WO2020228477A1 - 一种用于预防、缓解和/或治疗纤维化的药物、组合产品及其应用 - Google Patents

一种用于预防、缓解和/或治疗纤维化的药物、组合产品及其应用 Download PDF

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WO2020228477A1
WO2020228477A1 PCT/CN2020/085111 CN2020085111W WO2020228477A1 WO 2020228477 A1 WO2020228477 A1 WO 2020228477A1 CN 2020085111 W CN2020085111 W CN 2020085111W WO 2020228477 A1 WO2020228477 A1 WO 2020228477A1
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isovalerylspiramycin
fibrosis
metabolites
polymorphs
tautomers
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PCT/CN2020/085111
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English (en)
French (fr)
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何红伟
姜恩鸿
赫卫清
姜勋东
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沈阳福洋医药科技有限公司
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Priority to CA3142427A priority Critical patent/CA3142427A1/en
Priority to JP2021568677A priority patent/JP2022540291A/ja
Priority to KR1020217041077A priority patent/KR20220008895A/ko
Priority to EP20805604.4A priority patent/EP3970728A4/en
Priority to US17/611,847 priority patent/US20220211735A1/en
Priority to AU2020274548A priority patent/AU2020274548A1/en
Publication of WO2020228477A1 publication Critical patent/WO2020228477A1/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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • 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
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the invention belongs to the field of medicinal chemistry, and specifically relates to a medicine, a combination product and its application for preventing, relieving and/or treating fibrosis.
  • Fibrosis is a pathological change, manifested by fibroblast activation and proliferation, increased fibrous connective tissue in tissues and organs, and decreased parenchymal cells. Continued progress can cause structural damage and loss of function of tissues and organs. Fibrosis of important organs seriously affects the quality of life of patients, even life-threatening. Worldwide, tissue fibrosis is the main cause of disability and death from many diseases. According to relevant statistics in the United States, about 45% of the patients who died from various diseases in this country can be attributed to tissue fibrosis. At present, the treatment methods and drugs for this disease are still very lacking, and the prognosis is very poor. The development of new drugs that can effectively treat fibrosis is a very important and urgent task.
  • Carrimycin also known as Bitespiramycin and Shengjimycin
  • Carrimycin is produced by transgenic technology.
  • the 4′′-O-isovaleryltransferase gene (4′′-O-isovaleryltransferase gene) was cloned into Streptomyces spiramyceticus, and the spiramycin 4′′-OH was acylated and the isovaleryl group was added at the 4′′ position
  • R′ COCH 2 CH(CH 3 ) 2 is isovalerylspiramycin I;
  • R′ COCH 2 CH(CH 3 ) 2 is isovalerylspiramycin II;
  • the central structure is a 16-membered lactone ring, which is connected with one molecule of folofamine, one molecule of mycosaminoglycan and one molecule of mycaminophen. Its main components are isovalerylspiramycin I, II, III and spirulina
  • the difference in the structure of the mycine is that the group attached to the 4" position of mycaminophen is an isovaleryl group instead of a hydroxyl group.
  • the chemical structure contains more than ten kinds of components.
  • the finished product of climycin The composition standard is that the proportion of isovalerylspiramycin III ⁇ 30%, the total proportion of isovaleryl spiramycin I, II, and III ⁇ 60%, the proportion of total acylated spiramycin ⁇ 80%, and other unknown components The sum of ⁇ 5%.
  • the drug has good lipophilicity, strong tissue penetration ability, fast oral absorption, long maintenance time in the body, and continuous post-antibiotic effect. According to the relationship between pharmacodynamics and chemical conformation, after macrolide antibiotics are acylated at the 4′′ position, their lipophilicity and in vivo activity are improved, in vivo antibacterial activity and clinical therapeutic effect are significantly improved, and the stability of antibiotics in vivo As the carbon chain of the 4" hydroxy ester increases, it increases, that is, isovalerylspiramycin>butyrylspiramycin>propionylspiramycin>acetylspiramycin.
  • the technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a medicine for preventing, alleviating and/or treating fibrosis.
  • the medicine can effectively prevent, relieve and treat fibrosis and has significant social benefits and Economic benefits.
  • the present invention adopts the following technical solutions:
  • a medicament for preventing, relieving and/or treating fibrosis the active ingredients of the medicament include colimycin, isovalerylspiramycin I, isovalerylspiramycin II, isovalerylspiramycin One of III;
  • the medicine includes a medically acceptable carrier.
  • the medicine is prepared into medically acceptable tablets, capsules, pills, injections, sustained-release preparations and various particle delivery systems.
  • a medicine for preventing, alleviating and/or treating fibrosis is selected from one or more of the following ingredients:
  • a combined product for the treatment of fibrosis comprising a first medicament, and the active ingredients of the first medicament include climycin, isovalerylspiramycin I, and isovalerylspiramycin II One of isovalerylspiramycin III;
  • the combination product further includes a second agent, and the second agent includes at least one of related drugs for preventing, alleviating and/or treating fibrosis.
  • the related drugs for preventing, alleviating and/or treating fibrosis include corticosteroid hormones, colchicine, silymarin, and interferon.
  • a combined product for treating fibrosis comprising a first medicament, and the active ingredient of the first medicament is selected from one or more of the following ingredients:
  • the fibrosis described in the present invention includes pulmonary fibrosis, myocardial fibrosis, liver fibrosis, pancreatic fibrosis, kidney fibrosis, bone marrow fibrosis and skin fibrosis;
  • the pulmonary fibrosis includes pulmonary fibrosis caused by a novel coronavirus infection.
  • the medicine and combination products of the present application have ameliorating effect on pulmonary fibrosis caused by a novel coronavirus (SARS-CoV-2) infection.
  • Pulmonary fibrosis is caused by multiple causes of lung injury.
  • the interstitium secretes collagen to repair it. If it is over-repaired, it is formed by excessive proliferation of fibroblasts and massive accumulation of extracellular matrix.
  • Myocardial fibrosis is the excessive proliferation of cardiac interstitial fibroblasts, excessive collagen deposition and abnormal distribution.
  • Liver fibrosis is a pathological process in which various pathogenic factors cause abnormal proliferation of connective tissue in the liver and excessive precipitation of diffuse extracellular matrix in the liver. Many factors can cause liver fibrosis, such as viral infection, inflammation, oxidative stress, and alcoholism.
  • pancreatic fibrosis is that pancreatic acinar cells secrete a large amount of protein, while the fluid and bicarbonate secreted by pancreatic duct cells do not increase, resulting in the secretion of pancreatic acinar cells and GP2 (a kind of cast The concentration of protein) decreases and precipitates in the pancreatic duct, which leads to pancreatic fibrosis,
  • Renal fibrosis is a pathological process in which extracellular matrix and inappropriate connective tissue accumulate in the kidney, leading to changes in kidney structure and impaired function.
  • Bone marrow fibrosis is a myeloproliferative disease caused by the proliferation of collagen in the bone marrow hematopoietic tissue, and its fibrous tissue seriously affects the hematopoietic function.
  • Skin fibrosis is when fibroblasts divide, proliferate, migrate to the damaged part, produce extracellular matrix, and form scar tissue to repair the wound. Scar formation is a process of gradual fibrosis of granulation tissue.
  • the present invention also provides an application of any one of the above-mentioned drugs and combination products in the prevention, alleviation and/or treatment of fibrosis.
  • the present invention also provides the application of any of the above-mentioned drugs or combination products in inhibiting inflammation or lipid peroxidation, inhibiting the proliferation of fibroblasts, and promoting collagen degradation.
  • the present invention has the following benefits compared with the prior art:
  • the medicines and combination products provided by the invention have good therapeutic effects in the treatment of fibrosis, and have important social and economic benefits.
