CN1233189A - Treatment of asthma with TNFR-Ig - Google Patents

Treatment of asthma with TNFR-Ig Download PDF

Info

Publication number
CN1233189A
CN1233189A CN97194455A CN97194455A CN1233189A CN 1233189 A CN1233189 A CN 1233189A CN 97194455 A CN97194455 A CN 97194455A CN 97194455 A CN97194455 A CN 97194455A CN 1233189 A CN1233189 A CN 1233189A
Authority
CN
China
Prior art keywords
igg
tnfr
asthma
protein
goods
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.)
Pending
Application number
CN97194455A
Other languages
Chinese (zh)
Inventor
L·M·伦茨提
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.)
F Hoffmann La Roche AG
Original Assignee
F Hoffmann La Roche AG
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 F Hoffmann La Roche AG filed Critical F Hoffmann La Roche AG
Publication of CN1233189A publication Critical patent/CN1233189A/en
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Abstract

The present invention is directed to a method of combatting asthma with a composition containing an effective amount of a chimeric TNF alpha -binding protein which comprises the soluble part of the p55 TNF receptor and all domains except the first domain of the heavy chain constant region of a human IgG1 or IgG3 and to the use of such a chimeric TNF alpha -binding protein for the preparation of a medicament for the treatment of asthma.

