时间期限内给予至少一种剂量的(-)-表没食子儿茶素没食子酸酯类化合物和至少一种剂量的其他化合物，其中两种物质都显示药理学作用。所述的时间期限为一个给药周期，优选24小时以内，更优选12小时以内。可以同时或依次给予(-)-表没食子儿茶素没食子酸酯类化合物和其他抗纤维化药物。这种期限包括这样的治疗，其中通过相同给药途径或不同给药途径给予(-)-表没食子儿茶素没食子酸酯类化合物和其他抗纤维化药物。本发明所述联合的给药方式选自同时给药、独立地配制并共给药或独立地配制并相继给药。
在一些实施例中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为0.1-100mg/次。
优选的，在一些实施例中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为0.1-50mg/次。
更优选的，在一些实施例中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为0.1-30mg/次。
最优选的，在一些实施例中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为0.1-15mg/次。
示例性的，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量可选自0.1mg/次、1mg/次、1.5mg/次、2mg/次、2.5mg/次、3mg/次、3.5mg/次、4mg/次、4.5mg/次、5mg/次、5.5mg/次、6mg/次、6.5mg/次、7mg/次、7.5mg/次、8mg/次、8.5mg/次、9mg/次、9.5mg/次、10mg/次、10.5mg/次、11mg/次、11.5mg/次、12mg/次、12.5mg/次、13mg/次、13.5mg/次、14mg/次、14.5mg/次、15mg/次、15.5mg/次、16mg/次、16.5mg/次、17mg/次、17.5mg/次、18mg/次、18.5mg/次、19mg/次、19.5mg/次、20mg/次、25mg/次、30mg/次、35mg/次、40mg/次、45mg/次、50mg/次、55mg/次、60mg/次、65mg/次、70mg/次、75mg/次、80mg/次、90mg/次、100mg/次等。
现有文献报到的EGCG的口服给药剂量为400mg-600mg/次。在一些实施例中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为口服给药剂量的1/1000至1/10。
优选的，在一些实施例中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为口服给药剂量的1/600至1/10。更优选的，在一些实施例中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为口服给药剂量的1/200至1/10。
在一些实施例中，所述肺纤维化疾病为间质性肺疾病，所述肺纤维化疾病包括特发性间质性肺炎，自身免疫或结缔组织疾病引起的肺间质纤维化，接触或职业暴露相关的肺间质纤维化，治疗引起的肺间质纤维化，以 及结节病中的一种或多种。
在一些实施例中，所述特发性间质性肺炎包括特发性肺纤维化；所述自身免疫或结缔组织疾病引起的肺间质纤维化包括狼疮、硬皮病、多发性肌炎或皮肌炎、类风湿性关节炎相关的间质性肺疾病；所述接触或职业暴露相关的间质纤维化包括石棉肺、矽肺、过敏性肺炎；所述治疗引起的间质纤维化包括化疗、放疗和一些药物治疗引起的间质性肺疾病。
第二方面，本公开实施例提供一种预防和/或治疗肺纤维化疾病的可吸入药物组合物，其中，包括：(-)-表没食子儿茶素没食子酸酯类化合物作为有效成分，以及药学上可接受的辅料；所述(-)-表没食子儿茶素没食子酸酯类化合物为EGCG或其在药学上可接受的盐、酯、水合物或溶剂化物。
在一些实施例中，所述可吸入药物组合物中、所述(-)-表没食子儿茶素没食子酸酯类化合物的含量可为0.1-100mg、0.1-80mg、0.1-70mg、0.1-60mg、0.1-50mg、0.1-30mg、0.1-15mg等。
在一些实施例中，每剂量所述可吸入药物组合物中、所述(-)-表没食子儿茶素没食子酸酯类化合物的含量为0.1-50mg。
在一些实施例中，每剂量所述可吸入药物组合物中、所述(-)-表没食子儿茶素没食子酸酯类化合物的含量为0.5-30mg。
在一些实施例中，每剂量所述可吸入药物组合物中、所述(-)-表没食子儿茶素没食子酸酯类化合物的含量，示例性的为0.5mg,1mg，1.5mg，2mg，2.5mg，3mg，3.5mg，4mg，4.5mg，5mg，5.5mg，6mg，6.5mg，7mg，7.5mg，8mg，8.5mg，9mg，9.5mg，10mg，10.5mg，11mg，11.5mg，12mg，12.5mg，13mg，13.5mg，14mg，14.5mg，15mg，15.5mg，16mg，16.5mg，17mg，17.5mg，18mg，18.5mg，19mg，19.5mg，20mg，25mg，30mg。
在一个实施方式中，所述可吸入药物组合物是将所述(-)-表没食子儿 茶素没食子酸酯类化合物与所述药学上可接受的辅料采用常规或特殊的制剂工艺制备成吸入制剂，所述吸入制剂为吸入溶液、吸入混悬液、气雾剂、粉雾剂或其他吸入制剂。
进一步地，在一些实施例中，所述吸入溶液为雾化吸入溶液；所述吸入混悬液为雾化吸入混悬液。雾化吸入是指利用雾化装置将药物组合物分散成微小雾滴，使其悬浮于气体中，并吸入呼吸道及肺内。通过雾化吸入所述药物组合物可达到局部治疗和全身治疗的目的；如同时具有较强的抗纤维化作用和抗炎效果。
在一些实施例中，所述(-)-表没食子儿茶素没食子酸酯类化合物先经过干燥工艺被加工成粉末，在向受试者施用之前通过稀释剂将所述粉末与所述药学上可接受的辅料复溶后以雾化形式递送至受试者的肺部。所述干燥工艺可为冷冻干燥(freeze-drying)、喷雾干燥(spray-drying)、喷雾冷冻干燥(spray-freeze drying)或超临界流体技术(supercritical fluid technology)等，
在一些实施例中，所述可吸入药物组合物的剂型为吸入溶液，所述药学上可接受的辅料选自表面活性剂、pH调节剂、抗氧化剂、防腐剂、渗透压调节剂、金属离子络合剂、水和附加剂中的一种或多种。在另一些实施例中，所述可吸入药物组合物的剂型为吸入混悬液，所述药学上可接受的辅料选自表面活性剂、pH调节剂、抗氧化剂、防腐剂、渗透压调节剂、金属离子络合剂、水和附加剂中的一种或多种。进一步地，在一些实施例中，所述吸入溶液或吸入混悬液中几乎不含防腐剂。
在一些实施例中，所述吸入溶液和所述吸入混悬液中，所述(-)-表没食子儿茶素没食子酸酯类化合物的浓度为0.1-35mg/mL、为0.1-30mg/mL、0.1-25mg/mL、0.5-25mg/mL、0.5-15mg/mL、0.5-10mg/mL、0.5-5mg/mL等。
在一些实施例中，所述吸入溶液和所述吸入混悬液中，所述(-)-表没 食子儿茶素没食子酸酯类化合物的浓度,示例性地为1mg/mL,2mg/mL,3mg/mL,4mg/mL,5mg/mL,6mg/mL,7mg/mL,8mg/mL,9mg/mL,10mg/mL,11mg/mL,12mg/mL,13mg/mL,14mg/mL,15mg/mL,16mg/mL,17mg/mL,18mg/mL,19mg/mL,20mg/mL,21mg/mL,22mg/mL,23mg/mL,24mg/mL,25mg/mL。