  • Figure 1 shows the inhibitory effect of HB (Calinomycin) and HY (Iovalerylspiramycin I) on the activity of type I collagen ⁇ 1 promoter;
  • Figure 2 shows the IC50 values of HB (Calinomycin) and HY (Iovalerylspiramycin I) in HepG2 and LX2 cells;
  • Figure 3 shows the effect of Real-time PCR detection of HB (Calinomycin) and HY (Iovalerylspiramycin I) on the mRNA levels of major fibrosis markers in LX-2 cells induced by TGF ⁇ 1;
  • Figure 4 shows the effect of Western Blot detection of HB (Calinomycin) and HY (Iovalerylspiramycin I) on the protein levels of major fibrosis markers in LX-2 cells induced by TGF ⁇ 1;
  • Figure 5 shows the effect of HB (Calinomycin) on the pathological structure of rat liver tissue
  • sham is the H&E stained section of the liver tissue of the sham operation group
  • BDL is the H&E stained section of the liver tissue of the BDL model group
  • HB is the H&E stained section of the liver tissue of the climycin administration group result
  • Figure 6 shows the effect of HB (Calinomycin) on the degree of fibrosis in rats
  • sham is the result of Masson stained section of liver tissue of rats in the sham operation group
  • BDL is the result of Masson stained section of liver tissue of rats in the BDL model group
  • HB is the result of liver tissue of rats in the sham-operated group. Pine stained section results
  • Figure 7 shows the effect of Real-time PCR to detect the effect of Climycin and Isovalerylspiramycin I on the mRNA levels of the main fibrosis markers in lung fibroblasts MRC-5 induced by TGF ⁇ 1; among them, HB is Climycin, Isoamyl I is isovalerylspiramycin I;
  • Figure 8 shows the improvement of lungs before and after treatment with Climycin in Case 1;
  • Figure 9 shows the improvement of the lungs before and after treatment with Climycin in Case 2;
  • Figure 10 shows the improvement of lungs before and after treatment with Climycin in case 3;
  • Figure 11 shows the effect of Real-time PCR to detect the effects of Climycin (HB) and Isovalerylspiramycin I (YI) on the mRNA levels of the main fibrosis markers in CCC-ESF-1 induced by TGF ⁇ 1.
  • Drug C is one or more of Climycin or isovalerylspiramycin III or isovalerylspiramycin II or isovalerylspiramycin I, or its corresponding derivatives, pharmaceutically acceptable One or more of the salts, solvates, metabolites, stereoisomers, tautomers, polymorphs, and prodrugs of.
  • the main drug and auxiliary materials are respectively passed through a 100 mesh sieve, the prescription amount of drug C, microcrystalline cellulose and 1/2 prescription amount of sodium starch glycolate are mixed uniformly, and then 5% povidone K 30 aqueous solution is added.
  • Soft material granulated with 18 mesh sieve, dried wet granules at 60°C for 2h under ventilated conditions; after drying, granulate with 18 mesh sieve, then add 1/2 prescription amount of sodium starch glycolate and magnesium stearate to mix
  • the tablets were compressed with a dimple die with a diameter of 11 mm to obtain a tablet-containing core with a tablet weight of 350 mg and a hardness of 6.5 kg.
  • Preparation of coating solution Weigh the required Opadry II (white), add the required amount of water in the mixing container, add in portions, after all are added, reduce the stirring speed to make the vortex disappear, and continue stirring 30min, ready.
  • Preparation of film-coated tablets put the tablet core in a coating pan and determine the coating conditions.
  • the host speed is 20r/min
  • the inlet air temperature is 40°C
  • the outlet air temperature is 30°C
  • the spray pressure is 0.02MpC
  • the spray flow rate is 1ml /min for coating, after constant spraying for 1.5h, until the tablet surface is smooth and uniform in color, and it is qualified if it meets the film coating inspection standard.
  • the weight gain of the coating is about 5%.
  • Drug C is one or more of Climycin or isovalerylspiramycin III or isovalerylspiramycin II or isovalerylspiramycin I, or its corresponding derivatives, pharmaceutically acceptable One or more of the salts, solvates, metabolites, stereoisomers, tautomers, polymorphs, and prodrugs of.
  • Preparation process Weigh an appropriate amount of starch, dilute it to 15% concentration, and heat it to a paste to make an adhesive; the main ingredient drug C, auxiliary material starch, low-substituted hydroxypropyl cellulose, sodium carboxymethyl starch, stearic acid Magnesium was passed through a 100-mesh sieve, and the required main ingredients and auxiliary materials were weighed according to the prescribed amount; after drug C, starch, and low-substituted hydroxypropyl cellulose were thoroughly mixed uniformly, a starch paste with a starch concentration of 15% was used to make a soft material.
  • Example 3 Drug C capsules (calculated based on 10,000 capsules)
  • Drug C is one or more of Climycin or isovalerylspiramycin III or isovalerylspiramycin II or isovalerylspiramycin I, or its corresponding derivatives, pharmaceutically acceptable One or more of the salts, solvates, metabolites, stereoisomers, tautomers, polymorphs, and prodrugs of.
  • Preparation process After weighing the main ingredient medicine C and auxiliary material medicinal starch according to the technological formula, put it into the mixer and mix it for 1.5-2 hours; the data obtained by sampling and testing the content should be basically the same as the theoretical data (each capsule contains The weight is about 0.105g), the qualified medical No. 3 capsules and the mixed raw materials to be filled according to the operation requirements of the automatic capsule machine are respectively filled into the loader for filling, and the filled capsules are tested for difference ( Within ⁇ 10%, ⁇ 0.3g), the dissolution rate meets the requirements, put the capsules that meet the requirements after inspection into the lighter, add liquid paraffin for 15-20 minutes, and then take out the finished packaging box for inspection.
  • Example 4 Drug C dry syrup (calculated based on 10000 bags)
  • Drug C is one or more of Climycin or isovalerylspiramycin III or isovalerylspiramycin II or isovalerylspiramycin I, or its corresponding derivatives, pharmaceutically acceptable One or more of the salts, solvates, metabolites, stereoisomers, tautomers, polymorphs, and prodrugs of.
  • Preparation process The original powder of drug C, citric acid and sucrose are respectively pulverized by a high-speed jet mill into particles 85% passing through 300 meshes, 15% passing through 180 meshes, and then the pulverized powder is weighed according to the prescribed amount and mixed thoroughly 1- 1.5h, measure the content, calculate the filling quantity (theoretical filling quantity is 500mg per bag), then put the mixture into the bagging machine, pack the aluminum foil paper, and divide it according to the operation requirements of the filling machine, and the filling quantity difference is within ⁇ 5% , After installation, the outer packaging is carried out after passing the inspection.
  • Drug C is one or more of Climycin or isovalerylspiramycin III or isovalerylspiramycin II or isovalerylspiramycin I, or its corresponding derivatives, pharmaceutically acceptable One or more of the salts, solvates, metabolites, stereoisomers, tautomers, polymorphs, and prodrugs of.
  • Preparation process Pass the drug C, powdered sugar and dextrin through a 120-mesh sieve. Weigh the drug C, powdered sugar and dextrin according to the prescription and mix them evenly. Use 5% PVP-K 30 glue to make the above-mentioned materials into a soft paste. Material, rocking granule granulation, drying at 70°C, sizing, and packaging after passing inspection.
  • Preparation process Weigh 500mg of drug C and equimolar adipic acid, mix them uniformly, and dissolve in 5ml of water to obtain a light yellow clear solution with a pH between 4.6-5.6. Then 40mg of mannitol was added as a freeze-dried proppant, and after rapid freezing at low temperature for 9 hours, it was freeze-dried to obtain a light yellow loose mass, which was dissolved in 10 ml of sterile water before use.
  • Drug C is one or more of Climycin or isovalerylspiramycin III or isovalerylspiramycin II or isovalerylspiramycin I, or its corresponding derivatives, pharmaceutically acceptable One or more of the salts, solvates, metabolites, stereoisomers, tautomers, polymorphs, and prodrugs of.
  • the present invention uses human hepatic stellate cell line LX-2 as an in vitro research object, and uses Real-time PCR and Western Blot as research methods to confirm that drug C has an effect on the mRNA and protein levels of the main markers of fibrosis in LX-2 cells induced by TGF ⁇ 1 The inhibitory effect.
  • the present invention uses the common bile duct ligation rat fibrosis model and detects the pathological changes of rat liver tissue after drug C is orally administered. The results show that drug C can effectively relieve Pathological changes and degree of fibrosis in the rat liver caused by bile duct ligation.
