CN107320491A - Fullerene or its officinal salt are preparing the application in being used to treat the medicine of pulmonary fibrosis - Google Patents

Abstract

The application in being used to treat the medicine of pulmonary fibrosis is being prepared there is provided fullerene or its officinal salt the present invention relates to pulmonary fibrosis therapy field, wherein the fullerene or its officinal salt are present in the medicine with therapeutically effective amount.

Description

Fullerene or its officinal salt are being prepared for treating in the medicine of pulmonary fibrosis Using

Technical field

The present invention relates to pulmonary fibrosis therapy field, more particularly to fullerene or its officinal salt is being prepared for treating lung Application in the medicine of fibrosis.

Background technology

Pulmonary fibrosis (pulmonary fibrosis, PF) refers to cause lung's parenchyma to necrose due to inflammation, Final pathological outcome caused by the factor such as extracellular matrix abnormal increase and over-deposit of organizing, the lighter causes lung tissue fine Dimensionization, severe one causes lung tissue's structure destruction and occurs organ sclerosis.Pulmonary fibrosis can be betided in various environment, including not Stimulation, environment or the occupational exposure (such as asbestos, silica) of bright reason, by medicine cause (such as bleomycin) or secondary In other diseases, such as immune complex also can induce pulmonary fibrosis.

Idiopathic pulmonary fibrosis (idiopathic pulmonary fibrosis, IPF) is a kind of chronic, progressive, fibre Dimensionization interstitial pneumonia^[1], this kind of disease is damaged with chronic insubstantial and fibrosis is characterized, and pathogenesis ten is not distinguished still Chu.It is now recognized that IPF repeated the abnormal reparation after microlesion originating from alveolar epithelium, so cause cicatrization and Lung structure is destroyed^[2,^3].IPF morbidity can be divided into for three phases：(1) prodromal stage：Environmental factor (smoking, infection, gastroesophageal reflux thing Deng) and inherent cause (SFTPC gene mutations etc.) collective effect, cause alveolar epithelium repeatedly to damage and abnormal injury repair Reaction；(2) phase is originated：Er stress occurs for part epithelial cell so that part alveolar epithelium apoptosis, a large amount of cause fibers of secretion Change the factor, including platelet derived growth factor (PDGF), fibroblast growth factor (FGF), VEGF (VEGF), TGF-1 3 (TGF-13) family, interleukin-13 (IL-13) etc., the above-mentioned factor can induce epithelium The conversion of epithelium mesenchyma occurs for cell, can also recruit fibrocyte to lung, and promote fibroblast proliferation, flesh into fiber Cell differentiation, forms fibroblast stove；(3) progressive stage：Above-mentioned mesenchymal cell secretes a large amount of aberrant stromal albumen (mainly Collagen deposition) so that lung remodeling and scarring, ultimately form pulmonary fibrosis.IPF's is mainly shown as diffusivity lung Bubble is scorching, alveolar unit structure is disorderly and pulmonary fibrosis, ultimately results in the heavy damage of lung structure and function, the main table of its pathology It is now early stage Diffuse alveolar inflammation and later stage a large amount of interstitial cell hyperplasias, the aggregation of Collagen progressive so that replacing normal lung Institutional framework, has a strong impact on ventilation and the ventilatory of lung, ultimately results in respiratory failure and dead.

IPF is a kind of chronic, lethal disease, and survival rate is generally only 20% within 5 years.The effective curatives of current IPF Thing only has pirfenidone and Nintedanib.However, the side effect such as pirfenidone has hepatic disorder, dizzy and photosensitization^[4,5], Nintedanib can then make patient the adverse reactions such as liver enzyme rise, diarrhoea occur^[6], have impact on wide clinical application.Therefore, having must Develop for pulmonary fibrosis, the especially relatively low new types of therapeutic agents of IPF side effects.