在一些实施例中，所述可吸入药物组合物的剂型为气雾剂，所述药学上可接受的辅料选自助溶剂、表面活性剂、抛射剂和附加剂中的一种或多种。
进一步地，在一些实施例中，所述抛射剂为氢氟烷烃类化合物。较佳的，所述抛射剂为1,1,1,2－四氟乙烷(简称HFA134a)、1,1,1.2,3,3,3－七氟丙烷(简称HFA 227)中的一种或两种。更进一步的，在一些实施例中，所述附加剂包含溶剂，所述溶剂选自甘油、丙二醇、聚乙二醇、乙醇或油酸中的一种或多种；较佳的，所述溶剂为乙醇、丙二醇中的一种或两种。
在一些实施例中，所述可吸入药物组合物的剂型为粉雾剂，所述药学上可接受的辅料包括赋形剂、载体和附加剂。进一步地，在一些实施例中，所述赋形剂包括糖类、糖醇类、淀粉类、大分子聚合物、脂肪酸或其盐、蜡类、硫酸钙、碳酸钙、滑石粉、氧化铁以及轻质无水硅酸中的一种或多种。所述糖类包括乳糖、葡萄糖、白糖、海藻糖、蔗糖等中的一种或多种；所述糖醇类包括赤藓糖醇、甘露醇、山梨醇等中的一种或多种；所述大分子聚合物包括结晶纤维素、甲基纤维素、羟丙基纤维素、羧甲基纤维素钙、羟丙基甲基纤维素、羧甲醚纤维素钠、支链淀粉、糊精、阿拉伯胶、琼脂、明胶、黄蓍胶、海藻酸钠、聚乙烯吡咯烷酮、聚乙烯醇等中的一种或多种；所述脂肪酸可为软脂酸、硬脂酸、油酸等中的一种或多种。
较佳的，在一些实施例中，所述赋形剂选自糖类、糖醇类、大分子聚合物和碳酸钙中的一种或多种。所述糖类为乳糖或蔗糖；所述糖醇类为赤 藓糖醇、山梨醇或甘露醇；所述大分子聚合物为羧甲基纤维素钙、支链淀粉、聚乙烯吡咯烷酮或甲基纤维素。最佳的，所述赋形剂为乳糖或赤藓糖醇。
在一些实施例中，所述载体包括乳糖、葡萄糖、果糖、蔗糖、麦芽糖、葡聚糖、赤藓糖醇、山梨醇、甘露醇、硫酸钙、碳酸钙、滑石或氧化铁等。
在一些实施例中，所述食子儿茶素没食子酸酯类化合物的形态可为晶体或无定型态。
在一些实施例中，所述预防和/或治疗肺纤维化疾病时，所述可吸入药组合物通过吸入给药装置发射成的气溶胶液滴或微粉化颗粒递送至受试者的呼吸道和肺部。进一地，在一些实施例中，所述气溶胶液滴的平均粒径为0.5-10μm；示例性的，气溶胶液滴的平均粒径可为1-8μm、1-5μm、1-3μm、2-3μm等。进一步在一些实施例中，所述微粉化颗粒的平均粒径小于或等于20μm；优选小于或等于10μm，更优选1-9μm，最优选3-8μm；该粒径范围内微粉化颗粒能抵达受试者的呼吸道(如支气管)和肺部。
第三方面，本公开实施例提供一种预防和/或治疗肺纤维化疾病的方法，其中，所述方法包括：通过吸入给药向受试者施用所述(-)-表没食子儿茶素没食子酸酯类化合物，所述(-)-表没食子儿茶素没食子酸酯类化合物为(-)-表没食子儿茶素没食子酸酯或其药学上可接受的盐、酯、水合物或溶剂化物。
具体的，在一些实施例中，所述方法可包括通过吸入方式向哺乳动物的肺部递送治疗需要的有效量的所述(-)-表没食子儿茶素没食子酸酯类化合物。肺纤维化疾病可包括：特发性间质性肺炎，自身免疫或结缔组织疾病引起的肺间质纤维化，接触或治疗引起的肺间质纤维化，以及结节病等间质性肺疾病。其中，所述特发性间质性肺炎包括特发性肺纤维化。所述 自身免疫或结缔组织疾病引起的间质纤维化包括狼疮、硬皮病、多发性肌炎或皮肌炎、类风湿性关节炎相关的间质性肺疾病。所述接触或职业暴露相关的间质纤维化包括石棉肺、矽肺、过敏性肺炎。所述治疗相关的间质纤维化包括化疗、放疗和一些药物治疗引起的间质性肺疾病。
在一些实施例中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为0.01-2.0mg/kg。具体的，所述治疗方法中有效成分EGCG的受试动物的给药剂量可为0.01-2.0mg/kg，其换算为人类患者的拟临床用量为0.1-100mg，相应剂量参考临床或文献中报道的方法进行换算(E Boger,et al.2016；Ramon Hendrickx,et al.2018；Therese Ericsson,et al.2017)。考虑到患者的体重及其他生理病理情况，在一些实施方式中，人类的给药剂量可为0.1-80mg、0.1-70mg、0.1-60mg、0.1-50mg、0.1-30mg、0.1-15mg等。在一些实施方式中，人类的给药剂量示例性地可为1mg、1.5mg、2mg、2.5mg、3mg、3.5mg、4mg、4.5mg、5mg、5.5mg、6mg、6.5mg、7mg、7.5mg、8mg、8.5mg、9mg、9.5mg、10mg、10.5mg、11mg、11.5mg、12mg、12.5mg、13mg、13.5mg、14mg、14.5mg、15mg、15.5mg、16mg、16.5mg、17mg、17.5mg、18mg、18.5mg、19mg、19.5mg、20mg、25mg、30mg等。
现有文献报到的EGCG的口服给药剂量为400mg-600mg/次。在一些实施例中，所述治疗方法包括向患者肺部递送(-)-表没食子儿茶素没食子酸酯类化合物的剂量可为口服给药剂量的1/1000至1/10。较佳的，可为口服给药剂量的1/600至1/10。示例性的，可为口服给药剂量的1/200至1/10，可为口服给药剂量的1/50至1/10。
在一些实施例中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量可为0.1-80mg/次、0.1-70mg/次、0.1-50mg/次、0.5-30mg/次、0.5-15mg/次等。
最优选的，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为0.5-15mg/次。
示例性的，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量0.1mg/次、1mg/次、1.5mg/次、2mg/次、2.5mg/次、3mg/次、3.5mg/次、4mg/次、4.5mg/次、5mg/次、5.5mg/次、6mg/次、6.5mg/次、7mg/次、7.5mg/次、8mg/次、8.5mg/次、9mg/次、9.5mg/次、10mg/次、10.5mg/次、11mg/次、11.5mg/次、12mg/次、12.5mg/次、13mg/次、13.5mg/次、14mg/次、14.5mg/次、15mg/次、15.5mg/次、16mg/次、16.5mg/次、17mg/次、17.5mg/次、18mg/次、18.5mg/次、19mg/次、19.5mg/次、20mg/次、25mg/次、30mg/次、35mg/次、40mg/次、45mg/次、50mg/次、55mg/次、60mg/次、65mg/次、70mg/次、75mg/次、80mg/次、90mg/次、100mg/次等。
进一步地，在一些实施例中，所述治疗方法包括向患者的肺部递送0.1-100mg的(-)-表没食子儿茶素没食子酸酯类化合物；示例性的，0.1-80mg的(-)-表没食子儿茶素没食子酸酯类化合物，0.1-70mg的(-)-表没食子儿茶素没食子酸酯类化合物，0.1-60mg的(-)-表没食子儿茶素没食子酸酯类化合物，0.1-50mg的(-)-表没食子儿茶素没食子酸酯类化合物，或0.1-50mg的(-)-表没食子儿茶素没食子酸酯类化合物，0.1-30mg的(-)-表没食子儿茶素没食子酸酯类化合物等。示例性的，所述肺部递送剂量为1mg、1.5mg、2mg、2.5mg、3mg、3.5mg、4mg、4.5mg、5mg、5.5mg、6mg、6.5mg、7mg、7.5mg、8mg、8.5mg、9mg、9.5mg、10mg、10.5mg、11mg、11.5mg、12mg、12.5mg、13mg、13.5mg、14mg、14.5mg、15mg、15.5mg、16mg、16.5mg、17mg、17.5mg、18mg、18.5mg、19mg、19.5mg、20mg、25mg、30mg等。