  • Drug C (using Climycin and Isovalerylspiramycin I) inhibits the activity of COL1A1 promoter-luciferase reporter gene
  • the constructed monoclonal cell LX2-COL stably expressing the type I collagen ⁇ 1 promoter COL1A1P was spread on a 96-well white board with 2 ⁇ 10 4 cells per well. After the cell confluence is about 90%, different concentrations of drug C (using clinomycin and isovalerylspiramycin I) are added, and 4 replicate holes are set for each experiment.
  • drug C using clinomycin and isovalerylspiramycin I
  • 4 replicate holes are set for each experiment.
  • Bright-Glo TM Luciferase Assay System After 24 hours, aspirate and discard the medium, and add 50 ⁇ l/well of any medium. Add luciferase substrate 50 ⁇ l/well, and perform detection after 2min.
  • Figure 1 shows the inhibitory effects of cleritromycin (HB) and isovalerylspiramycin I (HY) on the activity of type I collagen ⁇ 1 promoter. The results indicate that cleritromycin and its single-component isovalerylspiramycin I have a significant inhibitory effect on the activity of the COL1A1 promoter.
  • Drug C (using Climycin and Isovalerylspiramycin I) inhibits the proliferation of human hepatocytes HepG2 and human hepatic stellate cells LX-2
  • Drug C (using Climycin and Isovalerylspiramycin I) inhibits the expression of the main markers of fibrosis in LX-2 cells induced by TGF ⁇ 1 at the mRNA and protein levels
  • TGF ⁇ 1 induces LX-2 cells and drug C (using Climycin (HB) and isovalerylspiramycin I (HY)) administration treatment:
  • LX-2 contains 10% fetal bovine serum, 1% Streptomyces penicillium Cultivation in DMEM, High Glucose, GlutaMAX TM (Gibco10566016) medium containing a mixture of vegetarian elements, 37°C and 5% CO 2 . 1 ⁇ 10 5 cells per well were plated on a 6-well plate. After 24 hours of culture, the original medium in the 6-well plate was removed by a vacuum pump and added with DMEM medium without 10% fetal calf serum.
  • TGF- ⁇ 1 (2ng/ml) induction After starvation for 24 hours, TGF- ⁇ 1 (2ng/ml) induction, adding different concentration gradients of cleritromycin and isovalerylspiramycin I at the concentrations of 10 ⁇ mol/L and 20 ⁇ mol/L respectively.
  • a control group was set up (without TGF- ⁇ 1 induction) , TGF- ⁇ 1 induction group (only TGF- ⁇ 1 induction) and TGF- ⁇ 1 induction administration group (both TGF- ⁇ 1 induction and drug C treatment).
  • RNA of LX-2 cells was extracted according to the TRIzol instructions.
  • the obtained cDNA, sterile water, Roche FastStart Universal Probe Master (Rox), and ABI TaqMan probes (GAPDH, COL1A1, TGFB1, ACTA2) were prepared into a 20 ⁇ l reaction system, and the ABI 7500 Fast Real-Time PCR System was used for detection. GAPDH was used as an internal control to analyze the results.
  • the rats were fasted with water for 12 hours, and the rats were sacrificed.
  • the liver tissue was taken, and the large hepatic lobe tissue was cut out and fixed in 10% formalin. After dehydration, paraffin embedding, slicing, and baking slices, paraffin sections are made.
  • Hematoxylin-eosin (H&E) staining solution was used for staining, and the pathological structure changes of rat liver tissue were observed under a microscope.
  • Drug C (using Climycin and Isovalerylspiramycin I) inhibits the expression of major fibrosis markers in lung fibroblasts MRC-5 induced by TGF ⁇ 1 at the mRNA and protein levels
  • TGF ⁇ 1 induces lung fibroblasts MRC-5 and administration of Climycin and Isovalerylspiramycin I: MRC-5 is treated with 10% fetal bovine serum, 1% penicillin streptomycin mixture, 1% non-essential Culture in amino acid MEM (Gibco 11095-080) medium at 37°C and 5% CO 2 . Plate 3 ⁇ 10 5 cells per well on a 6-well plate. After culturing for 24 hours, use a vacuum pump to remove the original medium in the 6-well plate and add MEM medium without 10% fetal bovine serum.
  • TGF - ⁇ 1 3ng/ml
  • concentration gradients of climycin and isovalerylspiramycin I the concentrations were 10 ⁇ mol/L, 20 ⁇ mol/L, 40 ⁇ mol/L, a total of control group (no TGF- ⁇ 1 induction), TGF- ⁇ 1 induction group (only TGF- ⁇ 1 induction), and TGF- ⁇ 1 induction administration group (both induction of TGF- ⁇ 1 and treatment with climycin or isovalerylspiramycin I) .
  • RNA of MRC-5 cells was extracted according to the TRIzol instructions.
  • the culture medium was discarded, and the total RNA of MRC-5 cells was extracted according to the TRIzol instructions.
  • the obtained cDNA, sterile water, Roche FastStart Universal Probe Master (Rox) and ABI TaqMan probes (GAPDH, COL1A1, TGFB1, ACTA2, MMP2) were prepared into a 20 ⁇ l reaction system, and the ABI7500 Fast Real-time PCR System was used for detection. GAPDH was used as an internal control to analyze the results.
  • the subjects were 18-75 years old and met the diagnostic criteria for pneumonia caused by novel coronavirus infection (Fifth Edition).
  • the patient meets any of the following: (1) Fever reappears, or clinical symptoms worsen, (2) Nucleic acid test of throat swab becomes negative, (3) Clinical symptoms are not improved or nucleic acid continues to be positive, (5) Chest CT shows pneumonia Or the progress of fibrosis. SOFA score: 1 point-13 points.
  • Light type Climycin tablets, 0.4g each time, once a day, orally after a meal for 7 consecutive days, and enter the follow-up observation period for 30 days after the treatment.
  • virus "fuyang" or treated patients There were 47 cases of virus "fuyang" or treated patients, including 11 cases of mild type, 27 cases of common type, 3 cases of severe type, and 6 cases of critical type. After pre-treatment, 40 patients were still positive for viral nucleic acid and 7 patients were negative for nucleic acid.
  • CT manifestations of pulmonary fibrosis include:
  • CT showed grid shadows
  • HRCT showed irregular thickening of the lobular septum, and the small blood vessels in the lobules became obvious due to the thickening of the wall.
  • CT shows extensive honeycomb shadows, with deformed lobular structure, which can cause bronchiectasis due to pulmonary fibrosis.
  • the extensive honeycomb shadows are most obvious under the middle and lower lungs;
  • Ground glass shadow It is an important sign of pulmonary fibrosis. Its existence means that the disease is in an active phase and requires active treatment. It can be an interstitial or substantial disease;
  • Honeycomb shadows Smaller cystic shadows, most from a few millimeters to ten millimeters, a few up to several centimeters, with thick and clear fibrous walls, which are more common in the periphery of the lung and under the pleura.
  • the normal structure of the part with obvious honeycomb shadow is distorted, the lobular structure is unrecognizable, and the pleura that is usually connected to the honeycomb shadow is slightly thickened, which is a manifestation of qualitative fibrosis in the late period.
  • Figure 8 is a CT image of the lungs of Case 1 before and after the treatment with cleritromycin. From the figure, it can be seen that the lung symptoms are significantly improved after 5 or 10 days of treatment with cleritromycin.
  • Figure 9 shows the changes in CT images of the lungs of Case 2.
  • A is the CT scan on the first day of illness
  • B is the CT scan on the 5th day of illness
  • C is the CT scan on the 6th day of illness Scanning image (starting day of climycin treatment)
  • D is the CT scan on the 8th day of illness
  • E is the CT scan on the 11th day of illness.
  • Figure 10 shows the changes in the CT image of Case 3. Specifically, the patient, female, 72 years old. After admission, he was given oxygen inhalation through a nasal cannula and orally 0.4 g of colimycin, once a day. On the second day after admission, the patient's general condition improved, coughing and dyspnea were significantly improved, and oxygen saturation increased to 98%; blood gas analysis oxygen partial pressure increased by 130mmHg, and throat swabs were performed on the 3rd and 6th days after the treatment with climycin The nucleic acid tests were all negative.