Description

Treat asthma with TNFR-Ig
Asthma is a kind of chronic inflammation disease of respiratory tract, it is characterized in that taking exercise because of contact specific allergen (IgE mediation reaction) cold air or nervous and recurrence increases the weight of.The inflammation characteristics relevant with asthma are to have activated acidophil, respiratory tract to the sensitivity of nonspecific stimulation increase (the respiratory tract anaphylaxis reaction: AHR), edema, Polyblennia and cough.Believe that this inflammatory process is partly mediated by the lymphocytic generation of Th2 type and the activation of the various cytokines of secretion.Cytokine TNF alpha relates to cause a kind of cytokine of symptoms of asthma.
The combination therapy agent is used for the treatment of asthma, uses to suck β 2Analeptic (alleviate acute bronchus spasm) and as the steroid (anti-inflammatory) of selective reagent.Yet, it is believed that it is enough effective not having a kind of therapeutic scheme, but the mortality rate of this disease is increasing gradually.One of the most urgent medical science needs are to need a kind of reagent that can reverse the pneumonia of the serious attack seen in acute/serious asthma rapidly.In addition, there is not a kind of therapeutic agent can be included into the disease alleviant.Must need a kind of medicine that can reverse the inflammatory response seen in acute/serious asthma rapidly and as the reagent of real disease alleviant.The effect of TNF α is to start symptoms of asthma, and the applicant determines that the effect that suppresses TNF α provides the approach of alleviating these symptoms.
The applicant has found a kind of method of resisting asthma in suffering from the patient of symptoms of asthma now, comprise to described patient and use the compositions that contains by one or more chimeric conjugated protein goods of forming of TNF α, each protein in the described goods is made up of the soluble fraction that is fused to the p55TNF receptor protein on the IgG, the IgG of wherein said fusion contains all the IgG domains except an IgG domain of IgG CH, described compositions contains the inert carrier in the treatment, and described compositions is administered to described patient so that provide the described chimeric protein goods of effective dose to resist described symptoms of asthma to patient.
Said composition can be administered to the patient who suffers from asthma effectively, and wherein the chimeric protein goods are used with the amount of the influence that is enough to alleviate described disease.Said composition also can be before asthma outbreak be administered to asthma patient with the amount that effectively prevents or delay described seizure of disease.
The present invention relates to contain the method for in suffering from the patient of symptoms of asthma, resisting asthma by the compositions of one or more chimeric conjugated protein goods of forming of TNF α by using to patient.Protein in these goods is formed (TNFR-Ig) by the soluble fraction that is fused to the p55 TNF receptor protein on the IgG (immunoglobulin G), and wherein this IgG contains all domains except first domain of IgG CH.Said composition also contains the inert carrier in the treatment.Said composition is administered to described patient so as the chimeric protein goods that effective dose is provided to patient to resist described symptoms of asthma.
The chimeric TNF α that is made up of the soluble fraction that is fused to the p55 TNF receptor protein on the IgG is conjugated protein, and to be used to prepare the medicine for the treatment of asthma be another object of the present invention, and wherein said fusion IgG contains all the IgG domains except an IgG domain of IgG CH.
Said composition effectively is administered to the amount of the protein articles of the influence that is enough to relieving asthma outbreak and is in the interparoxysmal patient of asthma.Said composition also can be before asthma outbreak be administered to asthma patient with the amount that effectively prevents or delay the protein articles of this seizure of disease.
Any TNFR-Ig, promptly the chimeric TNF α that is made up of the soluble fraction that is fused to the p55 TNF receptor protein on the IgG is conjugated protein can be used for goods of the present invention so that resist asthma among the patient of the described symptoms of asthma of paragraph on have, and wherein said fusion IgG contains all the IgG domains except an IgG domain of IgG CH.IgG can be human IgG1 or IgG3, preferred in the present invention IgG1.
The example of this TNFR-Ig is included in EP417 563; U.S. Patent number 5,447,851 and 5,395,760; Lesslauer etc., European Journal of Immunology, 21 (11): 2883,1991; Loetscher etc., journal of biological chemistry, 266 (27): 18324,1991; Ashkenazi etc., institute of NAS newspaper, 88:10535, disclosed protein in 1991, they also can be used on, and disclosed method obtains in these documents.
The goods of preferred TNFR-Ig molecule of the present invention are with the IgG1 preparation and comprise the protein that has the composite oligosaccharide that stops with one or more sialic acid residueses and have the N-acetyl-glucosamine of exposure, the mol ratio of sialic acid residues is that every mole of protein about 4 is to about 7 moles of sialic acides in the goods, especially about 5 to about 6, the mol ratio of the N-acetyl-glucosamine that exposes in the goods be every mole of protein from about 1 to about 2 moles of N-acetyl-glucosamines, the mol ratio of sialic acid residues and N-acetyl-glucosamine residue is from about 0.35 to about 0.5, especially about 0.4 to about 0.45 in the goods.The isoelectric point, IP of goods (pI) is from about 5.5 to about 7.5, and available chromatography focus measurement is also handled responsive to neuraminidase.
TNFR-Ig of the present invention can obtain with the conventional method of recombinant technique or protein synthesis.The DNA of coding p55 TNF receptor, the DNA of all domains of coding except an IgG1 or IgG3 CH, and these sequences are linked together to be used for expressing in suitable carriers the technical staff is known and is described in the literature.Suitable cloning vehicle and host cell be know and can select by the technical staff, and measured suitable condition of culture (see animal cell culture: practical exploration, the 2nd edition, Rickwood and Hames edit, the Oxford University Press, NY 1992).
Can use known protein matter to reclaim and purification process purification TNFR-Ig then.Although TNFR-Ig also can reclaim from the host cell pyrolysis product, preferably from culture medium, reclaim as excretory polypeptide.But centrifuge cell culture medium or pyrolysis product are to remove granular cell debris.Purification TNFR-Ig from soluble protein that impurity is arranged and polypeptide subsequently, following procedure is as the example of suitable purifying procedure: separate on the affine or ion exchange column in immunity; Ethanol precipitation; Reverse hplc; Chromatography at silicon dioxide or on such as the cation exchange resin of DEAE; Chromatofocusing; SDS-PAGE; Ammonium sulfate precipitation; For example use, Sephadex G-75 and protein A agarose column gel filtration are to remove the pollutant such as IgG.Also can be used for suppressing proteoclastic degraded during the purification such as the protease inhibitor of Phenylmethanesulfonyl fluoride (PMSF).Those skilled in the art can reckon with that the purification process needs that are suitable for desired polypeptides make amendment according to the variation of the feature of polypeptide expressed in the reconstitution cell culture.
TNFR-Ig of the present invention with specific pI and sialic acid and N-acetyl-glucosamine ratio can be by the condition of culture acquisition of using recombinant mammalian cells to express TNFR-Ig and produce TNFR-Ig by for example WO 96/39488 described control mammalian cell.The host cell of selecting should be able to be connected N-with O-the sialic saccharide that comprises is connected on their expressed protein.The example of a this host cell is a Chinese hamster ovary celI.Suitable condition of culture be know and depend on selected host cell.Increase the cell specific output at the glycoprotein production period and cause the mature protein sialic acid content to reduce, vice versa.The factor that influences the cell specific output is known in this area, and include but not limited to, influence the factor of DNA/RNA copy number, influence the factor of RNA, as stablize the factor of RNA, culture medium nutritional labeling and other fill-in, transcriptional enhancer concentration, the Osmolality of culture environment, the temperature of cell culture and pH value etc.These factors can be regulated to obtain the preferred content such as sialic glycoprotein with the method for knowing.By in cell culture with about 0.1mM to about 20mM, or 5 to 20mM concentration adds alkanoic acid, as sodium butyrate or its salt, and the Osmolality that guarantees cell culture is between about 250 to 600mOsm, is preferably in transition period and makes up the cell specific output that changes cell culture campaign mutually and produce the protein with different sialic acid amounts.Approximately the concentration of 6.0mM sodium butyrate and the condition of 350-400mOsm provide height of the present invention sialylated TNFR-Ig, and approximately the concentration of 12mM sodium butyrate provides low sialylated TNFR-Ig of the present invention.Back one concentration can make up with the condition of 450-550mOsm.
The technical staff will recognize that the Osmolality of culture medium depends on the active particle concentration of the infiltration in the culture fluid and many variablees of preparation mammal compound cells culture medium can influence Osmolality.The starting weight Morie osmolarity of the composition decision culture medium of culture medium.Use permeability manometer, Fisher Scientific for example, Pittsburgh, the permeability manometer that Pennsylvania sells with trade (brand) name OSMETTE (maybe can be from Compak Systems Ltd., Osmette 2007 types that NatickMA obtains) is measured Osmolality.In order to obtain the Osmolality of required scope, the concentration of each constituent in the scalable culture medium.Can add solute to increase its Osmolality in culture medium, this solute comprises protein, peptide, and aminoacid is such as the hydrolyzed animal protein of peptone, non-metabolism polymer, vitamin, ion, salt, saccharide (particularly glucose), metabolite, organic acid, lipid etc.In one embodiment, by during the fed-batch culture program, in cell culture, adding peptone and other medium component control Osmolality.
Can use three periods of cell culture, trophophase: wherein the growth of the cell of Xuan Ding mammalian host cell is the fastest; Then be the transition period, wherein selecting and using aforesaid is the cell culture parameter that obtains the required sialic acid content of ripe glycoprotein; Follow by campaign, wherein keep the parameter of transition period selection, produce also results glycoprotein product.About preferred purification in description of the present invention, can be purification technique and the method for selecting to be used for saccharide of the present invention.Can for example pass through, the HPLC of the soft gel chromatography of ion exchange or use cation or anion exchange resin at containing sialic molecule enrichment desired sugars form of the present invention, wherein collects acid stronger fraction.
The preferred TNFR-Ig of the present invention has one or more following features in purified composition: the pI scope of TNFR-Ig compositions is between about 5.5 to about 7.5, sialic acid and proteinic mol ratio are about 4 to about 7, especially about 5 to about 6, have the N-acetyl-glucosamine residue of every mole of protein about 1, have about 0.35 to about 0.5 and more preferably about 0.4 to about 0.45 the sialic acid and the mol ratio of N-acetyl-glucosamine to about 2 moles exposure.The only condition that obtains this preferred TNFR-Ig is that the sodium butyrate concentration that adopts is extremely approximately 6mM of about 0.1mM, and Osmolality maintains between about 300 to 450mOsm, the condition of culture of temperature between about 30 ℃ and 37 ℃.
The technical staff uses the technology of knowing can measure the above-mentioned feature of TNFR-Ig.For example, if desired, the routine techniques analysis that the compound saccharide part of the glycoprotein of producing with the inventive method is analyzed with saccharide easily.Therefore, for example,, disclosed terminal mannose or such as the ratio of other saccharide of galactose as the agglutinin trace in technology well known in the art.By use dehydration hydrazides or Enzymology method from protein discharge sugar and through ion exchange or size exclusion chromatography or other method well known in the art separate oligosaccharide susceptible of proof list-, two-, three-, four-Zhi oligosaccharide is by sialic termination.Also can handle with neuraminidase remove sialic acid before and measure the pI of glycoprotein afterwards.Neuraminidase is handled back pI to be increased and shows have sialic acid on glycoprotein.
Carbohydrate structure of the present invention appears on the protein that is expressed as that N-connects or the saccharide that O-connects.The saccharide main distinction that is connected with O-that N-connects is on its core texture.The glycosylation that N-connects refers to by GlcNAc sugar moieties is connected on the asparagine residue of peptide chain.The saccharide that N-connects also contains common core texture Man1-6 (Man1-3) Man β 1-4GlcNAc β 1-4GlcNAc β-R.Therefore, in described core texture, proteic asparagine residue is produced in the R representative.The proteinic peptide sequence of producing can contain agedoite-X-serine, agedoite-X-threonine, and agedoite-X-cysteine, and wherein X is the arbitrary amino acid except that proline.On the contrary, the saccharide that O-connects is characterized in that a common core texture, and it is the GalNAc that is attached on threonine or the serine hydroxyl.N-that connect with saccharide that O-is connected in, the most important thing is the compound saccharide that N-is connected with O-.This compound saccharide contains some branched structures.Single-, two-, three-and four-outboard structure be important for increasing terminal sialic acid.Outer side-chain structure like this provides the attachment site of specificity sugar and has comprised the key of saccharide of the present invention.
Can analyze the saccharide of gained to comprise known in the art any method of those methods as herein described.The certain methods that is used for the glycosylation analysis is known in this area and is useful in content of the present invention.This method provides the information about homogeny that is connected in the oligosaccharide on the peptide and composition.Useful in the present invention saccharide analytical method includes but not limited to the agglutinin chromatography; The HPAEC-PAD that uses high pH value anion-exchange chromatography is that oligosaccharide is separated on the basis with the electric charge; NMR; Mass spectrography; HPLC; GPC; The monosaccharide composition analysis; The order enzymic digestion.
In addition, the method for release oligosaccharide is known.These methods comprise 1) enzyme, use peptide-N-glycosidase F/ inscribe-beta galactosidase to carry out usually; 2) use the strong basicity environment to discharge main O-syndeton method of elimination; With 3) use the chemical method of dehydration hydrazides with the oligosaccharide that discharges N-and be connected with O-.
Can use the following step analysis:
1. dialyse sample to remove all buffer salts, lyophilizing subsequently with deionized water.
2. discharge complete oligomerization sugar chain with the dehydration hydrazides.
3. discharge one monosaccharide with the anhydrous oligomerization sugar chain that contains the HCl processes complete of methanol with the O-methyl-derivatives.
4. the N-acetylation of any first amino.
5. derivatization is to provide height-O-TMS methylglycoside.
6. separate these derivants by capillary GLC (gas liquid chromatography) on the CP-SIL8 post.
7. use from the holdup time of GLC and recently identify one glycosides derivatives mutually with known standard with mass spectral analysis.