在一些实施例中，所述通过吸入给药向受试者施用所述(-)-表没食 子儿茶素没食子酸酯类化合物，包括：先以较低剂量为起始剂量向受试者的肺部递送(-)-表没食子儿茶素没食子酸酯类化合物，然后根据患者病情以剂量递增方式向受试者的肺部递送(-)-表没食子儿茶素没食子酸酯类化合物。
在一些实施例中，所述方法还包括：通过吸入给药向受试者施用活性剂，所述活性剂与所述(-)-表没食子儿茶素没食子酸酯类化合物同时、独立或相继的施用。
在一些实施例中，所述活性剂选自吡非尼酮、尼达尼布、糖皮质激素、免疫抑制剂、前列环素及其类似物、CTGF抗体、Galectin-3抑制剂、整合素拮抗剂、重组血清淀粉样蛋白P(简称重组pentraxin-2，例如PRM-151)及其类似物、PDE抑制剂、LPA拮抗剂、JAK激酶抑制剂、多种细胞因子受体TKI中的一种或多种。进一步的，在一些实施例中，所述前列环素及其类似物可任选自曲前列环素和伊洛前列素；所述CTGF抗体可任选自Pamrevlumab；所述Galectin-3抑制剂可任选自GB0139；所述整合素拮抗剂为整合素(αvβ1、αvβ6)拮抗剂，所述整合素拮抗剂可任选自PLN-74809；所述重组Pentraxin-2可任选自PRM-151；所述PDE抑制剂可任选自BI 1015550；所述LPA拮抗剂可任选自BMS-986278。
在一些较佳的实施例中，所述活性剂选自吡非尼酮、尼达尼布、BI1015550、曲前列环素及其类似物、重组血清淀粉样蛋白P、LPA拮抗剂中的一种或多种。
在一些实施例中，所述施用的频次为1次/2天、1次/天或2次/天。也即，按照每2天1次、每天1次或每天2次向受试者施用所述(-)-表没食子儿茶素没食子酸酯类化合物。进一步地，在一些实施例中，每天向受试者施用的给药剂量小于或等于150mg；较佳的，每天向受试者施用的 给药剂量小于或等于100mg。示例性的，每天向受试者施用的给药剂量可为150mg、140mg、120mg、100mg、80mg、70mg、60mg、50mg、40mg、35mg、30mg、25mg、20mg、19.5mg、19mg、18.5mg、18mg、17.5mg、17mg、16.5mg、16mg、15.5mg、15mg、14.5mg、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等。
在一些实施例中，所述吸入给药是指采用载有所述药物的吸入给药装置将所述药物递送至所述受试者。
在一些实施例中，吸入给药装置为雾化发生器、压力定量吸入器、干粉吸入器或软雾吸入器。
应当理解的，本公开中所述的给药剂量可以是从吸入装置输出的剂量(即装置递送剂量)；也可以是按照本领域普遍认知的吸入给药装置的递送效率约为25％-75％来折算的递送至患者肺部的剂量(即肺部剂量)。
所述受试者为人类时，最优选的，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为1-15mg/次；吸入给药时向受试者施用的所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为3-50mg/次。
本公开实施例提供的以(-)-表没食子儿茶素没食子酸酯类化合物为有效成分的药物/药物组合物能以吸入给药的方式向受试者施用(如递送)，递送后(-)-表没食子儿茶素没食子酸酯类化合物可有效地沉积在受试者的肺部，使受试者的肺部的药物浓度更高，系统血药浓度更低，从而避免潜在的肝毒性等副作用。可以在疗效和毒性之间产生更大的跨度，使得吸入给药时、该药物具有很宽的治疗窗口。
且有，吸入给药本实施例的药物/药物组合物所提供的较高肺部浓度的 (-)-表没食子儿茶素没食子酸酯类化合物，可以快速且更有效地调节肺组织中的α-平滑肌肌动蛋白(α-SMA)、SNAI1、I型胶原蛋白(collagen I)、Fibronectin、磷酸化SMAD3(pSMAD3)、磷酸化SMAD2(pSMAD2)等肺纤维化和炎症相关的关键蛋白活性，产生更好的药效；而肺组织中的部分蛋白，在口服EGCG后是无法对其达到有效抑制程度。吸入给药本实施例的药物/药物组合物后，(-)-表没食子儿茶素没食子酸酯类化合物可以在受试者的肺部以较高药物浓度维持较长时间，为降低给药频次增加受试者的顺应性提供了可行性。此外通过吸入给药本实施例的药物/药物组合物，还避免了口服给药的EGCG与其他IPF治疗药物之间易发生药物-药物相互作用的问题。
总的来说，本公开实施例提供的以(-)-表没食子儿茶素没食子酸酯类化合物为有效成分的药物/药物组合物作为可吸入药物用于预防和治疗肺纤维化中，可极大降低给药剂量，提供更宽的治疗窗口、更低的不良反应、更低的给药频次，将为肺纤维化患者提供新的治疗选择，具有极大的社会效益和经济效益。
下面通过具体实施例，对本公开的技术方案进行详细说明。
实施例1气道雾化给药、静脉注射给药、灌胃给药EGCG后，大鼠血浆中的药代动力学研究
1.1准备
材料：EGCG，含量>98wt％。
气道雾化给药溶液的配制：精密称量EGCG化合物，加溶媒1配制成药物溶液，透明澄清液体；其中，溶媒1的组分为纯化水、枸橼酸和枸橼酸钠。
静脉注射给药、灌胃给药溶液的配制：精密称量EGCG化合物，加生理盐水配制成溶液。
仪器：液体肺部给药雾化器。
动物：清洁级雄性SD大鼠，由南京市江宁区青龙山动物繁殖场提供，体重范围160～200g。
1.2给药方法及给药剂量
气道内雾化：各气道雾化给药组鼠经异氟烷吸入麻醉后固定于鼠固定器上，麻醉咽喉镜压住动物舌根部暴露声门，将抽取一定量的EGCG溶液的肺部微型液体雾化器针头(钝性)轻柔地插入气管内，将供试品雾化进入肺脏，而后快速拔出针头，将鼠从固定器上取下，头部朝上左右旋转，使药液尽可能的均匀分布于各肺叶。气道雾化给药剂量为1.6mg/kg、3.2mg/kg、6.4mg/kg，每个剂量组5只。
灌胃：根据动物体重确定每只动物的给药量，采用合适规格的注射器和灌胃软管抽取每只动物药量，经口灌胃给药。剂量为60mg/kg，共5只。
静脉注射给药：大鼠尾静脉注射给药。剂量为3.2mg/kg，共5只。
1.3样本采集及处理方法
各实验组均采用眼底静脉丛取血方式，于给药前(0min)和给药后5min、15min、30min、1h、2h、4h、6h(共8个采血点)进行取血，每个采血点约50-100μL全血。
分别于固定时间点取血后，置肝素钠抗凝管中，离心，分取血浆于离心管中，冻存于-70℃待测。
1.4分析条件及药代动力学参数的计算方法
质谱条件：用UPLC-MS/MS进行测定。色谱条件：用HPLC进行测定。利用WinNonlin药代动力学专业软件，以统计矩方法进行计算，求得每只动物相应的药动学参数。
1.5实验结果及数据分析
气道雾化给药(inh,给药剂量分别为1.6mg/kg、3.2mg/kg、6.4mg/kg)、静脉注射给药(iv,给药剂量为3.2mg/kg)、灌胃给药(po,给药剂量为60 mg/kg)EGCG后，大鼠血浆中的药代动力学参数见如下表1。以不同给药剂量气道雾化给药(inh,给药剂量分别为1.6mg/kg、3.2mg/kg、6.4mg/kg)EGCG后，大鼠血浆和肺部药物浓度随时间的变化曲线对比如图1所示；静脉注射给药(iv,给药剂量为3.2mg/kg)EGCG后与灌胃给药(po,给药剂量为60mg/kg)EGCG后，大鼠血浆和肺部药物浓度随时间的变化曲线对比如图2所示。由表1和图1及图2所示的实验结果可知：吸入(如气道雾化)给药后，EGCG的生物利用度显著提高；当达到同等血浆暴露水平时，灌胃口服给药和吸入给药的剂量比相差37.5倍(po 60mg/kg：inh 1.6mg/kg)，表明吸入给药大大降低了EGCG的给药剂量。