  • CT image ( Figure 10) shows that on the 6th day of the disease course (1 day before taking climycin), bilateral lung texture increased, irregular ground-glass lesions were seen in the lower right lung field, and patchy shadows were scattered on the left side (arrow A) ); On the 9th day of the course of the disease (3 days after taking crimycin), bilateral lung texture is clear, and the irregular ground-glass lesions in the lower right lung field are obviously absorbed (indicated by the B arrow). After 5 days), the right lung lesion was obviously absorbed (shown by the C arrow) and a small amount of fibrosis formed. After continuing to take climycin treatment, the fibrosis condition improved.
  • Drug C (using Climycin and Isovalerylspiramycin I) inhibits the expression of main fibrosis markers in skin fibroblasts CCC-ESF-1 induced by TGF ⁇ 1 at the mRNA level
  • TGF ⁇ 1 induces skin fibroblasts CCC-ESF-1 and administration of Climycin and isovalerylspiramycin I: CCC-ESF-1 cells in a mixture containing 10% fetal bovine serum and 1% penicillin streptomycin Culture in DMEM, High Glucose, GlutaMAX TM (Gibco 10566016) medium at 37°C and 5% CO 2 . Spread 5 ⁇ 10 5 cells per well on a 6-well plate. After culturing for 24 hours, use a vacuum pump to remove the original medium in the 6-well plate and add DMEM medium without 10% fetal bovine serum.
  • TGF - ⁇ 1 (5ng/ml) induction
  • cleritromycin and isovalerylspiramycin I added at the concentrations of 20 ⁇ mol/L and 40 ⁇ mol/L, a total of control groups (without TGF- ⁇ 1 induction ), TGF- ⁇ 1 induction group (addition of TGF- ⁇ 1 induction only) and TGF- ⁇ 1 induction administration group (addition of TGF- ⁇ 1 induction and treatment with climycin or isovalerylspiramycin I).
  • RNA of LX-2 cells was extracted according to the TRIzol instructions.
  • the culture medium was discarded, and the total RNA of LX-2 cells was extracted according to the TRIzol instructions.
  • the obtained cDNA, sterile water, Roche FastStart Universal Probe Master (Rox) and ABI TaqMan probes (GAPDH, COL1A1, TGFB1, ACTA2, MMP2) were prepared into a 20 ⁇ l reaction system, and the ABI 7500 Fast Real-Time PCR System was used for detection.
  • GAPDH was used as an internal control to analyze the results, and the data showed that both clalimycin and isovalerylspiramycin I could significantly inhibit the expression of COL1A1, TGFB1, ACTA2 and MMP2 (Figure 11). Isovalerylspiramycin I is toxic to CCC-ESF-1 cells at a concentration of 40 ⁇ mol/L.

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Abstract

一种用于预防、缓解和/或治疗纤维化的药物，所述药物的有效成分包括可利霉素、异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ中的一种；或异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ中的两种或三种的组合。还涉及一种用于预防、缓解和/或治疗纤维化的组合物产品，所述组合产品包括第一药剂，所述第一药剂的活性成分包括可利霉素、异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ中的一种；或异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ中的两种或三种的组合。还涉及上述药物及组合物对预防、缓解和/或治疗纤维化中的应用。

Description

一种用于预防、缓解和/或治疗纤维化的药物、组合产品及其应用 技术领域
本发明属于医药化学领域，具体地说，涉及一种用于预防、缓解和/或治疗纤维化的药物、组合产品及其应用。
背景技术
纤维化是一种病理变化，表现为成纤维细胞激活增殖、组织器官内纤维结缔组织增多，实质细胞减少，持续进展可致组织、器官结构破坏和功能丧失。重要脏器的纤维化严重影响患者生存质量，甚至危及生命。在全世界范围内，组织纤维化是许多疾病致残、致死的主要原因，据美国有关统计资料证明，该国因各种疾病而致死的患者中，约45％可以归于组织纤维增生疾病。目前针对这一疾病的治疗手段和药物仍旧十分欠缺，预后情况很差，开发新的能够有效治疗纤维化的药物是一项十分重要和迫切的工作。
可利霉素(Carrimycin)，又称必特螺旋霉素(Bitespiramycin)、生技霉素(Shengjimycin)是由中国医科院生物技术研究所与本申请人合作，通过转基因技术将碳霉素产生菌的4″异戊酰基转移酶基因(4″-O-isovaleryltransferase gene)克隆至螺旋霉素产生菌(Streptomyces spiramyceticus)中，定向酰化螺旋霉素4″-OH，在4″位加入异戊酰基侧链所形成的以4″位异戊酰基螺旋霉素为主要组分的新型抗生素。
可利霉素主成分的结构示意图如式(1)所示，不代表构象：
Figure PCTCN2020085111-appb-000001
其中，当R＝H，R′＝COCH 2CH(CH 3) 2时为异戊酰螺旋霉素Ⅰ；
当R＝COCH 3，R′＝COCH 2CH(CH 3) 2时为异戊酰螺旋霉素Ⅱ；