8. use internal standard (13-O-methyl D-glucose) with the quantitative single derivant of FID.
Detect (HPAE-PAD saccharide system, Dionex company) with high performance anion exchange chromatography in conjunction with the pulse ammeter and can measure neutrality and amino sugar.For example, can discharge saccharide in 6 hours by 100 ℃ of hydrolysis in 20% (v/v) trifluoroacetic acid.Then by lyophilizing or with the dry hydrolyzate of Speed-Vac (Savant Instruments).Then residue is dissolved in the 1% sodium acetate trihydrate solution and (biochemical yearbook 195:269-280 (1991)) is described analyzes on the HPLC-AS6 post by Anumula etc.
Can in triplicate sample, measure sialic acid respectively with the direct color comparison method of (biochemical yearbook, 179:332-335 (1989)) such as Yao.In preferred embodiments, use Warren, L. journal of biological chemistry 238:(8) thiobarbituricacid (TBA) of (1959).
As selection, can carry out the analysis of immunoblotting saccharide.According to this method, use according to Haselbeck and Hosel[Hasebeck etc., carbohydrate conjugates magazine, 7:63 (1990)] the commercial polysaccharide detection system (Boehringer) of described oxidation western blotting method detects the saccharide of protein bound.According to the dyeing scheme of manufacturer's recommended, except moving on to, protein transduction replaces on the PVDF membrane the nitrocellulose filter, and the sealing buffer has comprised 5% bovine serum albumin in 10mM Tris buffer (pH7.4) and 0.9% sodium chloride.The anti-Digitoxin antibody that is connected with alkaline phosphatase junctional complex (Boehringer) that detection is used in dilution in 1: 1000 in the tris buffer saline carries out, the phosphatase substrate of use in containing the 100mM Tris buffer saline (pH9.5) of 100mM sodium chloride and 50mM magnesium chloride, 0.03% (w/v) 4-nitroblue tetrazolium chlorine and 0.03% (w/v) 5-bromo-, 4 chloro-3-indole-phosphoric acid.Usually in about 10 to 15 minutes, observe sacchariferous protein belt.
Also can be by analyzing saccharide with peptide-N-glycosidase F digestion.According to this method, residue is suspended in 14 μ l and contains 0.18%SDS, the 18mM beta-mercaptoethanol, and 90mM phosphate, 3.6mMEDTA is in the buffer of pH8.6,100 ℃ of heating 3 minutes.Behind the cool to room temperature, sample is divided into two parts of equal parts.Aliquot sample is not for further processing and with comparing.Second portion is adjusted to and contains about 1%NP-40 detergent, then adds the peptide-N-glycosidase F (Boehringer) of 0.2 unit.Two duplicate samples are analyzed with the SDS-polyacrylamide gel electrophoresis then 37 ℃ of insulations 2 hours.
TNFR-Ig of the present invention comprises the TNFR-Ig of PEGization, and it is meant covalently bound arriving such as the TNFR-Ig molecule in polyalkylene glycol (replacement or unsubstituted), the particularly polymer of Polyethylene Glycol.Connection can be directly, but preferably realizes by means of various coupling agents known in the art, for example at EP510346, and EP593838, U.S. Patent number 4,766,106,4,917,888,4,902,502,4,847,325,4,179,337,5,832,657, Veronese etc., applied biochemistry and biotechnology, 11:141 (1985) and Monfardini etc., disclosed those coupling agents among the biological coupling chemistry 6:62 (1995).The TNFR-Ig of PEGization can be used for treating asthma and prevention as described to TNFR-Ig.
TNFR-Ig goods of the present invention are useful for opposing asthma in asthma patient.When treatment had the patient of asthma, the influence that can palliate a disease of these goods as reversing the inflammation that has taken place, prevented further inflammation.When the mode with prevention provided, these goods can delay or prevent the outbreak of asthma, or when disease takes place, guaranteed that severity of disease reduces.
According to the present invention, these goods can be administered to patient in any conventional manner and be used for the treatment of or prevent.Therefore goods of the present invention can be used in conventional Pharmaceutical composition, and said composition comprises the inert carrier on any conventional therapy.This Pharmaceutical composition can contain activated additive on the inert and pharmacokinetics.Fluid composition for example can adopt, with the form of the blended sterile solution of water.In addition, also can be conventionally used as protection, stable, preserve moisture and the material of emulsifying agent and the material that changes osmotic pressure, as salt, change the material of pH value, as buffer and other additive.If desired, in Pharmaceutical composition, can comprise such as tocopherol N-methyl-γ-tocopheramine, the antioxidant of butylated hydroxyanisol or Yoshinox BHT.The pharmaceutically acceptable excipient or the carrier that are used for compositions comprise saline, buffer salt solution, glucose or water.Compositions also can comprise specific stabilizing agent, and for example sugar comprises mannose and mannitol, and the local anesthetic that is used for being fit to compositions (indictable composition), comprise, for example, lidocaine.Aforementioned carriers material and diluent can be the organic or inorganic materials, for example, and water, gelatin, lactose, starch, magnesium stearate, Pulvis Talci, arabic gum, polyalkylene glycol etc.Prerequisite is that all adjuvants and the material that are used to produce Pharmaceutical composition are nontoxic.
Goods of the present invention can suck through parenteral (for example, by subcutaneous, intravenous, intramuscular, in the eye socket, in the capsule, in the spinal column, intrasternal injection) or spraying and use.Can use any conventional medicinal products that is used for parenteral or spraying inhaled medication.The example of suitable Pharmaceutical composition is infusion or injection solution.Preferred application method is by intravenous injection.Another preferred application method is to use aerosol.
The goods antasthmatic of the present invention that can use any effective dose are to alleviate or to reverse the influence of disease or to prevent or to delay the outbreak of disease.Be used for the treatment of, prevent or the preferred dose that is used for the TNFR-Ig compositions of parenteral (injection) medication of the present invention that reverses asthma is the TNFR-Ig goods (mg/kg) of the every kg body weight of every patient about 0.1 to about 5.0mg.Particularly preferred dosage is to about 3.0mg/kg from about 1.0.Be used for identical purpose and be to account for 0.03% to 5.0% of TNFR-Ig product weight in the preferred dose of TNFR-Ig compositions of spraying medication.In arbitrary application method, therapeutic dose can be treated Japan-China medication once at each, or be divided into littler dosage during about 24 hours innerlich anwenden to reach total dosage of being given.
In order to treat asthma fast, can provide every patient every day 0.1 to 5.0, the particularly single dose of 1.0-3.0mg/kg through the parenteral medication.For through parenteral medication prevention of asthma, according to the different intervals of patient and state from every day, but preferably with from weekly twice to once in a week by every month once or other interim preferably provide 0.1 to 5.0, this dosage of 1.0-3.0mg/kg particularly.Equally, for through the spraying medication treat asthma fast, but suck single dose every day.For prevention, according to the different intervals of patient and state with every day, but preferably with from weekly twice to once or with other interim sucking this dosage by every month once in a week.The exact dose of using compositions can be according to the type of using, and the mode of use and patient's requirement change by technical staff's mensuration.The exact dose that is used for patient can be considered the seriousness of symptom by the technical staff, and used concrete prescription and the other medicines that may comprise are made concrete modification.
Spray composite of the present invention can be liquid and powder.They can pass through nose or through port (intranasal or oral cavity) medication.An example of spray composite be can at nose and mouthful spray, it is by by producing from aerosol apparatus the water of TNFR-Ig goods or the mechanism of saline solution.Another example is the atomisation agent, and wherein spray produces from this solution, can be sucked (not being that nose and oral cavity are entered in direct injection) by patient effectively then.If TNFR-Ig is pulverous, granule is should be enough big can not to suck the back by patient expired so that be kept in the respiratory tract.According to the medical scheme that is used for treatment fast or prevention, this spray can be by sucking one or many or frequency still less the every day that patient is introduced.For the spraying medication, can be by sucking treatment or preventing that the TNFR-Ig of the present invention of aforesaid asthma effective dose from coming the medication to patient.For asthma, make the affected part of respiratory tract (nasal cavity, trachea, rudimentary gas passage or bronchioles) accept the TNFR-Ig of appropriate amount by this way.
Sprayer formulation of the present invention preferably provides by weight from about 0.03% to 5.0%, more preferably about 0.03 to 0.5%, particularly about 0.1 to 0.3% of goods.Preferred spraying preparation is the aerosol preparation, in general, for spray, preferably than the low dosage scope, the preparation that for example atomizes, for aerosol, preferred higher dosage range.Can use any conventional aerosol preparation so that the TNFR-IG goods of effective dose to be provided in the present invention, the said products amount preferably is provided.By discharging the vaporific thing of aerosol of measuring dosage at patient's mouth when the patient respiration, spray is sucked the oral cavity and enters lung by respiratory tract use these goods most effectively.As mentioned above, effective dose can be once or several times with littler dosage in one day through the oral cavity or Nasacort, for the dosage medication of quick treatment with every day, for prevention with weekly to every month interval medication.
Suitable aerosol preparation comprises the medicinal activity compound (for example, TNFR-Ig goods) of suitable dosage and any conventional aerosol propellants of effective dose.Also can comprise any conventional surfactants.TNFR-Ig can with as the liquid in the solution or as the combination of the propellant of powder (for example, method lyophilizing) to know.Propellant can be suitably liquefaction.
Soluble or dispersible those surfactants in propellant are more effective.The surfactant that solubility is bigger in propellant is the most effective.In addition more importantly, surfactant should be nonirritating and avirulence.
Can use the acceptable any conventional propellant that is used for Pharmaceutical composition known to the skilled liquefaction or non-liquefaction.According to TNFR-Ig is powder type or liquid form, and propellant can be the propellant that is suitable for the propellant that uses with powder or is suitable for using with liquid actives.The liquefied propellant that adopts can be to be the propellant of gas down in room temperature (65) and atmospheric pressure (760mm mercury column), that is, under atmospheric pressure it can have boiling point and the avirulence that is lower than 65.Adoptable suitable liquefied propellant comprises and contains the nearly lower alkane of 5 carbon atoms, as butane and pentane.The example of liquefied propellant is fluorizated and the chlorating lower alkane of fluorine, as the propellant of selling with trade mark " Freon ".Can suitably adopt the mixture of above-mentioned propellant.
The present invention has been carried out general description now, will be more readily understood the present invention by the reference the following examples, it is not to plan to limit the present invention that these embodiment provide in the mode of illustration, except as otherwise noted.
Embodiment
Embodiment 1: the plasmid that is used to produce the coding TNFR-Ig of TNFR-Ig
A. cell line
Chinese hamster ovary (CHO) cell line as mammalian host cell line derives from CHO-K1 (ATCC number: CCL61 CHO-K1).CHO-K1 saltant dihydrofolate reductase (DHFR-) the deficient cell system that uses called after CHO-K1DUX-B11 (DHFR-) then is (available from doctor L.Chasin of Columbia University; Simonsen, C.C and Levinson, A.D., institute of (1983) NAS reports 80:2495-2499; UrlaubG., and Chasin, L., institute of (1980) NAS reports 77:4216-4220) by carrier (Sures etc. with the former precursor cDNA of insulin-containing; (1980) science, 208:57-59) transfection obtains the cell line that insulin requirements reduces.The selection clonal growth of called after dp12.CHO needs glycine, hypoxanthine and thymidine, thus confirm that they are DHFR -Genotype.
B. solubility 1 type TNFR-IgG 1Chimeric structure
By extracellular domain and IgG with people 1 type TNFR 1Heavy chain hinge region and C H2 and C HThe gene fusion of 3 domains (TNFR1-IgG hereinafter referred to as 1) make up solubility 1 type TNFR-IgG 1Chimera.As selection, also can use hinge region and the CH2 and the CH3 domain of IgG3 heavy chain.
The DNA sequence (see Loetscher etc., ibid) of coding people 1 type TNFR is from plasmid pRF-TNFR[Schall etc., cell, 61,361 (1990)] obtain.For making up this initial plasmid, 2.1kb Placenta Hominis cDNA clone (Schall etc., ibid) is inserted among the mammalian expression vector pRK5, it is structured in the EP publication number 307,247 and describes.This cDNA starts from 64 nucleotide places of the sequence that Loetscher etc. reported, initial methionine is positioned at 118bp place, downstream.
IgG 1The source of coded sequence is CD4-IgG expression plasmid pRKCD4 2F C1[Capon, D.J etc., nature, 337,525 (1989); Byrn etc., nature, 344,667 (1990)], this plasmid contains coding and human IgG 1The cDNA sequence of the hybridization polypeptide that the ripe human CD 4 protein matter 1-180 residue that sequence merges (2 terminal CD4 variable domains of N-) is formed, this IgG 1Sequence is at 216 aspartic acids initial (with 1st residue of 114 amino acids as CH) [Kabat etc., immunology protein of interest matter sequence, the 4th edition (1987)], i.e. IgG after relating to the bonded cysteine residues of weight-light chain 1The 1st residue of hinge) and in 441 residues stop to comprise IgG 1C H2 and C H3 Fc domains.Also see EP227110.As selection, can use pCD4-H γ 3 carriers (DSM5523, EP394,827).Remove CD4 cDNA to obtain required IgG3 sequence by Sst I enzyme action.
By producing plasmid pRK-TNFR and pRKCD4 2F C1Restriction fragment and connect them, use deletion mutagenesis to make 171 threonine residues and the IgG of mature T NFR ' s 1216 asparagicacid residues of heavy chain [Kabat etc., ibid] side by side make up TNFR1-IgG 1The plasmid pRKTNFR-IgG of gained contains TNFR 1-IgG 1Whole length of coded sequence.Then can be by this plasmid transfection being advanced in the host cell such as Chinese hamster ovary celI such as the conventional method of calcium phosphate precipitation.Available conventional method is cultivated the cell of gained to express TNFR-IgG, separates it with conventional method then.
Embodiment 2: have approximately: every protein 5-6M. sialic acid, every protein 0.4-0.45MN-acetylglucosamine, the production of the TNFR-Ig of the pI of 5.5-7.5
Cell culture
By transfection with coding solubility 1 type TNFR-IgG among the embodiment 1 1Gene import in the dp12.CHO cell.Use is finished transfection with the calcium phosphate technology that DNA imports in the mammalian cell.After the transfection 2 days, with trypsin digestion and cell and be applied to again and select culture medium (to add the no glycine-hypoxanthine of 2% dialysis serum and Ham ' the s F-12 DMEM of thymidine, 1: 1v/v).Subsequently, filter out TNF secretion R1-IgG 1Separated strain.The expression TNFR1-IgG that in methotrexate, increases 1Be cloned in and produce high-expression clone, be adapted to culture medium in the serum-free subsequently.These cells are under the Continuous Selection pressure and carry out the growth and the amplification of inoculum up to being transferred to non-selective culture medium.