表1气道雾化给药、静脉注射给药、灌胃给药EGCG后，大鼠血浆中的药代动力学参数
实施例2气道雾化给药、灌胃给药EGCG后大鼠血浆和肺部中的药物浓度变化的研究
由实施例1可知，吸入(如气道雾化)给药1.6mg/kg EGCG与灌胃口服给药60mg/kg EGCG后，药物在大鼠血浆中产生的AUC0-t相近。基于此，本实施例研究了单次气道雾化给药更低剂量(0.8mg/kg)与灌胃给药EGCG(60mg/kg)相比，在大鼠血浆和肺部药物浓度随时间的变化。吸入给药组和灌胃给药组均采用5只大鼠进行研究。
2.1采血方式：股动脉放血取血。于给药后30min、2h、4h、6h(共4个采血点)。采血后经气管注入0.9wt％生理盐水灌洗肺部两遍，然后摘 取整个肺部，滤纸吸干水分，置于-20℃冰箱中待检测。
2.2血浆样本处理方法：取1mL血浆样品与3mL乙酸乙酯溶液混匀，涡旋振荡，离心，取上清液挥干，后用复溶液复溶，涡旋振荡，取100μL进样测定血药浓度。
2.3肺组织样本处理方法：取肺组织样本，称重，加3倍生理盐水(w:v)匀浆，取1mL匀浆样品与3mL乙酸乙酯溶液混匀，涡旋振荡，离心，取上清液挥干，用复溶液复溶，涡旋振荡，取100μL进样测定肺部药物浓度。
2.4实验结果及数据分析
吸入给药(如气道雾化给药)EGCG溶液(给药剂量为0.8mg/kg)与灌胃给药EGCG溶液(给药剂量为60mg/kg)后，大鼠血浆和肺部的Cmax及AUC0-t见如下表2；气道雾化给药EGCG溶液(给药剂量为0.8mg/kg)及灌胃给药EGCG溶液(给药剂量为60mg/kg)后，大鼠血浆和肺部药物浓度随时间的变化曲线对比如图3所示。由表2和图3所示实验结果可知，吸入给药0.8mg/kg剂量与口服60mg/kg剂量的EGCG溶液后，在血浆中药物浓度水平及随时间变化的曲线接近；而吸入给药后，在肺部的药物浓度高达其在血浆中药物浓度的100倍，显示出与全身循环相比，可见吸入给药后肺部的药物浓度非常高吸入给药方式可以进一步拓宽EGCG抗肺部纤维化的安全有效窗口。
表2吸入给药0.8mg/kg及口服60mg/kg剂量的EGCG溶液后血浆和肺部的Cmax及AUC0-t
实施例3低剂量气道雾化给药、灌胃给药EGCG后大鼠血浆和肺部中的药代动力学研究
如实施例1中所述的给药方法，对大鼠灌胃给药或气道雾化给药不同剂量EGCG溶液，研究药物在大鼠肺部的药代动力参数。气道雾化给药剂量为0.05mg/kg、0.1mg/kg、0.2mg/kg、0.4mg/kg，每剂量组6-8只大鼠。灌胃给药剂量为60mg/kg，共6只大鼠。
采血方式及样本处理方法如实施例2中所述，本实验中的采血时间点为给药后5min、2h、6h、8h、10h、16h，每个采血点约采3.5mL全血。
3.1实验结果及数据分析
气道雾化给药低剂量EGCG(给药剂量分别为0.05mg/kg、0.1mg/kg、0.2mg/kg、0.4mg/kg)及灌胃给药(给药剂量为60mg/kg)EGCG溶液后，大鼠肺部药物浓度随时间的变化曲线对比如图4所示，由图4可知：(1)吸入给药剂量为0.2mg/kg以上时，大鼠肺部药物最高浓度Cmax在1μg/mL以上水平(如1-20μg/mL)；而灌胃给药后测得的肺部浓度以及血浆药物的平均浓度均小于100ng/mL；这表明吸入给药EGCG溶液后、肺部药物浓度非常高。(2)即使在给予很低的吸入剂量(0.05mg/kg)下，肺部药物浓度也可达到与口服给药(60mg/kg)后相当的药物浓度，此时吸入剂量仅相当于口服剂量的1/1200；这表明通过吸入方式递送EGCG，大大拓宽了药物的有效治疗剂量窗口。(3)气道雾化给予0.1、0.2、0.4mg/kg EGCG溶液后，肺部药物浓度在16小时内(大于50ng/mL)可维持在口服相应的峰浓度(32ng/mL)以上；这表明当吸入给药EGCG溶液后、可在肺部较长时间内维持较高的药物浓度水平。
气道雾化给药和口服给药EGCG溶液后、肺组织中的平均Cmax及AUC0-t见如下表3，表3中的平均Cmax及AUC0-t数据进一步表明，吸入给药EGCG溶液后、EGCG溶液在肺部具有很好的滞留效果，避免了给药后、药物迅速吸收进入血液循环，肺组织局部药物浓度大大提高。由此可以推断出EGCG抗 IPF活性很可能是由其在肺组织中的高暴露所驱动的；从而使得：与经口服给药施用相比，吸入给药施用EGCG的安全有效窗口显著扩大，同时吸入给药递送EGCG为延长给药间隔、实现更灵活的治疗方案提供了可能。
表3气道雾化给药和口服给药EGCG溶液后、肺组织中的平均Cmax及AUC0-t
实施例4吸入给药EGCG对博来霉素诱导的小鼠肺纤维化的作用
4.1实验动物
C57BL/6J小鼠，共56只，SPF级，雄性，7-10周龄，体重20-35g。购于斯贝福(北京)生物技术有限公司。
4.2主要试剂
注射用盐酸博来霉素(Bleomycin，BLM，15mg/支，批号20067411)：瀚晖制药有限公司)。
溶媒1：组分为纯化水、枸橼酸、枸橼酸钠，透明澄清液体。
溶媒2：氯化钠注射液(批号K21080307)，购自湖南科伦制药有限公司。
羟脯氨酸(HYP)试剂盒：购自默克Sigma-Aldrich。
4.3动物分组模型构建
随机分为假手术组(正常对照组)、肺纤维化模型组、灌胃给药组、气道雾化给药组1-4，每组8只。第(简写D)1天，正常对照组小鼠气道内雾化给予溶媒2(氯化钠注射液，1mL/kg)，其余各组小鼠均气道内雾化给 予博来霉素(2mg/kg，1mL/kg)，均上午、下午各一次，进行模型构建。
4.4给药剂量与频次
小鼠灌胃给药的剂量为100mg/kg(从大鼠口服剂量60mg/kg，根据FDA指导原则通过体表面积折算而得的小鼠口服等效剂量)，气道雾化给药分为0.2mg/kg、0.4mg/kg、0.8mg/kg、1.6mg/kg四种剂量。正常对照组及肺纤维化模型组气道内雾化溶媒1，1次/天，于造模后D10天开始给药。
4.5标本采集
各组小鼠分别于D22天经腹腔注射水合氯醛(50mg/mL，0.1mL/10g)麻醉，腹主动脉放血处死。所有动物均进行解剖，保存肺组织，称全肺重量(包括发现死亡、濒死安乐死动物)。剪取左肺，置于-60℃以下冰箱保存，用于羟脯氨酸含量检测。剩余右肺组织及细支气管置于10％中性缓冲福尔马林溶液中固定，并进行常规组织学处理：石蜡包埋、切片、制片、HE染色和Masson染色。
4.6检测指标
(1)肺系数
肺系数＝肺重(mg)/体重(g)，模型组早期炎症细胞大量渗出，晚期成纤维细胞大量增生，胶原过度沉积都可以导致肺系数增加，因此肺系数这一指标可以间接反应肺纤维化小鼠炎症和纤维化程度。
(2)小鼠体重
动物接收后，分组前，造模当天、首次给药及以后每3天称重1次；计划安乐死前，动物发现死亡或濒临死亡时也进行称重。小鼠体重的变化可以间接反应药物施用对小鼠的影响。通过使用双因素ANOVA和Tukey多重比较检验进行统计分析。
(3)病理检查
采用光学显微镜对HE染色切片进行肺组织炎症细胞浸润及纤维化程度等病变进行分级，Masson染色进行肺组织纤维化程度评价。对纤维化、炎 细胞浸润、肺泡的出血、扩张、上皮增生、纤维样渗出等情况，使用标准化术语按4分级法(轻微，轻度，中度，重度)进行诊断和分类。
(4)肺组织中胶原含量测定
即羟脯氨酸含量测定，根据试剂盒的操作说明书进行肺组织中羟脯氨酸检测。
(5)统计学分析