当R＝COCH 2CH 3，R′＝COCH 2CH(CH 3) 2时为异戊酰螺旋霉素Ⅲ。
可利霉素中主要活性成分异戊酰螺旋霉素(Ⅰ+Ⅱ+Ⅲ)总含量不低于60％，酰化螺旋霉 素的总含量不低于80％，于药学上是一种可接受的药物组合物。中心结构为16元内酯环，与一分子福洛胺糖、一分子碳霉胺糖和一分子碳霉糖连接而成，其主要成分异戊酰螺旋霉素Ⅰ、Ⅱ、Ⅲ与螺旋霉素结构不同之处在于碳霉糖4″位上连接的基团为异戊酰基而不是羟基。化学结构，如式(1)所示，共包含十余种组分。目前可利霉素成品组成标准为药物中异戊酰螺旋霉素Ⅲ≥30％，异戊酰螺旋霉素Ⅰ、Ⅱ、Ⅲ的比例总和≥60％，总酰化螺旋霉素的比例≥80％，其它未知组分的总和≤5％。
可利霉素属于16元大环内酯类抗生素，具有活性基团羧基、烷氧基、环氧基、酮基和醛基以及一对共轭的C＝C，分子量约为884～982。由于具有相似的化学结构，可利霉素与大环内酯类抗生素具有很多共性：易溶于酯类、丙酮、氯仿、醇类等大多数有机溶剂，微溶于石油醚，难溶于水；分子结构中含有两个二甲胺基而呈弱碱性，易溶于酸性水溶液；具有溶解度随温度的升高而降低的“负溶解度”性质。由于可利霉素主要组分异戊酰螺旋霉素4″位碳链较长，亲水性差，其水中溶解度比螺旋霉素及4″-乙酰螺旋霉素小。
该药具有亲脂性好，组织渗透能力强，口服吸收快，体内维持时间长，有持续的抗生素后效应。根据药效与化学构象的关系，大环内酯类抗生素4″位酰化后，其亲脂性和体内活性提高，体内抗菌活性与临床治疗效果均得到了显著提升，并且抗生素在体内的稳定性随着4″羟基酯的碳链增长而增强，即异戊酰螺旋霉素＞丁酰螺旋霉素＞丙酰螺旋霉素＞乙酰螺旋霉素。
初步体内外药效学试验表明，该药不仅对多数G +菌有较好抗菌活性，对部分G -菌也有一定作用，各项技术指标明显优于阿奇霉素、红霉素、乙酰螺旋霉素、麦迪霉素，尤其对肺炎支原体的抗菌活性最强，对红霉素耐药菌、淋球菌、肺炎球菌、金葡菌、绿脓假单胞菌、流感杆菌、流感嗜血杆菌、脆弱拟杆菌、军团菌、多行杆菌和产气荚膜梭菌也有一定抗菌活性，对临床耐红霉素的金葡球菌仅有极少交叉耐药性。可利霉素将主要用于治疗革兰氏阳性菌感染性疾病，尤其是上呼吸道感染，并可能用于泌尿系统感染等。
药代动力学研究结果表明，可利霉素中具有活性的有效组分主要为异戊酰螺旋霉素Ⅰ、Ⅱ、Ⅲ。可利霉素进入体内后代谢，以母体异戊酰螺旋霉素Ⅰ、Ⅱ、Ⅲ和代谢物螺旋霉素Ⅰ、Ⅱ、Ⅲ的AUC 0-t总和计算，其口服绝对生物利用度平均为91.6％。单次服药可利霉素消除较慢，T 1/2β在23-27小时之间。
2019年末至2020年1月，部分地区发生了不明原因肺炎，患者支气管肺泡灌洗标本送检新一代测序，发现新型冠状病毒。2020年2月12日，WHO将该新型冠状病毒(SARS-CoV-2) 导致的疾病命名为：Corona Virus Disease 2019，COVID-19。新型冠状病毒感染引起的肺部损伤，包括肺纤维化，寻找有效的治疗药物、改善肺部症状的药物刻不容缓。
迄今为止，未见有可利霉素治疗纤维化的记载和报道。
有鉴于此，特提出本发明。
发明内容
本发明要解决的技术问题在于克服现有技术的不足，提供一种用于预防、缓解和/或治疗纤维化的药物，该药物能有效预防、缓解和治疗纤维化，具有重大的社会效益和经济效益。
为解决上述技术问题，本发明采用如下技术方案：
一种用于预防、缓解和/或治疗纤维化的药物，所述药物的有效成分包括可利霉素、异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ中的一种；
或异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ中的两种或三种的组合。
进一步的，所述药物包括医学上可接受的载体。
进一步的，所述药物制备成医学上可接受的片剂、胶囊、丸剂、注射剂、缓释剂及各种微粒给药系统。
一种用于预防、缓解和/或治疗纤维化的药物，所述药物的有效成分选自以下成分中的一种或多种：
可利霉素的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互变异构体、多晶型物、药物前体；
异戊酰螺旋霉素Ⅲ的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互变异构体、多晶型物、药物前体；
异戊酰螺旋霉素Ⅱ的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互变异构体、多晶型物、药物前体；
以及异戊酰螺旋霉素Ⅰ的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互变异构体、多晶型物、药物前体。
一种用于治疗纤维化的组合产品，所述的组合产品包括第一药剂，所述第一药剂的有效成分包括可利霉素、异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ中的一种；
或者异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ中两种或三种的组合。
进一步的，所述的组合产品还包括第二药剂，所述第二药剂包括用于预防、缓解和/或治疗纤维化的相关药物中的至少一种。
进一步的，所述用于预防、缓解和/或治疗纤维化的相关药物包括皮质类固醇激素、秋水仙碱、水飞蓟素、干扰素。
一种用于治疗纤维化的组合产品，所述组合产品包括第一药剂，所述第一药剂的有效成分选自以下成分中的一种或多种：
可利霉素的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互变异构体、多晶型物、药物前体；
异戊酰螺旋霉素Ⅲ的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互变异构体、多晶型物、药物前体；
异戊酰螺旋霉素Ⅱ的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互变异构体、多晶型物、药物前体；
以及异戊酰螺旋霉素Ⅰ的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互变异构体、多晶型物、药物前体。
本发明中所述的纤维化包括肺纤维化、心肌纤维化、肝纤维化、胰腺纤维化、肾纤维化、骨髓纤维化和皮肤纤维化；
优选的，所述肺纤维化包括新型冠状病毒感染导致的肺纤维化。本申请的药物及组合产品对于新型冠状病毒(SARS-CoV-2)感染导致的肺部纤维化有改善作用。
肺纤维化是多种原因引起肺脏损伤时，间质会分泌胶原蛋白进行修补，如果过度修复，即成纤维细胞过度增殖和细胞外基质大量聚集而形成。
心肌纤维化是心脏间质成纤维细胞过度增殖、胶原过度沉积及异常分布。
肝纤维化是各种致病因子所致肝内结缔组织异常增生，肝内弥漫性细胞外基质过度沉淀的病理过程。多种因素均可引起肝纤维化，如病毒感染、炎症反应、氧化应激和酗酒等。
胰腺纤维化是胰腺腺泡细胞大量分泌蛋白质，而胰管细胞分泌的液体及碳酸氢盐并不增加，导致胰腺腺泡细胞分泌的胰石蛋白(Lithostathine)与GP2(一种可形成管型的蛋白)的浓度下降，并在胰管中沉淀而导致胰腺纤维化，
肾纤维化是细胞外基质和不适当结缔组织在肾聚集，导致肾结构改变和功能受损的病理 过程。
骨髓纤维化是由于骨髓造血组织中胶原增生，其纤维组织严重影响造血功能所引起的一种骨髓增生性疾病。
皮肤纤维化是在创伤等情况下，成纤维细胞分裂、增殖，向受损部位迁移，产生细胞外基质，形成瘢痕组织修复创伤。瘢痕的形成是肉芽组织逐渐纤维化的过程。
本发明还提供了一种上述任一所述药物及组合产品在预防、缓解和/或治疗纤维化中的应用。
本发明还提供了上述任一所述药物或组合产品在抑制炎症或脂质过氧化、抑制成纤维细胞的增生活化、促进胶原降解的应用。
采用上述技术方案后，本发明与现有技术相比具有以下有益：
本发明提供的药物、组合产品在治疗纤维化方面具有良好的治疗效果，具有重要的社会效益和经济效益。
下面结合附图对本发明的具体实施方式作进一步详细的描述。
附图说明
图1为HB(可利霉素)和HY(异戊酰螺旋霉素Ⅰ)对Ⅰ型胶原蛋白α1启动子活性的抑制作用；
图2为HB(可利霉素)和HY(异戊酰螺旋霉素Ⅰ)在HepG2和LX2细胞的IC50值；
图3为Real-time PCR检测HB(可利霉素)和HY(异戊酰螺旋霉素Ⅰ)对TGFβ1诱导LX-2细胞中纤维化主要标志物mRNA水平的影响；
图4为Western Blot检测HB(可利霉素)和HY(异戊酰螺旋霉素Ⅰ)对TGFβ1诱导LX-2细胞中纤维化主要标志物蛋白水平的影响；