Produce TNFR1-IgG in order to provide 1Cell culture, above-mentioned cell population goes down to posterity to cultivate and is expanded to the growth medium that does not contain methotrexate from the culture medium that contains methotrexate by carry out series in the container that constantly increases volume.For these steps of this method, non-selective growth medium is the preparation (for example, seeing United States Patent (USP) 5,122,469) based on DMEM/HAMF-12, the concentration of some of them composition changes to some extent, as, glucose, aminoacid, salt, sugar, vitamin, glycine, hypoxanthine, and thymidine; The recombinant human insulin, hydrolyzed protein peptone (Primatone HS or Primatone RL), cytoprotective such as Pluronic F86 (poly alcohol) or coordinate; Gentamycin; Lipid and trace element.
By using CO 2Gas (acid) and/or Na 2CO 3(alkali) is controlled at pH7.2+/-0.4 with culture.Cell growing period temperature is controlled at about 37 ℃.Make dissolved oxygen maintain more than 5% of air saturation by directly spraying into air and/or oxygen.Osmotic pressure maintains between about 250mOsm and the 350mOsm during the inoculum amplification.
Behind each trophophase of cultivating is the second phase or transition period, and wherein culture parameters is a working condition from the suitableeest growth alteration.Temperature in culture systems during this transition period generally is reduced between about 30 and 35 ℃.Add butyrate, thereby guarantee certain Osmolality scope.The sialic acid content of the product that analysis accumulated during this campaign.
In typical production routine, produce about 1.2 * 10 from the inoculum amplification of selecting the phase 6Individual cell, wherein the starting weight Morie osmolarity in trophophase growth is 300-450mOsm, preferably approximately 300.Growth medium is added trace element, recombinant human insulin and hydrolyzed protein peptone.Cell was grown two days with this understanding.Reduce the temperature of cell culture during beginning in the 3rd day.In the time of temperature change or afterwards, in cell culture, add about 1 to 6mmol sodium butyrate, preferred 6mmol and guarantee to produce required Osmolality by adding various medium components.The cultured cell growth is 9-10 days under these conditions.When needs, provide cell various medium components.
The pI of high sialic acid compositions is lower than the pI of low sialylated compositions.Compositions is carried out isoelectrofocusing.The pH gradient that the isoelectrofocusing gel uses the ampholyte of different pH to produce is separated the glycoprotein of compositions according to its isoelectric point, IP pI.The pH gradient of use 10 to 4 is carried out the compositions analysis.
Measuring isoelectric point, IP scope that above-mentioned composition shows by chromatofocusing is about 5.5 to about 7.5, and wherein pI handles responsive to neuraminidase.
The recovery of TNFR-IgG
As nature such as Capon, 337:525,1989 is described, can come the IgG purification fusion rotein by the protein A post by the centrifuge cell culture and with the culture supernatant of gained.Therefore, the cell culture of centrifugal above-mentioned acquisition and supernatant crossed the protein A post.This affinity chromatography by as by Capon etc., described TNFR1-Ig goods purification on the immobilization staphylococcus aureus protein A reaches surpasses 95% homogeneity but ibid.
Saccharide is analyzed
A. sialic acid content can be used Warren, L. (1959), and the method for journal of biological chemistry 234:1971-1975 is analyzed.
B. the complete neutrality and the release of amino sugar can detect in conjunction with the pulse ammeter by high pH anion-exchange chromatography and measure.Use following step to analyze:
1. carry out buffer-exchanged with TNFR1-IgG1 (about 50 μ g/ml) and suitable reference sample, so that final sample is contained in 1% acetic acid.
2. with about 90 μ g TNFR1-IgG 1And be frozen in dry ice and the ethanol bath with reference to sample, freezing sample lyophilizing is spent the night.
3. lyophilizing sample preparation and 120 ℃ of incubations 1 hour again in 500 μ l trifluoroacetic acids.
4. acid hydrolysis postcooling TNFR1-IgG 1With reference sample and evaporation drying.
5. samples with water is prepared again to final concentration and is approximately 0.6mg/ml.
By use Dionex CarboPac Pal (4 * 250mm) posts (and Dionex company, the U.S., the California, Sheng Niweier) the high pH anion-exchange chromatography that detects with the pulse ammeter at room temperature carries out the separation of monosaccharide.
7. by comparing the amount of determining every part of monosaccharide with reference monosaccharide.
The preparation of embodiment 3.TNFR-Ig
1. using the coprecipitation of calcium phosphate method is DP12 (EP307,247) with plasmid pSV16B.TNFR-IgG (coding TNFR1-IgG1) and pFD11 (encoding D HFR) transfection CHO cell.
2. the cell clone of transfection acquisition is expanded to 9 liters of suspension cultures with the selection culture medium that contains 2% diafiltration Ox blood serum and 100nmol/L methotrexate.Serum-free medium with no methotrexate is seeded to two 9 liters of cultures in 100 liters of fermentation tanks.With the washing of 1000 times production culture medium with after reducing residual serum composition, seed cells into 1000 liters produce culture vessels before, 100 liters of cultures are inoculated in 400 liters of cultures with serum-free medium.The production culture medium is supplemented with glucose, aminoacid, glycine, hypoxanthine, thymidine, hydrolyzed protein peptone, pluronic F68, gentamycin, lipid and trace element for the preparation based on DMEM/HAM F-12.
3. produce culture CO 2Gas and/or Na 2CO 3Maintain following 13 days of pH7.2+ or-0.2 condition.Reach 5 * 10 at cell concentration 6Behind cell/ml, cultivation temperature changes to 30 ℃ from 37 ℃.By adding the culture medium composition Osmolality of culture was adjusted to about 450mOsm/kg the 2nd day and the 4th day.Adding sodium butyrate to ultimate density is 12mM.By spray into to culture air and or oxygen make dissolved oxygen concentration maintain 60% gas saturation.
4.13 culture is collected in it back, separates from supernatant by the tangential filtration cell that flows.About 20 times of the Maxisette membrance concentration of the cell culture fluid of these results (HCCF) usefulness 10KD.By adding solid NaCl to 1.0M, filter by 0.22 micron Durapore filter subsequently and clarify residue and control its condition to carry out the protein A chromatography.
5. after filtering, will transfer the HCCF of consumption condition by fixed protein A.Last sample material washs several times before eluting.TNFR-IgG 0.05M sodium citrate/20% (w/v) glycerol, pH3.0 is eluting progressively.By adding the 1M sodium citrate eluting amalgamation liquid is adjusted to pH5.0.
6. after regulating pH, diluted protein A amalgamation liquid is also gone up sample to S-Sepharose Fast Flow.Sample is used 0.05M MOPS/0.05M NaCl pH7.2 stepwise elution after washing.
7. behind the stepwise elution, dilution S-Sepharose Fast Flow amalgamation liquid is gone up sample then to Q-Sepharose Fast Flow post.Use this post of linear gradient elution in 0.125M MOPS (pH7.2) from 0.0125M NaCl to 0.125M NaCl.
8. behind the linear gradient elution, (pH5.0 regulates Q-Sepharose Fast Flow amalgamation liquid to 0.1 sodium acetate/4.0M carbamide/2.0M ammonium sulfate by adding 1 volume, goes up sample then to on the phenyl Toyopearl650M post that 0.05M sodium acetate/2.0M carbamide/1.0M ammonium sulfate (pH5.0) balance is crossed.TNFR-IgG flows through this post under these conditions.Sample with the level pad flushing, merges effluent and flushing liquor subsequently, forms phenyl Toyopearl amalgamation liquid.
9. merge two phenyl toyophearl amalgamation liquids and use 10KD Filtron Centrassette membrance concentration.Concentrated solution by the G-25 post crossed with balance in 0.01M sodium citrate/0.023M glycine/0.023M mannitol (pH6.0) to produce the TNFR-Ig compositions.
Experiment showed, TNFIg relieving asthma outbreak effect of the present invention in the described body of following embodiment 4-10.But TNF receptor fusion protein TNFR-Ig obviously alleviates, even eliminates the reaction that antigen stimulation causes in the hypersensitivity pneumonitis animal model in some cases.The inhibiting degree of TNFR-Ig can be comparable to the degree that obtains with Broadspectrum antiphlogistic medicine dexamethasone.
Abbreviation: TNF α, tumor necrosis factor-alpha; TNFR-Ig, the TNF receptor IgG1 fusion rotein of the solubility of reorganization; BAL, the trachea bronchoalveolar lavage fluid; OA, ovalbumin; R L, lung resistance; HBSS, the Hanks balanced salt solution; Substance P, a kind of neuropeptide that is used to induce the acute bronchus spasm is called Substance P usually in the literature and (for example sees Selig and Tocker, European materia medica magazine 213 (3): 331-336 (1992)).
Data analysis: test at every turn all numerical value all calculate on average+/-the SEM value.Significant difference is checked by use student Newman-Keuls t-check or by the multiple comparisons of student t-check subsequently and is measured by the variance of two kinds of method repeated measure analysis ranked datas.It is remarkable statistically that P＜0.05 is thought.Calculating be used in the EXCEL5.0 of Microsoft that moves on the PC (softmart, Exton, PA, USA) and Sigma Stat (Jandel Scientific, SanRafael, CA, USA) software kit carries out.
Medicine: all medicines are all with the dosage medication of 1mg/kg.TNFR-Ig presses embodiment 3 described production and purification, and storage liquid is at sodium citrate (10mM), and glycine (23mM) and mannitol (230mM) (prepare in the pH6 buffer.Store and in Sterile Saline, dilute at-20 ℃ before the aliquot sample of storage liquid is used.(St.Louis, MO USA) buy and prepare in Sterile Saline following article: ovalbumin, aluminium hydroxide, urethane, Substance P acetas, (+/-) propranolol hydrochloride, dexamethasone 21-phosphate ester and Yi Wensilan from Sigma chemical company.
Embodiment 4: alleviate Cavia porcellus symptoms of asthma (trachea anaphylaxis) with TNFR-Ig
Present embodiment uses OA is produced Cavia porcellus hypersensitive, makes that contacting OA subsequently will induce the anaphylactoid symptoms of asthma of trachea.This symptom can be alleviated by TNFR-Ig, its effect also with dexamethasone, a kind of steroid of known relieving asthma compares.
Male guinea pig was at 0 day (10 μ g+1mg Al (OH) 3In 0.5ml saline) OA, subcutaneous injection) sensitization, the OA that accepted same dosage after the 14th day is as booster injection.Excite 30 minutes the 21st day animal with the 0.1%OA aerosol.This sensibilization causes Cavia porcellus asthma sample symptom, comprises the trachea allergy to Substance P.In order to measure the influence of TNFR-Ig to this symptom, before exciting, use TNFR-Ig, its symptom and unexcited animal are compared and be shown as alleviate.Use dexamethasone (30mg/kg, lumbar injection excite preceding 1 hour and back 4 hours) that trachea allergy is obtained similar effects.Dexamethasone is a kind of known treating asthma agent.
By sensitization, excite and the Cavia porcellus for the treatment of processing is used for following experiment.
Sensitization: male Hartley strain Cavia porcellus (the Charles River of heavy 250-300 gram, kingston, NY, USA) the 0th day and the 14th day with single agent subcutaneous injection (10 μ gOA+1mg aluminium hydroxide are in the 0.5ml Sterile Saline) energetically to the OA sensitization, between the 21st day and 28 days, be used for experiment.
Excite: the antigen stimulation scheme was existing in the past to be described (O ' Donnell etc., 1994).The animal of sensitization is placed in the lucite case, excites 30 minutes with 0.1% (w/v Sterile Saline) OA aerosol.Aerosol (is breathed service centre, Ephrata, PA, USA) 30L/ minute the air velocity transmission that drives with built-in fans with De Vilbiss Ultra-Neb 100 ultrasonic nebulizers.For the death toll that anaphylactic reaction is caused is reduced to minimum, when initial administration in 5 minutes, aerosol apparatus carries out 60 seconds switch circulation at interval, and the animal dead rate is approximately 5% under these conditions.
Drug therapy: preceding 18 and 1 hours of OA spraying, animal groups or accept carrier (seeing medicine), or accept TNFR-Ig (1 or 3mg/kg, lumbar injection).For the experiment of carrying out with dexamethasone, sprayed preceding 1 hour or 4 hours afterwards animal groups are accepted carrier (saline) or dexamethasone (30mg/kg, intraperitoneal) respectively at OA.Determine the optimal dose parameter of TNFR-Ig and dexamethasone according to preliminary observed result.
The result of present embodiment shows through contact OA OA Cavia porcellus hypersensitive 6 hour meters after OA excites is revealed trachea increased response to Substance P (1-10ug/kg intravenous injection).Intensified response to Substance P is the characteristic of asthma sample sensitization, shows that Cavia porcellus has by the inductive anaphylactoid symptoms of asthma of above-mentioned experiment condition.Anaphylaxis is suppressed by TNFR-Ig (3mg/kg, intraperitoneal excite preceding 18 and 1 hours).
Using method is as follows: trachea is measured in 6 hours sensitization unexcited and the Cavia porcellus that excites behind contact OA aerosol according to preceding method (Selig and Tocker, 1992) the reaction of Substance P.Animal is cooked carotid artery (blood pressure) and jugular vein (medicine injection) intubate with urethane (2g/kg, intraperitoneal) anesthesia with the PE-50 conduit.Trachea is with No. 15 needle cannula, animal put into the overall volume tracer (Modular Instruments, Malvern, PA, USA) in.Suppress spontaneous respiration with choline succinate dichloride dihydrate (1.2mg/kg, intravenous injection), use the tidal volume of 1ml/100g body weight and the frequency of 60 breaths/min to give animal mechanical ventilation (Harvard ApparatusModel 683; South Natick, MA, USA).The plethysmograph cumulative volume is 2L, and the volume of vent line is 10ml between animal and breathing equipment.Plethysmograph is equipped with the Fleisch pneumotachograph (Model#0000) that links to each other with Validyne dividing potential drop transmitter (DP45-14) and is used to measure air-flow.By being connected the tracheal intubation side arm and being inserted in the mensuration of the 2nd Validyne transmitter (DP45-28) between syringe needle transpulmonary pressure in the 5th and the 6th intercostal No. 16 pleuras.Use the Modular Instruments (Malvern, PA, USA) M-3000 data acquistion system record air-flow and the transpulmonary pressure that drive by IBM 486DX2 PC.Computer utilizes conventional software (BioReport, Modular Instruments) to carry out lung resistance (R according to the method for AmdurMead (1958) L) calculating.Reading carries out continuously with average interval more than 10 seconds.Proofread and correct R LValue is as the internal drag of trachea (0.11cm water column/ml/ second).Animals received propranolol (1mg/kg, intravenous injection) also stablized them 10 minutes before beginning application of substances P.
Provide large bolus injection to obtain the dose-response effect of Substance P (1,3,5 and 10 μ g/kg, intravenous injection) by interval with about 5-10 minute.Obtain each dosage R LMaximum change and represent with the percent of the basic value measured before the application of substances P.ED 200Value defined is for causing R LThe dosage of the Substance P of rising 200% is by measuring the linear regression of each animal log10 dose-response curve.
R LBasic value be about 0.30cm water column/ml/ second, do not have significant difference (P＞0.05) between group.Sensitization and unexcited Cavia porcellus is quite insensitive to Substance P, need the dosage of 30 μ g/kg to make R LRising at least 200% or bigger (table 1).On the contrary, after OA excited, trachea significantly raise to the reaction of Substance P.The R that obtains with the maximal dose of Substance P LWhen maximum raises about 5 times (tables 1), ED 200About 10 times (table 1) of value rising.
Table 1:TNFR-Ig and dexamethasone influence the Cavia porcellus of OA sensitization to Substance P aThe summary of trachea responsing reaction.Treatment b-logED 200 cMaximum responsing reaction dn e
(R LRising %) is not stimulated f4.86+/-0.08 139+/-28 6TNFR-Ig carrier 5.82+/-0.04 1811+/-228 71mg/kg 5.87+/-0.01 1344+/-195 63mg/kg 5.48+/-0.05 *714+/-69 *5 dexamethasone carrier 5.87+/-0.05 935+/-91 530mg/kg 5.35+/-0.04 *596+/-66 *6