可根据本领域已知的任何统计学检验方法如Students’t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定。采用SPSS(13.0)统计软件包进行检验分析。结果用平均值±标准误表示，评价整体性差异，组间均数采用One-Way ANOVA分析法进行方差齐性分析，并结合Dunnett’s test进行组间比较；当参数的正态检验失败时，则使用Kruskal-Wallis进行组间比较。采用Two-Way ANOVA和Tukey检验法进行组间的多重比较。被认为具有统计学意义的差异用星号表示(*P＜0.05；**P＜0.01；***P＜0.001)。
4.7实验结果及数据分析
4.7.1吸入给药EGCG对博来霉素诱导的肺纤维化小鼠肺系数的影响
与肺纤维化模型组小鼠对比，气道雾化给药EGCG溶液及灌胃给药EGCG溶液后对小鼠的肺重指数的影响(采用Kruskal-Wallis test检验，*:p＜0.05,即统计学上具有显著性差异；**：p＜0.01，即统计学上具有极显著性差异；***：p＜0.001，即统计学上具有极其显著的差异)如图5所示。造模后，模型组小鼠肺系数均明显高于正常对照组(**：P＜0.01)，由图5可知：与模型组小鼠肺系数相比，各气道雾化给药组和灌胃给药组小鼠肺系数均有明显降低，尤其是以给药剂量0.2mg/kg和0.4mg/kg气的道雾化给药组的疾病小鼠的肺系数(**：P＜0.01；***：P＜0.001)更是有显著降低；而灌胃给药组并未与模型组形成显著差异。
4.7.2吸入给药EGCG对博来霉素诱导的肺纤维化小鼠的体重的影响
与肺纤维化模型组小鼠对比，气道雾化给药EGCG及灌胃给药后对小鼠的体重的影响(通过使用双因素(Two-Way)ANOVA和Tukey多重比较检验进行统计分析)如图6所示。由图6可知：(1)博来霉素诱导后，各组小鼠体重明显下降。(2)当在D10天开始灌胃或吸入给予实验药物后，小鼠体重呈现上升趋势，但灌胃给药组的体重上升平缓，而0.2mg/kg和0.4mg/kg气道雾化给药组的小鼠体重在D22天时基本可以恢复至基线水平，且与模型组形成显著性差异(*P＜0.05；**P＜0.01；***P＜0.001)。这表明，相对于口服给药(如灌胃给药)，吸入给药适量的EGCG对肺纤维化小鼠的体重影响较小，也即吸入给药适量的EGCG可降低对肺纤维化小鼠的副作用小。
4.7.3吸入给药EGCG溶液对博来霉素诱导的肺纤维化小鼠肺组织病理结构改变的影响
D22天，分别对健康对照(control)组小鼠、肺纤维化模型(model)组小鼠、灌胃给药100mg/kg(即p.o 100)EGCG组小鼠、各剂量吸入给药组(inh.0.2，inh.0.4，inh.0.8，inh.1.6)小鼠的肺组织切片进行Masson’s染色和H&E染色，结果分别如图7a和图7b所示。
由图7a和图7b可知：健康对照/空白组(control)小鼠的肺组织结构清晰，肺泡间隔均匀，无充水水肿，无明显的肌成纤维细胞，无炎症及肺纤维化表现，肺泡腔内无明显渗出；肺纤维化模型组(model)小鼠的肺泡结构破坏严重，萎缩塌陷严重，肺泡间隔增厚，透明膜形成，并观察到明显的肺泡充血，大量炎症细胞浸润在小气道周围，血管内皮受损，成纤维细胞异常增生，肺间质中胶原纤维显著沉积，肺组织变实。
且有，如图7a所示的Masson’s染色结果表明：与模型组相比，气道雾化给药EGCG后，能显著降低胶原沉积，各吸入剂量呈现了明显的量效关系，尤其是吸入0.4mg/kg以上剂量时，小鼠的肺纤维化程度大幅降低，尤 其是当吸入0.8mg/kg、1.6mg/kg剂量后，几乎可以接近健康对照组小鼠。而口服EGCG后，小鼠肺部胶原沉积略有减少，但未与模型组形成显著差异，效果明显弱于气道雾化给药。如图7b所示的H&E染色结果表明：小鼠气道雾化吸入EGCG后，小鼠肺泡间质增生明显减轻，肺泡形态趋于完整，尤其是0.4mg/kg、0.8mg/kg剂量下效果最为优异。口服EGCG后小鼠肺组织性形态变化稍有改善，但明显弱于气道雾化给药，未与模型组形成显著差异。
4.7.4吸入给药EGCG溶液对博来霉素诱导的肺纤维化小鼠肺泡炎症和肺部纤维化的影响
与灌胃给药相比，气道雾化给药EGCG对肺纤维化小鼠肺泡炎细胞浸润、出血、肺泡扩张、肺泡纤维样渗出的影响如图8所示。由图8所示的各组的肺泡炎症评分(Score)对比可知：(1)与模型组相比，吸入给药EGCG溶液后在肺组织内均明显抑制了肺泡炎症的进一步产生，但灌胃给药组与肺纤维化模型组之间并未形成显著差异，甚至在肺泡出血、肺泡上皮增生方面还有加重趋势，表明口服给药EGCG溶液后对肺泡炎症的抑制效果不佳。(2)在抑制肺泡扩张方面，各剂量吸入给药组均与肺纤维化模型组均形成了显著差异(inh 1.6mg/kg的评分为*：p＜0.05，inh 0.2mg/kg和inh0.8mg/kg的评分均为**：p＜0.01，inh 0.4mg/kg的评分为****：p＜0.0001)，其中剂量为0.4mg/kg的吸入给药组的效果最优异(评分为****：p＜0.0001)。(3)抑制肺泡出血方面，剂量为0.2mg/kg、0.4mg/kg的吸入给药EGCG溶液组显著优于灌胃给药组。(4)抑制肺泡纤维样渗出方面，剂量为1.6mg/kg的吸入给药EGCG溶液组与肺纤维化模型组形成了显著差异。(5)在抑制炎细胞浸润方面，口服(如灌胃)给药EGCG溶液无抗炎效果，各剂量吸入给药组均降低了炎细胞的浸润，尤其是剂量为0.4mg/kg的吸入给药EGCG溶液组抗炎效果最优异(评分*：p＜0.05)。
肺纤维化模型组小鼠对比，气道雾化给药EGCG及灌胃给药后对小鼠纤维化评分的影响如图9所示，由图9所示的肺纤维化程度评分对比可知： 灌胃给药组和各剂量吸入给药组均能显著降低小鼠肺纤维化评分，当吸入0.4mg/kg剂量以上时，可以显著降低肺部纤维化(评分为*p＜0.05)，且剂量为0.2mg/kg、0.4mg/kg、0.8mg/kg吸入给药组呈现出了良好的量效关系。而灌胃给药组并未与肺纤维化模型组形成显著差异。
以上结果表明，EGCG溶液在吸入较低剂量(0.2mg/kg)时即可在肺部产生抗炎效果；随着吸入剂量的增加，抗纤维化效果也逐渐增强；剂量为1.6mg/kg的吸入给药EGCG溶液的抗纤维化效果尤为明显。可见，吸入给药EGCG的有效剂量范围非常宽。
4.7.5吸入给药EGCG溶液对肺纤维化小鼠肺组织中羟脯氨酸含量的影响
气道雾化给药EGCG与口服EGCG抑制博来霉素诱导的羟脯氨酸含量的数据见如下表4；与肺纤维化模型组小鼠对比，灌胃给药EGCG和气道雾化给药EGCG对小鼠肺组织中羟脯氨酸含量的影响(采用One-Way ANOVA分析法进行方差齐性分析，并结合Dunnett’s test分析方法进行组间比较)如图10所示。由表4可知：D22天，肺纤维化小鼠肺组织中羟脯氨酸的含量显著增加(***：p＜0.001)；气道雾化给药0.2mg/kg、0.4mg/kg、0.8mg/kg的EGCG溶液后，可以剂量依赖性地降低肺组织中的羟脯氨酸含量。由表4和图10可知：特别是吸入给药剂量为0.8mg/kg时，吸入给药EGCG溶液后、降低肺组织中的羟脯氨酸含量的效果显著优于口服(评分为***：p＜0.001)。而实施例2所述的吸入(如气道雾化)给药剂量为0.8mg/kg与大鼠口服(如灌胃)剂量为60mg/kg(相当于小鼠口服给药剂量为100mg/kg)的EGCG溶液后、药物在血浆中的暴露量相当。采用One-Way ANOVA分析法进行方差齐性分析，并结合Dunnett’s test分析方法进行组间比较。除特殊说明外，统计分析方法采用Students’T检验。*:p＜0.05,即统计学上具有显著性差异；**：p＜0.01，即统计学上具有极显著性差异；***：p＜0.001，即统计学上具有极其显著的差异。