图5为HB(可利霉素)对大鼠肝脏组织病理结构的影响；
其中，图5中：sham为假手术组大鼠肝脏组织H&E染色切片结果；BDL为BDL模型组大鼠肝脏组织H&E染色切片结果；HB为可利霉素给药组大鼠肝脏组织H&E染色切片结果；
图6为HB(可利霉素)对大鼠纤维化程度的影响；
其中，图6中：sham为假手术组大鼠肝脏组织马松染色切片结果；BDL为BDL模型组 大鼠肝脏组织马松染色切片结果；HB为可利霉素给药组大鼠肝脏组织马松染色切片结果；
图7为Real-time PCR检测可利霉素和异戊酰螺旋霉素Ⅰ对TGFβ1诱导肺成纤维细胞MRC-5中纤维化主要标志物mRNA水平的影响；其中，HB为可利霉素，异戊Ⅰ为异戊酰螺旋霉素Ⅰ；
图8为病例1可利霉素治疗前后肺部改善情况；
图9为病例2可利霉素治疗前后肺部改善情况；
图10为病例3可利霉素治疗前后肺部改善情况；
图11为Real-time PCR检测可利霉素(HB)和异戊酰螺旋霉素Ⅰ(YI)对TGFβ1诱导皮肤成纤维细胞CCC-ESF-1中纤维化主要标志物mRNA水平的影响。
需要说明的是，这些附图和文字描述并不旨在以任何方式限制本发明的构思范围，而是通过参考特定实施例为本领域技术人员说明本发明的概念。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对实施例中的技术方案进行清楚、完整地描述，以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1、药物C片
规格：200mg/350mg
片芯处方：
Figure PCTCN2020085111-appb-000002
包衣液处方：
Figure PCTCN2020085111-appb-000003
Figure PCTCN2020085111-appb-000004
药物C为可利霉素或异戊酰螺旋霉素Ⅲ或异戊酰螺旋霉素Ⅱ或异戊酰螺旋霉素Ⅰ中的一种或多种，或其对应的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互变异构体、多晶型物、药物前体中的一种或几种。
制备工艺：
片芯的制备：主药和辅料分别过100目筛，将处方量药物C、微晶纤维素与1/2处方量的羧甲淀粉钠混合均匀，然后加入5％聚维酮K 30水溶液制软材，以18目筛制粒，湿颗粒在60℃通风条件下干燥2h；干燥后以18目筛整粒，再加入1/2处方量的处方量羧甲淀粉钠与硬脂酸镁混合均匀后，用直径11mm的浅凹冲模压片，制得片重350mg、硬度6.5kg的含药片芯。
包衣液的配制：称好所需的欧巴代Ⅱ(白色)在配液容器中加入所需量的水，分次加入，待全部加入后，降低搅拌速度，使蜗旋消失，继续搅拌30min，即得。
薄膜包衣片的制备：将片芯置包衣锅内，确定包衣条件，主机速度为20r/min，进风温度40℃，出风温度30℃，喷雾压力0.02MpC，喷浆流量为1ml/min进行包衣，恒定后持续喷包1.5h，至片粒表面光滑、色泽均匀，符合薄膜衣检验标准为合格。包衣增重5％左右。
实施例2、药物C素片(按10000片计算)
处方：
Figure PCTCN2020085111-appb-000005
药物C为可利霉素或异戊酰螺旋霉素Ⅲ或异戊酰螺旋霉素Ⅱ或异戊酰螺旋霉素Ⅰ中的一种或多种，或其对应的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互变异构体、多晶型物、药物前体中的一种或几种。
制备工艺：称取适量淀粉，稀释至15％浓度，加热至糊状，制成粘合剂；主料药物C、 辅料淀粉、低取代羟丙基纤维素、羧甲基淀粉钠、硬脂酸镁分别过100目筛，按处方量，称取所需主料和辅料；药物C、淀粉、低取代羟丙基纤维素充分混合均匀后，用15％淀粉浓度的淀粉糊制成软材，14目筛制粒，50-60℃干燥，水份控制在3-5％，14目筛整粒，加羧甲基淀粉钠，硬脂酸镁混合，测定颗粒含量；根据颗粒含量，计算片重，压片(Φ9mm浅凹冲头)，检测片重差异；经检验合格后进行包装。
实施例3、药物C胶囊剂(按10000粒计算)
处方：
Figure PCTCN2020085111-appb-000006
药物C为可利霉素或异戊酰螺旋霉素Ⅲ或异戊酰螺旋霉素Ⅱ或异戊酰螺旋霉素Ⅰ中的一种或多种，或其对应的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互变异构体、多晶型物、药物前体中的一种或几种。
制备工艺：将主料药物C辅料药用淀粉按工艺配方量分别称取后，装入混合器充分混合后1.5-2小时；取样检测含量所得数据应和理论数据基本一致(每粒胶囊所装重量约为0.105g)，将经检验合格的药用3号胶囊及混合好的待装原料按全自动胶囊机操作要求，分别填入装料器进行填充，将填充好的胶囊进行差异检验(±10％以内，＜0.3g)，溶出度符合要求，将检验后符合要求的胶囊，放入打光机内加入液体石蜡进行15-20分钟的打光，然后取出进行成品包装盒检验。
实施例4、药物C干糖浆(按10000袋计算)
Figure PCTCN2020085111-appb-000007
药物C为可利霉素或异戊酰螺旋霉素Ⅲ或异戊酰螺旋霉素Ⅱ或异戊酰螺旋霉素Ⅰ中的一种或多种，或其对应的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互 变异构体、多晶型物、药物前体中的一种或几种。
制备工艺：药物C原粉，柠檬酸、蔗糖分别用高速气流粉碎机粉碎成颗粒85％通过300目，15％通过180目，然后将粉碎后的细粉按处方量称取后充分混合1-1.5h，测其含量，计算装量(理论装量为每袋500mg)，然后将混合物装入袋装机中，装好铝箔纸，按分装机操作要求分装，装量差异在±5％以内，装好后进行检验合格后外包装。
实施例5、药物C颗粒剂(按10000袋计算)
处方：
Figure PCTCN2020085111-appb-000008
药物C为可利霉素或异戊酰螺旋霉素Ⅲ或异戊酰螺旋霉素Ⅱ或异戊酰螺旋霉素Ⅰ中的一种或多种，或其对应的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互变异构体、多晶型物、药物前体中的一种或几种。
制备工艺：药物C、糖粉、糊精过120目筛，按处方量称取药物C、糖粉、糊精混合均匀，将混合均匀的上述物料用5％PVP-K 30胶浆制成软材，摇摆式颗粒剂制粒70℃干燥、整粒，送检合格后分装。
实施例6、冻药物C冻干粉针剂
制备工艺：称取药物C 500mg与等摩尔的己二酸混合均匀后溶解于5ml水中，得到淡黄色澄明溶液，pH在4.6-5.6之间。再加入甘露醇40mg作为冻干支撑剂，低温快速冷冻9h后，冷冻干燥，获得淡黄色疏松块状物，使用前用10ml无菌水溶解。
药物C为可利霉素或异戊酰螺旋霉素Ⅲ或异戊酰螺旋霉素Ⅱ或异戊酰螺旋霉素Ⅰ中的一种或多种，或其对应的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互变异构体、多晶型物、药物前体中的一种或几种。
实验例一药物C在抗肝纤维化中的作用
本发明利用人肝星状细胞系LX-2作为体外研究对象，以Real-time PCR、Western Blot为研究方法证实药物C对TGFβ1诱导LX-2细胞中纤维化主要标志物的mRNA和蛋白水平均的抑制作用。同时，为进一步确定药物C的抗纤维化作用，本发明利用胆总管结扎大鼠纤维 化模型，并将药物C口服给药后大鼠肝组织病理改变进行检测，结果显示，药物C能够有效缓解胆管结扎造成的大鼠肝脏病理改变和纤维化程度。
1、药物C(采用可利霉素和异戊酰螺旋霉素Ⅰ)抑制COL1A1启动子-荧光素酶报告基因的活性
将构建的稳定表达Ⅰ型胶原蛋白α1启动子COL1A1P的单克隆细胞LX2-COL铺于96孔白板，每孔2×10 4个细胞。待细胞汇合度约90％后，加入不同浓度的药物C(采用可利霉素和异戊酰螺旋霉素Ⅰ)，每组实验设4个复孔。按照Bright-Glo TM Luciferase Assay System说明书操作步骤，24h后吸弃培养基，加入任意培养基50μl/孔。加入荧光素酶底物50μl/孔，2min后进行检测。结果显示，随着药物C浓度的升高，荧光强度减弱，且在二者浓度为40μM时，荧光强度减弱最为明显(图1)。图1中显示了可利霉素(HB)和异戊酰螺旋霉素Ⅰ(HY)对Ⅰ型胶原蛋白α1启动子活性的抑制作用。该结果表明，可利霉素及其单组份异戊酰螺旋霉素Ⅰ对COL1A1启动子活性有明显的抑制作用。
2、药物C(采用可利霉素和异戊酰螺旋霉素Ⅰ)对人肝细胞HepG2和人肝星状细胞LX-2的增殖抑制
