CN102188707A - Application of IL-17 inhibitor in preparing medicament for treating influenza - Google Patents

Application of IL-17 inhibitor in preparing medicament for treating influenza Download PDF

Info

Publication number
CN102188707A
CN102188707A CN2011100459358A CN201110045935A CN102188707A CN 102188707 A CN102188707 A CN 102188707A CN 2011100459358 A CN2011100459358 A CN 2011100459358A CN 201110045935 A CN201110045935 A CN 201110045935A CN 102188707 A CN102188707 A CN 102188707A
Authority
CN
China
Prior art keywords
influenza virus
inhibitor
antibody
influenza
medicine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN2011100459358A
Other languages
Chinese (zh)
Other versions
CN102188707B (en
Inventor
蒋澄宇
王希良
李承刚
杨鹏辉
孙阳
邹镇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Basic Medical Sciences of AMMS
Institute of Basic Medical Sciences of CAMS
Original Assignee
Institute of Basic Medical Sciences of CAMS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Basic Medical Sciences of CAMS filed Critical Institute of Basic Medical Sciences of CAMS
Priority to CN201110045935.8A priority Critical patent/CN102188707B/en
Publication of CN102188707A publication Critical patent/CN102188707A/en
Priority to PCT/CN2012/071568 priority patent/WO2012113343A1/en
Application granted granted Critical
Publication of CN102188707B publication Critical patent/CN102188707B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Pulmonology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Genetics & Genomics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention relates to an application of an IL-17 inhibitor in preparing a medicament for treating influenza, in particular relating to an application of the IL-17 inhibitor in preparing a medicament for treating and/or preventing virus infection of influenza. The IL-17 inhibitor can be used for reducing or knocking out the expression of IL-17 in mammals. The IL-17 inhibitor is selected from specific anti-IL-17 antibody, siRNA or other any biochemical substances capable of reducing and/or knocking out expression of IL-17. The invention provides a medicament with new action mechanism for the treatment of influenza.