表4气道雾化给药EGCG与口服EGCG抑制博来霉素诱导小鼠肺部的羟脯氨酸含量
实施例5吸入给药EGCG对博来霉素诱导的大鼠肺纤维化的作用
5.1研究目的
与口服灌胃给药EGCG、气道内雾化给药吡非尼酮溶液对比，检测气道内雾化给药不同剂量的EGCG溶液对博来霉素诱导的肺纤维化模型大鼠肺组织中胶原含量的影响，以及病理组织切片的观察，来评价吸入给药EGCG对大鼠肺纤维化的治疗作用。
5.2主要试剂
EGCG雾化溶液的溶媒1、造模试剂及造模试剂的溶媒2如实施例4中所述。阳性对照药物为吡非尼酮(纯度为99％，药用级别)。阳性对照药品的溶媒3组分为NaCl、枸橼酸等，透明澄清液体。
5.3模型构建与分组给药
6至9周龄、体重约200至300g的SPF级SD大鼠，共88只。随机分为假手术组(正常对照组)(8只)、肺纤维化模型对照组(10只)、灌胃给药EGCG组1(10只)、灌胃给药EGCG组2(10只)、气道雾化给药EGCG溶液组1-4(每组10只，共40只)、气道雾化给药吡非尼酮溶液组(10只)。
D1天，正常对照组小鼠气道内雾化给予溶媒2(氯化钠注射液，1mL/kg)，其余各组小鼠均气道内雾化给予博来霉素(2.5mg/kg，1mL/kg)，均上午、下午各一次，进行模型构建。D8天对每组动物称重。于D10～D28，正常对照组及肺纤维化模型组气道雾化给予溶媒1，其余各组大鼠给予供试 品EGCG或对照品吡非尼酮，1次/天，各组给药剂量及给药方式如下所示：EGCG灌胃给药EGCG组1和组2的给药剂量分别为30mg/kg、60mg/kg；EGCG气道雾化组1-4的剂量分别为0.05mg/kg、0.4mg/kg、0.8mg/kg、1.6mg/kg；气道雾化对照药吡非尼酮溶液组的剂量为0.9mg/kg，该组剂量设计参照M.W.Surber,et al(ATS POSTER,2014)的已公开文献。
5.4标本采集
D29，所有动物安乐死并进行解剖，保存组织。尸检过程中，观察动物肺脏、气管及支气管是否异常。取左肺组织，于-60℃以下保存，用以进行肺组织中羟脯氨酸含量检测；取右肺组织，制备病理组织切片，并用HE染色和Masson染色，用以进行肺组织病理形态学观察及肺纤维化程度评价。
5.5吸入给药EGCG溶液对肺纤维化大鼠肺组织中羟脯氨酸含量的影响
口服灌胃给药EGCG、气道雾化给药EGCG溶液及气道雾化给药吡非尼酮溶液抑制博来霉素诱导大鼠肺部的羟脯氨酸含量的数据如下表5和图11所示：
各组均与模型组作对比，采用Kruskal-Wallis test检验进行统计学分析，*:p＜0.05,即统计学上具有显著性差异；**：p＜0.01，即统计学上具有极显著性差异；***：p＜0.001，即统计学上具有极其显著的差异；****：p＜0.0001，即统计学上具有极高度显著的差异。
D28天，与正常对照组相比，肺纤维化模型组大鼠肺组织中羟脯氨酸含量显著增加(****：p＜0.0001)，口服给药EGCG以及气道雾化给予对照药吡非尼酮溶液后，大鼠肺中羟脯氨酸含量虽有所降低，但均未与模型对照组形成统计学差异。而吸入给药EGCG后，大鼠肺中羟脯氨酸含量均显著降低(与模型对照组相比具有统计学差异，吸入给药EGCG 0.4mg/kg剂量组****：p＜0.0001，吸入给药0.8mg/kg和1.6mg/kg剂量组为***：p＜0.001)，且即使在很低的给药剂量0.05mg/kg下，也能显著降低肺纤维大鼠肺组织中的羟脯氨酸含量(*：p＜0.05)。结果表明，EGCG通过吸入方式给药后， 能显著降低模型动物的肺纤维化程度，可提供很宽的有效治疗剂量窗口。
表5口服灌胃给药EGCG、气道雾化给药EGCG溶液及气道雾化给药吡非尼酮溶液后抑制博来霉素诱导大鼠肺部的羟脯氨酸含量
实施例6 EGCG雾化溶液的制备
6.1处方
6.2制备方法：将EGCG原料药与辅料在配液罐中混合，加入注射用水至1000ml，搅拌溶解后冷却，再用枸橼酸和枸橼酸钠将溶液pH调节在3-4，然后将EGCG溶液通过预过滤后、再进行无菌过滤，无菌灌装后即得一种EGCG雾化溶液。
在另一种EGCG雾化溶液的制备例中，其与6.1所述的处方相比区别在于还含有适量的EDTA，制备方法同6.2所述。
实施例7健康受试者中EGCG雾化吸入溶液的单次剂量递增试验
在22例健康受试者中进行了一项随机、双盲、安慰剂对照的临床研究，目的是评价对健康受试者单次吸入给药EGCG雾化溶液后的安全性和耐受性，以及药代动力学特性，并与雾化溶液安慰剂、口服EGCG胶囊进行对照。
试验药物：
试验组：EGCG雾化吸入溶液(即如实施例6中所述含EDTA的EGCG雾化溶液)，初始浓度为10mg/mL，给药前根据不同剂量进行稀释。
安慰剂组：空白制剂
口服对照组：EGCG胶囊，每粒重150mg，其中EGCG含量约为总重量的94％。
分组及给药：
参见如下表6，口服对照组随机分配4例受试者，开盲给予600mg的EGCG胶囊(共4粒，共约含EGCG为564mg)。
EGCG雾化吸入给药共3组：(1)3mg剂量组，给药前将1mL起始药液加稀释液稀释至1mg/mL，取3mL加入雾化器进行给药。(2)10mg剂量组，给药前将1mL起始药液加稀释液稀释至3.33mg/mL，取3mL加入雾化器进行给药。(3)30mg剂量组，不稀释，直接取3mL加入雾化器给药。各雾化吸入剂量组随机分配6例受试者，从低剂量开始，每组中2例受试者设为哨兵(1:1随机，1例EGCG雾化吸入组，1例安慰剂组)，观察≧24小时，由研究者对受试者进行安全评估，经研究者许可后开始其余4例受试者给药(3:1随机，3例EGCG雾化吸入组，1例安慰剂组)。
表6口服对照组与EGCG雾化吸入给药组的分组信息
血液样本的采集：
通过留置静脉针的方式，在0h(给药前1h内)和给药后(按雾化吸入开始时或完成EGCG胶囊口服时开始计时)15min、30min、1h、1.5h、2h、3h、4h、6h、8h、10h、12h、24h共计13个采血点，采集受试者血液样品，每次采血4mL，提取血浆用于PK检测、备份和代谢物鉴定。
安全评估：
不良事件(Adverse Event，AE)监测；体检；生命体征(体温、血压、脉搏、呼吸)和血氧饱和度；心功能；肺功能。
受试者的筛选：入组标准
研究对象应符合以下标准：
·年龄在18-59岁(含两端值)的健康成年受试者，男女不限。
·理解研究程序和方法，自愿参加本试验，并书面签署知情同意书。
·体重指数(BMI＝体重/身高平方(kg/m2))：18≤BMI＜28；男性体重≥50.0kg且＜90.0kg，女性体重≥45.0kg且＜90.0kg。
·通过病史、体格检查、实验室检查、生命体征或心电图确认无临床显著异常，并且研究者判定为医学健康。
·同意在整个研究期间采取有效的避孕措施。具有生育能力的女性受试者，应在筛检前避孕至少一个月以上，并承诺整个研究期内采取避孕且持续到研究结束后3个月内；男性受试者承诺整个研究期内及持续到研究结束后3个月内，采取避孕且保证不捐献精子。
·1秒用力呼气量(forced expiratory volume in one second,FEV1)和用力肺活量(forced vital capacity,FVC)≥预测值的80％，FEV1/FVC≥0.7，同时胸片检查正常。
·同意在给药前48小时到给药后的48小时不摄入任何咖啡和茶水，以及含咖啡或茶成分的饮料和食品。
·能够在筛选期和研究期间正确有效地使用吸入装置的。
·给药前7天未使用药物，并且给药后的24小时也不使用其它药物。
受试者的筛选：排除标准
符合以下任一条标准的对象将排除于本研究：
·研究者认为可能对参与产生不利影响的重大病史。
·处于孕期或哺乳期的女性受试者。