Numerous studies find, oxidation/anti-oxidant unbalance plays an important role in IPF occurrence and development^[7], oxidation It stress develop with IPF generation closely related^[9-12].Oxidative stress refers to high active substance such as active oxygen (reactive in body Oxygen species, ROS) produce excessively, beyond the Scavenging activity of body, cause a kind of oxidation/anti-oxidant unbalance shape State.ROS is also known as reactive oxygen species material, and its generation is excessively to cause the direct factor of body oxidative stress, and active oxygen It is the important medium in pulmonary fibrosis process.When ROS prolonged high concentrations are present, easily oxygen is produced with surrounding biologic macromolecular substances Change reaction, damage the 26S Proteasome Structure and Function of cell, cause the generation of disease^[7,8].Research discovery, the bronchoalveolar lavage of IPF patient A large amount of MDAs can be detected in washing lotion (bronchial alveolar lavage fluid, BALF)^[9]；In lung Antioxidants are significantly reduced, and PFT is negatively correlated with oxidative stress^[10]；The degree of pulmonary fibrosis should with oxidation Sharp level is proportionate^[11].A large amount of ROS can make DNA single strand breaks, fracture, cause pulmonary epithelial cells damage necrosis；Activation is dead Die acceptor^[13], mitochondrial apoptotic pathway^[14], er stress apoptosis pathway^[15]Deng induction pulmonary epithelial cells apoptosis；Destroy tissue Middle protease/antiprotease balance^[16], promote collagen deposition, so as to promote IPF to develop.In addition, ROS can be by adjusting Section protein kinase and phosphoprotein phosphatase receptor pathway carry out Regulate signal transduction, promote TGF-β 1, TNF-α, interleukins, blood small The expression of the cell factors such as plate derivative growth factor^[17].Wherein, TGF-β 1 plays key work in pulmonary fibrosis forming process With, it is considered to be " master switch " of induction organ fibrosis (including pulmonary fibrosis)^[18], its quantity finally determines inflammation and fiber The degree of change^[19,20]；TNF-α can then mediate airway epithelial cell apoptosis, and raise rush brotic cells factor TGF-β 1 expression, is the important factor for promoting pulmonary fibrosis to develop^[21].These cytokine-expressings, which increase, can aggravate cell Apoptosis, aggravation pulmonary fibrosis development are damaged, but the up-regulation of these cytokine-expressings can also stimulate body generation more ROS, makes body be in high ROS environment, causes body Continuous Damage, forms a vicious circle.If can correct in vivo Oxidative stress status, it would be possible to mitigate the degree of pulmonary fibrosis^[12], therefore antioxidant is expected to turn into treatment pulmonary fibrosis A kind of new method.

Fullerene is except the allotrope of two kinds of naturally occurring elemental carbons：The outer mankind of graphite and diamond find the The allotrope of three kinds of elemental carbons^[22], it has closing cage structure, similar to the shape of football.The finder of fullerene Therefore Nobel chemistry Prize in 1996 is obtained.Fullerene molecule contains 32-1000 or more in various types of spheroids Carbon atom.Due to the unique chondritic of fullerene, on the surface full of pi-electron, therefore it has many special properties, At present increasing concern has been obtained in electronics, medical treatment, medical science and application for the purpose of improving health etc..

As the concern to fullerene increasingly increases, application of the fullerene in medicine and cosmetics was carried out in the past Numerous studies, it was also proposed that many schemes.For example, fullerene or fullerene mixture are compounded into cosmetics, (Japan is specially Sharp Unexamined Patent 6-192039), Sun block cosmetic composition is made using the ultraviolet radiation absorption effect of fullerene, and (Japan Patent is special Open flat 9-278625), optically make viral deactivated method (Japanese Patent Laid-Open 9- using fullerene as photosensitizing agents Application (Japanese Patent Laid-Open 2004-250690) 322767), using fullerene as antioxidant in skin preparations for extenal use etc., Application (Chinese patent application using fullerene as antioxidant in preventive/therapeutic composition for free radical disease CN101098684A) etc..

In numerous fullerenes, especially with Fullerene C20, C70, C82, C84 study it is the most abundant.Fullerene C20 is A kind of newfound Spectra of Carbon Clusters of the mid-80, is a kind of molecule being made up of 60 carbon atoms, and it is similar to football, therefore again Name football alkene, there are substantial amounts of double covalent bonds on its surface, easily with radical reaction^[23].Just because of this characteristic, fullerene C60 and its derivative can reach oxidation resistant effect by removing free radical, and suppress DNA oxidative damages^[23,24].Closely Nian Lai, fullerene is widely used in various life sciences as a kind of antioxidant, and research shows that Fullerene C20 can press down Tumour growth processed^[25,26], strengthen the chemotherapeutics such as Doxorubicin to the therapeutic effect of tumour and mitigate its side effect^[27].It is Chinese special Disclosed in the profit application lot of documents such as CN101098684A Fullerene C20, C70, C82, C84 because of its antioxidant properties by with Research in terms of tumour, antibacterial.

However, simultaneously no-trump fullerene is used to treat pulmonary fibrosis, any report of the pulmonary fibrosis of especially IPF inductions at present Road.

The content of the invention

Fowler is injected intraperitoneally as model in the mouse pulmonary fibrosis that the present invention is induced by taking Fullerene C20 as an example, using bleomycin Alkene is intervened it, by comparing the survival rate, changes of weight, lung of fullerene intervention group and model group in patients before and after intervention mouse Steep in terms of inflammation and fibrosis, collage synthesis, the pulmonary fibrosis that discovery fullerene can improve bleomycin induction is small The survival rate of mouse, slow down mouse weight downward trend, mitigate Pathomorphology co-fibration degree, significantly reduce expression of collagen in lung Deposition and ROS contents, it was demonstrated that alleviation and therapeutic action of the fullerene to IPF, this completes the present invention.

The application in being used to treat the medicine of pulmonary fibrosis is being prepared the invention provides fullerene or its officinal salt, its Described in fullerene or its officinal salt be present in therapeutically effective amount in the medicine.