Description

The purposes of IL-17 inhibitor in the medicine of preparation treatment influenza
Technical field
The present invention relates to IL-17 and treat and/or prevent purposes in the medicine of H 1 N 1 influenza A virus infection in preparation.
Background technology
(interleukin 17, interleukin-17, GeneID:3605 for IL17; Nucleotide:NM_002190.2; Protein:NP_002181.1) be a kind of important cytokine, no matter the people still is a muroid T lymphocyte, and microorganism (as spirillum, mycobacteria etc.) all can induce it to express IL-17.The long-term expression of the IL-17 of microorganism induction is the important medium of infection induced pathology immunity.By virus-mediated IL-17 gene transfer and high expressed IL-17, cause lungs TNF-α, IL-1 β, the expression of G-CSF is increased, and polymorphonuclear cell is raised, and bacteria clearance increases.
IL-17 is as the important medium of contact T cell and neutrophilic granulocyte, and bronchial epithelial cell, vascular endothelial cell and fibroblast are synthetic by stimulating, biotic factors such as release IL-8, GCP-2, MIP-2 and adhesion factor participate in neutrophilic granulocyte raising at air flue under the inflammatory conditions.The cell of IL-17 (+) removes T extracellular eosinophilic granulocyte and also expresses IL-17 significantly more than normal population among bronchial asthma patient's expectorant and the BALF; The eosinophilic granulocyte also expresses IL-17 in the peripheral blood; Plasma IL-17 concentration is higher than normal population.Experiment shows that splashing into hIL-17 in the rat trachea can significantly increase neutrophilic granulocyte quantity among the BALF, is dose dependent and may persist to splash into back 8h; The neutralizing antibody of hIL-17 this effect capable of blocking.Change the IL-17 gene over to airway of mice, make its overexpression, also optionally raise neutrophilic granulocyte.On the other hand, IL-17 also stimulates bronchial epithelial cell and fibroblast is synthetic, release neutrophilic granulocyte activation factor IL-6, and strengthens MPO and elastase activity.
Influenza is a kind of crowd of influence and commonly encountered diseases, frequently-occurring disease widely, and influenza virus strides species and infects that the situation is tense at present, the clinical symptoms that H 1 N 1 influenza A virus infection caused, and most of patients is lighter, shows as typical influenza-like symptom, can recover naturally.Common sympton comprises cough, heating, throat pain, headache and other senses of discomfort.The visible multiple focus of serious pneumonia patient x line rabat is soaked into, but rapid progress is ARDS, kidney or multiple organ dysfunction syndrome.The incidence rate of influenza A merging ARDS is 100 times of common influenza, and pulmonary damages and is mainly derived from systemic immune response out of control.Consistent with the ARDS that is secondary to viral pneumonia, comprise that DAD, bronchioles and blood vessel peripheral lymphoid cellular infiltration, outgrowth air flue change and bronchiolitis obliterans.
Clinical and pathological examination all points out patient with severe symptoms's pathological changes mainly at respiratory system.Consolidation appears in the visible patient with severe symptoms's of pathological examination pulmonary, often with hemorrhage, ooze out, pathological change such as abscess.Visible serosity or fiber disposition are oozed out in the alveolar space, and with hyaline membrane formation in various degree, prompting has diffuse lung tissue damage.Think that at present the lung basic lesion of influenza, SARS and the human and bird fluenza severe cases of lung tissue damage basic lesion and other types is similar due to the influenza A H1N1 influenza virus, be the diffusivity lung tissue that weight do not wait and damage.
Kohler in 1975 and Milstein at first report, use hybridoma technique, make through the splenocyte of sheep red blood cell (SRBC) (SRBC) mice immunized and the myeloma cell of mice and merge, and created first B cell hybridoma cell strain thus, obtained the monoclonal antibody of anti-SRBC.
The characteristics of monoclonal antibody be physicochemical character height homogeneous, biological activity single, with the bonded high specificity of antigen, be convenient to artificial the processing and quality control, and the source is easily.These advantages make it be paid much attention to once coming out, and are widely used in biology and medical research field.Can medicine etc. is directly crosslinked with monoclonal antibody, utilize its guide effect, make medicine etc. be positioned the specific therapy target position, this has not only improved curative effect, also can reduce Normocellular toxic reaction.
RNAi (RNA interference) is that RNA interferes, it is ubiquitous in vivo a kind of ancient biological phenomena of discovered in recent years, by double-stranded RNA (dsRNA) mediation, by the reticent phenomenon of the specific gene of certain enzyme participation, the expression of its blocking gene on transcriptional level, post-transcriptional level and translation skill.
Small interfering RNA (siRNA, siRNA) is a kind of small RNA molecular (～21-25 nucleotide), is processed by Dicer (in the RNAase III family double-stranded RNA being had specific enzyme).SiRNA is the main member of siRISC, excites the silence of complementary target mRNA with it.It is a kind of powerful experimental tool in laboratory that RNA interferes (RNAi), utilizes the silence of the special target gene of double-stranded RNA (dsRNA) induced sequence with homology, rapidly the blocking gene activity.SiRNA plays central role in the quiet passage of RNA, be to instruct key element to what specific messenger RNA (mRNA) was degraded.SiRNA is the intermediate product in the RNAi approach, is the necessary factor of RNAi performance effect.
RNAi maximum and final effect are metabolic process, the Physiology and biochemistry coefficient isophenous parameter generation obvious variation of cell.Recently it is also increasing that RNAi successfully is used to make up the report of transgenic animal model, indicates that RNAi will become the indispensable instrument of research gene function.Moreover, the RNAi technology also may become the new way of research cellular signal transduction path and gene therapy.
Though had the medicine of influenza so far, because also raising, still there are the demand to the treatment of influenza medicine of newtype in the aberration rate height of influenza virus, drug resistance.
Summary of the invention
Therefore, technical purpose of the present invention is to probe into IL-17 and prevents and/or treats purposes in the medicine of influenza at inhibitor that influenza developing effect takes place and probes into IL-17 in preparation.
Therefore, the inhibitor that a first aspect of the present invention provides a kind of IL-17 treats and/or prevents purposes in the medicine that mammal comprises that the human influenza virus infects in preparation, and wherein IL-17 is IL-17 albumen or its nucleotide coding sequence.Preferably, described influenza virus is an influenza A virus, preferably, described influenza virus is first type H1, H3, H5, H7 or H9 hypotype strain, and more preferably, described influenza virus is a first type H1 hypotype strain, more preferably, described influenza virus is an influenza A H1N1 influenza virus, and most preferably, described influenza virus is influenza A H1N1 influenza virus BJ501 strain.
Preferably, described IL-17 derives from mammal and comprises the people, and more preferably, the GeneBank of described IL-17 is numbered GeneID:3605, and nucleotide coding sequence is shown in NM_002190.2, and protein coding sequence is shown in NP_002181.1.
Preferably, the inhibitor of the described IL-17 antibody, siRNA or the specificity that are selected from the anti-IL-17 of specificity suppresses the chemical substance that mammal comprises that human il-17 is expressed.Preferably, the antibody of the anti-IL-17 of described specificity is selected from polyclonal antibody, monoclonal antibody, chimeric antibody, the surface of the anti-IL-17 of specificity and reinvents antibody, reshaped antibody, total man source antibody or its antigen-binding portion thereof, preferably, the antibody of the anti-IL-17 of described specificity is the monoclonal antibody of the anti-IL-17 of specificity.Preferably, the inhibitor of described IL-17 is selected from siRNA, and preferably, the just sequence of described siRNA is shown in SEQ.ID.NO.1:gaccucauuggugucacugUU, and antisense sequences is shown in SEQ.ID.NO.2:UUcuggaguaaccacagugac.