将人肝癌HepG2细胞和人肝星状细胞LX-2铺于96孔白板，每孔4×10 3个细胞，24小时后加入不同浓度的药物C(采用可利霉素(HB)和异戊酰螺旋霉素Ⅰ(HY))，每组实验设3个复孔。药物处理后24小时或48小时采用磺酰罗丹明(SRB)法进行染色，用酶标仪测定515nm的吸光值，计算半数抑制率(IC50)。结果显示可利霉素及其单组份异戊酰螺旋霉素Ⅰ对HepG2和LX-2细胞均有一定的增殖抑制作用，其IC50均在10μM到100μM之间(图2)。
3、药物C(采用可利霉素和异戊酰螺旋霉素Ⅰ)在mRNA及蛋白水平上抑制TGFβ1诱导LX-2细胞中纤维化主要标志物的表达
TGFβ1诱导LX-2细胞以及药物C(采用可利霉素(HB)和异戊酰螺旋霉素Ⅰ(HY))给药处理：LX-2在含10％胎牛血清、1％青链霉素混合液的DMEM，High Glucose,GlutaMAX TM(Gibco10566016)培养基中，37℃，5％CO 2条件下培养。按照每孔1×10 5个细胞铺于6孔板，培养24h后使用真空泵去除6孔板内原有的培养基并加入不含10％胎牛血清的DMEM培养基饥饿培养24h后，加入TGF-β1(2ng/ml)诱导，同时加入不同浓度梯度的可利霉素和异戊酰螺旋霉素Ⅰ，浓度分别为10μmol/L、20μmol/L，共设置对照组(不加入TGF-β1诱导)、TGF-β1诱导组(仅加入TGF-β1诱导)和TGF-β1诱导给药组(既加入TGF-β1诱导又给予药物C处理)。
继续培养24h后，弃去培养基，按照TRIzol说明书操作步骤提取LX-2细胞总RNA。按照 Roche Transcriptor First Strand cDNA Synthesis Kit说明书操作步骤将LX-2总RNA逆转录为cDNA。将所得的cDNA、无菌水、Roche FastStart Universal Probe Master(Rox)以及ABI TaqMan probes(GAPDH，COL1A1，TGFB1，ACTA2)配制成20μl反应体系，使用ABI 7500 Fast Real-Time PCR System进行检测。以GAPDH作为内参进行结果分析，数据显示，随着可利霉素和异戊酰螺旋霉素Ⅰ剂量的增加，COL1A1、TGFB1、ACTA2的表达量降低，并且在20μmol/L时在mRNA水平上抑制COL1A1、TGFB1、ACTA2的表达效果最佳(图3)。
TGFβ1诱导后，继续培养24h后，6孔板中每孔中加入1ml Ripa裂解液(含1％PMSF)提取蛋白，并用BCA法测定蛋白浓度。以每孔25μg/20μl上样，经电泳、转膜、5％脱脂牛奶封闭、抗体孵育后，使用天能5200成像系统获得所需蛋白条带。结果显示，随着可利霉素浓度的升高，COL1A1、TGFβ1、α-smooth muscle actin的表达量降低，在20μmol/L时可显著抑制上述标志物蛋白的表达(图4)。
4、SD大鼠胆总管结扎诱导纤维化模型制备
选体重在180-220g的SD雄性大鼠12只，随机分为假手术组、模型组、药物C给药组，每组各4只。动物实验前禁食不禁水12h后，用异氟烷麻醉动物进行手术。其中模型组和给药组行胆总管结扎(BDL)术，在无菌操作台中，于上腹正中切口，抬高肝缘，拨开十二指肠，分离总胆管2-3cm。在近十二指肠处和近肝门处分别用000号手术缝合线各结扎两道，从中间剪断胆总管。假手术组仅作上腹正中切口并缝合，不做胆总管结扎。动物麻醉清醒后，正常饮食，自由饮水。术后第2天开始灌胃给药，分别给予生理盐水(假手术组、BDL模型组)和可利霉素和异戊酰螺旋霉素Ⅰ 200mg/kg(药物C给药组)，每天1次，连续给药14天。
5、药物C(采用可利霉素)对BDL诱导的大鼠肝脏病理结构改变的改善作用
取样前禁食不禁水12h，处死大鼠，取肝脏组织，切下肝大叶组织块放入10％福尔马林中固定。经过脱水、石蜡包埋、切片、烤片等制作石蜡切片。使用苏木素-伊红(H&E)染色液进行染色，显微镜下观察大鼠肝脏组织病理结构变化情况。结果显示，假手术组大鼠肝脏组织肝细胞排列整齐，肝小叶结构完整，未见胆管增生；BDL术后大鼠肝脏组织病理结构发生明显改变，胆管增生十分明显，组织坏死明显增多；可利霉素给药组的大鼠肝组织结构病理改变缓解，胆管增生情况得到抑制，组织坏死程度显著降低(图5)。表明可利霉素能够明显改善BDL大鼠肝脏组织的病理改变。
6、药物C(采用可利霉素)对BDL诱导的大鼠纤维化的抑制作用
将石蜡切片用马松染液染色，通过观察大鼠肝脏纤维化改变情况。结果显示，BDL术后大鼠肝脏纤维化程度明显增加，胶原沉积严重。可利霉素给药后，纤维化和胶原沉积情况得 到明显抑制(图6)。表明可利霉素能够明显抑制BDL诱导的大鼠纤维化。
实验例二药物C在抑制肺纤维化方面的作用
一.药物C(采用可利霉素和异戊酰螺旋霉素Ⅰ)在mRNA及蛋白水平上抑制TGFβ1诱导肺成纤维细胞MRC-5中纤维化主要标志物的表达
TGFβ1诱导肺成纤维细胞MRC-5以及可利霉素和异戊酰螺旋霉素Ⅰ给药处理：MRC-5在含10％胎牛血清、1％青链霉素混合液、1％非必需氨基酸的MEM(Gibco 11095-080)培养基中，37℃，5％CO 2条件下培养。按照每孔3×10 5个细胞铺于6孔板，培养24小时后使用真空泵去除6孔板内原有的培养基并加入不含10％胎牛血清的MEM培养基饥饿培养24h后，加入TGF-β1(3ng/ml)诱导，同时加入不同浓度梯度的可利霉素和异戊酰螺旋霉素Ⅰ，浓度分别为10μmol/L、20μmol/L、40μmol/L，共设置对照组(不加入TGF-β1诱导)、TGF-β1诱导组(仅加入TGF-β1诱导)和TGF-β1诱导给药组(既加入TGF-β1诱导又给予可利霉素或异戊酰螺旋霉素Ⅰ处理)。
继续培养24小时后，弃去培养基，按照TRIzol说明书操作步骤提取MRC-5细胞总RNA。按照Roche Transcriptor First Strand cDNA Synthesis Kit说明书操作步骤将LX-2总RNA逆转录为cDNA。将所得的cDNA、无菌水、Roche FastStart Universal Probe Master(Rox)以及ABI TaqMan probes(GAPDH，COL1A1,TGFB1,ACTA2，MMP2)配制成20μl反应体系，使用ABI7500 Fast Real-time PCR System进行检测。以GAPDH作为内参进行结果分析，数据显示，随着可利霉素剂量的增加，COL1A1、TGFB1、ACTA2、MMP2的表达量降低，并且在40μmol/L时在mRNA水平上抑制COL1A1、TGFB1、ACTA2的表达效果最佳；随着异戊酰螺旋霉素Ⅰ剂量的增加，COL1A1的表达量降低，并且在40μmol/L时在mRNA水平上抑制COL1A1的表达效果最佳(图7)。
二、可利霉素对新型冠状病毒(SARS-CoV-2)感染导致的肺炎(Corona Virus Disease 2019，COVID-19，)的肺部纤维化的改善作用
受试者年龄18-75周岁，符合新型冠状病毒感染肺炎诊断标准(第五版)。患者符合以下任何一条：(1)再次出现发热、或临床症状加重，(2)咽拭子核酸检测阴转阳性，(3)临床症状无改善或核酸持续阳性，(5)胸部CT显示肺炎症或纤维化进展。SOFA评分：1分-13分。
1、治疗方法
轻型：可利霉素片，每次0.4g，每日1次，餐后口服，连续用药7天，治疗结束后进入随访观察期30天。
普通型：可利霉素片，每次0.4g，每日1次，餐后口服，10天，治疗结束后进入随访观察 期30天。
重型、危重型：可利霉素片，每次0.4g，每日1次，餐后口服，连续用药14天，不能口服者，鼻饲管给药。治疗结束后进入随访观察期30天。
2、主要疗效指标
(1)热退时间(天)。
(2)肺部症状消退时间(HRCT)(天)。
(3)治疗结束3天、7天咽拭子新冠病毒转阴率(％)。
3、病例总体情况
病毒“复阳”或经治患者47例，包括轻型11例，普通型27例，重型3例，危重型6例。经前期治疗后，病毒核酸仍阳性的患者40例，核酸阴性的患者7例。
4、主要疗效评价
(1)40例病毒核酸阳性的患者中，16例患者3天核酸阴转，13例患者7天阴转，1例患者15天阴转，其余10例患者刚入组，核酸未复查。
(2)入组时有肺部炎症的患者19例(核酸阴性7例，核酸阳性12例)，7天肺部炎症显著改善率为73.7％(14/19)。
(3)入组时发热的患者5例(核酸阴性3例，核酸阳性2例)，3天体温复常率为60％(3/5)，7天体温复常率为100％(5/5)。