·具有生育能力但在给药前至少30天未进行避孕的女性受试者；在参加本试验期间及给药后90日内，不愿意采取避孕措施的男性受试者(或不能保证不捐献精子)和有生育能力的女性受试者。
·对研究药物或研究药物中的任何成分过敏者。
·新冠肺炎、艾滋病毒、乙型肝炎和丙型肝炎检测阳性。
·筛选/基线访视丙氨酸氨基转移酶(Alanine aminotransferase，ALT)或者天门冬氨酸氨基转移酶(Aspartate Aminotransferase，AST)或γ-谷氨酰基转移酶(Gamma-glutamyltransferase，GGT)超过正常值上限(Upper limit of normal，ULN)者，或者总胆红素超过正常值上限(ULN)者。
·首次给药前6个月内抽烟或电子烟的。
·筛选期前三个月内有药物滥用史或酗酒史，酗酒定义为每周＞21个酒精单位(其中1个单位等于284mL啤酒或25mL 40％烈酒或125mL葡萄酒)。
·给药前3个月内献血/血浆≥400mL。
试验结果：
所有受试者的体格检查、生命体征、心功能及肺功能均未出现明显异常。安慰剂组和雾化给药组中仅个别受试者出现口咽部及呼吸道局部症状，包括咽痒、口干、口苦、咳嗽，所述症状轻微且停药后消失：10mg剂量的给药组1例/4例，安慰剂组1例/2例；30mg剂量的给药组2例/4例；3mg剂量组受试者均未出现上述症状。研究者判读，EGCG雾化吸入给药后，整体耐受性良好，安全系数高。
各组受试者给药后血浆中EGCG的平均药物浓度随时间的变化曲线如图12所示。由图12所示的试验结果可知：吸入给药后，血浆中药物暴露量与给药剂量之间存在良好的相关性。吸入给药后与口服给药后血浆中药物暴露量差异倍数远高于口服给药剂量和吸入给药剂量之间的差异倍数。
一般情况下，与口服给药方式相比，肺部给药吸收速度快，药物通过高通透性的肺毛细血管和肺泡表面迅速进入体循环，迅速达到很高的峰值。 然而令人惊讶的是，EGCG吸入给药后吸收进血液的药物浓度达峰时间Tmax较晚，且Cmax水平较低。在达到与口服给药等同或更优的肺部暴露量时，即产生等同或更优的治疗效果的剂量之下，吸入给药进入血液循环系统的药物浓度远低于口服给药后血浆中的药物水平，如10mg剂量组，吸入剂量为口服剂量的1/56，而吸入后血浆Cmax是口服后Cmax的约1/227。这也预示着EGCG雾化吸入给药后，可显著增加药物在肺部的滞留量和滞留时间，极大降低药物在血浆中的浓度。前述结果提示，雾化吸入EGCG，可在提高肺部药物治疗效果的同时大大减少系统性不良反应的风险。
综上所述，本公开提供的(-)-表没食子儿茶素没食子酸酯类化合物的应用，通过吸入而直接向肺组织施用EGCG，增加了药物在肺组织中的暴露，使得药物在肺组织中的浓度与血浆浓度的比大大增强，提高药效的同时降低了潜在的肝毒性；即使在很低的吸入给药剂量下，也能产生显著地抗炎和抗肺纤维化的效果，显著地扩大了EGCG对IPF的安全治疗窗口；当吸入给药EGCG后，药物可以在肺部以较高的有效浓度维持很长时间，而所述浓度高于口服给药后在肺组织中所能达到的药物浓度水平，为降低给药频次、增加患者顺应性、实现个体化给药方案提供了可行性。此外，通过吸入给药，还可避免口服EGCG与其他IPF治疗药物之间的药物-药物相互作用的问题。
应当理解的是，本公开的应用不限于上述的举例，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，所有这些改进和变换都应属于本公开所附权利要求的保护范围。

Claims (37)

  1. 一种(-)-表没食子儿茶素没食子酸酯类化合物在制备预防和/或治疗肺纤维化疾病的吸入药物中的应用，其中，所述(-)-表没食子儿茶素没食子酸酯类化合物为(-)-表没食子儿茶素没食子酸酯或其药学上可接受的盐、酯、水合物或溶剂化物。
  2. 如权利要求1所述的应用，其中，所述吸入药物被配制为可供吸入的剂型，所述剂型选自溶液、混悬液、气雾剂或粉雾剂。
  3. 如权利要求1所述的应用，其中，所述吸入药物是由所述(-)-表没食子儿茶素没食子酸酯类化合物经干燥工艺加工形成的粉末、再经稀释剂复溶形成。
  4. 如权利要求2所述的应用，其中，所述剂型为溶液时，所述吸入药物包括：作为活性成分的所述(-)-表没食子儿茶素没食子酸酯类化合物，稀释剂，以及pH调节剂、渗透压调节剂和抗氧化剂中的至少一种，所述(-)-表没食子儿茶素没食子酸酯类化合物完全溶解于所述稀释剂中；
    所述剂型为混悬剂时，所述吸入药物包括：作为活性成分的所述(-)-表没食子儿茶素没食子酸酯类化合物，稀释剂，以及表面活性剂、pH调节剂、张力调节剂中的至少一种，所述(-)-表没食子儿茶素没食子酸酯类化合物或所述所述(-)-表没食子儿茶素没食子酸酯类化合物与可适用于吸入给药的载体形成颗粒悬浮于稀释剂中；
    所述剂型为气雾剂时，所述吸入药物包括：作为活性成分的所述(-)-表没食子儿茶素没食子酸酯类化合物，稀释剂，抛射剂，以及表面活性剂、助溶剂和pH调节剂中的至少一种；
    所述剂型为粉雾剂时，所述吸入药物包括：作为活性成分的所述(-)-表没食子儿茶素没食子酸酯类化合物，可适用于吸入给药的载体，以及赋 形剂和表面活性剂中的至少一种。
  5. 如权利要求3或4的应用，其中，满足以下条件(1)-(3)中的至少一个：
    (1)所述稀释剂为水、乙醇和甘油中的一种或多种；
    (2)所述吸入药物的pH值为3.0-5.0。
    (3)所述吸入药物中、所述(-)-表没食子儿茶素没食子酸酯类化合物的浓度为0.1-25mg/mL。
  6. 如权利要求1所述的应用，其中，所述吸入药物包括所述(-)-表没食子儿茶素没食子酸酯类化合物，或者所述吸入药物包括所述(-)-表没食子儿茶素没食子酸酯类化合物和其他抗肺纤维化药物。
  7. 如权利要求6所述的应用，其中，所述其他抗肺纤维化药物选自吡非尼酮、尼达尼布、糖皮质激素、免疫抑制剂、前列环素及其类似物、CTGF抗体、Galectin-3抑制剂、整合素拮抗剂、重组血清淀粉样蛋白P及其类似物、PDE抑制剂、LPA拮抗剂、JAK激酶抑制剂、多种细胞因子受体TKI中的一种或多种。
  8. 如权利要求7所述的应用，其中，所述其他抗肺纤维化药物选自吡非尼酮、尼达尼布、BI 1015550、曲前列环素及其类似物、重组血清淀粉样蛋白P、LPA拮抗剂中的一种或多种。
  9. 如权利要求1所述的应用，其中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为0.1-100mg/次。
  10. 如权利要求9所述的应用，其中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为0.1-50mg/次。
  11. 如权利要求10所述的应用，其中，所述(-)-表没食子儿茶素没 食子酸酯类化合物的给药剂量为0.1-30mg/次。
  12. 如权利要求11所述的应用，其中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为0.1-15mg/次。