Preferably, the dosage form of described medicine is injection, spray, nasal drop, inhalant, oral agents.
A second aspect of the present invention relates to the inhibitor of a kind of IL-17, it is used as and treats and/or prevents the medicine that mammal comprises that the human influenza virus infects, wherein IL-17 is IL-17 albumen or its nucleotide coding sequence, preferably, the inhibitor of described influenza virus, IL-17, IL-17 is respectively as described in the relevant portion of first aspect present invention.
The compositions that a third aspect of the present invention relates to the inhibitor of a kind of IL-17 of containing treats and/or prevents purposes in the medicine that mammal comprises that the human influenza virus infects in preparation, wherein the inhibitor of IL-17 is the active component of described compositions, wherein IL-17 is IL-17 albumen or its nucleotide coding sequence, preferably, the inhibitor of described influenza virus, IL-17, IL-17 is respectively as described in the relevant portion of first aspect present invention.
In other words, the present invention utilizes influenza A H1N1 influenza virus attack mice proof IL-17 to infect in the process that causes the damage of chmice acute lung tissue pathology, death in influenza A H1N1 influenza virus BJ501 strain and plays a significant role, intervention at the IL-17 molecule is infected in the damage that is caused in treatment influenza A H1N1 influenza virus BJ501 strain, might play a significant role.The reuse influenza A H1N1 influenza virus was attacked described mice after the present invention utilized the monoclonal antibody immunity wild-type mice of the anti-IL-17 of specificity; presentation of results; anti-IL-17A antibody has been brought into play important protective effect to mice in infecting the acute lung tissue pathology damage that influenza A H1N1 influenza virus BJ501 strain causes, and antibody TNF-aRII infects in the process that causes dead mouse in influenza A H1N1 influenza virus BJ501 strain and do not bring into play remarkable therapeutical effect.Therefore, the present invention has proved that for the first time IL-17 plays a significant role and the treatment of specific inhibition of IL-6-17 can stop or slow down the serious consequence that influenza a virus infection causes in the influenza A pathological process.
Simultaneously; those skilled in the art know; when confirming the important facilitation of IL-17 performance in processes such as pathology damage that influenza infection causes, death; especially neutralize the intravital IL-17 of experimenter can protect the patient time at antibody, also can play identical or similar effect with the other technologies means of eliminating or reduce its influence at IL-17 with the anti-IL-17 of specificity.Those skilled in the art know, and RNAi technology and known in the art other that such technological means comprises in other the specificity and the antibody of IL-17, the reticent IL-17 of specificity express can reduce or eliminate the chemical substance of IL-17 expression.Antibody with IL-17 in the specificity can be to utilize the polyclonal antibody that obtains behind the IL-17 immunity mammal, monoclonal antibody, chimeric antibody, the surface that utilizes hybridoma technology to obtain to reinvent antibody, reshaped antibody, total man source antibody or its antigen-binding portion thereof, as long as such antibody or its part have kept antigen binding capacity and the described antigenic activity that can neutralize.Design at the siRNA of particular target gene is operation well known by persons skilled in the art, such sequence may length and at the position of target sequence on have nothing in common with each other, but must silence fall the IL-17 expression of gene.Simultaneously, those skilled in the art also can utilize known in the art can reduce other chemical substances that reticent IL-17 expresses or utilize technology screening known in the art to can reduce or other chemical substances of reticent IL-17 expression reduce or the expression of reticent IL-17, thereby realize the purpose of treatment swine flu, particularly influenza A H1N1.
Similarly, the compositions that comprises IL-17 inhibitor of the present invention also can be used for the treatment of swine flu, particularly influenza A H1N1.The active component of described compositions is the IL-17 inhibitor, and what also can comprise other simultaneously can play synergistic active component with described IL-17 inhibitor.Compositions of the present invention can also comprise medical substances such as antiseptic, stabilizing agent, buffer agent.
Description of drawings
Fig. 1: wild type C57BL/6 mouse infection TCID50 is 10 5.5Influenza A H1N1 influenza virus BJ501 strain after, preceding 24 hours respectively in infection, infected back first day, infect the 3rd day vein in back and give anti-IL-17 antibody or control antibodies.Write down survival condition in its 14 days, draw mortality curve.Every group of 10 mices.
Fig. 2: wild type C57BL/6 mouse infection TCID50 is 10 5.5Influenza A H1N1 influenza virus BJ501 strain after, preceding 24 hours respectively in infection, infected back first day, infect the 3rd day vein in back and give anti-IL-17 antibody or control antibodies.Write down body weight situation of change in its 14 days, draw the body weight change curve.Every group of 10 mices.
Fig. 3: wild type C57BL/6 mouse infection TCID50 is 10 5.5Influenza A H1N1 influenza virus BJ501 strain after, preceding 24 hours respectively in infection, infected back first day, infect the 3rd day vein in back and give anti-IL-17 antibody or control antibodies.After infecting the 5th day, get the mouse lung tissue and carry out the detection of lung tissue wet-dry ratio.Every group of 5 mices.
Fig. 4: wild type C57BL/6 mouse infection TCID50 is 10 5.5Influenza A H1N1 influenza virus BJ501 strain after, preceding 24 hours respectively in infection, infected back first day, infect the 3rd day vein in back and give anti-IL-17 antibody or control antibodies.After infecting the 5th day, get the mouse lung tissue and carry out the histopathology detection.Every group of 5 mices.
Fig. 5: wild type C57BL/6 mouse infection TCID50 is 10 5.5Influenza A H1N1 influenza virus BJ501 strain after, preceding 24 hours respectively in infection, infected back first day, infected the back the 3rd day and infected the back the 5th day, vein gives anti-TNF alpha RII antibody or control antibodies.Write down survival condition drafting mortality curve in its 14 days.Every group of 10 mices.
Fig. 6: wild type C57BL/6 mouse infection TCID50 is 10 5.5Influenza A H1N1 influenza virus BJ501 strain after, preceding 24 hours respectively in infection, infected back first day, infected the back the 3rd day and infected the back the 5th day, vein gives anti-TNF alpha RII antibody or control antibodies.Write down body weight situation of change in its 14 days, draw the body weight change curve.Every group of 10 mices.
Fig. 7: wild type C57BL/6 mouse infection TCID50 is 10 5.5Influenza A H1N1 influenza virus BJ501 strain after, preceding 24 hours respectively in infection, infected back first day, infected the back the 3rd day and infected the back the 5th day, vein gives anti-TNF alpha RII antibody or control antibodies.After infecting the 5th day, get the mouse lung tissue and carry out the detection of lung tissue wet-dry ratio.Every group of 5 mices.
The specific embodiment
To further specify the present invention by following non-limiting example below, as well known to those skilled in the art, without departing from the spirit of the invention, can make many modifications to the present invention, such modification also falls into scope of the present invention.
Following experimental technique is conventional method if no special instructions, and employed experiment material all can easily be obtained from commercial company if no special instructions.
Embodiment
Embodiment 1 anti-IL-17 antibody can reduce influenza A H1N1 influenza virus BJ501 strain infection and cause dead mouse
Experiment material:
1) main experimental apparatus: three-grade biological safety laboratory, three-grade biological safety cabinet, animal feeding cabinet, mice rearging cage, toy operating theater instruments, asepsis injector, pipettor, pipet etc.