The pulmonary fibrosis can be ultimately resulted in by any factor.Preferably, the pulmonary fibrosis is by idiopathic lung fiber Change causes.

Preferably, the fullerene is selected from Fullerene C20, C70, C82, C84 or its mixture.

Preferably, the fullerene includes the one or more fullerenes combined in oxygen-containing group, nitrogenous base, alkyl, institute Stating alkyl optionally has substituent.

Preferably, the officinal salt of the fullerene is selected from sodium salt, sylvite, magnesium salts, calcium salt, aluminium salt and/or sulfonate.

Fullerene available for the present invention is preferably Fullerene C20.Preferably, the Fullerene C20 can be to be insoluble in The Fullerene C20 of water, it can be by being dissolved in organic solvent applicable in vivo, including but not limited to edible oil such as corn oil, cotton Seed oil, soybean oil, sesame oil, peanut oil, coconut oil and/or olive oil etc. and be administered.Preferably, the Fullerene C20 For by the Fullerene C20 of water-soluble sex modification.

According to the present invention, there is the Fullerene C20 of the water-soluble sex modification following formula to represent：F(-X)_m, wherein

F is Fullerene C20 core；

X each stands alone as OH, (CH₂)_n-SO₃H or (CH₂)_n-SO₃- metal salt, wherein each n be independently 2-50 it Between integer；And

M is the integer between 2-40.

Preferably, the method for administration of the medicine is oral, intravenous, subcutaneous, intraperitoneal and/or intramuscular injection.

Preferably, the medicine includes pharmaceutically useful excipient.Preferably, the pharmaceutically useful excipient includes dilution Agent, adhesive, dispersant, surfactant, lubricant, coating material, flavor enhancement, colouring agent, control release preparation and/or sweet tea Taste agent etc..

Preferably, the pharmaceutical dosage form include tablet, pill, capsule, granule, powder, elata chewing gum agent, supensoid agent, Emulsion, injection solution and/or liposome injection suspension.

Inventor has found that fullerene can substantially reduce the content of ROS in pulmonary fibrosis mice lung tissue.Also, inventor It was observed that after fullerene is intervened, the content of TGF-β 1 and TNF-α is substantially reduced compared with BLM groups, points out fullerene to make TGF-β 1 Expression with TNF-α reduces (result is not shown).So, inventor speculates that fullerene may be by reducing ROS content, from And mitigate damage of the excessive active oxygen to lung tissue, and then the expression of the cell factors such as TGF-β 1, TNF-α is reduced, finally subtract The degree of subinflammation and fibrosis, reaches the effect for suppressing pulmonary fibrosis.

In addition, fullerene biological safety is good, toxic side effect is low, the Fullerene C20 used in the present invention is without the secondary work of poison With^[28,29], and oral Fullerene C20 can be by one times of the life of rat^[30].Therefore fullerene more likely turns into and faced A kind of new pulmonary fibrosis resistant medicine on bed.

Brief description of the drawings

Fig. 1 shows that bleomycin (BLM) model group and each dosage group of Fullerene C20 (1,10,100 and 500mg/kg) exist Mouse survival rate changes with time situation after administration.

Fig. 2 show blank control group (NS), bleomycin (BLM) model group and each dosage group of Fullerene C20 (1,10, 100 and 500mg/kg) upon administration mouse weight change with time situation.

Fig. 3 shows each treatment group mouse lung CT images in modeling the 28th day.A is normal mouse lung CT；B is BLM model group mouse lungs CT；C is 10mg/kg C60 treatment group mouse lungs CT after BLM inductions；D is that pyrrole is non-after BLM is induced Buddhist nun's ketone treatment group mouse lung CT.

Fig. 4 shows each treatment group mouse pathology HE (above) and MASSON (figure below) dyeing knot in modeling the 28th day Really.A is normal mouse lung pathologies；B is BLM model group mouse lung pathology；C is 10mg/kg C60 treatment groups after BLM inductions Mouse lung pathology；D is pirfenidone treatment group mouse lung pathology after BLM inductions.

Fig. 5 shows each treatment group Ashcroft appraisal results in modeling the 28th day.NS：Blank control group；BLM：It is rich Bleomycin model group；BLM+C60：10mg/kg C60 treatment groups after BLM inductions；BLM+pir：Pirfenidone is handled after BLM inductions Group.**p<0.01；**p<0.001.

Fig. 6 shows each treatment group mouse lung collagen immunization histochemical staining result in modeling the 28th day.A is normal small Mouse lung pathologies；B is BLM model group mouse lung pathology；C is 10mg/kg C60 treatment groups mouse lung disease after BLM inductions Reason；D is pirfenidone treatment group mouse lung pathology after BLM inductions.