5、主要病例肺部症状改善情况
肺纤维化CT表现有：
1、病变的分布呈外围性、胸膜下；
2、肺纤维化的CT表现在不同时期有明显不同，早期HRCT显示中下肺斑片状毛玻璃阴影，提示为活动性病变，是可逆性病变，这时的间质性病变改变不明显；
3、进展发展成为肺纤维化，CT表现为网格阴影，HRCT显示小叶间隔不规则增厚，小叶内的细小血管由于壁的增厚而变得明显。在晚期，CT呈广泛蜂窝状阴影，小叶结构变形，由于肺纤维化可致支气管扩张，广泛蜂窝状阴影以中下肺胸膜下最明显；
4、磨玻璃影：是肺纤维化的一个重要征象，它的存在意味着病变处于活动期且需要积极治疗，可以是间质性或实质性病变；
5、蜂窝状阴影：较小的囊状阴影，大多数几毫米至十毫米，少数可达几厘米，有厚的边缘清楚的纤维壁，多见于肺的外围和胸膜下。蜂窝状阴影明显的部位正常结构扭曲，小叶结构无法辨认，通常与蜂窝状阴影相连的胸膜轻度增厚，是晚期间质纤维化的表现。
新型冠状病毒感染肺炎患者可利霉素治疗后，肺部症状，纤维化情况得到改善。具体的：
图8为病例1在可利霉素治疗前后肺部CT图，从图中可以看出可利霉素治疗5、10天后肺部症状明显改善。
图9中是病例2的肺部CT图变化情况，(A)为患病第一天CT扫描图，(B)为患病第5天CT扫描图，(C)为患病第6天CT扫描图(开始可利霉素治疗日)，(D)为患病第8天CT扫描；(E)为患病第11天CT扫描图。后经过继续服用可利霉素治疗，肺部情况改善。
图10中所示为病例3的CT图变化情况。具体的，患者，女性，72岁。入院后给予鼻导管吸氧，口服可利霉素0.4g，每天1次。入院后第2天患者一般状态好转，咳嗽、呼吸困难明显好转，氧饱和度提升到98％；血气分析氧分压升130mmHg，可利霉素治疗后第3天、第6天2次咽拭子核酸检测均阴性。CT图(图10)显示病程第6天(服用可利霉素前1天)，双侧肺纹理增多，右肺下野可见不规则毛玻璃样病灶，左侧散在斑片状影(A箭头所示)；病程第9天(服用可利霉素后3天)，双侧肺纹理清晰，右肺下野不规则毛玻璃样病灶明显吸收(B箭头所示)，病程第12天(服用可利霉素后5天)，右肺病灶明显吸收(C箭头所示)少量纤维化形成。后经过继续服用可利霉素治疗，纤维化情况改善。
实验例三药物C在抑制皮肤纤维化方面的作用
1.药物C(采用可利霉素和异戊酰螺旋霉素Ⅰ)在mRNA水平上抑制TGFβ1诱导皮肤成纤维细胞CCC-ESF-1中纤维化主要标志物的表达
TGFβ1诱导皮肤成纤维细胞CCC-ESF-1以及可利霉素和异戊酰螺旋霉素Ⅰ给药处理：CCC-ESF-1细胞在含10％胎牛血清、1％青链霉素混合液的DMEM,High Glucose,GlutaMAX TM(Gibco 10566016)培养基中，37℃，5％CO 2条件下培养。按照每孔5×10 5个细胞铺于6孔板，培养24小时后使用真空泵去除6孔板内原有的培养基并加入不含10％胎牛血清的DMEM培养基饥饿培养24h后，加入TGF-β1(5ng/ml)诱导，同时加入不同浓度梯度的可利霉素和异戊酰螺旋霉素Ⅰ，浓度分别为20μmol/L、40μmol/L，共设置对照组(不加入TGF-β1诱导)、TGF-β1诱导组(仅加入TGF-β1诱导)和TGF-β1诱导给药组(既加入TGF-β1诱导又给予可利霉素或异戊酰螺旋霉素Ⅰ处理)。
继续培养24小时后，弃去培养基，按照TRIzol说明书操作步骤提取LX-2细胞总RNA。按照Roche Transcriptor First Strand cDNA Synthesis Kit说明书操作步骤将LX-2总RNA逆转录为cDNA。将所得的cDNA、无菌水、Roche FastStart Universal Probe Master(Rox)以及ABI TaqMan probes(GAPDH，COL1A1，TGFB1，ACTA2，MMP2)配制成20μl反应体系，使用ABI 7500 Fast Real-Time PCR System进行检测。以GAPDH作为内参进行结果分析，数据显示，可利霉素和异戊酰螺旋霉素Ⅰ均能显著抑制COL1A1、TGFB1、ACTA2和MMP2的表达量(图11)。异戊酰螺旋霉素Ⅰ在40μmol/L浓度下对CCC-ESF-1细胞的毒性明显。
以上所述仅是本发明的较佳实施例而已，并非对本发明作任何形式上的限制，虽然本发明已以较佳实施例揭露如上，然而并非用以限定本发明，任何熟悉本专利的技术人员在不脱离本发明技术方案范围内，当可利用上述提示的技术内容做出些许更动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明方案的范围内。

Claims (10)

  1. 一种用于预防、缓解和/或治疗纤维化的药物，其特征在于，所述药物的有效成分包括可利霉素、异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ中的一种；
    或异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ中的两种或三种的组合。
  2. 根据权利要求1所述的一种用于预防、缓解和/或治疗纤维化的药物，其特征在于，所述药物包括医学上可接受的载体。
  3. 一种用于预防、缓解和/或治疗纤维化的药物，其特征在于，所述药物的有效成分选自以下成分中的一种或多种：
    可利霉素的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互变异构体、多晶型物、药物前体；
    异戊酰螺旋霉素Ⅲ的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互变异构体、多晶型物、药物前体；
    异戊酰螺旋霉素Ⅱ的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互变异构体、多晶型物、药物前体；
    以及异戊酰螺旋霉素Ⅰ的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互变异构体、多晶型物、药物前体。
  4. 一种用于治疗纤维化的组合产品，其特征在于，所述的组合产品包括第一药剂，所述第一药剂的有效成分包括可利霉素、异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ中的一种；
    或者异戊酰螺旋霉素Ⅰ、异戊酰螺旋霉素Ⅱ、异戊酰螺旋霉素Ⅲ中两种或三种的组合。
  5. 根据权利要求4所述的一种用于治疗纤维化的组合产品，其特征在于，所述的组合产品还包括第二药剂，所述第二药剂包括用于预防、缓解和/或治疗纤维化的相关药物中的至少一种。
  6. 根据权利要求5所述一种用于治疗纤维化的组合产品，其特征在于，所述用于预防、缓解和/或治疗纤维化的相关药物包括皮质类固醇激素、秋水仙碱、水飞蓟素、干扰素。
  7. 一种用于治疗纤维化的组合产品，其特征在于，所述组合产品包括第一药剂，所述第一药剂的有效成分选自以下成分中的一种或多种：
    可利霉素的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互变异构 体、多晶型物、药物前体；
    异戊酰螺旋霉素Ⅲ的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互变异构体、多晶型物、药物前体；
    异戊酰螺旋霉素Ⅱ的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互变异构体、多晶型物、药物前体；
    以及异戊酰螺旋霉素Ⅰ的衍生物、药学上可接受的盐、溶剂化物、代谢产物、立体异构体、互变异构体、多晶型物、药物前体。
  8. 根据权利要求1或2所述药物或权利要求3所述药物或权利要求4-6任一所述组合产品或权利要求7所述组合产品，其特征在于，所述纤维化包括肺纤维化、心脏纤维化、肝纤维化、胰腺纤维化、肾纤维化、骨髓纤维化和皮肤纤维化；
    优选的，所述肺纤维化包括新型冠状病毒感染导致的肺纤维化。
  9. 一种权利要求1或2所述药物或权利要求3所述药物或权利要求4-6任一所述组合产品或权利要求7所述组合产品在预防、缓解和/或治疗纤维化中的应用。
  10. 一种权利要求1或2所述药物或权利要求3所述药物或权利要求4-6任一所述组合产品或权利要求7所述组合产品在抑制炎症或脂质过氧化、抑制成纤维细胞的增生活化、促进胶原降解的应用。
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