2) main experiment reagent: anti-IL-17A monoclonal antibody (AbM50016-1-PU, available from the BeiJing HuaDa protein Research Center Co., Ltd), antibody control (AbM59530-10-PU is available from the BeiJing HuaDa protein Research Center Co., Ltd), 1% (W/V) pentobarbital sodium solution, viral dilution liquid, disinfectant (2.5% iodine tincture and 75% ethanol) etc.
3) virus: influenza A H1N1 influenza virus BJ501 strain
http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi？mode＝Info
&id＝648856&lvl＝3&keep＝1&srchmode＝1&unlock&lin＝s。
4) laboratory animal:
SPF level wild type (WT) C57BL/6 mice (4 age in week): purchase in Jun Keyuan animal institute.
Experimental technique:
1) grouping: antibody control group, anti-IL-17A monoclonal antibody group;
2) mouse mainline antibody control or anti-IL-17A monoclonal antibody, every each intravenous injection 50 micrograms of mice are injected 3 times altogether, are respectively to infect preceding 1 day and infected the back the 1st day, infect the back the 3rd day;
3) safe fixedly mice is with the anesthesia of 1mL asepsis injector lumbar injection 1% (W/V) pentobarbital sodium solution;
4) keep the anesthetized mice head posture that upwards recedes, make its nasal cavity upwards, be convenient to the stable respiratory tract that enters of virus.(titre is 10 to splash into 10 μ L influenza A H1N1 influenza virus BJ501 strain virus liquid with the every side of pipet nostril 5TCID50) infect, the infective virus titre is 10 5TCID50/, every group is infected 10 mices;
5) kept this position of mice 15 seconds, make virus enter respiratory tract.Mice is placed in the mouse cage, treat its recover clear-headed after, give water and food;
6) infect the back and observe, in the 24h dead mice being taken place is non-specific death, will not count when carrying out mortality statistics;
7) observe 14d continuously, write down every group of dead mouse/survival number of elements and body weight change situation every day;
8) with GraphPad Prism 5 software statistics mouse death rates and body weight change situation.
Experimental result:
Wild type C57BL/6 mice is after intravenous injection antibody control or anti-IL-17A monoclonal antibody, and the infective virus titre is 10 5Mortality rate result and body weight change result after the influenza A H1N1 influenza virus BJ501 strain of TCID50, as shown in Figure 1 and Figure 2.
After infecting the BJ501 strain influenza A H1N1 influenza virus of identical titre, the mouse death rate of intravenous injection antibody control is apparently higher than the mice (Fig. 1) of the anti-IL-17A monoclonal antibody of intravenous injection.Body weight change (reduction) result consistent with the mortality rate result (Fig. 2).*P＜0.05
This presentation of results, IL-17 infects in the process that causes dead mouse in influenza A H1N1 influenza virus BJ501 strain and plays a significant role, intervention at the IL-17 molecule is infected in the damage that is caused in treatment influenza A H1N1 influenza virus BJ501 strain, might play a significant role.
Embodiment 2 anti-IL-17A antibody are alleviated the lungs edema that the back mice is infected in influenza A H1N1 influenza virus BJ501 strain
Experiment material:
1) main experimental apparatus: three-grade biological safety laboratory, three-grade biological safety cabinet, animal feeding cabinet, mice rearging cage, toy operating theater instruments, high temperature microstructure exsiccator, asepsis injector, pipettor, pipet etc.
2) main experiment reagent: anti-IL-17A monoclonal antibody (AbM50016-1-PU, available from the BeiJing HuaDa protein Research Center Co., Ltd), antibody control (AbM59530-10-PU is available from the BeiJing HuaDa protein Research Center Co., Ltd), 1% (W/V) pentobarbital sodium solution, viral dilution liquid, aseptic PBS, disinfectant (2.5% iodine tincture and 75% ethanol) etc.
3) virus: influenza A H1N1 influenza virus BJ501 strain
http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi？mode＝Info
&id＝648856&lvl＝3&keep＝1&srchmode＝1&unlock&lin＝s。
4) laboratory animal:
SPF level wild type C57BL/6 mice (4 age in week): purchase in Jun Keyuan animal institute.
Experimental technique:
1) grouping: antibody control group, anti-IL-17A monoclonal antibody group;
2) mouse mainline antibody control or anti-IL-17A monoclonal antibody, every each intravenous injection 50 micrograms of mice are injected 3 times altogether, are respectively to infect preceding 1 day and infected the back the 1st day and infected the back the 3rd day;
3) safe fixedly mice is with the anesthesia of 1mL asepsis injector lumbar injection 1% (W/V) pentobarbital sodium solution;
4) keep the anesthetized mice head upwards, the posture that recedes, make its nasal cavity upwards, be convenient to the stable respiratory tract that enters of virus.The influenza A H1N1 influenza virus BJ501 strain virus liquid inductance that splashes into 10 μ L with the every side of pipet nostril dyes mice, and the infective virus titre is 10 5TCID50/, every group is infected 5 mices;
5) kept this position of mice 15 seconds, make virus enter respiratory tract.Mice is placed in the mouse cage, treat its recover clear-headed after, give water and food;
6) infect the back and observe, in the 24h dead mice being taken place is non-specific death, will not count;
7) 5d behind the infective virus makes dead mouse with the excessive narcotic method of lumbar injection;
8) mice is fixed in the toy operating-table, removes skin of chest and skeleton, expose the thoracic cavity, the complete lungs of mice are taken out, remove surperficial blood and unnecessary connective tissue, weighing is also write down the lungs weight in wet base;
9) to place 55 ℃ of high temperature microstructure exsiccators to do roasting for lungs, takes out behind the 24h, treats that temperature reduces to weighing and record that room temperature is carried out the lungs dry weight;
10) calculate mice lungs weight in wet base/dry weight ratio (Wet/dry ratio), carry out statistical analysis.
Experimental result:
Detect mice lungs wet-dry ratio, can reflect the degree of mice generation acute lung edema.As can be seen from Fig. 3,4 the week age wild type C57BL/6 mice in intravenous injection after the anti-IL-17A monoclonal antibody, infect influenza A H1N1 influenza virus BJ501, its lungs wet-dry ratio is compared antibody control treatment group mice and is significantly reduced, and illustrates that anti-IL-17A antibody can significantly alleviate the lungs edema that the back mice is infected in influenza A H1N1 influenza virus BJ501 strain.*P＜0.05。
This result further specifies, and the IL-17 molecule infects in influenza A H1N1 influenza virus BJ501 strain and causes mice to take place to have brought into play important function in the pathological process of acute lung tissue damage.
Embodiment 3 anti-IL-17 antibody can be alleviated influenza A H1N1 influenza virus BJ501 strain infection and cause the damage of mouse lung histopathology
Experiment material:
1) main experimental apparatus: three-grade biological safety laboratory, three-grade biological safety cabinet, animal feeding cabinet, mice rearging cage, toy operating theater instruments, vertical DL device, histotome, tissue embedding machine, microscope, asepsis injector, pipettor, pipet etc.
2) main experiment reagent: anti-IL-17A monoclonal antibody (AbM50016-1-PU, available from the BeiJing HuaDa protein Research Center Co., Ltd), antibody control (AbM59530-10-PU is available from the BeiJing HuaDa protein Research Center Co., Ltd), 1% (W/V) pentobarbital sodium solution, viral dilution liquid, paraformaldehyde fixative, aseptic PBS, disinfectant (2.5% iodine tincture and 75% ethanol) etc.
3) virus: influenza A H1N1 influenza virus BJ501 strain
http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi？mode＝Info
&id＝648856&lvl＝3&keep＝1&srchmode＝1&unlock&lin＝s。
4) laboratory animal:
SPF level wild type C57BL/6 mice (4 age in week): purchase in Jun Keyuan animal institute.
Experimental technique:
1) grouping: antibody control group, anti-IL-17A monoclonal antibody group;
2) mouse mainline antibody control or anti-IL-17A monoclonal antibody, every each intravenous injection 50 micrograms of mice are injected 3 times altogether, are respectively to infect preceding 1 day and infected the back the 1st day and infected the back the 3rd day;
3) safe fixedly mice is with the anesthesia of 1mL asepsis injector lumbar injection 1% (W/V) pentobarbital sodium solution;
4) keep the anesthetized mice head posture that upwards recedes, make its nasal cavity upwards, be convenient to the stable respiratory tract that enters of virus.(titre is 10 to splash into 10 μ L influenza A H1N1 influenza virus BJ501 strain virus liquid with the every side of pipet nostril 5TCID50) or contrast (viral dilution liquid) infect, the infective virus titre is 10 5TCID50/, every group is infected 3 mices;