aThe dose-response effect of Substance P is 6 hours mensuration after exciting 30 minutes with the 0.1%OA aerosol.Before checking the responsing reaction of trachea, use propranolol (1mg/kg, intravenous injection) pretreatment animal 10 minutes to Substance P.
bUse the dosage of 1ml/kg, with carrier separately, TNFR-Ig (OA excites preceding 18 and 1 hours) or dexamethasone (OA excites preceding 1 hour and 4 hours afterwards) are through intraperitoneal pretreatment animal.
cValue is represented lung resistance (R L) increased the negative logarithm (average+/-S.EM. value) of 200% required Substance P dosage (with g/kg) of basic value.
dValue (average+/-S.E.M. value) is represented the basic R that is caused by Substance P (10 μ g/kg, intravenous injection) LRising percent.
eEvery treated animal number.
fThe dose-response effect curve of Substance P is measured in the sensitization animal groups that does not excite with the OA aerosol.
*Value between respective carrier and the drug treating group has the significantly difference of (P＜0.05) statistically.
Use TNFR-Ig (3mg/kg) remarkable (P＜0.05) for the sensitization animal and suppress of the anaphylaxis of the inductive respiratory tract of OA Substance P.The ED of Substance P 200Value reduces about 3 times, R LMaximum reduces about 60% (table 1).The TNFR-Ig of smaller dose (1mg/kg) does not have influence (table 1) to the sensitivity of material.In the sensitization Cavia porcellus, handle also to suppress the inductive respiratory tract anaphylaxis reaction of OA with heavy dose of similarity degree (table 1) that TNFR-Ig was obtained remarkable (P＜0.05) with dexamethasone (30mg/kg).
Embodiment 5: with the Cavia porcellus symptoms of asthma (inflammatory cell inflow) of TNFR-Ig alleviation
Present embodiment use embodiment 4 described to OA Cavia porcellus hypersensitive so that will induce symptoms of asthma when contacting OA subsequently, promptly inflammatory cell flows into.This symptom is alleviated by TNFR-Ig, and also with itself and dexamethasone, a kind of steroid of known relieving asthma compares.
In embodiment 4, make male guinea pig excite 6,24, the 48 and 72 hours quantitative respiratory inflammation cells in back to accumulate to the OA sensitization and by described exciting to induce symptoms of asthma, to comprise.In embodiment 4, in order to measure the influence of TNFR-Ig, before exciting, use TNFR-Ig to these symptoms, with unexcited animal relatively, these symptoms and demonstrate sx.Also use TNFR-Ig and be used for inflammation before exciting, the result shows that reversing inflammatory cell flows into.Obtained the cumulative similar influence of inflammatory cell with 24 hours 6 with dexamethasone (30mg/kg, intraperitoneal excite preceding 1 hour and 4 hours afterwards).
Using method is as follows: OA excite back 6 and 24 hours according to before described method (Selig and Tocker, 1992) measure the respiratory inflammation cell by BAL and flow into.Briefly, Cavia porcellus makes incision of trachea with urethane (2g/kg, intraperitoneal) anesthesia with No. 15 intubate.Do not contain Ca with 3 * 5ml 2+And Mg 2+Aseptic Hank ' s balanced salt solution (HBSS) (Gibco, GrandIsland, NY, USA) lavation lung.Sample is at 25 ℃, and centrifugal 10 minutes of 200g with distilled water (1ml, 30 minutes) molten erythrocyte of opening from the precipitation of gained, adds 10mlHBSS then and recovers Osmolality.Centrifugal (under 25 ℃, 200g, 10 minutes) sample for the second time, the precipitation of gained is suspended among the 1ml HBSS again.With hematimeter from the aliquot sample of cell suspending liquid with platform expect orchid (Sigma chemical company, St.Louis, MO, USA) repelling attack is measured total cell number.Be the classification cell counting, with the aliquot sample of cell suspending liquid centrifugal in Cytospin (5 minutes, 1300rpm; Shandon Southern Instruments, Sewickey, PA, USA), and smear, fixing, with improvement Wright's stain (Leukostat; FisherScientific, Pittsburgh, PA, USA) dyeing.Under light microscopic, adopt the standard type criterion at least 300 cells of classification.Data are expressed as BAL cell * 10 6/ animal.
Handle (18 and 1 hours and pretreatment in 1 hour, for BAL, at 6 and 24 hours, significantly (P＜0.05) suppressed behind the OA accumulation of neutrophil cell and total cell number among 6 and 24 hours BAL respectively for 1-3mg/kg, intraperitoneal) with TNFR-Ig.TNFR-Ig (3mg/kg, intraperitoneal) is eosinophilic granulocyte's number of 2 time point BAL of remarkable (P＜0.05) minimizing also, and low dosage (1mg/kg, intraperitoneal) does not have influence.This experimental result shows that also the neutrophilic granulocyte composition is mediated by TNF in the inflammatory reaction of allergen-induced.Be apparent that most that behind antigen stimulation 24 hours, TNFR-Ig almost eliminated the inflow of neutrophilic granulocyte of the BAL of sensitization Cavia porcellus, wherein the neutrophilic granulocyte number accounts for about 2% of total cellular score among the BAL.This external TNFR-Ig rather than dexamethasone are handled, and are exciting back 6 hours, cause that the Cavia porcellus neutrophilic granulocyte descends in a large number.By TNFR-Ig or similar antagonist blocking-up TNF, can cause the known neutrophilic granulocyte into indirect decline of the factor of lung that refluxes that helps.
Sensitization, before forming, the cell of unexcited Cavia porcellus BAL is described (Selig and Tocker, 1992).Total cell counting average out to about 1 * 10 in these animals 6/ animal, wherein approximately 2-3% is eosinophilic granulocyte and neutrophilic granulocyte.OA handled back 6 hours, contained at least 40 and 20% eosinophilic granulocyte and the neutrophilic granulocyte that contain total cell count respectively in the animal of the vehicle treated of TNFR-Ig or dexamethasone.Eosinophilic granulocyte's percent kept constant at 24 hours among the BAL, and the neutrophilic granulocyte counting then drops to about 10% of total cell number.Inflammatory cell number and total cell number are at the zero difference (P＞0.05) between the vehicle group separately of TNFR-Ig and dexamethasone.
Inflammatory cell flows into sensitization, and the Cavia porcellus BAL that excites also is subjected to the inhibition of dexamethasone (30mg/kg), and OA handled back 6 hours, and eosinophilic granulocyte and total cell number have significantly the decline of (P＜0.05) among the BAL, and this and the caused situation of TNFR-Ig are similar.During 6 hours time points the neutrophilic granulocyte number there is not influence.OA handled back 24 hours, eosinophilic granulocyte among the BAL, and neutrophilic granulocyte and total cell number are significantly suppressed by dexamethasone (P＜0.05).Compare with TNFR-Ig, the animal eosinophilic granulocyte that dexamethasone is handled reduces about 5 times (P＜0.05).On the contrary, reduced the neutrophilic granulocyte number among the BAL although dexamethasone is handled, these cell percentage ratios remain unchanged (P＞0.05) in different cells, are 8% of total approximately cell number.
In the sensitization Cavia porcellus, check the ability of TNFR-Ig retrotransposon activity inflammatory reaction.Make animal to the OA sensitization, excite as mentioned above then.After exciting 30 minutes, uses OA TNFR-Ig (3mg/kg, intraperitoneal).Exciting back 24,48 and 72 hours by BAL detection inflammation.For 48 and 72 hours time points, TNFR-Ig administration every day.Excite the back to handle the inflow that obviously reverses inflammatory cell among the sensitization Cavia porcellus BAL with TNFR-Ig.
Embodiment 6:TNFR-Ig is alleviated the symptoms of asthma (pulmonary edema) of Cavia porcellus
Present embodiment uses embodiment 4 described, will induce symptoms of asthma, i.e. pulmonary edema to OA Cavia porcellus hypersensitive so that contact OA subsequently.This symptom is alleviated by TNFR-Ig, and also with itself and dexamethasone, a kind of steroid of known relieving asthma compares.
In embodiment 4, make male guinea pig excite back 6 hours quantitative edema to the OA sensitization and by described exciting to induce symptoms of asthma, to be included in.In embodiment 4, in order to measure the influence of TNFR-Ig, use TNFR-Ig before exciting to these symptoms, these symptoms and unexcited animal are compared and show to some extent alleviate.
Using method is as follows: described method (Wasserman etc. before OA handles and used in back 6 hours, prostaglandin, thromboxane, leukotriene progress, 23:271-273,1995) with the Yi Wensilan dyeing liquor respiratory tract blood capillary seepage of standard measure of exosmosing as the pulmonary edema sign.The jugular vein intubate is anaesthetized and done to Cavia porcellus with urethane (2g/kg, intraperitoneal).Then animals received during 60 seconds in the Yi Wensilan dyeing liquor (30mg/kg, intravenous injection) of administration.Open the thoracic cavity after 10 minutes and conduit (PE-240 pipe) is inserted aorta by left ventricle.Intersect and to clamp ventricle, cut right atrium and by making medication tube pump (Masterflex; Cole-Parmer, Chicago, IL USA) discharges blood with 100ml saline with 100ml/ minute speed perfusion animal.By at the pulmonary artery interpolation pipe and cut left atrium, the pulmonary circulation of reuse 50ml perfusion of saline.The whole lung that takes out downcuts trachea (terminal 5mm) and main bronchus, blots between filter paper and weighs.In Methanamide, extract Yi Wensilan dyestuff (37 ℃, 24 hours) also by (NJ USA) surveys absorptance and carries out quantitatively under 620nm for BeckmanInstruments Model DU-64, Somerset with spectrophotometer.Tissue stain's content is by obtaining on Yi Wensilan dye strength (the 0.5-10 μ g/ml) standard curve and representing with the ng/mg tissue wet.
When using the Yi Wensilan dyestuff as trachea blood capillary infiltration labelling, sensitization, the basic value average out to 10-20ng/mg tissue wet of unexcited guinea pig trachea and main bronchus.OA excites back 6 hours, and Yi Wensilan dyestuff content has raise about 5 times in trachea and the main bronchus.Handle exciting back 6 hours with TNFR-Ig (1 or 3mg/kg) or dexamethasone (30mg/kg) and to have weakened the inductive respiratory tract infiltration of OA in (P＜0.05) trachea and the main bronchus.
TNFR-Ig (1 or 3mg/kg. intraperitoneal) has eliminated the inductive respiratory tract edema of OA in sensitization guinea pig trachea and the main bronchus (tissue content with the Yi Wensilan dyestuff comes quantitatively).
Embodiment 7: alleviate brown Norway rat symptoms of asthma with TNFR-Ig
Similar to the Cavia porcellus of using among the embodiment 4, in rat, induce inflammatory cell accumulation (a kind of symptoms of asthma) and treat with TNFR-Ig.This model is that with the difference of Cavia porcellus the anaphylaxis of brown Norway rat is mediated by IgE.
Using method is as follows: make male brown Norway rat at the 0th, 1 and 2 day to OA (1mgOA+100mgAl (OH) 3Be dissolved in the 0.5mg saline intraperitoneal) sensitization.At the 21st day, excited animal 30 minutes with the 1%OA aerosol.Excite back 24 hours and come quantitative inflammatory cell accumulation with BAL.Sensitization excites scheme with BAL similar in appearance to above described to Cavia porcellus, but following exception is arranged.Male brown Norway rat (the Charles River of body weight 200 to 250g, Kingston is NY) the 1st, 2, activated its sensitization be used for experimentizing (Elwood etc., 1992) with single agent peritoneal injection (the 1mgOA+100mg aluminium hydroxide is dissolved in the 1ml Sterile Saline) in 3 days at the 21st day to OA.OA sprayed preceding 1 hour, and each is organized rat and accepts separately carrier respectively, TNFR-Ig (1 or 3mg/kg, intraperitoneal) or dexamethasone (0.3mg/kg, intraperitoneal).
Test the same day, rat excites 30 minutes with the 1%OA aerosol.Excite back 24 hours, carry out BAL by HBSS lavation lung with 2 * 1ml/100g.With 0.5ml dissolved in distilled water erythrocyte, recover Osmolality with 5ml HBSS.
Handling above-mentioned rat with TNFR-Ig (3mg/kg, intraperitoneal excite preceding 1 hour) has eliminated the accumulation of neutrophilic granulocyte among the BAL basically and has caused eosinophilic granulocyte's number and total cell number significantly descends.Obtain similar result with dexamethasone (0.3mg/kg, intraperitoneal excite preceding l hour).TNFR-Ig (1mg/kg, intraperitoneal) than low dosage has also obviously reduced the neutrophilic granulocyte number among the BAL, but eosinophilic granulocyte and total cell number are not had influence.
In more detail, sensitization, unexcited brown Norway rat total cell count basic value in BAL is about 1 * 10 6/ animal, wherein 1-2% is eosinophilic granulocyte and neutrophilic granulocyte in proportion.OA excites back 24 hours, and total cellular score has raise about 3 times among the animal BAL of vehicle treated, and wherein eosinophilic granulocyte and neutrophilic granulocyte account at least 40% and 25% of total cellular score respectively.Inflammatory cell counting and total cell number are at zero difference (P＞0.05) between the vehicle treated group separately among the BAL.
All reduced eosinophilic granulocyte among the BAL with TNFR-Ig (3mg/kg, intraperitoneal) or dexamethasone (0.3mg/kg, intraperitoneal) processing, the accumulation of neutrophilic granulocyte and total cell number with similar degree remarkable (P＜0.05).(P＜0.05) has suppressed neutrophilic granulocyte number among the BAL with handle also significantly than the TNFR-Ig (1mg/kg, intraperitoneal) of low dosage, but the accumulation of eosinophilic granulocyte among the BAL or total cell number is not had influence.
Embodiment 8: the minimizing of neutrophilic granulocyte in the injured induced lung
Eosinophilic granulocyte and neutrophilic granulocyte number rise and granuloma forms, this and being similar to seen in the chronic asthma in the inductive pneumonia of rats models show of Sephadex (polydextran gel) the granule lung.This model is used to show that TNFR-Ig alleviates this chronic sympton.
Using method is as follows: (7.5mg/kg .iv.) induces the accumulation of inflammatory cell in lung in male Sprague-Dawley rat by using Sephadex (polydextran gel) particle suspension.Used Sephadex (polydextran gel) back 24 hours and 72 hours, total white blood cells significantly raises in BAL liquid.In the time of 24 hours, the neutrophilic granulocyte number accounts for about 50% of total white blood cells, then drops to about 10% of sum during by 72 hours.Eosinophil count maintains about 10% of total white blood cells.
With TNFR-Ig (1 and 3mg/kg, .ip., excite preceding 1 hour) or dexamethasone (0.1 and 0.3mg/kg, intraperitoneal) pretreatment suppressed neutrophilic granulocyte in back 24 hours using Sephadex (polydextran gel), although TNFR-Ig pair cell sum does not have obviously influence.Used Sephadex (polydextran gel) back 72 hours, TNFR-Ig (1 and 3mg/kg, intraperitoneal, every day) obviously reduces the neutrophilic granulocyte that flows into BAL liquid, but the eosinophilic granulocyte is counted the unrestraint effect.On the contrary, dexamethasone (0.1 and 0.3mg/kg, intraperitoneal, every day) is eliminated neutrophilic granulocyte and the eosinophilic granulocyte infiltration to BAL liquid basically.TNFR-Ig (1 and 3mg/kg, intraperitoneal) or dexamethasone (0.1 and 0.3mg/kg, intraperitoneal) obviously reduce total cell count at 72 hours time points.
Embodiment 9: the alleviation of primates idiosyncrasy asthma
Employing shows the effect that idiosyncrasy asthma primates model that the macaque that the field of natural airway sensitivity catches sets up is used to prove the reaction of TNFR-Ig relieving asthma symptoms respiratory tract anaphylaxis to Ascaris suum antigen.Repeat to contact the symptoms of asthma of antigen induction respiratory tract, especially increased lung resistance (R L).Monkey shows R when handling with TNFR-Ig LDescend.
When repeating to contact A.suum antigen, the open-air wild Ascaris responsive type macaque that catches shown enhanced to the methacholine that sucks respiratory response and increased the respiratory tract eosinophilic granulocyte.Therefore use the anaphylaxis and the respiratory tract anaphylaxis reactivity of the symptoms of asthma of generation subsequently of this antigen induction in these monkeys.Following scheme is used to check the influence of TNFR-Ig to the respiratory tract anaphylaxis reaction.