  13. 如权利要求9所述的应用，其中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量选自0.1mg/次、1mg/次、1.5mg/次、2mg/次、2.5mg/次、3mg/次、3.5mg/次、4mg/次、4.5mg/次、5mg/次、5.5mg/次、6mg/次、6.5mg/次、7mg/次、7.5mg/次、8mg/次、8.5mg/次、9mg/次、9.5mg/次、10mg/次、10.5mg/次、11mg/次、11.5mg/次、12mg/次、12.5mg/次、13mg/次、13.5mg/次、14mg/次、14.5mg/次、15mg/次、15.5mg/次、16mg/次、16.5mg/次、17mg/次、17.5mg/次、18mg/次、18.5mg/次、19mg/次、19.5mg/次、20mg/次、25mg/次、30mg/次、35mg/次、40mg/次、45mg/次、50mg/次、55mg/次、60mg/次、65mg/次、70mg/次、75mg/次、80mg/次、90mg/次或100mg/次。
  14. 如权利要求1所述的应用，其中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为口服给药剂量的1/1000至1/10。
  15. 如权利要求14所述的应用，其中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为口服给药剂量的1/600至1/10。
  16. 如权利要求1-15中的任一项所述的应用，其中，所述肺纤维化疾病为间质性肺疾病，所述肺纤维化疾病包括特发性间质性肺炎，自身免疫或结缔组织疾病引起的肺间质纤维化，接触或职业暴露相关的肺间质纤维化，治疗引起的肺间质纤维化，以及结节病中的一种或多种。
  17. 如权利要求16所述的应用，其中，所述特发性间质性肺炎包括特发性肺纤维化；
    所述自身免疫或结缔组织疾病引起的肺间质纤维化包括狼疮、硬皮病、多发性肌炎或皮肌炎、类风湿性关节炎相关的间质性肺疾病；
    所述接触或职业暴露相关的间质纤维化包括石棉肺、矽肺、过敏性肺炎；
    所述治疗引起的间质纤维化包括化疗、放疗和一些药物治疗引起的间质性肺疾病。
  18. 一种预防和/或治疗肺纤维化疾病的可吸入药物组合物，其中，包括：(-)-表没食子儿茶素没食子酸酯类化合物作为有效成分，以及药学上可接受的辅料；所述(-)-表没食子儿茶素没食子酸酯类化合物为EGCG或其在药学上可接受的盐、酯、水合物或溶剂化物。
  19. 如权利要求18所述的药物组合物，其中，每剂量所述可吸入药物组合物中、所述(-)-表没食子儿茶素没食子酸酯类化合物的含量为0.1-50mg。
  20. 如权利要求19所述的药物组合物，其中，每剂量所述可吸入药物组合物中、所述(-)-表没食子儿茶素没食子酸酯类化合物的含量为0.5-30mg。
  21. 如权利要求18-20中的任一项所述的药物组合物，其中，所述可吸入药物组合物的剂型为吸入溶液、吸入混悬液、气雾剂或粉雾剂。
  22. 如权利要求21所述的药物组合物，其中，所述可吸入药物组合物的剂型为吸入溶液时，所述药学上可接受的辅料选自表面活性剂、pH调节剂、抗氧化剂、防腐剂、渗透压调节剂、金属离子络合剂、水和附加剂中的一种或多种；
    所述可吸入药物组合物的剂型为吸入混悬液时，所述药学上可接受的辅料选自表面活性剂、pH调节剂、抗氧化剂、防腐剂、渗透压调节剂、 金属离子络合剂、水和附加剂中的一种或多种；
    所述可吸入药物组合物的剂型为气雾剂时，所述药学上可接受的辅料选自助溶剂、表面活性剂、抛射剂和附加剂中的一种或多种；
    所述可吸入药物组合物的剂型为粉雾剂时，所述药学上可接受的辅料包括赋形剂、载体和附加剂。
  23. 一种预防和/或治疗肺纤维化疾病的方法，其中，所述方法包括：通过吸入给药向受试者施用所述(-)-表没食子儿茶素没食子酸酯类化合物，所述(-)-表没食子儿茶素没食子酸酯类化合物为(-)-表没食子儿茶素没食子酸酯或其药学上可接受的盐、酯、水合物或溶剂化物。
  24. 如权利要求23所述的方法，其中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为0.01-2.0mg/kg。
  25. 如权利要求23所述的方法，其中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为0.1-80mg/次。
  26. 如权利要求25所述的方法，其中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为0.1-50mg/次。
  27. 如权利要求26所述的方法，其中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为0.1-30mg/次。
  28. 如权利要求27所述的方法，其中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为0.1-15mg/次。
  29. 如权利要求25所述的方法，其中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量选自0.1mg/次、1mg/次、1.5mg/次、2mg/次、2.5mg/次、3mg/次、3.5mg/次、4mg/次、4.5mg/次、5mg/次、5.5mg/次、6mg/次、6.5mg/次、7mg/次、7.5mg/次、8mg/次、8.5mg/次、9mg/次、9.5mg/次、10mg/次、10.5mg/次、11mg/次、11.5mg/ 次、12mg/次、12.5mg/次、13mg/次、13.5mg/次、14mg/次、14.5mg/次、15mg/次、15.5mg/次、16mg/次、16.5mg/次、17mg/次、17.5mg/次、18mg/次、18.5mg/次、19mg/次、19.5mg/次、20mg/次、25mg/次、30mg/次、35mg/次、40mg/次、45mg/次、50mg/次、55mg/次、60mg/次、65mg/次、70mg/次、75mg/次、80mg/次、90mg/次或100mg/次。
  30. 如权利要求23所述的方法，其中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为口服给药剂量的1/1000至1/10。
  31. 如权利要求30所述的方法，其中，所述(-)-表没食子儿茶素没食子酸酯类化合物的给药剂量为口服给药剂量的1/600至1/10。
  32. 如权利要求23所述的方法，其中，还包括：通过吸入给药向受试者施用其他抗肺纤维化药物，所述其他抗肺纤维化药物与所述(-)-表没食子儿茶素没食子酸酯类化合物同时、独立或相继的施用。
  33. 如权利要求32所述的方法，其中，所述其他抗肺纤维化药物选自吡非尼酮、尼达尼布、糖皮质激素、免疫抑制剂、前列环素及其类似物、CTGF抗体、Galectin-3抑制剂、整合素拮抗剂、重组血清淀粉样蛋白P及其类似物、PDE抑制剂、LPA拮抗剂、JAK激酶抑制剂、多种细胞因子受体TKI中的一种或多种。
  34. 如权利要求33所述的方法，其中，所述其他抗肺纤维化药物选自吡非尼酮、尼达尼布、BI 1015550、曲前列环素及其类似物、重组血清淀粉样蛋白P、LPA拮抗剂中的一种或多种。
  35. 如权利要求23-34中的任一项所述的方法，其中，所述施用的频次为1次/2天、1次/天或2次/天。
  36. 如权利要求23-34中的任一项所述的方法，其中，所述吸入给药 是指采用载有所述药物的吸入给药装置将所述药物递送至所述受试者。
  37. 如权利要求36所述的方法，其中，吸入给药装置为雾化发生器、压力定量吸入器、干粉吸入器或软雾吸入器。
PCT/CN2023/132299 2022-11-24 2023-11-17 (-)-表没食子儿茶素没食子酸酯类化合物的应用 WO2024109652A1 (zh)

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