5) kept this position of mice 15 seconds, make virus enter respiratory tract.Mice is placed in the mouse cage, treat its recover clear-headed after, give water and food;
6) infect the back and observe, in the 24h dead mice being taken place is non-specific death, will not count;
7) 5d behind the infective virus makes dead mouse with the excessive narcotic method of lumbar injection;
8) mice is fixed in the toy operating-table, removes skin of chest and skeleton, expose the thoracic cavity, the mice lungs are taken out simultaneously together with heart,, place fixedly 48h of paraformaldehyde fixative room temperature with aseptic PBS flush away surface blood;
9) sample after fixing is carried out processing such as embedding, section, HE dyeing by Pathology Lab;
10) pathological section places microscopically to observe, and record.
Experimental result:
(* 200 times, HE dyeing) pathology photo shows among Fig. 4: infection titer is 10 5After the influenza A H1N1 influenza virus BJ501 strain of TCID50, in the wild type C57BL/6 mouse lung tissue in 4 ages in week of intravenous injection antibody control serious pathology damage appears.The lung tissue normal configuration is destroyed, and the lung tissue texture disorder is followed pathology damage such as hemorrhage, inflammatory exudation and a large amount of erythrocyte, inflammatory cell infiltration.
The mouse lung that infects the anti-IL-17A monoclonal antibody of identical titre virus intravenous injection is organized and is not seen remarkable pathology damage, do not have hemorrhage significantly, ooze out or pathological change such as inflammatory cell infiltration lung tissue clean mark, structural integrity.
Presentation of results, anti-IL-17A antibody has been brought into play important protective effect to mice in the acute lung tissue pathology damage that infection influenza A H1N1 influenza virus BJ501 strain causes.
Embodiment 4 anti-TNF-aR II antibody infect the influence that causes dead mouse to influenza A H1N1 influenza virus BJ501 strain
Experiment material:
1) main experimental apparatus: three-grade biological safety laboratory, three-grade biological safety cabinet, animal feeding cabinet, mice rearging cage, toy operating theater instruments, asepsis injector, pipettor, pipet etc.
2) main experiment reagent: anti-TNF-aRII antibody (the benefit match is general, available from Shanghai CP Guojian Pharmaceutical Co.,Ltd), antibody control, 1% (W/V) pentobarbital sodium solution, viral dilution liquid, disinfectant (2.5% iodine tincture and 75% ethanol) etc.
3) virus: influenza A H1N1 influenza virus BJ501 strain
http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi？mode＝Info
&id＝648856&lvl＝3&keep＝1&srchmode＝1&unlock&lin＝s。
4) laboratory animal:
SPF level wild type (WT) C57BL/6 mice (4 age in week): purchase in Jun Keyuan animal institute.
Experimental technique:
1) grouping: antibody control group, anti-TNF-aRII antibody group;
2) injected in mice antibody control or anti-TNF-aRII antibody, every mice per injection 50 micrograms are injected 4 times altogether, be respectively infect preceding 1 day, infect the back the 1st day, infect the back the 3rd day, infected the back the 5th day;
3) safe fixedly mice is with the anesthesia of 1mL asepsis injector lumbar injection 1% (W/V) pentobarbital sodium solution;
4) keep the anesthetized mice head posture that upwards recedes, make its nasal cavity upwards, be convenient to the stable respiratory tract that enters of virus.(titre is 10 to splash into 10 μ L influenza A H1N1 influenza virus BJ501 strain virus liquid with the every side of pipet nostril 5TCID50) infect, the infective virus titre is 10 5TCID50/, every group is infected 10 mices;
5) kept this position of mice 15 seconds, make virus enter respiratory tract.Mice is placed in the mouse cage, treat its recover clear-headed after, give water and food;
6) infect the back and observe, in the 24h dead mice being taken place is non-specific death, will not count when carrying out mortality statistics;
7) observe 14d continuously, write down every group of dead mouse/survival number of elements and body weight change situation every day;
8) with GraphPad Prism 5 software statistics mouse death rates and body weight change situation.
Experimental result:
Behind wild type C57BL/6 injected in mice antibody control or the anti-TNF-aRII antibody, the infective virus titre is 10 5Mortality rate result and body weight change result after the influenza A H1N1 influenza virus BJ501 strain of TCID50 are as Fig. 5, shown in Figure 6.
After infecting the BJ501 strain influenza A H1N1 influenza virus of identical titre, injection of antibodies contrasts the mouse death rate (Fig. 5) of anti-TNF-aRII antibody and body weight change (reduction) (Fig. 6) does not have a significant difference.
This presentation of results, TNF-aRII infect in the process that causes dead mouse in influenza A H1N1 influenza virus BJ501 strain does not bring into play remarkable therapeutical effect.
Embodiment 5 anti-TNF-aR II antibody infect the influence of the lungs edema of back mice to influenza A H1N1 influenza virus BJ501 strain
Experiment material:
1) main experimental apparatus: three-grade biological safety laboratory, three-grade biological safety cabinet, animal feeding cabinet, mice rearging cage, toy operating theater instruments, high temperature microstructure exsiccator, asepsis injector, pipettor, pipet etc.
2) main experiment reagent: anti-TNF-aRII antibody (the benefit match is general, available from Shanghai CP Guojian Pharmaceutical Co.,Ltd), antibody control, 1% (W/V) pentobarbital sodium solution, viral dilution liquid, aseptic PBS, disinfectant (2.5% iodine tincture and 75% ethanol) etc.
3) virus: influenza A H1N1 influenza virus BJ501 strain
http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi？mode＝Info
&id＝648856&lvl＝3&keep＝1&srchmode＝1&unlock&lin＝s。
4) laboratory animal:
SPF level wild type C57BL/6 mice (4 age in week): purchase in Jun Keyuan animal institute.
Experimental technique:
1) grouping: antibody control group, anti-TNF-aRII antibody group;
2) injected in mice antibody control or anti-TNF-aRII antibody, every mice per injection 50 micrograms are injected 4 times altogether, be respectively infect preceding 1 day, infect the back the 1st day, infect the back the 3rd day, infected the back the 5th day;
3) safe fixedly mice is with the anesthesia of 1mL asepsis injector lumbar injection 1% (W/V) pentobarbital sodium solution;
4) keep the anesthetized mice head upwards, the posture that recedes, make its nasal cavity upwards, be convenient to the stable respiratory tract that enters of virus.The influenza A H1N1 influenza virus BJ501 strain virus liquid inductance that splashes into 10 μ L with the every side of pipet nostril dyes mice, and the infective virus titre is 10 5TCID50/, every group is infected 5 mices;
5) kept this position of mice 15 seconds, make virus enter respiratory tract.Mice is placed in the mouse cage, treat its recover clear-headed after, give water and food;
6) infect the back and observe, in the 24h dead mice being taken place is non-specific death, will not count;
7) 5d behind the infective virus makes dead mouse with the excessive narcotic method of lumbar injection;
8) mice is fixed in the toy operating-table, removes skin of chest and skeleton, expose the thoracic cavity, the complete lungs of mice are taken out, remove surperficial blood and unnecessary connective tissue, weighing is also write down the lungs weight in wet base;
9) to place 55 ℃ of high temperature microstructure exsiccators to do roasting for lungs, takes out behind the 24h, treats that temperature reduces to weighing and record that room temperature is carried out the lungs dry weight;
10) calculate mice lungs weight in wet base/dry weight ratio (Wet/dry ratio), carry out statistical analysis.
Experimental result:
Detect mice lungs wet-dry ratio, can reflect the degree of mice generation acute lung edema.As can be seen from Fig. 7,4 the week age wild type C57BL/6 mice after having injected antibody control or anti-TNF-aRII antibody, infect influenza A H1N1 influenza virus BJ501, the wet dried significant difference that do not have of its lungs illustrates that the lungs edema that anti-TNF-aRII antibody infects the back mice to influenza A H1N1 influenza virus BJ501 strain does not have remarkable mitigation.
Figure IDA0000047968490000011