The 1st day: measure the dosage of the tracheospasm derivant methacholine (MCh) of a kind of similar substance P, it produced 100% (PC 100) the lung resistance (R of rising L).
The 3rd day: with producing the R that doubles at least LAn Ascaris antigen inhalation dose excite monkey.
The 5th day and the 8th day: repeat the 3rd day experiment.
The 10th day: repeat the 1st day experiment, measure PC between the 1st day and the 10th day 100The variation of logarithm value.
The 1st, 3, used TNFR-Ig (3mg/kg, intravenous injection) and weakened the anaphylaxis of respiratory tract in 5 and 8 days MCh.
Embodiment 10:TNFR-Ig is alleviated the mouse allelgic respiratory inflammation
Use the muroid model proof TNFR-Ig of OA inducing mouse hypersensitivity pneumonitis to reduce inflammation symptoms of asthma that cell flows into.The quick variation mice that contacts OA repeatedly forms gradually that the eosinophilic granulocyte flows into increase among respiratory tract anaphylaxis and the BAL.This model and the rising of IgE level interrelate and show typical Th2 cytokine feature (raising as IL-4 and IL-5 level).In this model, estimated the ability that TNFR-Ig weakens the irritated inflammatory reaction of activeness.
The muroid model of hypersensitivity pneumonitis is similar to primates, and for inducing the inflow of respiratory tract anaphylaxis reaction and inflammatory cell, repeatedly contact allergy is former to need the animal of sensitization.(10 μ g contained 1mg Al (OH) to female C57BL/6 mice with OA at the 0th day 3Gel, 0.1ml, intraperitoneal) sensitization, then, from 14-20 days, excited 30 minute with the 1%OA aerosol every day.Respiratory tract to the anaphylaxis of MCh and BAL the last time OA excited finish in back 24 hours.Fix some and come autosensibilization, the lung that excites is used for histological examination, and other is used to measure the TNF level by homogenate.
In sensitization, inflammatory cell accumulation and TNF level peak excite (the 20th day) for the last time with the OA aerosol after in the mice that excites.In this experiment, use TNFR-Ig (3mg/kg, intraperitoneal) immediately after contacting OA the last time.TNFR-Ig has weakened the inductive anaphylaxis of OA, but inflammatory cell among the BAL is flowed into the nothing influence.Use TNFR-Ig every day at duration of exciting the BAL cell counting is not had influence yet.Obviously reduced sensitization yet use TNFR-Ig (3mg/kg, intraperitoneal, the 20th day), the eosinophilic granulocyte's number in the mouse lung tissue that excites and removed the TNF level in the lung tissue.
Embodiment 11: aerosol composition
Be the embodiment that contains TNFR-Ig goods of the present invention below
Embodiment A-aerosol
TNFR-Ig (granular size scope 1-5 micron) 3.0%
Span 85 (sorbitan trioleates) 1.0
Freon 11 (the single fluoromethane of trichlorine) 30.0
Freon 114 (dichlorotetra-fluoroethanes) 41.0
Freon 12 (dichlorodifluoromethane) 25.0
Embodiment B-aerosol
TNFR-Ig 0.5％
Span 85 0.5％
Propellant B 199.0%
1Propellant B is by 10% freon-11,50.4% Chlorfluoranum, and 31.5% Freon 12 and 8.0% butane are formed.
Embodiment C-aerosol
TNFR-Ig 1.00％
Span 85 0.25% freon-11s, 5.0% freon W 193.75% 1Freon W is made up of 61.5% Chlorfluoranum and 38.5% Freon 12.Embodiment D-aerosol TNFR-Ig 0.50%Span 85 0.50% propellants (C) 199.0% 1Propellant C is made up of 30.0% freon-11 and 70% freon W.Embodiment E-aerosol TNFR-Ig 0.88% sodium sulfate (anhydrous), powder 0.88%Span 85 1.00% propellants, by 50% Freon 12,97.24%25% freon-11 and 25% Chlorfluoranum are formed embodiment F-aerosol TNFR-Ig 0.06%Span 85 0.05% freon-11 20.0% Freon 12s/Chlorfluoranum (20/80) 78.9% embodiment 12: be an example that contains the injectable composition of TNFR-Ig goods of the present invention below the injectable composition.
The every ml of composition contains
TNFR-IgG1 *
Anhydrous citric acid, USP 1.92mg
Glycine, USP 1.70mg
Mannitol, no thermal source, USP 41.90mg
Sodium hydroxide is an amount of, pH6.0
Hydrochloric acid is an amount of, pH6.0
Injection water, USP is an amount of, to 1.0ml *
*The TNFR-IgG1 concentration of using in this prescription can be 1.0mg/ml to 20mg/ml.The final quantity of TNFR-IgG1 is that the basis is selected with every bottle composition concentration and volume in this prescription.
*Shift by lyophilizing.
1mg medicine bottle (with the 1.0mg/ml 1ml that fills a prescription)
2.5mg medicine bottle (with the 2.5mg/ml 1ml that fills a prescription)
5.0mg medicine bottle (with the 5.0mg/ml 1ml that fills a prescription)
10mg medicine bottle (with the 5.0mg/ml 2ml that fills a prescription)
10mg medicine bottle (with the 10.0mg/ml 1ml that fills a prescription)
20mg medicine bottle (with the 8.0mg/ml 2.5ml that fills a prescription)
20mg medicine bottle (with the 5.0mg/ml 4ml that fills a prescription)
50mg medicine bottle (with the 20mg/ml 2.5ml that fills a prescription)
List of references
Ackerman, V., Marini, M., Vittori, E., Bellini, A., Vassali, G., and Mattoli, S.The detection in cytokine and cell thereof source in the asthma patient bronchial biopsy specimen.The specific reaction situation of respiratory tract anaphylaxis reaction, the relation of symptom and level.Division of chest disease 105 (3): 687-96,1994.
Amdur, M.O. and Mead, J.Anaesthetized guinea pig respiratory mechanism not.U.S. physiology magazine 192:364-372,1958.
Amrani, Y., Martinet, N. and Bronner, C.Tumor necrosis factor strengthens Kallidin I and the inductive calcium signal of carbachol in people's trachea smooth muscle.Britain pharmacology magazine 114 (1): 4-5,1995.
Anderson, JA., Miller, F.N., Sims, D.E. and Edwards, M.J.Tumor necrosis factor does not rely on neutrophilic granulocyte and mastocyte causes the blood capillary proteinaceous effluent.Surgery research magazine 56 (6): 485-90,1994.
Ashkenazi, A., Marsters, S.A., Capon, D.J, Chamow, S.M., Figsri, I.S., Pennica, D.Goeddel, D.V., Palladino, MA. and Smith, D.H.Tumor Necrosis Factor Receptors immune adherence albumen prevents endotoxin shock.Institute of NAS reports 88:10535-10539, and 1991.
Bloemen, P.G, van den Tweel, M.C., Henricks, P.A., Engels, F., Wagenaar, S.S., Rutten, A.A. and Nijkamp, F.P.The expression of human bronchial epithelial cell adhesion molecule and adjusting.Cellular elements magazine biology 9 (6) is breathed by the U.S.: 586-593,1993.
Boschetto, P., Rogers, D.F., Fabri, L.M., and Bames, P.J..The coritcosteroid of respiratory tract blood capillary seepage suppresses.U.S.'s respiratory tract disease looks back 143 (3): 605-609,1991.
Bradding, P., Roberts, JA., Britten, K.M., Montefort, S., Djukanovic, R., Mueller, R., Heusser, C.H., Howarth, P.H. and Holgate, S.T.Interleukin 4 in the normal and asthma respiratory tract ,-5 and-6 and tumor necrosis factor-alpha: reference's mastocyte is the source of these cytokines.Cellular elements magazine biology 10 (5) is breathed by the U.S.: 471-480,1994.
Broide, D.H., Lotz, M., Cuomo, A.J., Coburn, D.A., Federman, E.C. and Wasserman, S.I.Cytokine in the symptomatic asthma respiratory tract.Anaphylaxis clinical immunology magazine 89 (5): 958-967,1992.
Cembrzynska-Nowak, M., Szklarz, E., Inglot, A.D. and Teodorczyk-Injeyan, JA.Bronchial asthma patient's bronchus blood capillary leukocyte promotes the release of tumor necrosis factor and r-interferon.U.S.'s respiratory tract disease looks back 147 (2): 291-295,1993.
Costa, J.J., Matossian, K., Resnick, M.B., Beil, W.J., Wong, D.T., Gordon, J.R., Dvorak, A.M., Weller, P.F. and GAlli, S.j.The people eosinophilic granulocyte can express cell factor tumor necrosis factor-alpha and engulf inflammatory protein-1 α.Journal of Clinical Investigation 91 (6): 2673-2684,1993.
Cromwell, O., Hamid, Q., Corrigan, C.J., Barkans, J., Meng, Q., Collins, P.D. and Kay, A.B.Interleukin 8, the enhancing of IL-6 and granulocyte-macrophage colony stimutaing factor and IL-1b and tumor necrosis factor are expressed and produced to bronchial epithelial cell.Immunology 77:330-337,1992.
Devalia, J.L., Campbell, A.M., Sapsford, R.J., Rusznak, C., Quint, D., Godard, P., Bousquet, J. and Davies, R.J.The synthetic influence of inflammatory cytokine that external nitrogen dioxide is expressed human bronchial epithelial cell.Cellular elements magazine biology 9 (3) is breathed by the U.S.: 271-278,1993.
Elwood, W., Lotvall, J.O., Barnes, P.J. and Chung, K.F.Dexamethasone and ring spore A are to the sensitization brown Norway rats breathing road anaphylaxis of allergen-induced and the influence of inflammatory cell reaction.U.S.'s respiratory disease looks back 145 (6): 1289-1294,1992.
Finotto, S., Ohno, I., Marshall, J.S., Gauldie, J., Denburg, JA., Dolovich, J., Clark, DA. and Jordana, M.Upper respiratory tract infection (nasal polyp) eosinophilic granulocyte produces tumor necrosis factor.Immunology 153 (5): 2278-2289,1994.
Gater, P.R., Wasserman, MA., Paciorek, p.M.. and Renzetti, L.M.TNFR-Ig, the Tumor Necrosis Factor Receptors fusion rotein is to the inhibition of the inductive induced lung damage of Sephadex.Cellular elements magazine biology 1996 (waiting to publish) are breathed by the U.S..
Gordon, J.R and Galli, S.J.Mastocyte is as the inborn and source derivable TNF α of immunology/evil matter disease.Nature (London), 346:274-276,1990.
Gosset, P., Tsicopoulos, A., Wallaert, B., Vannimenus, C., Joseph, M., Tonnel, A.B. and Capron, A.Form the secretion that consecutive pulmonary alveolar macrophage has increased tumor necrosis factor and interleukin-6 with LAR.Anaphylaxis clinical immunology magazine 88:561-571,1991.
Gosset, P., Tsicopoulos, A., Wallaert, B., Joseph, M., Capron, A. and TonnelA.B.After relying on the IgE stimulation, the generation of irritated asthma patient mononuclear phagocyte tumor necrosis factor and interleukin-6.U.S.'s respiratory tract disease is looked back 146:768-774,1992.
Gundel, RH., Wegner, C.D., Torcellini, CA. and Letts, L.G.The effect of chronic tracheitis iuntercellular adhesion molecule-1.The clinical experiment anaphylaxis, 22 (5): 569-574,1992.
Kips, J.C., Tavernier, J. and Pauwels, R.A.Tumor necrosis factor causes the bronchus of rat anaphylaxis.U.S.'s respiratory tract disease looks back 145 (2): 332-336,1992.
Kwon, O.J., Au, B.T., Collins, P.D., Adcock, I.M., Mak, J.C., Robbins, R.R., Chung, K.F. and Barnes, P.J.The human respiratory epithelial cell of tumor necrosis factor inducing culture is expressed interleukin 8.U.S. physiology magazine 267 (4): L398-405,1994.
Lesslauer, W., Tabuchi, H., Reiner, G., brockhaus, M., Schlaeger, E.J., Grau, G., Piguet, P.F., Pointaire, P., Vassali, P. and Loetscher, H.What the opposing of recombinant soluble tumor necrosis factor receptor protein protection mice was lipopolysaccharide-induced causes death.Europe Journal of Immunology 21 (11): 2883-2886,1991.
Levine, S.J., Larivee, P., Logun, C., Angus, C.W., Ognibene, F.P. and Shelhamer, J.H.Tumor necrosis factor is induced mucinous excessive secretion of human respiratory epithelial cell and MUC-2 gene expression.Cellular elements magazine biology, 12:196-204,1995 are breathed by the U.S..
LI, X.Y., Donaldson, K., Brown, D. and MacNee, W.The effect of tumor necrosis factor in rising acute pneumonia airway epithelial cell permeability.Cellular elements magazine biology 13 (2) is breathed by the U.S.: 185-195,1995.
Loetscher, H., Grentz, R., Zulauf, M., Lustig, A., Tabuchi, H., Schlaerger, E.J., Brockhaus, M., Gallati, H., Manneberg, M. and Lesslauer, W.Reorganization 55-KDa Tumor Necrosis Factor Receptors: with the bonded Chemical Calculation of TNF α and TNF α and the active inhibition of TNF.Journal of biological chemistry 266 (27): 18324-18329,1991.
Lukacs, N.W., Strieter, RM., Chensue, S.W., Widmer, M., and Kunkel, S.L.TNF α is in the neutrophilic granulocyte of respiratory inflammation phase mediation and replenishing of eosinophilic granulocyte.Journal of Immunology 154 (10): 5411-5417,1995.
Matsumoto, T., Ashida, Y. and Tsukuda, R.Cavia porcellus is regulated with the materia medica that postpones the infiltration of respiratory response and leukocyte immediately.Pharmacology experiment treatment magazine 269 (3): 1236-1244,1994.
Myatt, N., Coghill, G., Morrison, K., Jones, D. and Cree, I.A.Use the tumor necrosis factor in situ hybridization detection type sarcosis and the tuberculosis granuloma.Clinical pathology magazine 47 (5): 423-426,1994.
Nash, J.R., Mclaughlin, P.J., Hoyle, C. and Roberts, D.The immunolocalization of tumor necrosis factor in the dead patient's lung tissue of the respiratory tract anxious syndrome of growing up.Histopathology 19 (5): 395402,1991.
O ' Donnell, M., Garippa, R.J., Rinaldi, N., Selig, W.M., Simko, B., Renzetti, L., Tannu, SA., Wasserman, MA., Welton, A. and Bolin, D.R.A kind of novelty, long active vasoactive intestinal peptide antagonists.I part: external and body entobronchus expander research.Pharmacology experiment treatment magazine 270 (3): 1282-1288,1994.
Ohkawara, Y., Yamauchi, K., Tanno, Y., Tamura, G., Ohtani, H., Nagura, H., Ohkuda, K. and Takishima, T.The IgE receptor triggers human lung mast cell and pulmonary macrophage generation tumor necrosis factor in the back sensitization lung tissue.Cellular elements magazine biology 7 (4) is breathed by the U.S.: 385-392,1992.
Pilewski, J.M., Panettieri, R.A., Jr., Kaiser, L.R. and Albelda, S.M.The expression of endotheliocyte adhesion molecule in the human bronchial heteroplastic transplantation.Crisis medical treatment magazine 150 (3) is breathed by the U.S.: 795-801,1994.
Robinson, D., Hamid, Q., Ying, S., Bentley, A., Assoufi, B., Durham, S. and Kay, A.B.Meticortelone treatment asthma and bronchovesicular perfusion cell interleukin 4, interleukin 5 is relevant with the adjusting of r-interferon cytokine gene expression.U.S.'s respiratory tract disease looks back 148 (2): 401-406,1993.
Schleimer,R.P。Glucocorticoid is used relevant to the curative effect of inflammatory cell with its treatment in respiratory tract.U.S.'s respiratory tract disease is looked back 141:5%69,1990.
Selig, W. and Tocker, J.Interleukin-1 receptor antagonist is to the influence of the inductive Guinea pig lung reaction of antigen.Europe materia medica magazine 213 (3): 331-336,1992.
Smart, S.J. and Casale, T.B.It is that IL-8 is dependent that the inductive neutrophilic granulocyte of TNF α shifts through endothelium.U.S. physiology magazine 266 (3): L238-245,1994.
Thmas, P.S., Yates, D.H. and Barnes, P.J.Tumor necrosis factor increases normal volunteer's respiratory response and apoplexy due to phlegm granulocyte.Crisis medical treatment magazine is breathed by the U.S..152(1):76-80,1995。
Turner, C.R., Andresen, C.J., Smith, W.B. and Watson J.W.Cyclopentyloxy anisyl ketopyrrolidine contacts the influence of responsing reaction to feeling anxious property and chronic antigen.Crisis medical treatment magazine is breathed by the U.S..149(5):1153-1159,1994。
Wasserman, M.A., Welton, A.F. and Renzetti, L.M.LTD 4The synergism that in reducing the respiratory tract blood capillary seepage of antigen induction, shows with paf receptor antagonists.Prostaglandin, thromboxane, leukotriene research 23:271-273,1995.
Watson, M.L., Smith, D., Bourne, A.D., Thompson, R.C. and Westwick, J.Cytokine helps the respiratory tract functional disorder of antigen stimulation Cavia porcellus: the interleukin-1 receptor antagonist pretreatment suppresses the respiratory tract anaphylaxis reaction, and lung eosinophilic granulocyte accumulation and tumor necrosis factor produce.Cellular elements magazine biology 8 (4) is breathed by the U.S.: 365-369,1993.
Wershil, B.K., Furata, G.T., Lavigne, J.A., Choudhury, A.R., Wang, Z-S. and Galli, S.J.Dexamethasone and ring spore A suppress mastocyte-leukocytic cytokine cascade.Journal of Immunology 154:1391-1398,1995.
Windsor, A.C., Mullen, P.G., Walsh, C.J., Fisher, B.J., Blocher, C.R., Jesmok, G., Fowler, A.A. and Sugerman, H.J.Delay the tumor necrosis factor blocking-up and alleviate pulmonary function imbalance and the metabolic acidosis relevant with experimental gram-negative sepsis.Arch.Surg.129(1):80-89,1994。
Ying, S., Robinson, D.S., Varney, V., Meng, Q., Tsicopoulos, A., Moqbel, R., Durham, S.R., Kay, A.B. and Hamid, Q.The expression of tumor necrosis factor mRNA α in the irritated inflammation.Clinical experiment anaphylaxis 21:745-750,1991.
Zuany-Amorim, C., Haile, S., Leduc, D., Dumarey, C., Huerre, M., Vargaftig, B.B. and Pretolani, M.The cell that interleukin 10 suppresses antigen induction replenishes the mouse breathing road of into sensitization.Journal of Clinical Investigation 95 (6): 2644-2651,1995.