Fig. 7 shows the statistical analysis of the result of collagen immunization histochemical staining shown in Fig. 6.NS：Blank control group；BLM：It is rich next Mycin model group；BLM+C60：10mg/kg C60 treatment groups after BLM inductions；BLM+pir：Pirfenidone is handled after BLM inductions Group.*p<0.05；**p<0.01.

Fig. 8 shows in modeling the 28th day ROS contents in each treatment group mouse lung tissue.NS：Normal group；BLM： Bleomycin model group；C60 10mg/kg：C60 10mg/kg treatment groups.*p<0.05；***p<0.001.

Embodiment

Below, will the present invention will be described in more detail in conjunction with specific embodiments, but the scope of the present invention is not limited to This.

Unless otherwise defined, all technologies used herein and scientific terminology have with it is of the art common The identical meaning that technical staff is generally understood that.All publications specifically mentioned herein, be include description and openly Institute of the possibility reported in the publication including the present invention is combined the chemical substance used, instrument, statistical analysis and method Purposefully, it is expressly incorporated herein by reference with entire contents.All bibliography quoted in this manual should be considered as Indicate the technical merit of this area.Any content herein is not necessarily to be construed as recognizing no right of the invention earlier than passing through These disclosures formerly invented.

Fullerene is insoluble in water in itself, can be administered into vivo by being dissolved in organic solvent applicable in vivo.Give for convenience Medicine, fullerene can pass through water-soluble sex modification, it is possessed water solubility.Preparing the method for water-soluble fullerene is in the art Known, its preparation method for details, reference can be made to United States Patent (USP) No.5,994,410, No.5,177,248, No.5,294,732, Dugan L.L.et al, Proc Natl Aad Sci.USA 94:9434-9439 etc., these prior art documents are whole with its Body is incorporated herein by reference.

As used herein, " pharmaceutically acceptable " is included in rational medical judgment scope, is suitable to and human and animal Tissue contact and without excessive toxicity, excitant, allergic reaction or with rational benefit/risk than the other problems that match simultaneously Send out those compounds, material, composition and/or the formulation of disease.

In the specification and in the claims, term " comprising " and "comprising" are open terms, and should be explained To mean " to include but is not limited to ".These terms cover more restricted term " substantially by ... constitute " and " by ... constitute ".It should also be noted that term "comprising" and " comprising ", " being characterised by " can be with used interchangeablies with " having ".

When herein and claims in use, without it is specific amount of censure include its odd number or plural reference Thing, unless it is not such that context, which is clearly described,.In addition, without specific amount of denotion, term " one or more " and " at least one It is individual " can be with used interchangeably.

When the scope of offer value, it should be appreciated that each value between two parties between the bound of the scope and be worth between two parties Any combinations or sub-portfolio, and in the range of the statement any other statement or value between two parties, all covered in Within the scope of the value described.

As used herein, " administration " refers to compound importeding into the body of subject, preferably imported into by In the system circulation of examination person, just as described in more detail below.Example include but is not limited to orally, surface, cheek, sublingual, lung, Transdermal, transmucosal and subcutaneous, intraperitoneal, the injection of intravenous and intramuscular, or it is taken through gastral liquid or solid medicine The form of agent.

As used herein, " therapeutically effective amount " of compound is enough in treatment or the management in disease or illness Therapeutic benefit, or delay or the amount for minimizing the one or more symptoms related to the disease or illness are provided.Compound " therapeutically effective amount " mean individually or when combine with other therapies, the offer in the treatment or management of the disease or illness The amount of the healing potion of therapeutic benefit.Term " therapeutically effective amount " can cover improvement overall therapeutic, disease is reduced or avoided Illness symptom or the cause of disease or improve another healing potion therapeutic efficacy amount." therapeutically effective amount " will be with chemical combination Thing, morbid state to be treated, the seriousness of disease to be treated, the age of subject and relative healths, method of administration and form, Cure mainly judgement and the other factors of medical treatment or animal doctor practitioner and become.

As used herein, term " treatment " take into account the drop taken when patient suffers from and specifies disease or obstacle The low disease or the seriousness of obstacle or delay or the action for slowing down the disease or the process of obstacle.

As used herein, " subject " includes mammal and nonmammalian." mammal " refers to lactation Any member of guiding principle, the including but not limited to mankind, non-human primate such as chimpanzee and other apes and monkey species；Farm Animal such as ox, horse, sheep, goat and pig, domestic animal such as rabbit, dog and cat；Laboratory animal include rodent such as rat, Mouse and cavy etc..The example of nonmammalian includes but is not limited to birds etc..Term " subject " do not indicate given age or Sex.The targeted main subject of the present invention is with hemodialysis or receives the mankind of haemodialysis.Term is " tested Person " herein can be with term " patient " or " individual " used interchangeably.

As used herein, " peroral dosage form " can include the capsule (solid port being made up of shell and filler Oral dosage form), wherein shell is by single hermetically sealed case or is combined together and is constituted sometimes with two and half overcoats of tape seals, and And wherein capsule shell can be made up of gelatin, starch or cellulose or other suitable materials, can be soft or hard, And filled with the solid or liquid component that can be toppled over or extrude.Peroral dosage form can also be capsule or be coated spherolite, wherein In hard or soft soluble container that drug encapsulation is made in the gelatin by being adapted to form or " shell ".Medicine in itself may be used In the form of taking particle, the particle has been applied in different amounts of coating, or takes delayed release coating capsule form, its Middle medicine is encapsulated in the hard or soft soluble container that are made up of the gelatin for being adapted to form or " shell ".In addition, can To be covered in capsule in the coating specified, it is compared with one or more medicines with existing as regular dosage form, at least to permit Perhaps the mode of reduction administration frequency discharges one or more medicines.

Peroral dosage form can also be sustained release capsule, wherein medicine be encapsulated in by the gelatin for being adapted to form be made it is hard In matter or soft soluble container, and its other times release medicine (or multi-medicament) after not being to be administered immediately, Therefore enteric coating product is delayed release dosage forms.Medicine is encapsulated in the sustained release in hard or flexible container or " shell " Spherolite capsule, is also useful.In these cases, medicine takes the form for the particle for being applied in enteric coating in itself, because This is by the hangover of medicine untill it is passed into intestines.Sustained release capsule and film coating sustained release capsule, be also Useful.

In addition, capsule is covered in the film coating specified, it is using the one or more with existing as regular dosage form Medicine is compared, and at least allows the mode for reducing administration frequency to discharge one or more medicines.Capsule (the wherein medicine of gelatine glaze It is encapsulated in the solid dosage forms in the hard or soft soluble container being made up of the gelatin for being adapted to form；By adding band mistake Journey, capsule is coated with other gelatin layer, to form complete seal), the capsule of liquid filling (a kind of solid dosage forms, its Middle medicine is encapsulated in be plasticized by adding polyhydric alcohols such as D-sorbite or glycerine, and is therefore had than hard-shell capsule summary In the Soluble Gelatin shell of micro- thicker denseness).

Generally, active component can be dissolved or suspended in liquid medium, can be particle (small particles or particulate), ball Grain (with or without the small sterile solid material being made up of highly purified medicine of excipient, its by particle formation or Manufactured by suppressing and moulding) or the spherolite of delayed release coating (a kind of solid dosage forms, wherein medicine take particle in itself Form, the particle has been applied in different amounts of coating, and it is using one or more medicines with existing as regular dosage form Compare, it is allowed to which the mode for reducing administration frequency discharges one or more medicines).

Other forms include pill (being intended to the small rounded solid formulation containing medicament for oral administration), pulvis (the fine powder medicine and/or the intimate mixture of chemical substance that may be intended to the drying of for oral administration or external application), elixir (contain dissolving Bright, pleasant aroma the sweetened aqueous alcohol liquid of medicament；It is intended to be administered orally), elata chewing gum agent (various different shapes Sweetened and seasoning insoluble plastic material, drug substance is discharged into oral cavity by it when being chewed), syrup (contain height The oral administration solution of concentration sucrose or other carbohydrates；The term also be used to being included in prepared in sweet taste and resisting medium it is any its His liquid dosage form, including oral suspension), tablet (solid dosage forms containing the suitable diluent of medicine, with or without), nozzle Chewable tablet (is used for the solid dosage forms of the diluent suitable containing medicine, with or without chewed, it produces mouth in the oral cavity The pleasant residue of taste, the residue is easy-to-swallow and does not leave bitter taste or undesirable pleasant impression), coated tablet or prolong Slow release tablet, dispersible tablets, effervescent tablet, delayed-release tablet, film coating tablet or film coating delayed-release tablet Agent, wherein tablet are formulated such that included medicine can be used in long period upon intake.

In other forms, solution tablet, suspension tablet, multilayer tablet, sustained release multilayer tablet can be provided, Compared with wherein tablet is formulated into the medicine with as regular dosage form existing, at least allow to reduce administration frequency.Orally disintegrating tablet Agent, oral delayed-release tablet, soluble tablets, sugar coated tablet, osmotic tablet etc., are also suitable.

Oral dosage form composition can include active pharmaceutical ingredient and one or more inactive drug ingedient such as figurations Agent, diluent, solubilizer, alcohols, adhesive, controlled release polymer, enteric polymer, disintegrant, excipient, colouring agent, Flavor enhancement, sweetener, antioxidant, preservative, pigment, additive, filler, suspending agent, surfactant (such as anion Type, cationic, both sexes or non-ionic) etc..The surface active substance of various FDA approvals can be in the " inactive of FDA Found in compositional data storehouse " (The Inactive Ingredients Database), the database contains manufacturer and is intended to use In the active substance of this purpose, wherein according to the definition of the active component provided in 21CFR 210.3 (b) (7), it is inactive into Active component can also be taken as in some cases by dividing.Alcohol is depending on product formula, can be taken as active or nothing The good example of the composition of activity.

As used herein, the formulation of injectable and infusion includes but is not limited to injectable liposome, and it is by lipid Body constitutes or formed liposome (the double-layer of lipoid vesica being generally made up of phosphatide, it be used to encapsulate active drug substance).Bag The parenteral solution that parenteral is used is intended to containing sterile preparation, is also suitable.Including the breast being made up of sterile, apyrogeneity preparation Liquid, it is intended to which the injection emulsion or lipid complex parenteral solution of parenteral, are also suitable.

Other forms include being used for the pulvis that solution is injected, and it is sterile preparation, it is intended to which reconstruct is used for parenteral to be formed The solution used；The pulvis injected for suspension, it is sterile preparation, and manufacture reconstructs to be formed for hanging that parenteral is used Liquid；The pulvis for the freeze-drying injected for Liposomal suspensions, it is the preparation of sterile freeze-drying, it is intended to reconstructed for intestines Stomach is used outside, its be formulated into allow to be formed after reconstitution liposome (the double-layer of lipoid vesica being generally made up of phosphatide, its by with In active drug substance is encapsulated in double-layer of lipoid or in aqueous space)；The pulvis for the freeze-drying injected for solution, its It is that described freeze is related under extremely low pressure by freezing the formulation being intended to for solution prepared by (" freeze-drying "), The process of water is removed under freezing state from product.

This is intended to meet the solution that injection is required in all respects for subsequent adding liquid to produce；For injection suspension Freeze dried powder, the injection suspension be intended to that parenteral uses containing being suspended in suitable fluid media (medium) and in all sides Face meets the liquid preparation of sterile suspension requirement；It is intended to prepare by lyophilized (" freeze-drying ") for the medicament of suspension, institute State the lyophilized process for being to be related under extremely low pressure, removing water from product in a cold or frozen state；Solution is injected, it is containing dissolving The liquid preparation of one or more medicines the suitable solvent or the mixture of mutual solvents suitable for injection；Injection is molten Liquid concentrate, it is the sterile preparation used for parenteral, is produced after the suitable solvent of addition and meets note in all respects Penetrate the solution of requirement.

Injection suspension includes the liquid preparation suitable for injection, and it is by being dispersed in particle insoluble in whole liquid phase therein Solids constitute, the liquid phase can also be by the oil phase being dispersed in whole aqueous phase or the water being dispersed in whole oil phase Property is mutually constituted.Liposome injection suspension includes the liquid preparation for being suitable for injection, and it is by the oil phase that is dispersed in whole aqueous phase Constitute, to form liposome, (the double-layer of lipoid vesica being generally made up of phosphatide, it be used to active drug substance being encapsulated in In double-layer of lipoid or in aqueous space).Ultrasonic injection suspension includes the liquid preparation for being suitable for injection, and it is by being dispersed in particle not The solids being dissolved in whole liquid phase therein are constituted.In addition, ultrasound is carried out to product when by gas sparging by suspension, This causes to form microballoon by solids.

Parenteral vehicles system includes the excipient being adapted in one or more pharmacy, such as solvent and cosolvent, solubilising Agent, wetting agent, suspending agent, thickener, emulsifying agent, chelating agent, buffer, pH adjusting agent, antioxidant, reducing agent, anti-micro- life Thing preservative, extender, protective agent, ooze degree of rising conditioning agent and special additive.It is suitable for the formula of parenteral easily Sterile oiliness or aqueous formulation comprising active component, its blood preferably with acceptor are isotonic.

Embodiment

Embodiment 1：

Material and method

1. experimental animal：The male C 57 BL/6 J mouse of 6-8 week old, 22~25 grams of body weight ties up tonneau China real purchased from Beijing Zoo technical Co., Ltd is tested, is fed at Beijing Chaoyang Hospital Attached to Capital Medical Univ.'s basic medical research center.

2. medicine and reagent

3. Animal Model

The mouse pulmonary fibrosis model of conventional bleomycin induction when the present invention is using research pulmonary fibrosis and IPF^[31]。 Weigh mouse weight and with 1% yellow Jackets (50mg/kg) intraperitoneal injection of anesthesia, mouse is fixed on operating desk in dorsal position On, routine disinfection, row neck median incision, blunt separation tracheae side muscle, exposure tracheae；With 1ml syringes by tracheal rings Gap slowly saline injection or bleomycin (3.5mg/kg^[32]) (control group saline injection, remaining each group injection is rich Bleomycin), immediately by mouse 3~5min of vertical rotary, decoction is uniformly distributed in both sides intrapulmonary；Skin suture^[33]。

4. animal packet, medication and sample disposal

Explore optimal dose：Each dosage group, be respectively：Control group (NS), model group (BLM), 1mg/kg C60 intervention groups, 10mg/kg C60 intervention groups, 100mg/kg C60 intervention groups, 500mg/kg C60 intervention groups, daily to be injected intraperitoneally once, each group Put to death in 21 days, record death time and the changes of weight of each group mouse.