Claims (9)

1. the inhibitor of an IL-17 treats and/or prevents purposes in the medicine that mammal comprises that the human influenza virus infects in preparation, and wherein IL-17 is IL-17 albumen or its nucleotide coding sequence.
2. the inhibitor of IL-17 according to claim 1 treats and/or prevents purposes in the medicine that mammal comprises that the human influenza virus infects in preparation, it is characterized in that described influenza virus is an influenza A virus, preferably, described influenza virus is first type H1, H3, H5, H7 or H9 hypotype strain, more preferably, described influenza virus is a first type H1 hypotype strain, more preferably, described influenza virus is an influenza A H1N1 influenza virus, most preferably, described influenza virus is influenza A H1N1 influenza virus BJ501 strain.
3. the inhibitor of IL-17 according to claim 1 and 2 treats and/or prevents purposes in the medicine that mammal comprises that the human influenza virus infects in preparation, it is characterized in that described IL-17 derives from mammal and comprises the people, more preferably, the GeneBank of described IL-17 is numbered GeneID:3605, nucleotide coding sequence is shown in NM 002190.2, and protein coding sequence is shown in NP 002181.1.
4. treat and/or prevent purposes in the medicine that mammal comprises that the human influenza virus infects according to the inhibitor of each described IL-17 of claim 1 to 3 in preparation, it is characterized in that the inhibitor of described IL-17 is selected from the antibody of the anti-IL-17 of specificity, siRNA or specificity and suppresses the chemical-biological material that mammal comprises that human il-17 is expressed.
5. the inhibitor of IL-17 according to claim 4 treats and/or prevents purposes in the medicine that mammal comprises that the human influenza virus infects in preparation, the antibody that it is characterized in that the anti-IL-17 of described specificity is selected from polyclonal antibody, monoclonal antibody, chimeric antibody, the surface of the anti-IL-17 of specificity and reinvents antibody, reshaped antibody, total man source antibody or its antigen-binding portion thereof, preferably, the antibody of the anti-IL-17 of described specificity is the monoclonal antibody of the anti-IL-17 of specificity.
6. the inhibitor of IL-17 according to claim 4 treats and/or prevents purposes in the medicine that mammal comprises that the human influenza virus infects in preparation, the inhibitor that it is characterized in that described IL-17 is selected from siRNA, preferably, the just sequence of described siRNA is shown in SEQ.ID.NO.1:gaccucauuggugucacugUU, and antisense sequences is shown in SEQ.ID.NO.2:UUcuggaguaaccacagugac.
7. treat and/or prevent purposes in the medicine that mammal comprises that the human influenza virus infects according to the inhibitor of each described IL-17 of claim 1 to 6 in preparation, the dosage form that it is characterized in that described medicine is injection, spray, nasal drop, inhalant, oral agents.
8. the inhibitor of an IL-17, it is used as and treats and/or prevents the medicine that mammal comprises that the human influenza virus infects, wherein IL-17 is IL-17 albumen or its nucleotide coding sequence, preferably, described influenza virus is as described in the claim 2, described IL-17 is as described in the claim 3, or the inhibitor of described IL-17 is as claim 4 to 6 as described in each.
9. the compositions of an inhibitor that contains IL-17 treats and/or prevents purposes in the medicine that mammal comprises that the human influenza virus infects in preparation, wherein the inhibitor of IL-17 is the active component of described compositions, wherein IL-17 is IL-17 albumen or its nucleotide coding sequence, preferably, described influenza virus is as described in the claim 2, described IL-17 is as described in the claim 3, or the inhibitor of described IL-17 is as claim 4 to 6 as described in each.
CN201110045935.8A 2011-02-25 2011-02-25 The purposes of IL-17 inhibitor in the medicine of preparation treatment influenza Active CN102188707B (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201110045935.8A CN102188707B (en) 2011-02-25 2011-02-25 The purposes of IL-17 inhibitor in the medicine of preparation treatment influenza
PCT/CN2012/071568 WO2012113343A1 (en) 2011-02-25 2012-02-24 Use of il-17 inhibitor in preparation of drug for treating and/or preventing lung injury