Claims (11)

  1. One kind in suffering from the patient of symptoms of asthma the opposing asthma method, comprise to described patient and use the compositions that contains by one or more chimeric conjugated protein goods of forming of TNF α, each protein in the described goods is made up of the soluble fraction that is fused to the p55 TNF receptor protein on the IgG, the IgG of wherein said fusion contains all the IgG domains except an IgG domain of IgG CH, described compositions contains the inert carrier in the treatment, and described compositions is administered to described patient so that provide the described chimeric protein goods of effective dose to resist described symptoms of asthma to patient.
  2. 2. the process of claim 1 wherein that described patient suffers from asthma, and the chimeric protein goods are used with the amount of the influence that is enough to alleviate described disease.
  3. 3. the process of claim 1 wherein that described compositions is administered to asthma patient with the amount that effectively prevents or delay described seizure of disease before asthma outbreak.
  4. 4. any one method in the claim 1 to 3, wherein the IgG of Rong Heing is a human IgG 1
  5. 5. any one method in the claim 1 to 4, wherein the protein in described protein articles has the composite oligosaccharide that stops with one or more sialic acid residueses and has the N-acetyl-glucosamine of exposure, in described goods the mol ratio of sialic acid residues be every mole of protein from about 4 to about 7 moles of sialic acides, the mol ratio of the N-acetyl-glucosamine that in described goods, exposes be every mole of protein from about 1 to about 2 moles of N-acetyl-glucosamines, the mol ratio of sialic acid residues and N-acetyl-glucosamine residue is from about 0.35 to about 0.5 in described goods, and the isoelectric point, IP of described goods is from about 5.5 to about 7.5.
  6. 6. the method for claim 5, wherein the mol ratio of sialic acid and N-acetyl-glucosamine is from about 0.4 to about 0.45, and sialic acid and proteinic mol ratio are from about 5.0 to about 6.0.
  7. 7. any one method in the claim 1 to 6 wherein is administered to described patient with every kilogram of dosage from 0.1mg to 5.0mg of every patient body weight every day through injection with described goods.
  8. 8. the method for claim 7, wherein dosage be every day every kilogram of every body weight from 1.0mg to 3.0mg.
  9. 9. any one method in the claim 1 to 8, wherein said goods are with the mode medication of oral cavity or nasal spray.
  10. 10. the method for claim 9, wherein said spraying is to contain the liquid spray composition of weight ratio from about described goods of 0.03% to 5.0%.
  11. 11. by the conjugated protein application in the medicine of preparation treatment asthma of chimeric TNF α that the soluble fraction that is fused to the p55 TNF receptor protein on the IgG is formed, wherein said fusion IgG contains all the IgG domains except an IgG domain of IgG CH.
CN97194455A 1996-05-08 1997-05-02 Treatment of asthma with TNFR-Ig Pending CN1233189A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US1684296P 1996-05-08 1996-05-08
US60/016,842 1996-05-08

Publications (1)

Publication Number Publication Date
CN1233189A true CN1233189A (en) 1999-10-27

Family

ID=21779275

Family Applications (1)

Application Number Title Priority Date Filing Date
CN97194455A Pending CN1233189A (en) 1996-05-08 1997-05-02 Treatment of asthma with TNFR-Ig

Country Status (15)

Country Link
EP (1) EP0910413A2 (en)
JP (1) JP2000510113A (en)
KR (1) KR20000010825A (en)
CN (1) CN1233189A (en)
AR (1) AR007020A1 (en)
AU (1) AU725408B2 (en)
BR (1) BR9708928A (en)
CA (1) CA2253557A1 (en)
CO (1) CO4820397A1 (en)
HR (1) HRP970224A2 (en)
MA (1) MA24169A1 (en)
PE (1) PE70698A1 (en)
TR (1) TR199802238T2 (en)
WO (1) WO1997041895A2 (en)
ZA (1) ZA973843B (en)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002508934A (en) * 1997-12-29 2002-03-26 リジェネロン・ファーマシューティカルズ・インコーポレイテッド Novel nucleic acids and polypeptides having homology to the TNF receptor
TR200504220T2 (en) 1998-12-17 2007-04-24 Biogen Idec Ma Inc. Active lymphotoxin-beta receptor immunoglobulin chime A method for high level expression and purification of purified protein proteins and a method for purification of active lymphotoxin-beta receptor immunoglobulin chimeric proteins.
US20040220103A1 (en) 1999-04-19 2004-11-04 Immunex Corporation Soluble tumor necrosis factor receptor treatment of medical disorders
US20010021380A1 (en) * 1999-04-19 2001-09-13 Pluenneke John D. Soluble tumor necrosis factor receptor treatment of medical disorders
CA2515539A1 (en) * 2003-02-28 2004-09-10 Ares Trading S.A. Liquid formulations of tumor necrosis factor-binding proteins
TWI374935B (en) * 2004-08-27 2012-10-21 Pfizer Ireland Pharmaceuticals Production of α-abeta
KR20090071652A (en) 2006-10-20 2009-07-01 바이오겐 아이덱 엠에이 인코포레이티드 Treatment of demyelinating disorders with soulble lymphotoxin-beta-receptor
US8338376B2 (en) 2006-10-20 2012-12-25 Biogen Idec Ma Inc. Compositions comprising variant LT-B-R-IG fusion proteins

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10399023I2 (en) * 1989-09-12 2006-11-23 Ahp Mfg B V TFN-binding proteins
EP0620739A4 (en) * 1992-09-15 1997-01-15 Immunex Corp Method of treating tnf-dependent inflammation using tumor necrosis factor antagonists.
US5684222A (en) * 1993-01-22 1997-11-04 Ontario Cancer Institute Mutant mouse having a disrupted TNFRp55
CA2119089A1 (en) * 1993-03-29 1994-09-30 David Banner Tumor necrosis factor muteins
CZ283219B6 (en) * 1994-07-22 1998-02-18 F. Hoffmann-La Roche Ag Use of chimeric protein binding tnf in the preparation of a pharmaceutical preparation for treating auto-immune diseases
US5705364A (en) * 1995-06-06 1998-01-06 Genentech, Inc. Mammalian cell culture process
IL116436A (en) * 1995-12-18 2006-12-31 Yissum Res Dev Co Fc?Á-PE CHIMERIC PROTEIN FOR TARGETED TREATMENT OF ALLERGY RESPONSES AND

Also Published As

Publication number Publication date
WO1997041895A2 (en) 1997-11-13
ZA973843B (en) 1997-11-10
PE70698A1 (en) 1998-11-18
EP0910413A2 (en) 1999-04-28
MA24169A1 (en) 1997-12-31
CO4820397A1 (en) 1999-07-28
AU725408B2 (en) 2000-10-12
AU2776497A (en) 1997-11-26
BR9708928A (en) 2000-05-09
HRP970224A2 (en) 1998-04-30
TR199802238T2 (en) 1999-02-22
KR20000010825A (en) 2000-02-25
JP2000510113A (en) 2000-08-08
AR007020A1 (en) 1999-10-13
WO1997041895A3 (en) 1998-03-12
CA2253557A1 (en) 1997-11-13

Similar Documents

Publication Publication Date Title
CN1071760C (en) N-terminally chemically modified protein compositions and methods
CN1259973C (en) Preventives or remedies for psoriasis containing as the active ingredient IL-6 antagonist
JP6325585B2 (en) Application of interleukin-22 in the treatment of viral hepatitis
EP0811065B1 (en) Antagonists of interleukin-15
EP1366062B1 (en) Efficient recovery of correctly refolded proteins
WO1996022308A2 (en) Appetite suppression factor and related methods
FI104903B (en) A method for producing a primate IL-3 protein and a recombinant DNA sequence encoding the protein
WO1996022308A9 (en) Appetite suppression factor and related methods
CN1129224A (en) Glucagon-like insulinotropic peptide analogs, compositions, and methods of use
CN1323225A (en) Polymer conjugates of interferon beta-1a and their uses
CN1157290A (en) Recombinant obese (ob) proteins
CN1198929C (en) Antibodies against interferon alpha/beta receptor
CN1894275A (en) Human EPO mimetic hinge core mimetibodies, compositions, methods and uses
CN1146442C (en) Lymphotoxin-
CN1145693C (en) Amino-terminally truncated MCP-2 as chemokine antagnosits
CN1233189A (en) Treatment of asthma with TNFR-Ig
CN1284134A (en) Methods of producing anti-angiogenic proteins: endostation, angiostation or restin, using pichia yeast expression system
CN103641896B (en) The purposes of gelatin-like unit
JP2914672B2 (en) BSF (2) Antagonist
JP2003525592A (en) Interferon tau mutant and method for producing the same
CN1684978A (en) Glycosylated human granulocyte colony-stimulating factor (G-CSF) isoform
CN1229138C (en) Method for accelerating rate of mucociliary clearance
CN1088107C (en) Method for preparing human granulocyte macrophage colony stimulating factor and its expressing carrier and engineering bacteria
CN1194986A (en) Anti-obesity proteins
CN1295332C (en) Recombination human alkaline fiber forming cell growth factor gene and its nonfusion expression product, production method and application

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C02 Deemed withdrawal of patent application after publication (patent law 2001)
WD01 Invention patent application deemed withdrawn after publication
REG Reference to a national code

Ref country code: HK

Ref legal event code: GR

Ref document number: 1056076

Country of ref document: HK

REG Reference to a national code

Ref country code: HK

Ref legal event code: WD

Ref document number: 1022424

Country of ref document: HK