Treatment group, be respectively：Control group (NS), model group (BLM), 10mg/kg C60 intervention groups are injected intraperitoneally one daily It is secondary, pirfenidone (300mg/kg/d, bid) group.Each group started treatment in 14 days, put to death within 28 days, and the of record each group mouse CT, pathology and the collagen content analysis of 28 days.

5. lung tissue paraffin section H＆E is dyed

1) toast：Paraffin section is positioned in 60 DEG C of insulating boxs and toasts 2h；

2) dewaxing and aquation：Section is placed on each 20min in dimethylbenzene I, dimethylbenzene II, absolute ethyl alcohol I, absolute ethyl alcohol II In each 5min, 95% ethanol 1min, 70% ethanol 1min, 50% ethanol 1min, 30% ethanol 1min, originally wash 2 times after, put Enter in deionized water 2 minutes；

3) dye：Brazilwood extract dyeing 5min, flowing water is rinsed 3 times, and 1% hydrochloride alcohol differentiation 30sec, flowing water is rinsed 3 times, ammonia Water returns indigo plant, up and down under 3, washes 3 times, 1% eosin stains 30sec, and flowing water is rinsed 3 times；

4) serial dehydration and transparent：Section is positioned over the 30sec of 95% ethanol I, the 2min of 95% ethanol II, absolute ethyl alcohol I, nothing Each 5min in each 5min in water-ethanol II, dimethylbenzene I, dimethylbenzene II；

5) neutral gum mounting：Note slice, thin piece wiped clean during mounting, while preventing bubble from producing；

6) pictorial information is gathered：The desk-top slide scanner collection pictorial informations of Aperio.

6. lung tissue paraffin section immunohistochemical staining

1) toast：Paraffin section is placed on metallochromy frame, is positioned in 60 DEG C of insulating boxs and is toasted 2h；

2) dewaxing and aquation：Section is placed on each 20min in dimethylbenzene I, dimethylbenzene II, absolute ethyl alcohol I, absolute ethyl alcohol II In each 5min, 95% ethanol 1min, 70% ethanol 1min, 50% ethanol 1min, 30% ethanol 1min, originally wash 2 times after, leaching Enter in 0.001mol/L EDTA (pH8.0) buffer solution and (notice that the lung tissue on paraffin section need to be completely immersed in EDTA solution)；

3) hot high pressure repairs antigen：Edta buffer liquid is heated in boiling water, Stainless steel pot cover is covered, but can not be locked, 210w heats 10min, locks pot cover, and pressurization, 160w heating 8min unscrew valve, treat that it is decompressed to normally, by edta buffer liquid Being placed on ventilating and cooling place makes its natural cooling；

4) activity of endogenous peroxydase is eliminated：After after edta buffer liquid natural cooling, paraffin section is taken out, is washed After 2 times, the moisture around tissue is blotted as far as possible, 3% hydrogen peroxide is organizationally added dropwise, and wet box places 15min, originally washes three After secondary, 10min is balanced in PBS liquid；

5) primary antibody is incubated：After antibody operation instructions recommendation ratio dilution primary antibody, primary antibody is added drop-wise to the group of slide Knit and (tissue need to be completely covered), 4 DEG C of overnight incubations；

6) HRP marks secondary antibody to be incubated：After primary antibody incubation terminates, PBS is rinsed, 2min × 3 time, is incubated using HRP mark secondary antibodies 1h is educated, PBS is rinsed, 2min × 3 time；

7) DAB develops the color：The reaction time is controlled under color development at room temperature, mirror, typically between 5-20min, running water color development stopping；

8) nuclear targeting：Running water is fully rinsed after color development stopping, and haematine is slightly redyed；

9) dehydration, transparent, mounting：As described in H＆E dyeing；

10) pictorial information is gathered：The desk-top slide scanner collection pictorial informations of Aperio, Aperio software statistics Relative expression situation of the destination protein in lung tissue different parts.

7.MASSON dyeing, ROS are determined to be operated by kit specification.

8. statistical analysis

Statistical analysis is carried out using the softwares of GraphPad Prism 6, experimental result is represented with mean ± standard deviation, is compared Difference between group is examined with t.With p<0.05 is that difference is statistically significant.

Embodiment 2：Dosage choice

The comparison of each group survival rate and changes of weight：The 21st day after modeling, Normal group mouse all survives, BLM Group and low dosage intervention group survival rate are 30%, 10mg/kg C60 intervention groups survival, 66.7% (such as Fig. 1).It is small by each group The changes of weight curve (such as Fig. 2) of mouse understands that Normal group mouse weight is significantly raised, and BLM group body weight is decreased obviously, and The changes of weight of 10mg/kg C60 intervention groups is substantially better than BLM groups.This explanation 10mg/kg Fullerene C20 can improve lung fiber Change the survival rate of mouse, slow down mouse weight downward trend.Therefore, subsequent experimental is using 10mg/kg Fullerene C20s as selected agent Amount.