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201110045935.8A CN102188707B (en) 2011-02-25 2011-02-25 The purposes of IL-17 inhibitor in the medicine of preparation treatment influenza

Publications (2)

Publication Number Publication Date
CN102188707A true CN102188707A (en) 2011-09-21
CN102188707B CN102188707B (en) 2015-08-05

Family

ID=44598139

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201110045935.8A Active CN102188707B (en) 2011-02-25 2011-02-25 The purposes of IL-17 inhibitor in the medicine of preparation treatment influenza

Country Status (2)

Country Link
CN (1) CN102188707B (en)
WO (1) WO2012113343A1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012113343A1 (en) * 2011-02-25 2012-08-30 中国医学科学院基础医学研究所 Use of il-17 inhibitor in preparation of drug for treating and/or preventing lung injury
WO2015120792A1 (en) * 2014-02-11 2015-08-20 王宾 Immunotherapeutic composition, therapeutic method and diagnostic method
CN105420237A (en) * 2015-09-08 2016-03-23 中国农业科学院兰州兽医研究所 Sequence siRNA-180 achieving targeted inhibition of mouse interleukin-17A gene
CN105420238A (en) * 2015-09-08 2016-03-23 中国农业科学院兰州兽医研究所 Sequence siRNA-136 achieving targeted inhibition of mouse interleukin-17A gene

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106729634A (en) * 2017-01-05 2017-05-31 中国科学院微生物研究所 Interleukin-17 is resisting the application during influenza virus is infected

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101001871A (en) * 2004-06-10 2007-07-18 津莫吉尼蒂克斯公司 Soluble zcytor14, anti-zcytor14 antibodies and binding partners and methods of using in inflammation
CN101370525A (en) * 2005-08-19 2009-02-18 艾博特公司 Dual variable domain immunoglobin and uses thereof
WO2009136976A2 (en) * 2008-02-21 2009-11-12 Amgen Inc Il-17ra-il-17rb antagonists and uses thereof
WO2010034443A1 (en) * 2008-09-29 2010-04-01 F. Hoffmann-La Roche Ag Antibodies against human il 17 and uses thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102188707B (en) * 2011-02-25 2015-08-05 中国医学科学院基础医学研究所 The purposes of IL-17 inhibitor in the medicine of preparation treatment influenza