Embodiment 3：Mouse lung CT results contrasts

Modeling the 28th day, each group mouse lung CT performance such as Fig. 3.Model group lung CT shows that mediastinum window has substantial amounts of reality Matter is filled, and 10mg/kg C60 treatment groups and pirfenidone treatment group are obviously improved compared with model group.

Embodiment 4：HE is compared with Masson dyeing

Om observation finding, control group lung tissue structure is clear, and alveolar wall, which has no, to be thickened, and alveolar epithelial cells structure is complete It is whole；The destruction of BLM groups alveolar structure is notable, and alveolar space is full of fibr tissue, even complete fibrosis；10mg/kg C60 and Based on pirfenidone intervention group is thickened with continuous alveolar septum, pathology substantially mitigates (such as Fig. 4) compared with BLM groups；Pathology is contaminated Color carries out Ashoft scorings^[34](such as Fig. 5), 10mg/kg C60 and pirfenidone intervention group substantially reduce (p compared with BLM groups< 0.01), but 10mg/kg C60 and pirfenidone intervention group do not have obvious difference.

Embodiment 5：Mouse lung collagen content compares

The immunohistochemical staining (such as Fig. 6) of collagen is carried out to each treatment group's lung tissue, and it is counted (such as Fig. 7), It can be seen that model group collagen deposition showed increased, and the content of collagen substantially drops in 10mg/kg C60 and pirfenidone intervention group It is low, and 10mg/kg C60 and pirfenidone intervention group do not have obvious difference.

Embodiment 6：Mouse lung ROS comparision contents

As shown in figure 8, ROS is significantly raised in intratracheal injection bleomycin mouse lung tissue, however, using 10mg/kg C60 then significantly reduces (p after intervening<0.05), prompting Fullerene C20 can reduce the active oxygen in pulmonary fibrosis mice lung tissue Content.

Although it should be understood that having combined preferred particular implementation of the invention, invention has been described, above Description and embodiment thereafter be intended to illustrative and not limiting the scope of the present invention.It will be recognized by one of ordinary skill in the art that In the case of without departing from the scope of the invention, it can be variously modified and available equivalents are replaced, and it is in addition, it is to be understood that other Aspect, advantage and modification will be apparent to those skilled in the art.Except reality as described herein Apply outside mode, the present invention covers and claimed by the feature of invention cited herein and cited prior art reference text Those inventions produced by the combination for the feature (it supplements the feature of the present invention) offered.Similarly, it is to be understood that any material Material, feature or article can be applied in combination with any other material, feature or article, and these combinations are considered as the present invention's In the range of.

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Claims (10)

1. fullerene or its officinal salt are preparing the application in being used to treat the medicine of pulmonary fibrosis, wherein the fullerene or Its officinal salt is present in the medicine with therapeutically effective amount.

2. application according to claim 1, wherein the pulmonary fibrosis is caused by idiopathic pulmonary fibrosis.

3. application according to claim 1, the fullerene is selected from Fullerene C20, C70, C82, C84 or its mixture.

4. application according to claim 1, wherein the fullerene includes one combined in oxygen-containing group, nitrogenous base, alkyl Plant or a variety of fullerenes, the alkyl optionally has substituent.

5. application according to claim 1, wherein the officinal salt of the fullerene is selected from sodium salt, sylvite, magnesium salts, calcium Salt, aluminium salt and/or sulfonate.

6. application according to claim 3, wherein the fullerene is Fullerene C20；Preferably, the Fullerene C20 To be insoluble in the Fullerene C20 of water or the Fullerene C20 of water-soluble sex modification.

7. application according to claim 6, wherein the Fullerene C20 for being insoluble in water applicable in vivo has by being dissolved in Machine solvent and be administered；Preferably, the organic solvent being applicable in vivo is selected from：Corn oil, cottonseed oil, soybean oil, sesame Oil, peanut oil, coconut oil and/or olive oil.

8. application according to claim 6, wherein there is the Fullerene C20 of the water-soluble modification following formula to represent：F(-X )_m, wherein

F is Fullerene C20 core；

X each stands alone as OH, (CH₂)_n-SO₃H or (CH₂)_n-SO₃- metal salt, wherein each n is independently between 2-50 Integer；And

M is the integer between 2-40.

9. application according to claim 1, wherein the method for administration of the medicine is oral, intravenous, subcutaneous, intraperitoneal And/or intramuscular injection.

10. application according to claim 1, wherein the medicine includes pharmaceutically useful excipient；Preferably, it is described can medicine Excipient include diluent, adhesive, dispersant, surfactant, lubricant, coating material, flavor enhancement, colouring agent, Control release preparation and/or sweetener；Preferably, the formulation of the medicine include tablet, pill, capsule, granule, dissipate Agent, elata chewing gum agent, supensoid agent, emulsion, injection solution and/or liposome injection suspension.