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101001871A (en) * 2004-06-10 2007-07-18 津莫吉尼蒂克斯公司 Soluble zcytor14, anti-zcytor14 antibodies and binding partners and methods of using in inflammation
CN101370525A (en) * 2005-08-19 2009-02-18 艾博特公司 Dual variable domain immunoglobin and uses thereof
WO2009136976A2 (en) * 2008-02-21 2009-11-12 Amgen Inc Il-17ra-il-17rb antagonists and uses thereof
WO2010034443A1 (en) * 2008-09-29 2010-04-01 F. Hoffmann-La Roche Ag Antibodies against human il 17 and uses thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
20090925 Christopher R. Crowe等 Critical Role of IL-17RA in Immunopathology of Influenza Infection 5301-5310 1－9 第183卷, *
CHRISTOPHER R. CROWE等: "Critical Role of IL-17RA in Immunopathology of Influenza Infection", <J IMMUNOL.>, vol. 183, no. 8, 15 October 2009 (2009-10-15), pages 5301 - 5310 *
CHRISTOPHER R. CROWE等: "Critical Role of IL-17RA in Immunopathology of Influenza Infection", <THE JOURNAL OF IMMUNOLOGY>, vol. 183, 25 September 2009 (2009-09-25), pages 5301 - 5310 *
陈文杰等: "大鼠急性肺损伤肺组织中白细胞介素17的含量变化及异丙酚的影响", <中国呼吸与危重监护杂志>, vol. 9, no. 5, 30 September 2010 (2010-09-30), pages 532 - 536 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012113343A1 (en) * 2011-02-25 2012-08-30 中国医学科学院基础医学研究所 Use of il-17 inhibitor in preparation of drug for treating and/or preventing lung injury
WO2015120792A1 (en) * 2014-02-11 2015-08-20 王宾 Immunotherapeutic composition, therapeutic method and diagnostic method
CN105420237A (en) * 2015-09-08 2016-03-23 中国农业科学院兰州兽医研究所 Sequence siRNA-180 achieving targeted inhibition of mouse interleukin-17A gene
CN105420238A (en) * 2015-09-08 2016-03-23 中国农业科学院兰州兽医研究所 Sequence siRNA-136 achieving targeted inhibition of mouse interleukin-17A gene

Also Published As

Publication number Publication date
WO2012113343A1 (en) 2012-08-30
CN102188707B (en) 2015-08-05

Similar Documents

Publication Publication Date Title
Wang et al. A critical role of IL-17 in modulating the B-cell response during H5N1 influenza virus infection
Gu et al. Cytokine signature induced by SARS-CoV-2 spike protein in a mouse model
Wu et al. Innate immune response to H3N2 and H1N1 influenza virus infection in a human lung organ culture model
Damjanovic et al. Negative regulation of lung inflammation and immunopathology by TNF-α during acute influenza infection
Kim et al. Chitinase 3‐like 1 protein plays a critical role in respiratory syncytial virus‐induced airway inflammation
Han et al. Toll-like receptor 2–expressing macrophages are required and sufficient for rhinovirus-induced airway inflammation
Kohan et al. Enhanced osteopontin expression in a murine model of allergen‐induced airway remodelling
CN102188707B (en) The purposes of IL-17 inhibitor in the medicine of preparation treatment influenza
CN102215862A (en) Globo h and related anti-cancer vaccines with novel glycolipid adjuvants
Zhang et al. Inhibition of Rac1 activity alleviates PM2. 5-induced pulmonary inflammation via the AKT signaling pathway
JP2016538276A (en) Compositions and methods for inhibiting NF-κB and SOD-1 for the treatment of amyotrophic lateral sclerosis
Cai et al. Salidroside suppresses group 2 innate lymphoid cell-mediated allergic airway inflammation by targeting IL-33/ST2 axis
Zhang et al. Suppressing syndecan-1 shedding ameliorates intestinal epithelial inflammation through inhibiting NF-κB pathway and TNF-α
Shao MiR-216a-5p ameliorates learning-memory deficits and neuroinflammatory response of Alzheimer’s disease mice via regulation of HMGB1/NF-κB signaling
Xu et al. Interaction of Mycoplasma synoviae with chicken synovial sheath cells contributes to macrophage recruitment and inflammation
Liu et al. Chemokine receptor antagonist block inflammation and therapy Japanese encephalitis virus infection in mouse model
CN105816854A (en) Application of antibacterial peptide CRAMP to prevention and treatment of viral myocarditis
Asif et al. Interleukin-6 expression after infectious bronchitis virus infection in chickens
Huang et al. Met-CCL5 represents an immunotherapy strategy to ameliorate rabies virus infection
Razzuoli et al. The swine IFN system in viral infections: Major advances and translational prospects
Levast et al. Synthetic cationic peptide IDR-1002 and human cathelicidin LL37 modulate the cell innate response but differentially impact PRRSV replication in vitro
Chen et al. A valid protective immune response elicited in rhesus macaques by an inactivated vaccine is capable of defending against SARS-CoV-2 infection
Longhi et al. CD59a deficiency exacerbates influenza‐induced lung inflammation through complement‐dependent and‐independent mechanisms
KR20150064147A (en) Compounds for treating the remyelination blockade in diseases associated with the expression of herv-w envelope protein
CN103417967B (en) Application of inhibitor of CXCL-10 in preparation of drug for treatment and/or prevention of lung injury

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant